Neuropathogenesis-on-chips for neurodegenerative diseases

Developing diagnostics and treatments for neurodegenerative diseases (NDs) is challenging due to multifactorial pathogenesis that progresses gradually. Advanced in vitro systems that recapitulate patient-like pathophysiology are emerging as alternatives to conventional animal-based models. In this review, we explore the interconnected pathogenic features of different types of ND, discuss the general strategy to modelling NDs using a microfluidic chip, and introduce the organoid-on-a-chip as the next advanced relevant model. Lastly, we overview how these models are being applied in academic and industrial drug development. The integration of microfluidic chips, stem cells, and biotechnological devices promises to provide valuable insights for biomedical research and developing diagnostic and therapeutic solutions for NDs.

As life expectancy increases with advances in medical care, humanity faces a new crisis related to the rising ageing population 1 .This leads to a concomitant increase in the incidence of incurable neurodegenerative diseases (NDs) [2][3][4] .These diseases generally affect the brain activity in the elderly by diminishing their cognitive and behavioural functions [5][6][7][8] .Tremendous efforts have been made for decades to investigate the underlying mechanisms of these fatal NDs using both in vivo and in vitro systems, despite the complexity of the human brain structure and the limitations of real-time observation.Notably, several pathogenic features, such as specific neuronal loss, gliosis, neuroinflammation, oxidative stress, mitochondrial dysfunction, and early vascular damage tend to overlap in common NDs, including Alzheimer's disease (AD) 9,10 , Parkinson's disease (PD) 6,11 , amyotrophic lateral sclerosis (ALS) 7,12 , and Huntington's disease (HD) 13,14 .Although the primary initiating factor remains elusive, hypotheses suggest that the accumulation of several dysfunctional proteins is closely related to the onset of each ND 15 .
Despite advances in our understanding of NDs, the development of disease-modifying treatments (DMT) that target the underlying mechanisms of their pathogenesis remains challenging.Only a few DMTs, such as aducanumab 16 and lecanemab 17 for AD and riluzole 18 and edaravone 19 for ALS, have recently been approved.A substantial number of drug trials for NDs that were effective in preclinical studies have yet to demonstrate efficacy in clinical trials, and the limitations of current experimental models are responsible for this failure.Notably, animal models, which have been heavily relied upon for the drug development of NDs, differ fundamentally from humans in terms of their immune systems 20 and the ratio, distribution, morphology, and gene expression of their brain cells 21 .Moreover, current animal models of NDs cannot fully replicate the complex pathogenic lesions of human NDs, and often overlook sporadic cases, which account for most cases 22,23 .To investigate the pathological processes of NDs, advanced in vitro models that can accurately replicate various physiological conditions, multicell types, and interactive cell-to-cell environments, and provide real-time monitoring modulated by different biochemical and biophysical cues are in high demand.
Over the past decades, stem cell biology and advances in cell culture techniques have rapidly developed a stem cell-based 3D culture platform that generates an "organoid" for disease modelling and drug screening 24 .Organoids have several advantages over conventional 2D cell cultures, including patient-specific origins and intrinsically complicated 3D compositions that mimic tissues.Several brain organoid subregions have been developed for brain studies, including the cerebral cortex 25 , hippocampus 26 , hypothalamus 27 , forebrain 27 , midbrain 27,28 , functional choroid plexus 29 , and cerebellum 30 .However, current methods for organoid growth have several limitations that need to be addressed, including insufficient neuronal functionality and cellular microenvironment, lack of vascular and immunological components, low reproductivity and uniformity, and difficulties in longterm maintenance [31][32][33] .
Microfluidic organs or organoids-on-chips have provided unique opportunities for researchers to develop an inventive experimental design by allowing the reproduction of critical elements of each organ, such as disease-specific miniaturised environments with high controllability and tunability, biochemical, and biophysical cues, continuous medium flow, and complex interactions between cells relevant to the in vivo system 34 .Moreover, integrated microfluidic chips are ideal platforms for drug screening and basic life science studies in biomedical research 35 .Despite the challenges in determining the brain's critical structural and functional units owing to its complex morphology and physiology, many commercialised and customised microfluidic chips have simplified the complex characteristics of the brain into miniaturised systems 22 .Existing brain-onchips mimic diverse levels of brain units, such as the axonal chip 36 , neuron-glia chip 37 , blood-brain-on-a-chip 38,39 , and neurovascular-uniton-a-chip 40 .These have been applied in studies on brain tumours 41 , vascular diseases 40 , brain injury 42 , neuroinflammation 43 , and NDs 44 .These microfluidic chip designs can be repurposed for desired disease conditions according to the experimental targets, allowing researchers to understand neuropathogenesis at a more profound level.
Recently, the idea of taking advantage of microfluidic chips to overcome the limitations of organoid formation was proposed, with promising results in the development of organoids-on-chips for the pancreas 45 , liver 46 , kidney 47 , stomach 48 , and brain [49][50][51] .This approach provides better in vivo mimicry properties than conventional models, preventing necrosis by controlling organoid expansion with chip size, integration with the vascular bed to provide sufficient nutrients, allowing co-culturing with systemic microglia, applying mechanical and physical cues, delivering a drug through a physiologically relevant barrier, and achieving mature characterisation with continuous fluid flow.However, there is still room for improvement in both culture systems to accelerate their application in drug screening and personalised medicine.In this review, we aim to highlight the potential of integrated microfluidic technologies for advancing research on the neuropathogenesis of NDs (AD, PD, ALS, and HD) to make substantial breakthroughs in disease modelling and drug development.

Current understanding of neurodegenerative diseases
Numerous studies have identified the interconnected pathogenic features that contribute to ND progression over several decades.These include dysfunctional protein-related pathogeneses, gliosis, neuroinflammation, metabolic alterations, oxidative stress, mitochondrial dysfunction, and genetic alterations.However, the primary aetiologies of selective neuronal cell death and synaptic loss in NDs remain to be determined.This has hindered the development of disease-modifying drugs and early diagnostic tools.In this section, we review the current progress in the investigation of AD, PD, HD, and ALS.These diseases share some commonalities and distinctions in their pathogeneses (Fig. 1).
Alzheimer's disease AD has become a major concern worldwide, accounting for 80% of all dementia cases.The prevalence of AD is expected to rapidly increase over the next 30 years 52 .In AD, symptoms of cognitive and behavioural impairment occur as the first, but memory loss presents much earlier, before clinical diagnosis, especially in the predominant age group (>65 years) 53,54 .This results from progressive neuronal and synaptic loss mainly in the cortex and hippocampus (Fig. 1a).Unfortunately, when AD is first diagnosed in the clinical setting, several irreversible pathological changes occurring in the brain are already in progress 55 .One of the main pathological characteristics of AD is the deposition of amyloid plaques and neurofibrillary tangles (NFT) in the brain tissue.On a macroscale, moderate atrophy of the cortex, amygdala, and hippocampus as well as enlargement of the temporal horn of the lateral ventricle have been observed in AD.This ultimately leads to a loss of whole brain volume 56 .Gene mutations linked to amyloid protein processing pathways, such as APP 57 , PSEN1 58 , and PSEN2 59 are responsible for early-onset AD (EOAD).Recent genome-wide association studies (GWAS) have revealed many genetic factors associated with late-onset AD (LOAD), such as BIN1, APH1B, PTK2B, PILRA, CASS4, CCDC6, TSPAN14, NCK2, and SPRED2 60,61 .However, most LOAD cases are attributed to APOE4 62 and TREM2 R47H variants 63,64 , which regulate brain inflammation, cholesterol, and glucose metabolism, and microglia function 65,66 .In this section, we describe the current leading hypotheses of AD, such as amyloid-β (Aβ) and tau protein dysfunction, neuroinflammation, blood-brain barrier (BBB) dysfunction, gut-brain axis, mitochondrial dysfunction, oxidative stress, and metabolism alterations.
Amyloid-β and hyperphosphorylated tau-related pathogenesis.The most prominent histological hallmark of AD is the deposition of misfolded amyloid aggregates or plaques in the extracellular space.The core element of amyloid plaques is the pathological Aβ peptide (~4 kDa), which is produced by altered cleavage of the amyloid precursor protein (APP).APP is a type I transmembrane protein that is typically involved in cell interactions, migration, synapse formation, and neuroprotection.It is mainly produced by neurons, astrocytes, platelets, and vascular endothelial cells 67,68 .However, in the familial AD brain, the APP cascade is altered due to mutation in protein processing genes (APP, PSEN1, and PSEN2), causing the formation of Aβ peptides 67,68 .Among Aβ peptides of various lengths, Aβ42 tends to aggregate and form plaques, but Aβ40 is the most abundant.In addition, this abnormal aggregation is induced by the impairment of protein regulation systems, such as the endoplasmic reticulum system, ubiquitin-proteasome system (UPS), and autophagy-lysosomal pathway (ALP) 69 .Importantly, dysfunction of several regulating proteins, such as LRP1 70 , SNX6 71 , GGA3 72 , and SORLA 73 affect APP endocytosis, endosomal trafficking, and Aβ production, which are closely associated with the onset of AD.
Studies have focused on examining differences in the effects of various forms of Aβ on cells, and they reported that intermediate forms of Aβ, such as oligomers and protofibrils, are more neurotoxic than mature fibrils 10,74,75 .Several in vitro studies have reported that the pathogenicity of oligomers varies depending on their size and structure.These oligomers are known to cause neurotoxicity by binding to neuronal receptors (PrPc 76 , NMDAR 77 , β2-AR 78 , p75NTR 79 , and α7nACR 80 ), altering cell membranes 81 , causing mitochondrial dysfunction 82 , Ca +2 imbalance 83 , as well as inducing tauopathy 84 .
AD progression may be linked to Aβ propagation among neurons.In the human AD postmortem models, the positions of Aβ plaques exhibit a regional-specific outer to inner pattern, initially detected at the frontal, and medial parietal cortex, followed by the allocortical region and midbrain.Then, in late clinical phase, Aβ accumulation is found in the brainstem and cerebellum 85 .Furthermore, positron emission topography (PET) imaging of the AD brain has revealed that Aβ dynamically accumulates in synaptic contact areas, which may be a result of Aβ propagation 86 .Also, an in vitro study has demonstrated prion-like propagation of Aβ oligomers between neighbouring cells 87,88 , which is explained by the "seeding-nucleation" theory observed in an in vitro study as a three-step process (seeding, nucleation, and elongation), where Aβ oligomers formed from monomers elongate into fibrils.These fibrils then accelerate the formation of oligomers (nuclei), resulting in increased aggregations 89,90 .Moreover, current hypotheses have shown three Aβ propagation ways: direct cell contact 91 , exosomes [92][93][94] , and tunnelling nanotubes between cells [95][96][97] .
Abnormal intracellular accumulation of tau proteins, NFTs, is another primary pathological marker that is considered to be more closely related to cognitive decline 98 .Tau, a microtubule-associated protein, is mainly located in the axons and supports axonal transport, maintenance, and generation of microtubules.However, alterations in post-translational modification promote its aberrant aggregation 99 .
Among the several types of post-translational modifications, the relationship between hyperphosphorylated tau (pTau) and AD pathogenesis has been well studied by discovering several phosphorylation sites (Thr175, Ser202, Thr205, Thr212, and Ser422) and its toxic promotion of misfolding and self-aggregation 100 .Furthermore, other neuroprotective phosphorylated states (Ser214, Ser262, and Ser305) inhibit tau aggregation 101,102 .However, hyperphosphorylation depends on the starting site, which may accelerate other multisite phosphorylation states 103 .The leading cause of tau hyperphosphorylation is unknown despite the discovery of several tau-associated kinases and phosphatases, such as GSK-3β, CDK5, ERK2, PKA, and PP2A [104][105][106][107] .Recently, one group found that a 12-amino-acids-long peptide derived from CDK5 disrupts hyperactivated CDK5 (CDK5-p25 complex).This enzyme leads to decreased DNA disruption, pTau levels, and neurodegeneration, and increased behavioural ability in an AD mouse model 108 .
The spread of toxic tau shows similar characteristics to Aβ, such as prion-like behaviour and propagation between cells via direct Motor cortex

Motor Neuron
Mutant TDP-43 Mutant HTT  and other proteins, degeneration of motor neurons in the motor cortex and spinal cord, and muscle atrophy with dysfunctional proteins.d HD is characterized by including mutant Huntingtin protein (mHTT) and degeneration of medium spiny neurons in the basal ganglia, and corpus striatum of the brain.AD Alzheimer's disease, ALS amyotrophic lateral sclerosis, BDNF brain-derived neurotrophic factor, EAL endosomal-autophagic-lysosomal pathway, GABA gamma-aminobutyric acid, HD Huntington's disease, PSEN presenilin 1, SNCA synuclein alpha.contact 109 , exosomes 110 , and nanotubules 111 .In vivo and in vitro studies have shown that tau propagation from the entorhinal cortex (EC) to the hippocampal region is mediated by exosomes released from microglia 112 .Similarly, one in vitro study has also shown that microglia secreted extracellular vesicles (EVs) containing Aβ propagate through axons, causing synaptic alterations 113 .Despite the protective effect of microglia exosomes and EVs by promoting clearance of Aβ and tau, studies suggest that these may be a factor in propagation among cellcell interactions in the AD brain, which makes them an exciting aim for further research.
The relation between Aβ and NFT in AD pathogenesis remains controversial.In the early phase, NFTs were observed in neurons of the more profound parts of the EC and CA1 regions of the hippocampus, followed by other parts of the brain, including the parietal, medial, and lateral occipital cortices, in a reversed pattern of amyloid plaques 86 .A histological-level study also found that NFT begins to form and spread under the influence of Aβ deposition 114 .Recent automatic PET imaging analysis has revealed that tau accumulation first appears at the rhinal cortex, independent from Aβ 115 .Subsequently, tau spreads robustly throughout neocortex when the Aβ level reaches at certain threshold.This finding has been termed 'Ca-tau-strophe' and highlights the importance of the tau protein in earlier diagnosis and therapeutic choice 115,116 .However, the detailed mechanisms underlying tau protein toxicity have not been fully elucidated.However, substantial research findings have proposed an association between Aβ and other proteins (such as Pin1, HSP, Fyn kinase, α-synuclein, and PASCIN1) regarding tau hyperphosphorylation 117,118 .Aβ accumulation induces tau aggregation and propagation trans-synaptically in vitro studies 119 .Notably, some studies have suggested that NFT may protect against amyloid plaques 117 .This emerging evidence has indicated the importance of synergy of Aβ and tau in AD pathogenesis 120 .
APP-C99, a C-terminal fragment of APP, has recently been suggested as an early AD marker associated with endosomal-autophagiclysosomal (EAL) malfunction 121 .C99 accumulation in neurons causes lysosomal and synaptic distortion and cognitive changes by aggregating within EAL vesicle membranes 122 .
Neuroinflammation and systematic inflammation.Compelling evidence for the association of immune-regulating genes with AD suggests that systemic immune-mediated neurodegeneration is an alternative hypothesis for the pathogenesis of AD.As ageing, circulating proinflammatory cytokines (IL-1β, IL-6, TNFα, and IL-18) induced by infection, chronic diseases, stresses, and cellular senescence may initiate or contribute to the neuropathogenesis of AD 123 .When these cytokines cross the BBB, they can activate glia and astrocytes, leading to Aβ and tau phosphorylation, oligomerization, neuroinflammation, and neurotransmitter toxicity 123 .In pathological studies, increased levels of proinflammatory proteins in the cerebrospinal fluid (CSF) have been associated with brain volume loss and thinning of the white matter, corresponding to cognitive dysfunction 124 .
As Aβ plaques form, the presence of activated microglia and astrocytes also increase 125 .Therefore, extensive research has focused on the role of astrocytes and microglia in AD progression, including their contribution to Aβ and pTau depositions, neuroinflammation, and Ca +2 imbalance.Microglia exert their neuroprotective activities by clearing misfolded proteins 126 .However, as AD progresses, microglia transform into a deteriorating state, enhancing pTau-related kinase expression and production of the inflammasome NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3).Increased production of NLRP3 accelerates the activity of caspase-1 and secretion of proinflammatory cytokines, such as IL-1β and IL-18, and promotes neuronal and glial death via apoptosis 127,128 .In addition, GWAS identified multiple genes associated with LOAD (CR1, SPI1, MS4As, TREM2, ABCA7, CD33, and INPP5D) that are expressed in microglia, underscoring the important role of microglia in AD pathogenesis 129 .Specifically, studies have suggested that a mutation of TREM2, lipid sensing transmembrane glycoprotein expressed in microglia, causes the loss of its phagocytosis function 130 , and diminishes its migration towards Aβ plaques and uptake of Aβ 40 and Aβ 42 131,132   .Notably, the observation of APOE as a ligand for TREM2 has revealed new insights into their association with the pathogenesis on Aβ and pTau 133,134 .Yeh et al. 134 demonstrated that microglia expressing TREM2 variants exhibited reduced internalisation of Aβ and the apolipoprotein or lipoprotein complex.Gratuze et al. 135 recently suggested that microglial activation could worsen tauassociated neurodegeneration in the presence of APOE4 through a TREM2-independent pathway.One group recently developed a successful therapeutic approach based on TREM2 function in microglia activation.They introduced a high-affinity human TREM2 antibody with a monovalent transferrin receptor (TfR) binding site to enable efficient delivery to the brain.This antibody promotes glucose and lipid metabolism in microglia, shifting them to a metabolically responsive state distinct from that triggered by amyloid pathology 136 .
Reactive astrocytes play a critical role in the progression of AD.It has been suggested that astrocytes become reactive to surrounding plaques, but there are limited data on the exact morphological and functional alteration 137,138 .A recent PET study revealed a correlation between reactive astrocytes and protein biomarkers (Aβ, and tau) in the CSF of patients with AD, suggesting their contribution to progression 139 .The expression of APOE in many cell types has gained attention because APOE4 is considered the primary genetic risk factor for sporadic AD 140 .Astrocytes expressing APOE4 have demonstrated diminished functions, such as autophagy, clearance, and internalisation of Aβ, and support for neurons for their durability and synapse formation 141,142 .Moreover, a recent report suggests that astrocytes expressing APOE4 immoderately provide cholesterol to neurons by expanding their lipid membrane, increasing APP deposition, and promoting Aβ formation 143 .Conversely, oligodendrocytes expressing APOE4 exhibit dysfunction in myelination 144 , coinciding with increased neurotransmitter production and Aβ42 secretions in APOE4 neurons 145 .Studies have demonstrated an association between APOE4 expression and tauopathy.The deletion of APOE4 leads to the reversal of pathologies, including increased maintenance of the myelin sheath and decreased disease-associated neural subpopulations, and facilitates the severity of tauopathy 146 .
At the molecular level, using single-cell and single-nucleus RNA sequencing, researchers have recently revealed the specific transcriptional and functional characteristics of astrocytes and microglia, which have been categorised as disease-associated astrocytes (DAA) and microglia (DAM) 147,148 .The DAA subtype of astrocytes is associated with the early stages of AD and progressively increases over time.In AD mouse models, the level of homoeostatic astrocytes declines, while intermediate and glial fibrillary acidic protein highly expressed DAAs in the hippocampus and cortex region 147 .DAM showed the same transitioning pattern from a homoeostatic to an activated state.Moreover, one study indicated that phagocytic DAM subtypes can internalise Aβ proteins through a two-step sequential process that involves the downregulation of microglia-regulating genes and upregulation of phagocytic and lipid metabolism pathways 148 .According to a recent study using 3D in situ RNA sequencing, spatiotemporal variations in glial cells have been observed in the brains of AD mice.This study has revealed a layered position of cells surrounding Aβ plaques, with the first layer being DAM, followed by DAA and oligodendrocytes 149 .
Blood-brain barrier dysfunction.Research on the role of the BBB in AD is important because of the strong hypothesis of its association to the circulation of misfolded proteins originating from the brain or body system, and other pathogenic features.BBB disruption can be observed during the early stages of AD before regional atrophy occurs, and is emerging as a leading pathogenesis 150  misfolded proteins and neuroinflammation, and directly contribute to neurodegeneration 151 .Conversely, according to animal studies, pathological Aβ and tau proteins may cause disruption in blood vessels in vulnerable areas of AD during disease progression 152,153 .BBB disruption begins with changes in permeability, which are associated with a decrease in tight junction proteins in endothelial cells and reduced pericytes 151,154 .However, Aβ 155 and tau accumulation 156 disrupt the BBB integrity by altering the level of tight junction proteins.Moreover, microglia-driven neuroinflammation damages the BBB integrity and contribute to disease development 157 .Therefore, investigating brain vascular pathology is a promising avenue for gaining insight into the initial systemic mechanisms of all NDs.
Glymphatic system dysregulation.The dynamics of CSF and tissue interstitial fluid are supported by the perivascular network known as the glymphatic system 158 .Bidirectional aquaporin channels (AQP) facilitate continuous fluid flow to assist in circulating proteins and metabolites.The contribution of AQP to AD pathogenesis, associated with the clearance of pathological proteins, has been investigated 159,160 .In the absence of AQP4, APP mouse had decreased influx of CSF tracer, and accumulation of Aβ and APOE4 proteins in neurons, as well as phagocytic activation of microglia, suggesting the substantial role of AQP4 during disease progression 160 .Moreover, AQP4 knockout tauexpressing mice demonstrate increased tau aggregation and neurodegeneration, suggesting a highly relevant AQP4-dependent extracellular tau clearance pathway 161 .
AQP4 is primarily expressed in the endfeet processes of perivascular astrocytes in the BBB and tripartite synapses 162,163 .It is also found in the epithelial cells of the choroid plexus and the ependymal cells of the ventricular lining in the brain.Human postmortem AD models have shown decreased levels of perivascular AQP4 in the frontal cortex, which is associated with cognitive dysfunction and amyloid and tau pathology 164,165 .At the cellular level, the effect of mislocalisation of AQP4 channels on Aβ accumulation have been also investigated with deletion of α-syntrophin (SNTA1), important for perivascular localisation 166 .Several approaches have been proposed to inhibit AQP4 mislocalisation as a treatment for central nervous system (CNS) injury and stroke, with the aim of stabilising the BBB and disrupting the blood-spinal cord barrier disruption 167,168 .One group tested a calmodulin inhibitor targeting the mislocalisation of AQP4 to the calmodulin-mediated cell surface.This approach has successfully demonstrated reduced oedema and recovery of spinal cord injury.Moreover, the same calmodulin inhibitor decreases the expression of AQP4 proteins and reduces oedema, while increasing glycogen metabolism in the early acute phase in a post-stroke mouse model 168 .Cerebral oedema is the main hallmark of CNS injury and the main underlying factor for NDs, treating oedema by regulating AQP4 could be the treatment of choice for AD.
Gut-brain axis.The brain and gut communicate via neural, immune, endocrine, and metabolic pathways.Several hypotheses have proposed that the gut microbiome substantially contributes to brain disorders, including AD 169 .Considerable evidence suggests a connection between changes in gut microbiota and various features of AD, including Aβ, tauopathy, and neuroinflammation, and other pathogenesis-inducing neurodegeneration 170,171 .The current hypothesis is that gut microbiota indirectly affects BBB homoeostasis by activating peripheral immune cells.Alternatively, it can occur through the direct release of metabolites and hormones that travel through the vagus nerve, leading to neuroinflammation and contributing to proteinopathy 171 .Notably, a study has reported the antimicrobial effect of Aβ, revealing its protective role in immunity and inflammation 172 .This antimicrobial function has been consistent in all forms of Aβ peptide from oligomers to fibrils, not only in in vitro cultures, but also in AD mouse brains.Bacterial infection has accelerated the seeding and formation of Aβ deposition, which is in close proximity to the pathogen.These findings suggest that the inflammatory effects of infection and microbial changes may possibly initiate amyloid pathogenesis 172 .
A recent study demonstrated the impact of the gut microbiota on tauopathy of human APOE isoforms in a tau-transgenic mouse model 173 .In bacterial-free tauopathy mice, the level of pTau in the hippocampal area decreased, and microglia and astrocytes remained in a homoeostatic state in a sex-and APOE-dependent manner.In particular, when testing the differences in APOE isoforms, only APOE3 expressed in the tau male model showed improvement after treatment with antibiotics, but not the APOE4 isoforms of both sexes.Moreover, short-chain fatty acids in bacterial metabolites play a role in tauinduced neurodegeneration in mice.These findings strongly suggests that the importance of the gut-brain axis on AD pathogenesis, association with Aβ-and tau-induced pathogenesis, and neuroinflammation.
Brain-derived neurotrophic factor deficiency. Brain-derived neurotrophic factor (BDNF) is a critical factor in hippocampal neurogenesis that sustains synapses and neuronal circuits.A deficiency in BDNF is associated with several neuropathogenesis, including Aβ deposition, phosphorylated tau formation, neuroinflammation, and apoptotic cell death 174 .Moreover, the deficiency in neurogenesis observed in the early stages of AD can be explained by alterations in BDNF signalling; however, the underlying mechanism remains unknown.One study claimed that Aβ monomers maintain the production and release of BDNF by activating IGF receptors 175 .In addition, when BDNF was restored in a tau mouse model, behavioural changes, neuronal loss, and synaptic loss were reduced, but not hyperphosphorylation 176 .
Mitochondrial dysfunction and oxidative stress.In the early stages of AD, changes in mitochondrial structure, function, and accumulation have been observed 177 .Specifically, impairment of the mitophagy process is the major change currently attributed to cholesterol metabolism alterations related to APOE, mitophagy regulation of Aβ, tau protein, and APP-C99 accumulations on mitochondrial-associated membrane [177][178][179] .Astrocytes expressing APOE4 exhibit reduced fission and mitophagy 180 .
The principal characteristic of most NDs is the different vulnerable neurons 181 .During the progression of AD, neurons of the ECII, pyramidal neurons of the CA1, cholinergic neurons of the basal forebrain, noradrenergic neurons of the locus coeruleus, and rostral neurons are more vulnerable at the onset of the disease, whereas sensory cortical neurons begin to degenerate only at later stages.A recent study identified the molecular basis of vulnerable neuronal subtypes by profiling mRNA and successfully built a data source for modelling AD with specific neuronal circuits 182,183 .Vulnerability is attributed to oxidative stress, because oxygen-sensitive glutamatergic cells are selectively affected 184 .When oxidative stress increases, the vulnerable cells tend to experience DNA damage and mitochondrial fragmentation 185 .
Altered glucose and lipid metabolism.Understanding the complex roles of glucose and lipid metabolism is crucial for understanding neurodegeneration in the brain.According to a recent cohort study, high blood glucose and cholesterol levels increase the risk of AD 186 .Additionally, as the disease progresses, glucose levels in the brain decrease, whereas cholesterol levels increase 187,188 .Brain imaging studies have revealed that glucose metabolism is impaired in the hippocampal and cortical areas before the onset of cognitive decline or pathological changes 188,189 .Reduced glucose uptake is associated with accelerated neurodegeneration and Aβ depositions [189][190][191] .A fourmonth insulin treatment via the intranasal route improved cognitive function in APOE2-and APOE3-male mouse models, but not APOE4expressed female mice 192 .
Others suggest that Aβ depositions cause a deficiency of GLUT-1 and GLUT-3 in vulnerable regions 193 .One group has proposed that BBB dysfunction, which is apparent at the onset of AD, is due to decreased GLUT-1 expression in BBB endothelial cells 194 .These results suggested a complex association between glucose metabolism and AD pathogenesis.
In the oldest investigation by Alois Alzheimer, lipid accumulation was highlighted as the main pathological feature in AD 195 .Subsequently, many high-risk genes associated with AD were found to be involved in lipid metabolism, including APOE, TREM2 CLU, and ABCA7.This suggests that lipids are essential for AD progression; however, the complex link between changes in lipid metabolism and AD pathology remains unclear.Cholesterol and sphingomyelin of lipid rafts regulate APP processing in lipid rafts, suggesting their role in the formation of Aβ 196,197 .During ageing, disruption of the lipid composition of the transmembrane membrane could be one of the earlier pathogeneses of LOAD.Furthermore, studies have indicated the role of glial cells in lipid metabolism, particularly cholesterol 143,144 .Astrocytes expressing APOE4 increase cholesterol input to neurons, causing an expansion of lipid rafts that ameliorate Aβ formation 143 .Meanwhile, oligodendrocytes expressing APOE4 show decreased myelinating function due to altered cholesterol metabolism 144 .Moreover, other studies have suggested a connection between lipid and glucose metabolism in AD owing to reduced glucose uptake caused by high levels of oxidised cholesterol 198 .These findings demonstrate that the initial pathogenesis of AD, particularly LOAD, may be related to disruption of metabolism in the brain.

Parkinson's disease
PD currently affects over six million patients worldwide and is the second most prevalent ND 164 .PD progresses slowly with complex clinical features, affecting not only the motor systems (tremor, bradykinesia, rigidity, and gait disturbance), but also non-motor systems (rapid eye movement sleep behaviour disorder), constipation, hyposmia, and cognitive impairment 199,200 .These symptoms correlate with neuronal degeneration in certain brain regions, most commonly in the substantia nigra pars compacta (SNpc), where dopamine neurons reside 201 (Fig. 1b).Subsequently, patients begin to experience cognitive dysfunction in various extra-nigral regions, including the basal forebrain, thalamus, amygdala, and cortex, which initiates atrophy.Unfortunately, it is often diagnosed in a clinic much later, after the brain has lost approximately 50% of dopaminergic cell death 202 .
Most cases of PD are sporadic and occur later in life.Less than 10% of the cases related to genetic inheritance occur at an earlier age 203 .Currently, owing to GWAS, more than 200 genes associated with both early-and late-onset PD have been identified [204][205][206][207] .There were 90 loci related to familial cases, including SNCA, LRRK2, VPS35, PRKN, PINK1, DJ-1 and GBA.Among these genes, point mutations and multiplications discovered in SNCA strongly intensified with the development of PD 208 .Other genes that govern important biological pathways in dopaminergic neurons, including mitochondrial function and protein degradation (LRRK2, PARK2, PARK7, PINK1, and PRKN), have also been suggested as significant risk factors for PD.Despite notable findings, the early pathogenesis of PD remains unclear because of its complexity.Here, we focus mostly on misfolded α-syn related pathogenesis, and dopaminergic neuron vulnerability, as well as mitochondrial dysfunction occurring in PD.
Accumulation of misfolded α-synuclein.One of the main conspicuous histopathological hallmarks of PD is the abnormal protein deposition called Lewy bodies (LBs) and Lewy neurites, which consist chiefly of aggregated α-syn 209 and are found mostly in the extracellular space of SNpc 210 .A similar inclusion can be found in synucleinopathies, such as dementia with LBs, Parkinson's disease dementia, and multiple system atrophy 211,212 .Physiological α-syn (14 kDa) is an intracellular protein, normally expressed by the SNCA gene, and is abundantly found in presynaptic terminal of neurons and erythrocytes.Although the physiological function of α-syn is not fully understood, several studies have suggested that it is associated with the modulation of synaptic functions 213 and may have neuroprotective role 214 .The α-syn structure is generally divided into three regions: amphipathic domain (N terminus, residues 1-60), central non-amyloid component (NAC region, residues 61-95), and carboxyl acidic tails (C terminus, residues 96-140) 215 .The N terminus of α-syn consists of four 11-amino acid imperfect repeats and consensus sequence, KTKEGV, which is responsible for forming the α-helical structure when binding to the cellular membrane, such as apolipoproteins 216 .The hydrophobic NAC region is essential for forming β-sheet rich α-syn fibrils, which were first discovered in amyloid plaques and induce the formation of aggregated forms of α-syn 217 .However, the toxicity of LBs is still controversial, as some suggest that it is merely an accumulated trace of the cytoprotective response against PD progression 218 .Misfolded α-syn aggregation is considered the culprit of complicated pathogenesis of PD.Misfolded oligomeric α-syn is responsible for triggering neuronal toxicity by interfering with cell-to-cell signal transmission, mitochondrial pathways, and disruption of protein degradation pathways rather than mature fibrils [219][220][221][222] .Moreover, the physiological conformation of α-syn in vivo is an α-helical tetramer (58 kDa), rather than an unfolded α-helical monomer structure, that is resilient against aggregation 223,224 .
One of the notable theories of aggregation pathways is that misfolded α-syn aggregates may operate similarly to prions and selfpropagate by converting normal prion proteins into abnormal ones through a seeding effect 87 .In the process of prion-like aggregation, αsyn creates a stable seed or nucleus as it combines into insoluble aggregates.This, in turn, accelerates formation of aggregated α-syn by misfolding α-syn and recruiting it into itself.The resulting aggregated α-syn can then be fragmented into small seeds, thereby perpetuating a vicious cycle of aggregation 225 .Preformed synthetic α-syn aggregates can accelerate the fibrillation of α-syn in buffer conditions 226,227 .Additionally, administrating preformed α-syn aggregates into mouse brains and cultured neurons facilitates the accumulation of α-syn aggregates in neurons, ultimately leading to the development of PD [228][229][230] .α-syn originates in the olfactory bulb or dorsal nucleus of the vagus nerve before propagating to other regions 15,231 .Notably, some groups have proposed the bottom-up hypothesis of prion-like progression of α-syn pathology, which suggests that the propagation is caused by the transmission through the autonomic nervous system 232 .This is supported by the earlier gastrointestinal symptoms prior to the onset of motor symptoms in patients with PD.This current research suggests that prion-like behaviour of α-syn may explain the progression of PD.
The factors that contribute to the development of abnormal α-syn aggregates are not yet fully understood.However, familial PD risk genes play a role in triggering aberrant aggregation.Specifically, multiplications (duplication and triplication) of the SNCA gene results in an overexpression of α-syn by disrupting the proteostasis process, including the synthesis, degradation, and clearance of α-syn 233,234 .In addition, several missense mutations in the SNCA gene, including A53T, A30P, E46K 235 and H50Q 236 , disturb the folding dynamics of αsyn by distorting its intrinsic structure, which can lead to the early onset of PD 237,238 .
Dysfunction in protein degradation pathways.As LBs accumulate during PD progression, researchers have investigated protein degradation pathways to confirm the link between the impairment of these pathways and the onset of the disease.Under normal physiological conditions, protein dynamics are regulated via protein degradation pathways, such as UPS 239 and ALP 240 .However, when these degradation pathways are disrupted owing to ageing, oxidative stress, inflammation, or other unknown causes, they can lead to the abnormal deposition of misfolded proteins in neurons, causing neuronal toxicity, which is related to various NDs 241,242 .
In PD, LBs are highly ubiquitinated and the cellular components of the UPS are abundantly identified, suggesting that protein clearance via the UPS is closely related to the pathogenesis of PD 243,244 .In vitro and in vivo studies have shown that introducing proteasomal inhibitors to disrupt the UPS led to accumulation of misfolded α-syn and neuronal death due to an impaired 26S proteasome pathway [245][246][247] .Conversely, overexpression of α-syn in dopaminergic neurons can cause dysfunction in the UPS by disrupting the 26S proteasome.This precedes the onset of behavioural impairment and neurodegeneration 248 .
Another protein degradation pathway, the ALP pathway, plays an important role in clearing large cellular debris that cannot be removed by the UPS.The ALP system functions through lysosomal acidification via three delivery pathways: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy.Perturbations of these lysosome-mediated protein degradation pathways are associated with pathogenesis of PD 249 .The introduction of genetic mutations (ATP13A2 and LAMP2) into the components of lysosomal degradation pathways induces the accumulation of α-syn aggregates 250,251 .In contrast, overexpression, or modified forms of α-syn could impair the clearance of misfolded proteins by hampering the CMA-mediated protein degradation.This could eventually lead to an increase in the pathogenesis of α-syn 252,253 .
Degeneration of highly vulnerable dopaminergic neurons.Motor dysfunction in PD is explained by dopamine deficiency caused by the degeneration of dopaminergic neurons in the SNpc.Mainly dopaminergic cells are found to be the most vulnerable to pathogenic protein toxicity, leading to impaired dopaminergic signalling from the basal ganglia to the whole brain area, such as the nigrostriatal pathway.Corresponding to dopamine depletion, several pathological processes that regulate dopamine synthesis occur in the brain 254 .Compensatory overexpression of postsynaptic dopamine receptors occurs when there is a considerable loss of dopaminergic neurons 255 .Activated microglia are also observed on-site in dopaminergic neurons, promoting degeneration by releasing reactive oxygen species (ROS) 256 and neurotoxic cytokines, and inducing glutamate excitotoxicity 257 .Furthermore, a deficiency in the dopaminergic circuitry is associated with reduced neurogenesis in the subventricular and subgranular zones 6,258 .However, molecular evidence for the selective vulnerability of dopaminergic neurons to degeneration is lacking.Recently, researchers profiled the genomics of PD-derived dopaminergic neurons and found that AGTR1 expressed by a specific subgroup in the ventral tier of the pars compacta was highly upregulated in degeneration targets 259 .Mitochondrial dysfunction.Mitochondria, which control cell death and energy production, have been linked to the pathogenesis of various NDs 260 .The chemical toxin 2-methyl-4-phenyl-1,2,3,6-tetrahydrophiridine (MPTP) was first found to cause acute Parkinsonism by disrupting mitochondrial pathways 261 .Once MPTP enters dopaminergic neurons through the dopamine transporter, it is oxidised by enzymes and converted into 1-methyl-4-phenylpyridinium ions (MPP + ).It damages dopaminergic neurons by interfering with mitochondrial metabolism, specifically complex I of the electron transport chain, leading to increased oxidative stress 262,263 .As this environmental factor causes mitochondrial dysfunction, which is implicated in Parkinsonism, accumulating reports have suggested a substantial correlation between mitochondrial dysfunction and PD 264,265 .
α-syn plays a central role in pathogenesis of PD by inhibiting mitochondrial pathways 226 .One study identified α-syn null mice that were resistant to Parkinsonism caused by the administration of MPTP, implying that α-syn is essential for causing toxicity to dopaminergic neurons in conjunction with MPP + 266,267 .Additionally, α-syn aggregates negatively influence mitochondrial function, including mitochondrial fission and fusion, mitochondrial fragmentation, and complex I deficiency 268 .Exposure of preformed α-syn fibrils induces the recruitment of phosphorylated acetyl-CoA carboxylase 1 (ACC1) at the mitochondrial membranes, causing mitochondrial fragmentation.Oligomeric α-syn specifically compromises the mitochondrial complex I pathway and generates ROS in cybrid cell lines, leading to increased cell death 269 .Dopaminergic neurons generated from patients with PD carrying the A53T mutation or triplication of the SNCA gene show accumulated α-syn inclusions, as well as impaired cellular morphology and membrane potential 270 .In addition to impacts of environmental agents and misfolded α-syn on mitochondrial dysfunction in PD, GWAS have discovered that various genetic variants related to mitochondria are implicated in the early onset of PD 271,272 .Among the genetic factors, autosomal recessive PARK2 and PARK6, which express E3 ubiquitin ligase (Parkin), and PTEN-induced kinase 1 (PINK1), respectively, are strongly associated with mitochondrial dysfunction.Under normal conditions, PINK1 and Parkin are associated with the mitochondrial quality control system, mitophagy, which degrades and recycles damaged mitochondria to maintain mitochondrial homoeostasis 273 .However, when genetic mutations occur in these genes, the PINK1/Parkin system is unable to prevent oxidative stress and an imbalance in ATP production in mitochondria, eventually leading to the degeneration of dopaminergic neurons [274][275][276] .
The impairment of DJ-1, which protects neurons from oxidative stress by acting as a redox sensor and antioxidant, is also associated with the pathogenesis of PD, particularly in familial cases 277,278 .In PD, due to DJ-1 dysfunction, ROS production increases in the mitochondria, causing damage to neurons.In fact, DJ-1 is localised in the mitochondria and overexpression of DJ-1 relieves oxidative stress, preventing neuronal death [279][280][281][282] .However, when genetic mutations of the DJ-1 gene disrupt its biological pathways, which can lead to neurodegeneration and a monogenic form of Parkinsonism by compromising antioxidant activity 277 .
Environmental factors and neurotoxicity.Given the increasing impact of environmental pollution, it is crucial to consider the influence of neurotoxic chemicals on the onset and development of agerelated NDs.Neurotoxic heavy metals, pesticides, and metal-based nanoparticles contribute to ND pathogenesis 283 .Chronic exposure to lead, the main component of polluted air, is associated with cognitive dysfunction 284 .Common neurotoxic mechanisms have been observed in BBB disruption, oxidative stress, mitochondrial dysfunction, and protein aggregation during ND progression 285 .
Liu et al. 286 recently reported that polystyrene nanoplastic particles induce the α-syn mediated neurodegeneration by invading the brain through BBB.They hypothesised that charged nanoplastics disrupt the lysosome degrading function, and trigger the α-syn protein misfolding, resulting in the intracellular α-syn fibril accumulation in neurons.This study has demonstrated that there is a notable area to investigate concerning the detrimental effects of nanoplastics on the other NDs.

Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis is a chronic, progressive disorder that specifically degenerates upper and lower motor neurons in the nerve skeletal muscles (Fig. 1c).General clinical symptoms begin with motor dysfunction, such as muscle weakness, dysarthria, and dysphagia, and gradually disturb cognitive and behavioural activities, usually leading to immobility due to respiratory failure.Mutations in more than 30 susceptibility genes are associated with familial and sporadic ALS pathogenesis.These corresponding genes were commonly clustered under functions, such as protein homoeostasis, RNA homoeostasis, axonal outgrowth, cellular transport, mitochondrial alteration, oxidative stress, and neuroinflammation 287,288  are due to mutations in the four main genes (SOD1, C9ORF72, TARDB and FUS) associated with RNA metabolism 289 .Despite the existence of known genetic risk factors, the initial cause and interconnected pathogenetic mechanism of sporadic cases, which account for 90-95% of all cases, have not yet been clearly identified 290 .
Vulnerable motor neuron degeneration.Neuronal degeneration of ALS often occurs in the corticospinal fibres, partial upper motor cortex, ventral horn of the spinal cord, and precentral gyrus.However, in most cases no significant whole-brain changes were observed 291 .As the disease progresses, pathological damage in cortical regions mirrors cognitive dysfunction examined in 50-65% of patients 292 .Histologically, with motor neuron degeneration, astrogliosis appears in the anterior horn of the spinal cord and motor nuclei of the brainstem, and spongiosis and microvacuolation are predominantly observed in the motor cortex.At the lower motor neuron level, the tripartite synapse (neuromuscular junction) consists of muscle fibres and synapsecovering glial cells (Schwann cells), and the axonal terminal of the presynaptic motor neuron is prone to damage, which often progresses to the upper neurons 293 .The higher vulnerability of these neurons is explained by their fast fatigue and high metabolism 185 .In particular, long-extruding axons are more sensitive to metabolic impairment.Recent evidence shows that the significance of non-motor (systematic) symptoms progressively increases with the severity of ALS, which may be related to the spreading pathology, similar to other NDs 294 .
Dysfunctional protein-related pathogenesis.Similar to other representative NDs, the main hallmark of both familial and sporadic ALS is mislocated protein accumulations, including commonly hyperphosphorylated TPD43, and dipeptide repeat (DPR) protein depositions following protein superoxide dismutase (SOD1) deposits found in cytoplasm 295 .These proteins are found predominantly in motor neurons.These deposition could be explained by the disturbance of the protein degradation mechanism.However, efforts are needed to reveal the consequences of protein aggregation on motor neuron function.
More than 200 mutations in SOD1 are known to cause neurotoxicity in several ways, including protein misfolding, proteasome impairment, excitotoxicity, oxidative stress, endoplasmic reticulum stress, impaired axonal transport, axonopathy, inflammation, altered RNA processing, and mitochondrial dysfunction 296 .Mutated SOD1 proteins are associated with motor neuron toxicity through the formation of SOD1 aggregations 297,298 .Furthermore, a recent molecular study on the effects of different mutations in SOD1 indicated that altered conformational variants of SOD1 are more toxic than the full form of SOD1 when associated with clinical phenotypes 299 .SOD1 aggregates cause selective motor neuronal death 300 .SOD1 aggregates are found not only in the cytoplasm of motor neurons, but also in astrocytes, microglia, and oligodendrocytes 301 .Moreover, similar to other NDs, evidence gathered from studies suggests exosome transfer along anatomical pathways [302][303][304] and the potential role of oligodendrocytes in the uptake and prion-like propagation of SOD1 aggregates 305 .Regardless of this evidence, other components of SOD1linked ALS need to be studied further because of reported cases of sporadic and familial ALS without SOD1 inclusion in some brain areas 306 .
Approximately 95% of patients with familial ALS have motor neurons with mutant TDP-43 inclusions in the cytoplasm 307,308 .Under normal physiological conditions, TDP-43 is localised in the nucleus and regulates gene expression and RNA metabolism.However, hyperphosphorylation, ubiquitination, and generation of C-terminal fragments cause aggregation in the cytoplasm of glia and neurons, leading to mitochondrial dysfunction, ROS production, and nucleoplasmic transport impairment.The double spiral-fold structure of TDP-43 suggests several underlying mechanisms associated with macromolecules, prion-like aggregation, and propagation that need further investigations 309 .Correlating with an increase in cytoplasmic aggregation, lower levels of nucleus TDP-43 have led to abnormal splicing, especially in UNC13A transcripts, which are crucial for neurotransmitter-meditated communication [310][311][312] .However, the pathological functions of UNC13A remain unclear.Notably, TDP-43, which is found in the cytoplasm of myotubes, exerts a protective effect by integrating into muscle formation and regeneration 313 .
Lastly, hexanucleotide repeat expansion in C9ORF72 is found in 3-7% of sporadic ALS cases (40% of familial ALS) and is a common genetic cause of ALS 314 .RNA transcripts of these extended regions form five different DPRs and RNA foci deposited in the neurons of the ALS brain exhibit toxicity in several models [315][316][317] .DPRs influence transport from the nucleus to the cytoplasm by causing defects in the nuclear membrane and inducing TDP-43 dislocation 318 .Following this investigation, therapeutic options have been developed.One group recently suggested an antisense oligonucleotide (ASO) that can suppress the expression of repeat groups in transgenic mice, which is considered a primary step towards gene therapy 319 .Notably, these distinct aggregations may be positive for other phenotypes.DPR aggregates are found along with TDP-43 aggregates in the ALS brain; therefore, it is important to further demonstrate their relationship and differences 320 .
Neuroinflammation.Imaging and postmortem cell culture studies have shown the critical roles of microglia, astrocytes, other nervous and immune cells, and inflammatory cytokines in ALS pathophysiology 321,322 .When motor neurons have degenerated, microglia are induced and secrete ROS and proinflammatory cytokines, such as TNF-α, IL-1, and IL-6.However, as the disease progresses, microglial activation becomes detrimental to motor neurons 323 .Whereas mutated SOD1-expressed astrocytes induced selective toxicity in spinal motor neurons 324 .Studies have suggested several pathways of astrocyte-induced neurotoxicity such as glutamate toxicity 325 , lactate impairment 326 , secretion of inflammatory mediators 327,328 , and necroptosis 329 .However, the contribution of mutant proteins to astrocyte dysfunction and motor neuron-specific toxicity remains unknown.Likewise, C9ORF72 and SOD1-expressed oligodendrocytes have also exhibited motor neuron neurotoxicity in soluble and direct ways 330 .
Mitochondrial dysfunction.Motor neurons are highly energetic; therefore, they tend to be sensitive to age-related metabolic changes and mitochondrial dysfunction.In ALS pathophysiology, motor neurons derived from SOD1, TDP-43, C9ORF72, and FUS show impaired mitochondrial structure, dynamics, and functions that lead to death due to the activation of ROS and intrinsic apoptotic signalling pathways [331][332][333] .A study of sporadic ALS patient-derived induced pluripotent stem cells (iPSCs) reported several changes in mitochondrial parameters, including increased ROS, depolarisation of the inner membrane potential, reduced ATP production, impairment of oxidative phosphorylation, and the protein import system 334 .
Disturbance in RNA metabolism.Many genes linked to ALS contribute to regulation of RNA metabolism, such as SOD1, TDP-43, FUS, and C9ORF72 335 .As mentioned above, the abnormal expansion of C9ORF72 is highly related to the production of abnormal RNA nuclei loci, which contribute to neurotoxicity.The RNA-binding protein encoded by TDP-43 has a regulatory role in RNA splicing, stability, and transport, especially in the expression of ND-associated (FUS, tau, ATXN2 and progranulin), synaptic, and neurotransmitter-regulating proteins, which suggest an important role in ALS pathogenesis 336,337 .

Huntington's disease
HD is a rare disease with an autosomal dominant inherited pattern caused by a mutation in the Huntingtin gene (HTT) 338  loss of striatal and cortical neurons leads to chorea and deterioration in cognitive and behavioural abilities, mostly as a result of the toxic effects of inherited mutant HTT (mHTT)-encoded large HTT proteins.At an early stage, caudate nucleus atrophy occurs and the neurons of the basal ganglia (corpus striatum) and corticostriatal circuit begin to degenerate when patients experience clinical symptoms 339 (Fig. 1d).As the disease progresses, the cortical, occipital, and parietal regions become atrophied, corresponding to the clinical characteristics of motor and cognitive alterations 339,340 .Although the exact triggers of striatal neuron degeneration remain elusive, the majority of studies have focused on earlier pathogenesis features, such as the toxicity of mHTT and impaired DNA regulation of the HTT gene, which inspired several drug candidates, but all have failed at different stages.In addition, other HTT-dependent and HTT-independent pathways have been investigated, including tau dysregulation, mitochondrial dysfunction, excitotoxicity, impaired BDNF, and neuroinflammation.These pathways may have a decisive impact on HD pathogenesis, similar to mHTT.
CAG repeat and mutant Huntingtin protein.HTT, the main cause of HD, HTT gene resides on chromosome 4 and encodes the large Huntingtin protein HTT (348 kDa) with a segment of polyglutamine 341 .It is normally found in the cytoplasm or nucleus of neurons; however, its specific function has not yet been determined.Some reports have claimed that it may be related to BDNF production, axonal transport, neuroblast migration, and the overall development and communication of the nervous system 342,343 .However, in its mutated form, the HTT gene is composed of an aberrant number of cytosine, adenosine, and guanine (CAG) repeats ranging from 40 to 250 repeats, which ranges from 6 to 35 in healthy individuals 341 .Over 1000 CAG repeats have been reported previously 344 .The instability of CAG repeats was first discovered in the striatal neurons of mice two decades ago and later validated in a human autopsy model 345 .The number of CAG repeats correlates well with the severity of clinical HD symptoms 346 .Therefore, longer and more unstable repeats generally result in an earlier onset 13 .Early-onset HD (Westphal variant) is associated with dystonia, Parkinsonism, and psychiatric symptoms, whereas late-onset HD often correlates with 40-55 CAG repeats, which initially occur with chorea.However, the toxicity of full-length HTT proteins, especially in striatal neurons, is not fully understood, and whether they are the earliest causative factors is still debated.
Current HD pathogenesis has been broadly described as the polyglutamine tract of mHTT generated from CAG expansion and the abnormal post-translational process of HTT, which eventually leads to a cascade of pathological process 343,347,348 .The deleterious effect of mHTT was first demonstrated in mouse brains, exhibiting intracellular inclusion, striatal shrinkage, and compensatory ventricular enlargement accompanied by progressive motor alteration 349 .In addition, earlier in vivo studies provided evidence that a decrease in mHTT expression correlates with the recovery of behavioural and pathological alterations 350 .
However, it remains controversial whether full-length Huntingtin proteins or fragments are toxic to neurons 351 .When the number of the CAG repeats increases, translated large Huntingtin proteins tend to divide into fragments that aggregate, causing the death of striatal neurons 352,353 .Evidence has been gathered regarding the pathogenicity of different mHTT protein fragments.The most toxic fragment is the short HTT exon 1 protein generated by either aberrant splicing 354 or proteolysis 355 .This protein aggregates and causes neurotoxicity in the striatal region, resulting in motor and transcriptional dysregulation in mouse models 354 .This is similar to the toxicity of full-length HTT, but tends to cause more severe neuronal death 356 .
In contrast, oligomers are more toxic than large inclusions found in cells 353,[357][358][359] .Current evidence suggests that the N17, polyproline, and polyserine (polyS)-rich domains are crucial for aggregation and neuronal toxicity [360][361][362] .In contrast, some reports indicated that monomer mHTT induces apoptotic death, whereas mHTT aggregates exhibit a protective role by withholding neuron necrosis 357,363 .Additionally, a recent report claimed that HTT aggregates were not linked to degeneration of the cortical and striatal regions 364 .
Several in vitro studies have demonstrated the ability of mHTT to spread from cell to cell via tunnelling nanotubes 365 , endocytosis 366 , and prion-like behaviours to form wild-type HTT aggregate 367 .Importantly, the malfunction of the proteasome system on the clearance of mHTT has been observed in several in vitro and animal models 368,369 .Moreover, aggregated polyS and polyleucine-containing proteins exhibit propagation behaviour, which was recently observed in vivo and in vitro studies 362 .
CAG repeats are not only associated with neurotoxicity at the protein level, but mRNA-containing CAG repeats in the nucleus may also be neurotoxic.Mutant HTT mRNA generates clusters in the nucleus of cortical and striatal neurons in HD mice and in postmortem models 370 .However, it remains unclear whether the formation of mRNA clusters exerts neurotoxic or preventative effects 370 .
Tau dysregulation.Recent studies have reported a significant association between tau protein and HD pathogenesis 371 .Studies have identified tauopathy features, including misfolding, hyperphosphorylated tau aggregates, NFTs, and mHTT oligomers, in the postmortem HD brain.These features correlate with severe cognitive alterations but not with motor dysfunction 372 .Furthermore, several cellular-level studies have confirmed tau dysregulation in HD models, including altered splicing 373 and hyperphosphorylation 374,375 of tau proteins induced by mHTT aggregates.Further studies are needed to investigate the advanced protein crosstalk mechanisms between tau and mHTT proteins to understand the pathogenesis of HD.
DNA instability and oxidative stress.Recently, the focus has shifted from polyglutamine tract proteins, the product of HTT gene, to CAG repeats and, most importantly, dysfunction of the DNA repair system, which seems to correlate with much earlier and more severe pathogenesis 376 .This was shown in a GWAS, where uninterrupted expansion of CAG repeats in DNA, but not polyglutamine, was related to earlier and more severe pathophysiology in patients with HD 377 .Dysfunction of DNA repair proteins that induce CAG expansion was observed in the highly clustered DNA repair genes MSH3, MLH1, PMS1, PMS2, MLH3, and FAN.Therefore, these genes may be effective targets for the treatment 376,377 .Small single-stranded RNA, ASOs, restrict MSH3 expression, which lowers the rate of CAG expansion 378 .In addition, a recent study showed the influence on CAG expansion by inactivating FAN1 with a mutation in an HD model using human iPSCs(hiPSCs) 379 .This finding shows a promising approach for delaying onset and severity with genetic treatment, since FAN1 is predominantly clustered in the earlier onset of HD.
Notably, the normal HTT protein itself plays a role in transcriptional regulation by binding to support the DNA repair enzymes activated by oxidative stress 380 .mHTT directly affects the transcriptional processes of cells, mostly in the caudate nucleus of the brain and in some peripheral tissues 381 .Moreover, there is evidence on the relationship between HTT and several other transcriptional regulators, such as p53, the CREB-binding protein, CBP, and miRNAs.A recent study investigated the miRNA profiles of patients with HD and found that overexpression of hsa-mir-10b-5p inhibited GTPBP10 expression, which may correlate with the gradual loss of mitochondrial dysfunction 382 .Furthermore, epigenetic and chromatinmodifying factors are suggested to be involved in transcriptional dysregulation.Alterations in transcriptional regulation were observed in an early HD mouse model with downregulated epigenetic regulatory genes 383 .
Vulnerable medium spiny neurons and excitotoxicity.In patients with HD, neurons and other cells exhibit CAG expansion 384 .Notably, typically inhibitor GABAergic medium spiny neurons (MSN) of the corticostriatal tract, specifically those in the caudate putamen and globus pallidus, are preferentially degenerated 385 .The causes of this vulnerability and degeneration still need to be better understood.However, oxidative stress and the toxic effects of mHTT aggregates on synapse formation 386,387 , and excitotoxicity 388 , similar to other NDs, may play a role.Morphologically, MSN have many spines on their axons that substantially increase in number and size at an early stage.As the disease progresses, MSN as well as other types of neurons begin to be affected 13 .Patient-derived MSN neurons displayed ageassociated characteristics, such as DNA damage and mitochondrial dysfunction, as well as severe death in the presence of endogenous mHTT aggregates 387 .
Overproduction of glutamate neurotransmitters generates excitotoxicity, followed by a cascade of alterations in neurons of the corticostriatal pathway 388,389 .In a proteomic analysis of late-stage HD mouse brain tissue, convincing impairment of proteins corresponding to glutamate signalling, neurotransmitter balance, synaptic transmission, glycolysis, and ATP production was revealed 390 .Notably, astrocytes display alterations in the glutamate-GABA-glutamine regulatory system, leading to decreased GABA production and increased glutamine release.In addition to MSN, cortical neurons exhibit decreased synaptic activity, increased excitability, and reduced complex dendritic arborisation during disease progression.However, this can be reversed in the second postnatal week and HD symptoms reappear.Treatment with ampakine CX516 stopped reappearing by stabilising glutamate transmission 391 .
Mitochondrial dysfunction.Various mitochondrial changes occur in neuronal and non-neuronal cells, including changes in mitochondrial structure, ATP production, motility, and biogenesis, which tend to be interdependent on the mHTT protein [392][393][394][395] .The interaction of mHTT with the inner and outer mitochondrial membranes causes abnormalities in Ca +2 homoeostasis and mitochondrial protein dynamics 396 .Changes in the structure and localisation of mitochondria have been observed in HD models, such as decreased number and fusion, increased fragmentation and fission, and accumulation in the soma with disrupted motility 397,398 .
Impaired brain-derived neurotrophic factor synthesis and transport.As with other NDs, BDNF impairment is another pathophysiological feature of HD, which correlates with its anatomically vulnerable site.Cortical neurons of the corticostriatal pathway provide sufficient BDNF to the MSN, which has physiologically low BDNF mRNA 399 .Additionally, altered BDNF formation and transmission in cortical neurons with mHTT aggregates may lead to a reduction in BDNF in MSN 400 .In addition, evidence suggests that mHTT affects the BDNF transcription factor of neurons 401 and astrocytes 402 .
Neuroinflammation.Whether neuroinflammation is a causative factor or a response to other pathophysiological features in HD remains unclear.Inevitably, owing to their role in extracellular glutamate clearance, glial cells are critical for studying their influence on the highly vulnerable glutamatergic MSN.There is evidence on astrocyte and microglial activation, increased proinflammatory cytokines, and hypothesised systemic inflammation, all of which are associated with disease progression 403 .Many animal and human models have indicated that astrogliosis and striatal astrocyte dysfunction are considerable components of HD pathogenesis 404,405 .In an in vivo study, glial cells expressing mHTT induced a more severe disease phenotype and caused hyperexcitability in striatal neurons 406 .In the HD postmortem model, C3 expressed-A1 astrocytes contributed to the death of neurons and oligodendrocytes while losing some functions, such as synapse formation and function, and phagocytic capacity, which confirmed the neurotoxic function of astrocytes 407 .Importantly, A1 astrocytes were induced by IL-1A, TNF, and C1q cytokines of reactive microglia and were present in other NDs, such as AD, ALS, PD, and multiple sclerosis, which makes them critical candidates for further research.In other studies, mHTT in astrocytes caused astrogliosis and impaired BDNF release 402 and glutamate uptake 408 , leading to striatal neuronal death when co-cultured with astrocytes 409 .In a recent study, it was discovered that mHTT aggregates promote proinflammatory cytokines and ROS by activating microglia 410 .

Neuropathogenesis-on-chips for neurodegenerative diseases
Recently, several alternative culture systems have emerged as substitutes for traditional in vitro models to provide more reliable and representative human systems.These techniques include 3D cell culture, tissue engineering with 3D bioprinting, and microfluidics 411,412 .Among these, microfluidics stands out for its capability to replicate critical elements of organs with precise control over biochemical and biomechanical aspects, offering a high-throughput, physiologically relevant, and cost-effective solution compared with conventional methods.The representative lung-on-chip replicates the alveolus, which is the key functional unit of lungs 413 .This two-chamber chip design allows externally controllable mechanical forces to mimic the alveolus movement, and direct monitoring of the interaction between epithelial and endothelial cells separated by semi-permeable membrane 413 .Moreover, this design has been applied to pulmonary disease 414 as well as other organ units with similar mechanical conditions, such as gut-on-a-chip 415 and glomerular-on-a-chip 416 .
The possibilities of chip-based in vitro modelling are expanding in collaboration with advanced biological and engineering techniques, such as iPSCs, 3D culture, genome editing, hydrogels, 3D bioprinting, integrated biosensors, monitoring and analysis methods, various designs, and fabrication techniques.In this section, we highlight the benefits of using 2D and 3D microfluidic chips to model NDs.First, we provide practical biological and technological guidance for modelling NDs using various designs and introduce existing examples of NDs-onchips.Furthermore, we present a more reliable and physiologically relevant approach: a 3D culture-on-a-chip (organoid-on-a-chip) design that can be adapted for studying NDs.

Strategy for modelling neurodegenerative diseases on a microfluidic chip
Choosing cell source.Approximately 86 billion neurons are interconnected in a highly ordered manner to form neuronal networks within the human brain 417 .These networks exhibit distinct activities with support from non-neuronal cells (~9.8 billion) such as astrocytes, microglia, and oligodendrocytes 417 .Cerebrovascular cells comprise 0.3% of all brain cells and play a crucial role in disease progression.Recent single-cell sequencing data revealed the existence of different subtypes within each cell type, based on their spatial location and pathological conditions at the molecular level, suggesting the complexity of brain cells 418 .Owing to the limited access to the human brain, there is a demand for the development of methods for source cells with region-specific characteristics.This is particularly important for modelling NDs because they exhibit pathogenesis specific to certain regions.
Owing to the existing animal-based in vivo and in vitro models of NDs, current knowledge was accumulated.These models offer valuable insights into cellular characteristics, disease progression, systemic and age-related changes, and the impact of the environment on the complex pathogenesis of NDs, as described in the preceding section [419][420][421][422] .In particular, mice and rats are widely used because of their closely identical genomes (~85%) 423 424 .Transgenic mice that overexpressed or knock-in of human genes associated with familial NDs have been mostly developed.AD mouse models are created using transgenic, knock-in, and injection methods, focusing on Aβ pathology (PDAPP, Tg2576, and APP23), tau, and neuroinflammation (JNPL3, rTg4510, PS19, and 3xTg) 419 .Currently, over 170 mouse models with AD mutations mostly exhibit characteristics of EOAD.Recently, a new sporadic AD mouse model has been generated 425 .This model includes humanised Aβ by knock-in method without expressing the FAD mutation and exhibits age-dependent impairment of cognition, synaptic, immune response, formation of periodic-acid-schiff granules, as well as transcriptomic changes in energy, metabolism, and neuroplasticity-related genes.
However, the external approach is widely used for NDs, using various chemicals and recombinant pathological proteins.In patients with PD, drugs (reserpine and haloperidol), neurotoxins (6-OHDA, MPTP, and lipopolysaccharide), and agrochemicals (rotetone, paraquat, and manab) induce PD-like characteristics, such as motor deficits, mitochondrial dysfunction, neuroinflammation, and nigrostriatal dopaminergic cell death 420 .Numerous studies have used exogenous recombinant human and mouse proteins to characterise misfolded protein pathologies both in vitro 362 and in vivo 426 .α-syn preformed fibrils generated from monomeric recombinant α-syn can induce hyperphosphorylation of endogenous α-syn and enhance the spread in vitro 427 and in vivo 428 .
Non-mammalian species, such as Caenorhabditis elegans, Drosophila, and yeasts are simpler and more cost-effective alternative models 429 .Cytotoxicity, aggregation, and propagation of the α-syn protein has been investigated in these models with overexpressed SNCA regarding PD.Despite the genetic variations in humans, these short-lifespan species are ideal for investigating the effects of ageing on ND pathogenesis 429 .
Human embryonic stem cells (hESCs) have also been used to model NDs and to provide genetic and molecular information relevant to humans.Human neural progenitor cells (hNPCs) derived from hESCs can differentiate into several subtypes, such as cholinergic, dopaminergic, serotonergic, noradrenergic, and medium spiny striatal neurons, expressing neuron-specific markers 430 , among which H9 and H1 are dominantly used in research 431 .However, ethical concerns surrounding hESCs led to the need for alternative solutions.Methods to induce neural differentiation, such as neural induction and direct reprogramming, have been successfully developed since the discovery of iPSCs from somatic cells with transfection factors 432,433 .Current differentiation protocols utilising genetic or chemical methods have demonstrated promising results in the generation of diverse types of neural cells, including cortical, basal forebrain, dopaminergic, cholinergic, glutamatergic, GABAergic, MSN, and functional motor neurons.Additionally, these protocols can generate other types of brain cells, such as oligodendrocytes, astrocytes, pericytes, vascular endothelial cells, and microglia 434,435 .HiPSCs have been extensively used in PD studies owing to the availability of current protocols that mainly focus on the induction of dopaminergic neurons.However, for further applications, there is a need to improve and standardise the derivation methods 436 .
Familial and sporadic patient-derived iPSCs are another candidate cell source.This approach allows the generation of humanrelevant pathological phenotypes that cannot be achieved using other animal models.iPSC lines from patients with PD with SNCA, LRRK2 Parkin, CHCCHD2, PARK2, and PINK1 mutations exhibit important phenotypes including mitochondrial dysfunction, oxidative stress, and α-syn accumulation in induced midbrain-like dopaminergic neurons 437,438 .
High reliance on genetically modified models often overlooks sporadic cases and fails to fully consider other important factors, such as ageing and patient-specific traits.Additionally, there are inevitable limitations due to the differences between human and animal brains at the cellular and tissue levels 20,21 .While genetically engineered animalderived cells continue to play a fundamental role in in vitro studies of NDs 439 , human patient-derived cells show promise as reliable in vitro models for basic scientific research and the development of personalised medicine.
Choosing microfluidic chip design.First chip application on brain research began with a notable compartmentalised in vitro system referred to as the 'Campenot chamber' 440 .This device has two fluidically separated chambers and is used to study the effects of nerve growth factors on axonal growth.Subsequently, researchers have converted the complex characteristics of the brain into simple and miniaturised systems by mimicking diverse levels of units of the brain, including axons, neuron-glia cells, the BBB, and the neurovascularunit 22 .
Typical microfluidic chips consist of two or more compartments (chambers) for cell co-culture.These compartments were connected by microchannels, porous membranes, and phase guides.This allows for direct or indirect interactions between homogeneous (neural circuits) or heterogeneous (neuron-glia) cell populations which are loaded into fluidically isolated chambers.
The earliest design of microfluidic chips for the CNS involved the separation of a neuronal soma from its neurites using microchannels.This design allowed for directional control of neurite growth 36 .By incorporating multiple compartments for different neuronal subpopulations, the connectivity and size of the circuits can be regulated, enabling the modelling of neural circuits in a highly ordered manner.Current neuronal chips have been designed with compartments positioned in various geometries 441 and different diameters and numbers 442,443 , microchannels with patterned shape 444,445 , and controlled fluidic flow 446 .These features effectively manipulate neurite growth and can create both indirect and direct as well as asymmetric and symmetric neuronal connections.Moreover, valves can be established on the chip to act as a controllable barrier between compartments using external factors, such as air, oil, gel, liquid, temperature, or pressure [447][448][449] .Regulated fluid flow can be achieved using extra pump systems and passive hydrostatic pressure, which applies controllable shear stress to the cells.This allows for a gradient of chemicals with varying concentrations throughout the cell compartments, which is useful for disease modelling.In ND research, gradients-on-a-chip used for investigation of pathological protein dynamics and toxicity, synaptic and neurochemical interactions, vesicle transportation, and neurite ingrowth 450,451 .
Another approach involves the use of a porous membrane-based chip that acts as an interface between two or more compartments.This chip enables indirect interactions mediated by soluble chemicals and direct physical contact.The size, position, and number of the pores were customised according to the research objectives.The thickness of the membrane was adjusted to facilitate the experimental interactions.Based on this design, BBB mimicry has been achieved on chips 452,453 .It mimics the walls of blood vessels by culturing a monolayer of endothelial cells on a porous membrane, facilitating soluble interactions with brain chambers 452 .In another example, a porous membrane was placed horizontally between two chambers to culture vascular and brain cells separately 453 .This design enables more dimensional interactions than a planar culture.
Monitoring and analysing steps.Microfluidic chips offer several advantages over conventional models for monitoring and analysis.These include allowing optical clarity for real-time monitoring under various chemical or physical cues, gas permeability, and continuous flow to balance nutrients or waste for longer viability; fluidic isolation for multi-omics analysis of soluble biomarkers from distinct cell populations; and microengineering to integrate biochemical, electrochemical, and electrophysiological biosensors.Most microfluidic chips are composed of clear optical materials (polystyrene and polydimethylsiloxane).Additionally, most designs include a pre-determined image acquisition region within their structures, which enables consistent and accurate monitoring.Specific biomarkers can be detected directly from the chip using fluorescence and confocal microscopy by introducing a very small amount of staining dyes and antibodies through the fluidic channel inlet.However, issues of contamination, accumulated cell debris, and air bubbles in tiny channels can pose challenges in maintaining cultures and imaging.These can be prevented by carefully processing the fabrication steps, using trapped air bubble methods, or force-out procedures 454 .
The integration of various types of biosensors on microfluidic chips enables the continual measurement of the biochemical and electrophysical properties of cell cultures.Various parameters, such as oxygen, pH, and soluble metabolite levels can be analysed using immune-and enzyme-based sensors, allowing the evaluation of cellular survival and metabolism in response to induced pathophysiological conditions 454 .Moreover, the incorporation of automated electrochemical biosensors in microfluidic chips enables the continuous monitoring and analysis of dynamic cellular activity 455 .
In the case of the brain-on-a-chip, a transendothelial electrical resistance system, integrating electrodes 38,456 , and planar electrophysical multielectrode arrays (MEA) 457 are often incorporated to measure the integrity and permeability of the BBB and assess neuronal electrical signals, respectively.Off-chip analysis tools, such as mass spectroscopy, liquid chromatography, and biochemical assays, such as ELISA, can be utilised to analyse small amounts of samples isolated from microfluidic chip cultures.Alternatively, whole-cell culture samples can be lysed and collected for further genomic, transcriptomic, and proteomic analyses.In certain cases, rather than opting for the commonly used closed approach, an open-chip design offers various benefits.The open chambers provide easier exposure to additional chemical components, imaging, transfer for analysis, and sample collection.Notably, most open designs tend to lose their dynamic fluid flow, which can limit the controllability and relevance.However, the benefits of using open chips have also been reported in recent studies.Salman et al. 458 developed an open chip for modelling the human brain endothelial microvessel.It enables the unidirectional exchange of media, gas, and nutrients within the collagen gel through a cylindrical hollow lumen.The top opening increases oxygen availability.Furthermore, it simplifies the harvesting of cells and exosomes for subsequent transcriptomic and proteomic analyses and allows for standard and advanced imaging by carefully optimising the dimensions and geometry.

Application in modelling neurodegenerative disease pathogenesis.
Based on this research objective, relevant anatomical and physiological units, including the neuromuscular junction (NMJ), corticostriatal pathway, substantia nigra, BBB, glymphatic system, neurovascular unit (NVU), and gut-brain axis, can be replicated using matched chip designs.However, most existing ND studies using microfluidic chips still lack physiological relevance owing to the choice of cell source and designs under 2D and static conditions.These drawbacks limit the wide applicability of microfluidic chips.The limitations of 2D systems include their inability to generate complex interactions, waste removal, and oxygen diffusion.A planar environment reduces morphology, proliferation, differentiation ability, cellular signalling, and gene expression which affects the in vivo relevance of hiPSCs 459 .
Conversely, the implementation of a 3D extracellular matrix (ECM) gel provides mechanostructural and biochemical cues to cells which have improved relevance in cellular morphology, migration behaviour, cellular signalling, interactions, and substantial gene expressions 460 .Moreover, it allows time-dependent differentiation and neurodegenerative changes 461 .The important factor of NDs, ageing, alters properties of the brain ECM 460 .Aβ induced accumulation and aggregation of pTau has been observed in 3D in vitro condition 462,463 .This evidence indicates the importance of ECM in modelling NDs.
To characterise a 3D microenvironment that provides not only mechanical, but also biochemical cues, the properties of the ECM, such as pore size, solute retention, attachment motifs, biodegradability, and stiffness, must be considered 460 .Alterations in these properties can affect the behaviours of various brain cells as well as disease pathogenesis 464,465 .During AD, owing to altered functional connectivity, vulnerable regions of the brain exhibit altered stiffness, which is correlated with structural changes 466 .Furthermore, evidence shows that ECM stiffness directly affect the aggregation and propagation of pathogenic molecules, such as Aβ and tau oligomers 460 .
Various hydrogels have been integrated into microfluidic chips to demonstrate direct interactions while creating a more physiologically relevant brain tissue-like environment 467,468 .One example of this design utilises a phase guide in the central channel to confine ECM gelembedded cells from dispersing into side flow channels filled with medium, while still allowing interaction without a barrier 461,468 .In the next section, we provide an overview of the use of custom and commercial microfluidic chips to model NDs pathogenesis.

Existing examples of neurodegenerative disease-on-chips
Alzheimer's disease-on-chips.Existing studies using microfluidic chips have investigated substantial AD pathogenesis, including Aβ and tauopathy, mitochondrial dysfunction, and neuroinflammation, providing valuable new insights that were not observed using conventional culture methods (Table 1).In particular, numerous neurons-ona-chip are used for tau propagation [469][470][471][472][473] , and Aβ toxicity studies [474][475][476] .Neurons-on-a-chip allows the separation of the soma and neurites, facilitating the real-time visualisation of proteinopathy.Some groups have found evidence of tau transfer via exosomes 110 and synapses 477,478 using 2-3 compartment chips, suggesting prion-like propagation of tau.
Aβ-related pathology is also being investigated mainly using microfluidic chips consisting of two to four compartments.Substantial evidence of Aβ toxicity has been gathered, including the induction of hyperphosphorylation of tau proteins, degeneration of axons 474 , and disruption of BDNF retrograde signalling of axons 475 .Some studies have cultured neurons as well as co-cultured microglia with primary hippocampal neurons in the presence of exogenous Aβ proteins.Ruiz et al. 479 studied the glial clearance of different forms of Aβ, in a 4-compartment circular open microfluidic system.Each open compartment allowed for four parallel experiments and easy seeding cocultures of different populations, such as microglia and neurons, with various cell densities under gravity-induced dynamic conditions.Aβ oligomers have a higher toxicity to hippocampal neurons compared to that of fibrils.However, only the microglia changes in Aβ exposure; the neuronal aspect is not included.Choi et al. 451 developed a microfluidic chip that can generate dynamic fluid conditions for gradient formation of Aβ aggregates, similar to in vivo studies, by integrating an osmotic pump to induce slow laminar flow (0.25 µL/min), similar to the interstitial flow rate (Fig. 2a).Under dynamic conditions, the cultured primary rat neurons showed a higher level of viability (7.5%).However, when exposed to Aβ oligomers, the neurons lost their synaptic activity and eventually experienced apoptosis.In addition, the fibril form of Aβ was less toxic to neurons than oligomers.
Several on-chips studies have been conducted using human neural progenitor cells transduced with familial AD-specific mutated genes, such as FAD 670N/M671L/V717, APP, and PSEN1.In AD pathogenesis, astrocytes play an essential role in the progression of AD, with "a double-edged sword" effect on neuroinflammation, owing to their neuroprotective and neurotoxic effects 138,480,481 .However, the dynamics of astrocytes and pathogenic proteins involved in neuroinflammation are not well understood.Park et al. 482 designed a simple circular chip that has two chambers (central and radial) which allow microglia migration towards to the centre where AD neurons derived chemokines and cytokines (TNFα, IL-1, and IL-6) are located (Fig. 2b).This design created a gradient of Aβ and neuroinflammatory molecules in a 3D environment and showed great potential for further molecular studies, including co-culture with glial cells.Additionally, numerous studies have been conducted on the migration of human neutrophils 483 and microglia 484 towards exogenous Aβ monomers and oligomers with chemotaxis chips in 2D conditions.
By incorporating various types of cells, a molecular understanding of the neuroinflammatory phenomenon can be obtained using microfluidic chips.Recently, research was focused on welldefined brain features, such as the BBB and the neurovascular unit, in AD neuropathogenesis.Shin et al. 452 developed a BBB-on-a-chip comprising a cell chamber for AD-mutated hNPCs and a tube-shaped endothelial cell chamber separated by a central barrier.As shown in Fig. 2c, the accumulation of Aβ and pTau proteins were observed in the brain channel.Upon removal of the barrier by Matrigel application, the proteins are transferred to endothelial cells, where they are deposited in the cytoplasm.They cause damage to tight junctions, indicating BBB disruption, which is observed in the early stages of AD.These promising results suggest that BBB-on-a-chip can be used as a drugscreening tool in the future.
A microfluidic chip composed of 50 cylindrical wells was developed to culture neurospheroids and integrate them with an osmotic micropump to provide a continuous medium supply similar to the interstitial flow rate 485 (Fig. 2d).Medium with exogenous Aβ flows over neurospheroids continuously, causing axonal degeneration, synaptic dysfunction, and neuronal death.The spherical wells of this chip generate more homogeneous spheroids, and continuous flow leads to increased viability, which directly affects cell-cell interactions.This chip demonstrated that integrating microfluidic chips with 3D cultures can create more in vivo-like conditions that are unavailable in other conventional systems.
Unique characteristics of the glymphatic clearance system can be explored with microfluidic chips by allowing dynamic glymphatic flow in the space between astrocytes and endothelial cell co-culture 163 .Lee et al. 486 recently replicated the gliovascular unit using a microfluidic chip in AD studies.The glymphatics-on-a-chip design allows for the coculturing of human astrocytes and blood vessels in 3D gel-embedded parallel microchannels.LPS-induced neuroinflammation causes altered fluid drainage and Aβ clearance through matrisome-and celldependent fluid transport pathways.
Parkinson's disease on-chips.The majority of existing PD-on-a-chip studies have focused on recapitulating α-syn related pathogenesis (Table 2).However, only a few have used dopaminergic neurons to increase the relevance of these models.The first on-a-chip PD model was developed using simple single-linear microchannels with a gradient of neurotoxins to model the apoptosis of dopaminergic neurons caused by 6-OHDA.Depending on the concentration, this neurotoxin can induce apoptosis or necrosis 487 .By generating a back-and-forth continuous flow, the model achieved low and high concentration gradients of 6-OHDA, which resulted in PC12 cell line apoptosis by oxidatively damaging the mitochondria.
The microfluidic chip with two chambers connected through microchannels is the most common method for investigating α-syn inclusion and propagation.This chip design allows for clear observation of the process of α-syn fibril uptake, and its anterograde or retrograde transport through axons to other neurons by exposing neurons to exogenous α-syn 488,489 .Van Laar et al. 490 used the same microfluidic chip to emulate the dynamics of mitochondrial alterations observed in the earlier stages of PD.In PD, neural degeneration begins in long, less myelinated and distant axons 491 .This was confirmed by their response to rotenone; mitochondrial biogenesis increased in distal axons, followed by cell bodies and dendrites.Prots et al. 492 also used the same chip to investigate the toxicity of α-syn oligomers in neurons derived from iPSCs from patients with PD with mutant α-syn (E46K and E57K).They observed α-syn oligomerization led to the impairment of anterograde mitochondrial transport through axons, and structural synaptic loss.This result confirmed the role of mutant The effect of Aβ oligomers on neurons was observed in gradient concentration condition 451 .b 3D static neuroinflammation-on-a-chip.Microglia migrated in response to inflammatory cytokines across microchannels to the centre chamber where AD neurons and astrocytes were cultured 482 .c BBB-on-achip.Formation of amyloid plaques, neurofibrillary tangles, Aβ, and increased permeability of brain ECs, were fully recapitulated in a 3D environment 452 .d Dynamic neurospheroid-on-a-chip.A flow of exogenous Aβ caused by an osmotic pump resulted in axonal degeneration and cell death 485 .PD-e Navie and GFP-tagged α-syn expressing neuroglioma cells were co-cultured to monitor the spreading of αsyn 494 .f Gradient chip.It modulated the intracellular α-syn expression of singularly trapped yeasts in system with galactose gradient 450 .g Dopaminergic neurons-on-achip.In a 3D condition, it recapitulated mitochondrial abnormalities and degeneration of dopaminergic neurons with the PD-related mutation 461,577 .h Substantia nigra and vascular barrier chip.hiPSCs-derived midbrain dopaminergic neurons, primary glia cells and a brain microvascular EC line were separated by a porous membrane to assess BBB on-a-chip dysfunction, progressive neuronal loss, neuroinflammation, and astrogliosis 453 .ALS-i 3-chamber-chip allowed metabolic interaction between SOD1-mutated astrocytes and cortical neurons through microchannels in a glutamate gradient condition 502 .j ALS chip.Applying chemotactic and volumetric gradients successfully formed interactions between FUSmutated motor neurons and mesangioblast-derived myotubes through microchannels 503 .k An open compartmentalized NMJ device was used to co-culture optogenetic motor neuron and SOD1-mutated astrocytes as a spheroid to assess the muscle denervation pathology of ALS 505 .l TDP-43 mutated motor neuron spheroid and muscle fibres were co-cultured in a 3D condition between two separate reservoirs, which recapitulate ALS pathologies 506 .HD-m Impaired BDNF transport due to mHTT expression through cortical axons resulted in striatal neurons degeneration 509 .n The synaptic connection between cortical axons and striatal dendrites was formed by the different lengths of microchannels and a separate synaptic channel, assessed the early progression pathology 507 .AD Alzheimer's disease, ALS amyotrophic lateral sclerosis, BBB blood-brain barrier, EC endothelial cell, HD Huntington's disease, hiPSC human-induced pluripotent stem cell, NMJ neuromuscular junction, PD Parkinson's disease.
α-syn oligomers in the early pathogenesis of PD.Additionally, one group cultured preformed primary neurons with exogenous α-syn fibrils for an α-syn transfer study based on evidence that exogenous recombinant α-syn can promote misfolding of physiological α-syn, leading to the formation of LB-like aggregates in neurons 493 .In this experiment, a three-chamber chip was used to demonstrate the toxic effect of α-syn aggregation and uptake by inhibiting regulator protein of protein trafficking.They also investigated how endogenously released α-syn are toxic to separately cultured neurons in a paracrine way.An example of a customised chip with micro-valves that allows control fluid dynamics has been developed to demonstrate α-syn uptake and spread 494 (Fig. 2e).By co-culturing cells expressing GFPtagged α-syn and normal cells in two chambers connected through microchannels, the researchers were able to observe α-syn successfully spreading throughout two different populations.Another group reconstructed a human corticocortical neuronal network by culturing hiPSCs on a microfluidic chip 495 .In this design, neurons in the two-cell chambers interact with each other in a narrow central chamber by projecting their axons through asymmetric microchannels.They indicated that prion-like propagation of physiological α-syn occurs between human neurons.Two-, and three-, or multichambered chips recapitulate neuron to neuron α-syn spread.A microfluidic chip with two to six interlocked chambers that can be adjusted in several different ways successfully recapitulated the complex neuronal network without barriers and demonstrated phosphorylated α-syn propagation 441 .A well-known design of microfluidics, the gradient-generator chip, can provide a reliable chemical concentration by creating nine different doses in microchannels 450 (Fig. 2f).They induced intracellular α-syn with exogenous α-syn monomer and fibrils inside Saccharomyces cerevisiae which were trapped individually in the chip with different chemical gradient environments.Recently, another group cultured live yeast on a microfluidic chip integrated with automated feedback control, which can regulate the concentration of α-syn aggregation in S. cerevisiae expressing A53T α-syn combined with GAL-inducible promoter with GFP in real-time 496 .Over a galactose gradient, they determined the threshold of α-syn mutant protein expression that induces the formation of inclusions and their clearance, which is strictly based on autophagy at the single-cell level.This approach can be applied to other quantitative studies of human neuronal cell lines.
As dopaminergic neurons are selectively damaged during the progression of PD, Bolognin et al. 461 studied the effect of the LRRK2-G2019S mutation on dopaminergic neurons (Fig. 2g).In this experiment, they used a "Mimetas" culture chip with a phase guide that allows culturing cells in 3D ECM conditions.The dopaminergic neurons successfully formed structurally relevant networks on the chip.Notably, substantial changes due to the PD mutation, including neurodegeneration and changes in the number and complexity of mitochondria, were only observed after six weeks in the 3D condition.This result demonstrates the importance of mechanical cues in demonstrating physiologically relevant pathogenesis.
Considering the significance of the BBB in PD pathogenesis, one group has successfully demonstrated the BBB dysfunction caused by the toxicity of exogenous human α-syn fibrils 453 using a 3D microfluidic chip (Fig. 2h).The design of the chip allows the culturing of brain and vascular cells in tissue-specific ECM cocktails in two separate channels connected through a horizontal semi-permeable membrane.This mimicry of BBB has exhibited formation of intracellular inclusion by internalised α-syn, which leads to a progressive loss of dopaminergic neurons, mitochondrial dysfunction, and neuroinflammation associated with glia cell activation.Brain endothelial cells were cultured in support of pericytes, microglia, and astrocytes, therefore, the BBB was formed with higher maintenance and functionality.Moreover, dopaminergic neurons, the main cells of the substantia nigra, stably secrete dopamine, indicating that functional maturation is not considerable in conventional cultures.
More recently, de Rus Jacquet explored astrocytic activity in both BBB dysfunction and inflammation during PD pathogenesis 497 .The significance of the chip design is that the ECM gel (collagen) acts as a barrier between the brain vascular channels which allows direct interaction.The phenotypes of vascular changes in vitro which were observed in sporadic PD and human postmortem models were successfully demonstrated and vascular changes depended on LRRK2-G2019S-expressing astrocytes, which exhibit proinflammatory activity.In this model, LRRK2-G2019S-expressing patient-derived astrocytes failed to support BBB integrity and inflammation, particularly angiogenesis, as confirmed by RNA-seq data.Moreover, mutated astrocytes regulated BBB integrity via the MEK1/2 signalling pathway, providing evidence for the importance of astrocytes in PD pathogenesis.
Amyotrophic lateral sclerosis on-chips.Numerous rodent models have been developed to investigate ALS based on newly discovered genetic factors.These models have become indispensable tools for investigating disease mechanisms, although they mostly reflect familial case of ALS 498 .In vitro modelling, mostly using cells sourced from transgenic mice with familial gene mutations, such as TARDBP, SOD1, and C9ORF72, has also revealed important dysfunctions of cellular processes related to ALS which are also shared with sporadic ALS 498 .These dysfunctions appeared in protein transport, homoeostasis, RNA transcripts, and mitochondria 499 .Although such models provide defined mutation-related information, a specific model for sporadic ALS has not been developed 421 .In ALS, several connected mechanisms lead to motor neuron death, causing diverse phenotypes that are difficult to model.Mutations in RNA-binding proteins, such as TDP-43, FUS, NRPNA1, MATR3, and SETX, alter RNA metabolism 500 .
Therefore, a unified model that reflects a patient's phenotype is required.
Previous ALS studies have investigated toxic protein aggregation and propagation using simple 2-chamber microfluidic chips (Table 3).Westergard et al. 501 determined that C9ORF72-linked DPRs spread between cortical neurons via exosomes and non-exosomes via axonal anterograde and retrograde transmissions.This study once again proved that the spread of toxic proteins is a common underlying mechanism of progressive NDs.
Similar to other NDs, neuroinflammation is a pathogenic feature of ALS.Accordingly, astrocytes were simulated using microfluidic culture systems.In one of the earliest studies, Kunze et al. 502 co-cultured primary cortical neurons and SOD1-mutated astrocytes without direct contact to demonstrate the effect of astrocytes on neuronal death under a glutamate gradient condition (Fig. 2i).They observed neuronal death and decreased synaptic protein expression in neurons in the presence of astrocytes from patients with ALS, indicating SOD1 toxicity.
Considering that NMJ degeneration is the main hallmark of ALS, the co-culture of myocytes and motor neurons has been mostly recapitulated for ALS studies, taking advantage of the compartmentalised elements of microfluidic chips.Several groups have used different types of culture systems, including monolayer cultures and spheroids derived from genetically modified ESCs and hiPSCs [503][504][505][506] .Radial-shaped two-chamber microfluidic chips demonstrated mitochondrial toxicity, protein synthesis dysfunction, and axonal phosphorylated TDP-43 deposition associated with the activity of C9ORF72-modified hiPSCs neurons 504 .Another research group successfully established NMJ impairment in ALS by culturing hiPSC-derived FUS-mutated neurons and human primary mesangioblast 503 (Fig. 2j).They generated NMJ by creating a volumetric gradient of BDNF, GDNF, and CNTF in microfluidic chambers to induce axonal growth in the muscle cell chambers through a microchannel.They found that FUS neurons exhibited a lower axonal growth rate, leading to fewer NMJ.However, an HDAC6 inhibitor reversed axonal growth in FUS neurons.Machado et al. 505 developed an open microfabricated chip composed of three compartments, which enabled the analysis of interactions between spheroids containing ESC-derived neurons and SOD1 astrocytes as well as primary myofibres (Fig. 2k).They investigated how astrocytes with mutated SOD1 induce neuronal loss, followed by myofibril dysfunction.However, these advanced models lose their physiological relevance when primary rodent myofibres are used, despite the use of human ALS-specific motor neurons.An exceptional example of such a model is co-cultured patient-derived motor neuron spheroids and hiPSC-derived skeletal myoblasts on a custom-made 3D microfluidic chip 506 (Fig. 2l).Once skeletal muscle fibres are formed, motor spheroids are introduced into a 3D gel to form NMJ.Moreover, optogenetic technology has been applied to motor neuron spheroids by transfecting them with a light-sensitive ion channel (channelrhodopsin-2), allowing the non-invasive control of motor neuronal activity.The chip design also includes several important features, such as a separate reservoir allowing for the co-culturing of cells.The region for neurite elongation was designed to be longer to achieve a physiologically relevant neurite length.The pillars in these designs provide functions, such as limiting the size of the spheroid, preventing core necrosis, and measuring the strength of muscle contraction by changes in pillar displacement.By generating NMJ, they successfully recapitulated TDP-43 aggregation, neurotoxicity, and muscle atrophy due to synaptic loss.
Huntington's disease on-chips.Compared with other NDs, there have been relatively few microfluidic chip applications for HD (Table 4).These chips replicate the corticostriatal network, a vulnerable region in HD pathophysiology, with two- 406 and three-chambers 507,508 .To understand the toxic effects of mHTT on the corticostriatal neural circuit, on-a-chip studies commonly use transgenic mouse neurons.One group investigated how mHTT reduced the cortical axonal transport of BDNF and detected a positive effect of the TCP-1 ring complex (TRiC) on decreasing mHTT levels 509 (Fig. 2m).Chip contains two separate chambers for cortical and striatal neurons, connected by microchannels.Design allows the observation of striatal neuron degeneration, and decreased synapses.In their three-chamber chip (presynaptic, synaptic, and postsynaptic), Virlogeux et al. 508 established laminin and poly-D-lysine gradients from presynaptic and synaptic chambers.By culturing mHTT-transduced primary neurons, deficiencies in synapses, BDNF, and glutamatergic trafficking, and a decline in mitochondrial transport and vesicle synthesis were observed.Later, this group used the chip in a drug target study by recapitulating the restoration of Huntingtin-dependent BDNF vesicle transport by activating palmitoylation 510 .They observed improved axonal BDNF trafficking, synapse formation, and viability in the chip channels.Based on an in vivo study of HD knock-in mice that showed improvements in motor deficiency and behavioural changes, the authors claimed that ML348, which inhibits acyl-protein thioesterase 1 (APT1), could be used as a therapy against disease progression.Using the same chip, Migazzi et al. 507 identified distortion in the anterograde axonal transport of HTT-meditated vesicles and neuronal damage due to arginine methylation-defective HTT(Fig.2n).They identified how PRMT6 expression could restore HTT-mediated axonal trafficking.To model the corticostriatal pathway, they reconstituted two separate cell chambers connected through microchannels of different lengths and the middle "synaptic" chamber.
In another study, basal ganglia circuits were composed of the cortex, striatum, globus pallidus internal, substantia nigra reticularis, compacta, and thalamus using a chip with five chambers linked to microchannels 511 .Notably, the size and location of each regional chamber were designed to mimic a similar ratio of cells and interactions as the 3D anatomical structure of the basal ganglia circuits.They demonstrated the direct neuronal connectivity between five different cell populations using rodent primary neurons.The striatum and its communications with connected regions are important neuronal circuits in HD studies.
Moreover, by integrating an MEA with 256 electrodes, this microfluidic chip analysed the neuronal network functionality in real time.Although the experiment has not yet characterised the diseasespecific pathophysiology, it is possible to detect the effect of Huntingtin or α-syn proteins in neural circuit function using this in vivo relevant MEA-integrated regional-neuron network chip.Microfluidic chips can be modified in several ways.One research group recently proposed an electrostimulation microfluidic chip by modifying the chip design for current HD disease modelling.They created holes with electrodes in cell chambers to directly stimulate neurons.This design recapitulated and successfully measured the neuronal activity of the corticostriatal circuit but has not been applied to HD studies yet 512 .
In the pathophysiological progression of NDs, crosstalk between distinct regions is crucial point to investigate.For such a recapitulation, combining two or more brain region-specific organoids may be useful.By addressing this, Pasca et al. 526 first generated combined organoids termed "assembloid" to better recapitulate the regional interactions, including non-neuronal cells, such as microglia and endothelial cells from different embryonic layers.Following this, brain assembloids have been developed for various regions, including the cortical-striatal, cortical-spinal 527 , cortical-perictyes 528 , corticospinal-skeletal muscle 529 , and disease models, such as Timothy disease 530 and AD 531,532 .Moreover, to overcome the limitation of vascularisation, Kong et al. 531 recently developed a fused cortical vessel assembloid treated with Aβ proteins externally.When exposed to SARS-CoV-2 infection, this assembloid showed an increased number of astrocytes and microglia, indicating how systematic inflammation accelerates neuroinflammation in AD.In another study, instead of regional characterisation, Rickner et al. 532 generated a neuro-astrocyte assembloid (asteroids) composed of neurons cultured with tau oligomers and astrocytes that showed rapid maturation.Assembloid successfully demonstrated several pathological changes, such as tau misfolding, aggregation, and reactive astrogliosis.When treated with an HSP inhibitor, the pathological and neurodegenerative changes declined.
However, the use of organoid models to study NDs remains controversial for several reasons.The current method for developing organoids has only recently achieved anatomical and functional characterisations closer to those of the foetal neonatal human brain after approximately nine to 10 months of culture 533,534 .Therefore, it is considered more suitable for modelling neurodevelopmental disorders and has been applied to diseases, such as autism spectrum disorder 535 , epilepsy 536 , and microencephaly 25,537 .Nonetheless, encouraging results of organoid maturation have also been observed in terms of cellular composition, anatomical characteristics, such as distinct cortical layers, functional choroid plexus, and brain convolution formation using genetic or mechanical methods 538,539 .
Recently, various organoid culture platforms have been designed with promising results (Fig. 3).These platforms aim to improve in vivo characterisation, maturation, and uniformity of organoids while reducing the culturing time and labour required.They also have the potential to cooperate with immune and vascular components, bioimaging, and biosensors for effective mimicry and analysis 412 .
Conventional organoid protocols involve several steps that are likely to lead to contamination, damage, sample loss, and dissimilarity.This could be solved with a "one-stop" microfluidic platform that enables all steps of organoid culturing and long-term maintaining process, while monitoring and analysing without any manual additional transferring process 540,541 .Ao et al. 540 designed a simple device to study the effects of cannabis on prenatal brain organoids (Fig. 3a).This simple "one-stop" chip allows organoids to start from embryonic bodies (EB).With a physical barrier and an air-liquid interface, organoids are prevented from random merging and core necrosis, which leads to better uniformity and maturation.A similar "one-stop" approach has been employed to the micropillar array device, resulting in greater uniformity and reproducibility 541 (Fig. 3b).However, organoids eventually exhibit nutrient deficiency in their cores.Furthermore, these models should be transferred to separate analysis tools such as MEA because the design does not include integrated analysis tools.Some research groups have used spinning bioreactors to provide an equal distribution of nutrients to cultured organoids 542 .
To enhance organoid viability, a dynamic medium flow can be achieved by integrating either a rocking platform or a syringe pump 49,543 .Qian et al. 542 .developed a 3D-printed bioreactor that resulted in improved cortical characterisation, such as a human-like outer radial glial layer, compared with static conditions (Fig. 3c).Furthermore, microfluidics can help achieve more human-like characteristics, such as surface wrinkles 544 and functional myelinated axons of peripheral neurons 545 .One group induced mechanical force using a simple chip device to produce wrinkled brain organoids 544 (Fig. 3d).Compressed organoids are manipulated to an oblate shape with a surface-wrinkling effect that is not observed in conventional methods.They observed contraction of the cytoskeleton in the core and nuclear expansion in the outer area of the brain organoids.Another crucial component of 3D culture is the surrounding scaffolds.Supporting biomaterials, such as Matrigel, decellularised ECM (dECM), and other biocompatible hydrogels have been used to accelerate maturation and structural development by providing a brain-like environment 50,546 .Cho et al. 50generated organoids with a brain-derived decellularized ECM on a microfluidic-based platform integrated with a rocking platform (Fig. 3e).Compared to the commonly used Matrigel, dECM induces rapid growth and desirable neuron-glia interactions.The microfluidic chip provides an optimal environment for dynamic fluid flow and gas nutrient transfer, thereby accelerating cell viability and growth.Additionally, this platform allows for indirect interactions between organoids which can be applied in coculture studies of organoids from different regions.There are a few The four-step organoid maturing process was performed on one platform 540 .b High-throughput micropillar array.Brain organoids were directly generated from dissociated hiPSCs in a controllable micropillar array with restrained space in the microarray 541 .c Miniaturised multi-well spinning bioreactor device.The device is cost-effective, high throughput, and has increased maturation and reproducibility 542 .d With the mechanical stimulation of a microfluidic chip, surface folding appeared in the brain organoid 544 .e Organoids in a human brain-derived decellularized ECM gel, under the dynamic condition of a microfluidic device, showed higher survival, and uniformity 50 .f Organoids were encapsulated via microfluidic electrospray, and transferred to multi-layered microfluidic chip to form brain-region-specific assembloid 547 .g A tumour spheroid was integrated with a perfusable vascular network in a three-line chamber microfluidic chip 548,549 .h Neuroimmune interaction of cortical organoid and microglia in a 3D-printed organoid culturing device 550 .i NK cell interaction with tumour organoid in a microfluidic chip 552 .j Microfluidic device for clearing intact 3D microtissue in 1 d for confocal microscopy 553 .k Acoustofluidic chip for forming uniform spheroids in a fast and high-throughput manner 554 .l Hanging drop device with plug-in multi biosensors 555 .PTFE polytetrafluoroethylene, EB embryonic bodies, ECM extracellular matrix, HESC human embryonic stem cell, hiPSC human-induced pluripotent stem cell, HUVEC human umbilical vein endothelial cell, IDT interdigital transducer pattern, NK natural killer.
examples of the use of microfluidic chips for assembloid development.
However, only recently one group reported a microfluidic-based assembloid-forming device 547 (Fig. 3f).They designed a three-layered microfluidic chip (micropillar, microhole, and culture top layer) to generate an assembloid by combining encapsulated organoids.However, before loading onto the chip, EB and different brain regional organoids should be separately generated via a microfluidic encapsulation method.The interaction between the cortical-hippocampalthalamic regions has been well characterised using assembloid modelling.
Vascularisation and immune interactions are critical for further characterisation of brain organoids.Using vasculogenesis-or angiogenesis-inducing factors, some groups have attempted to vascularise 3D organoids using microfluidic platforms.Nashimoto et al. 548,549 created a blood vessel network merged with tumour spheroids in a fibroblast hydrogel in the middle chamber by culturing human umbilical vein endothelial cells in two side channels (Fig. 3g).The vascular network exhibited high perfusability and viability.In other designs, instead of following the commonly used closed-well approach, Salmon et al. 51 established an open-well organoid chip using 3D printing and successfully generated perfusable neurovascular organoids by simultaneously differentiating iPSCs into vascular and neuronal lineages.One group developed an assembly chip device for the interaction of circulating microglia and brain organoids using a tubular porous scaffold 550 (Fig. 3h).Moreover, this chip design was repurposed in different studies to recapitulate organoid vascularisation 506,551 .Repurposed chips can also be used in brain studies.Tumour spheroid interactions with natural killer cells were recapitulated in chips with vascular-mimicked channels 552 (Fig. 3i).
Microfluidic chips can be integrated into bioimaging devices and biosensors for precise monitoring and analysis.However, because of their density and size, 3D cultures have limited imaging properties.One group has proposed on-a-chip clearing techniques for 3D spheroids for better imaging.Sequential clearing steps were performed on the device channels.This approach was 20 times faster than conventional clearing methods 553 (Fig. 3j).For precise manipulation of 3D cultures, the surface acoustic wave has been applied to the microfluidic chip, which allows high-throughput production of uniform spheroids and assembling spheroids 554 (Fig. 3k).Also, biosensorintegrated microfluidic chips have also been developed for 3D cultures 555 .This hanging drop chip allowed the monitoring of secreted glucose and lactate levels with electrodes functionalized with oxidase enzymes 555 (Fig. 3l).

The outlook of in vitro model in the pharmacological industry
The challenges in neurodegenerative disease drug development Despite substantial advances in our understanding of disease mechanisms and technological innovations, the development of effective treatments for NDs remains challenging.Recent breakthroughs have been made in the treatment of AD, including the use of aducanumab 16 , lecanemab 17 , and donanemab 556 .However, the effectiveness of the new drugs only appears at certain stages of AD 116,556 .Moreover, Aβ-against antibodies are controversial in human cognition although it exhibits a considerable decrease of the plaque.While these recent approvals are beneficial in certain groups of patients, there is still much work to be done to expand the effectiveness of these drugs, considering the multifactorial and heterogeneous characteristics of NDs.
In addition, the treatment development process requires a costeffective and time-saving approach.The AD drug development field has invested approximately $42.5 billion in clinical trials over the past 30 years, which requires a long, dedicated effort, but has yielded few promising results 116,557 .Several factors have been attributed to drug failure, including intervention at an inappropriate stage of disease progression, insufficient biomarkers, and the use of incorrect research models 116 .Specifically, preclinical studies conducted on familial genetically engineered mouse models have limitations as they do not fully replicate all the features of NDs, especially sporadic cases.These models exhibit varied clinical and pathological phenotypes that restrict their effectiveness in translation to human physiology 558 .Moreover, long-term preservation of animals with age-related characteristics can be costly and time-consuming 559 .Translation to a more physiologically relevant, human-cell-sourced, in vitro model that incorporates most features of certain NDs for studying disease mechanisms and developing drugs has the potential to reduce costs and time for development and produce more representative and reliable test results.

Microfluidic chip in pharmaceutical industry
Over the past two decades, the development of advanced in vitro models has increased in the academic field.However, the pharmaceutical industry has not yet adopted microfluidic chips 560,561 .
Following the recent FDA policy on the shift towards the use of more human-relevant models in drug testing, both academic and pharmaceutical industries have begun to use organ-on-a-chip and organoid-based models 562 .Several large pharmaceutical companies (AstraZeneca, Bayer, and Roche) have been using organoid and microfluidic chips to a limited degree to enhance the screening accuracy of potential drug candidates 561,563 .Moreover, global efforts (USA, Europe, and Japan) have been made to translate academic achievements to the pharmaceutical industry using a standardised and reproducible predictive drug-screening approach [564][565][566] .Tissue chip testing centres established by the NIH National Center for Advancing Translational Sciences initiated grantees to validate various types of tissue chip platforms for drug testing 566,567 .Under these grants, three independent organisations validated microphysiological systems that replicated the kidney, liver, and BBB for drug transport and toxicity studies, with the goal of increasing the reliability, reproducibility, robustness, and throughput of its effective application 568 .

Microfluidic chip for drug screening at the academic level
Despite ongoing efforts to address reproducibility and scalability in the pharmaceutical industry, drug screening and toxicity studies of potential ND drug components have also been conducted on-a-chip in the academic field.Numerous companies, such as Emulate, TissUse, Mimetas, Nortis, and CN Bio offer various designs of microfluidic chips to researchers for conduct physiologically relevant studies of disease mechanism, drug screening, and toxicity 563 .Some research groups have performed drug screening and toxicity studies on previously rejected drug candidates using microfluidic chips.Clinical trials on ASOs to prevent and alleviate the progression of AD, PD, ALS, and HD are currently underway.However, these clinical trials demonstrated toxicity.Nieskens et al. 569 verified ASOs toxicity by testing varying doses on a kidney proximal tubule-on-a-chip using human renal proximal tubule epithelial cells.In addition, one group reported the dose-dependent efficacy of NMJ-blocking drugs for ALS using a commonly used micro-channelled two-compartment design.This chip allows the interaction between motor neurons and muscles only through the axons.Functional ability was revealed by muscle contraction when motor neurons were stimulated using silver electrodes.They tested the several doses of botulinum neurotoxin, Curare, and α-bungarotoxin (α-BTX), and quantified the dose-dependent NMJ dysfunction as dose-response curve 570 .
Monolayer culture approaches have often been applied to drug discovery and preclinical screening.However, as described in the previous section, 3D cell culture is expected to provide closer in vivo characteristics, increasing the predictability of clinical results.In addition, replacing animal-based drug screening with patient-origin organoids is considered a substantial development in the pharmaceutical field which will increase human reliability and reduce costs and ethical concerns by overcoming the drawbacks of animal models.Recently, several attempts have been made to apply organoids in drug discovery studies in an academic field.Osaki et al. 506 tested the muscle atrophy-reversing effect of bosutinib and rapamycin on spheroids on chips, confirming drug efficacy with caspase-3/7 staining at day 14.Moreover, neuroprotective treatments positively influenced the optogenetic stimulation of muscle contraction.This chip design is beneficial because it evaluates not only the single or combined drug effects on functional 3D co-cultures, but also drug delivery across the EC barrier.This chip design significantly increased throughput by simultaneously screening single and combined drugs.Park et al. 515 .introduced a novel organoid-culture drug-screening platform that employed mathematical network analysis.Researchers have investigated the effects of current FDA-approved drug candidates on AD patient-origin cerebral organoids and confirmed the structure, viability, and Aβ and tau depositions.This study demonstrates the potential of patient-origin organoids for drug screening of NDs.

Potentials of advanced microfluidic chip in pharmaceutical industry
Through collaborative global efforts, the integration of microfluidic chips with advanced technologies has the potential to revolutionise the pharmaceutical industry.The organoid-on-a-chip and multiorgan-on-a-chip systems can play crucial roles in various stages of drug discovery, ranging from target identification to preclinical screening (Fig. 4).In addition, we believe that such an advanced model can be useful even in the clinical stage when deciding on the effective DMT for each patient subtype by sourcing patient-derived cells.However, to achieve this, many crucial industrial and technological challenges must be considered, such as reliability, robustness, reproducibility, compatibility with analysis techniques, material choice, scalability, and standardisation.Moreover, there is a great need for defined metrics for validation and benchmarks for modelling each ND.
An advanced on-a-chip system has the capability to not only validate the effects of drugs on neuropathological features, but also measure functional changes through real-time electrophysiological measurements.Efforts have been made to measure brain waves in real time from brain organoids using invasive and non-invasive electrophysiological biosensors integrated on a high-throughput microfluidic chip 534 .This integration will provide a clinically relevant assessment of NDs.Moreover, once drug candidates are identified for NDs, their mechanism of action, safety, dose-response relationship, potential side effects, and pharmacokinetics can be tested on even more complex inclusive systems, such as multiorgan-on-a-chip platforms (composed of the brain, kidney, liver, gut, and heart) 571 .The use of multiorgan-on-a-chip models reduces the dependence on animal models during this stage, while potentially providing more representative, cost-effective, and accurate results, thereby facilitating successful clinical testing.In addition, patient-derived cell sources can facilitate the development of effective treatments for certain patient subsets.Advanced on-a-chip systems could be beneficial for patient stratification testing to identify effective DMT with fewer side effects, especially in challenging multifactorial diseases, such as AD 572 .
In the future, microfluidic chips will substantially contribute to the translation of brain organoids and assembloids for the development of ND drug discovery and personalised medicine.Notably, in the development of microfluidic chips, there are biological, technical, and commercial challenges for the entire application to the pharmaceutical industry and personalised medicine 573 .Despite these hurdles, we believe that advanced microfluidic chips and molecular biophysical techniques will advance our understanding of the complexities of ND pathogenesis and challenge our dependence on animal-based tools.

Fig. 1 |
Fig. 1 | The similarities and differences in the main hallmarks of neurodegenerative diseases from the organ to molecular levels.a AD is characterised by the inclusion of misfolded amyloid-β (Aβ) and neurofibrillary tangles in pyramidal neurons, primarily in the hippocampus and cortex regions of the brain.b PD is characterised by Lewy body aggregates composed of misfolded α-synuclein and degeneration of dopaminergic neurons in the substantia nigra region of the brain.c ALS is characterised by including mutant TAR DNA-binding protein 43 (TDP-43) Review article https://doi.org/10.1038/s41467-024-46554-8Nature Communications | (2024) 15:2219

Fig. 2 |
Fig.2| Examples of microfluidic chips for NDs modelling.AD -a Gradient chip with interstitial flow.The effect of Aβ oligomers on neurons was observed in gradient concentration condition451 .b 3D static neuroinflammation-on-a-chip.Microglia migrated in response to inflammatory cytokines across microchannels to the centre chamber where AD neurons and astrocytes were cultured482 .c BBB-on-achip.Formation of amyloid plaques, neurofibrillary tangles, Aβ, and increased permeability of brain ECs, were fully recapitulated in a 3D environment452 .d Dynamic neurospheroid-on-a-chip.A flow of exogenous Aβ caused by an osmotic pump resulted in axonal degeneration and cell death485 .PD-e Navie and GFP-tagged α-syn expressing neuroglioma cells were co-cultured to monitor the spreading of αsyn494 .f Gradient chip.It modulated the intracellular α-syn expression of singularly trapped yeasts in system with galactose gradient450 .g Dopaminergic neurons-on-achip.In a 3D condition, it recapitulated mitochondrial abnormalities and degeneration of dopaminergic neurons with the PD-related mutation461,577 .h Substantia nigra and vascular barrier chip.hiPSCs-derived midbrain dopaminergic neurons, primary glia cells and a brain microvascular EC line were separated by a porous membrane to assess BBB on-a-chip dysfunction, progressive neuronal loss,

Fig. 3 |
Fig.3| Representative designs of organoid on-a-chip.a One-stop microfluidic organoid-culture platform.The four-step organoid maturing process was performed on one platform540 .b High-throughput micropillar array.Brain organoids were directly generated from dissociated hiPSCs in a controllable micropillar array with restrained space in the microarray541 .c Miniaturised multi-well spinning bioreactor device.The device is cost-effective, high throughput, and has increased maturation and reproducibility542 .d With the mechanical stimulation of a microfluidic chip, surface folding appeared in the brain organoid544 .e Organoids in a human brain-derived decellularized ECM gel, under the dynamic condition of a microfluidic device, showed higher survival, and uniformity50 .f Organoids were encapsulated via microfluidic electrospray, and transferred to multi-layered Review article https://doi.org/10.1038/s41467-024-46554-8Nature Communications | (2024) 15:2219

Fig. 4 |
Fig. 4 | The future outlook of NDs-on-a-chip for drug development.a Patientderived pluripotent stem cells can provide relevant insights into ND pathophysiology.b 3D culture-on-a-chip combines the in vivo relevant characteristics of patient-derived 3D cultures (neurospheroids, organoids, and assembloids) and the unique engineering possibilities of microfluidic chips that can model interconnected brain regions and multi-organs.c Microfluidic-based, patientderived in vitro ND models will contribute to drug discovery and preclinical drug development studies by replacing the animal models with the promise of personalised treatment.ND neurodegenerative disease.
. Initial defects in brain vessels may initiate various interconnected pathways such as . Over 70% of familial ALS cases . The progressive and the presence of agedependent behavioural phenotypes that can be compared with

Table 3 |
Summary of the amyotrophic lateral sclerosis-on-chipsAst astrocyte, C close, CHR chamber, D dynamic, ESC embryonic stem cell, G gradient, h human, hiPSC human-induced pluripotent stem cell, MC microchannel, MN motor neuron, NMJ neuromuscular junction, O open, P primary, pCoNeu primary cortical neuron, Pt patient, S static.