Cholesterol imbalance and neurotransmission defects in neurodegeneration

The brain contains the highest concentration of cholesterol in the human body, which emphasizes the importance of cholesterol in brain physiology. Cholesterol is involved in neurogenesis and synaptogenesis, and age-related reductions in cholesterol levels can lead to synaptic loss and impaired synaptic plasticity, which potentially contribute to neurodegeneration. The maintenance of cholesterol homeostasis in the neuronal plasma membrane is essential for normal brain function, and imbalances in cholesterol distribution are associated with various neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. This review aims to explore the molecular and pathological mechanisms by which cholesterol imbalance can lead to neurotransmission defects and neurodegeneration, focusing on four key mechanisms: (1) synaptic dysfunction, (2) alterations in membrane structure and protein clustering, (3) oligomers of amyloid beta (Aβ) protein, and (4) α-synuclein aggregation.


INTRODUCTION
Cholesterol is a lipid that is critical for the structure and function of cell membranes in the brain, where it participates in neuronal signaling and synaptic transmission.The brain contains the highest concentration of cholesterol in the human body, accounting for 20-25% of the total cholesterol 1,2 .The blood-brain barrier (BBB) is impermeable to peripheral cholesterol 3 ; therefore, most cholesterol in the brain is generated by de novo synthesis, mainly in the glia and to a lesser extent in neurons 3 .The high cholesterol concentration suggests an important role for cholesterol in brain physiology.
Cholesterol is involved in neurogenesis and synaptogenesis 4,5 .Age-related reductions in cholesterol levels in the plasma membrane lead to synaptic loss 6,7 and impaired synaptic plasticity 8 , suggesting that cholesterol imbalance in the neuronal plasma membrane affects neuronal activity and contributes to neuronal degeneration 9,10 .The maintenance of cholesterol homeostasis is essential for normal brain functions [11][12][13] .An imbalance in cholesterol distribution can cause the pathological changes observed in various neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), suggesting that neurodegenerative diseases are associated with dysregulated cholesterol distribution 11,12,14 .
The aim of this review is to examine the molecular and pathological mechanisms by which cholesterol imbalance causes neurotransmission defects.This review focuses on four molecular mechanisms to explain how cholesterol imbalance in neurons results in neurodegeneration: (1) synaptic dysfunction, (2) membrane structure and protein clustering, (3) amyloid beta (Aβ) aggregation, and (4) α-synuclein (α-syn) aggregation.

MAIN TEXT
The molecular mechanisms of neurodegeneration induced by cholesterol imbalance Cholesterol has a complex and multifaceted role in neurodegeneration.Given that cholesterol is an essential component of cell membranes and is involved in various physiological processes in the brain, an imbalance and dysregulation of cholesterol homeostasis can contribute to the pathogenesis of neurodegenerative diseases 11,12,14 .Table 1 summarizes the links between cholesterol and different neurodegenerative diseases.Several mechanisms have been proposed to explain how cholesterol imbalance may contribute to neurodegeneration: 1. Synaptic dysfunction: cholesterol is critical for the formation and function of synapses, the connections between neurons that facilitate communication in the brain.Altered cholesterol levels can affect synaptic transmission and plasticity, impairing neuronal signaling and contributing to the cognitive deficits observed in neurodegenerative diseases.The plasma membrane is enriched with cholesterol, ~80% of which is cellular cholesterol 15 .Therefore, reduced cholesterol levels in the plasma membrane, i.e., cholesterol imbalance, impairs synaptic transmission and plasticity and thus induces neurodegeneration 9,10,16 .Depletion and imbalance of cholesterol in the plasma membrane cause deficits in neurotransmission; e.g., cholesterol depletion reduces Ca 2+ -dependent exocytosis of large dense-core vesicles (LDCVs) 17 , cortical secretory vesicles 18 , and synaptic vesicles in hippocampal neurons 9,19 , cortical synaptosomes 20 , ribbon synapses 21 , and motor nerve terminals 22 .However, unveiling the molecular pathology of cholesterol imbalance in neurodegeneration is challenging because cholesterol is involved in various cellular signaling processes and neuronal functions.The reconstitution system of vesicle fusion with purified native vesicles, including LDCVs and synaptic vesicles, can be a good model for elucidating the molecular mechanisms by which cholesterol deficiency affects vesicle fusion 23 (Fig. 1).This reconstitution of vesicle fusion shows that cholesterol has little effect on Ca 2+ -independent basal fusion of synaptic vesicles but is required for Ca 2+ -dependent fusion of LDCVs and synaptic vesicles 23 .It is surprising that cholesterol reduction and imbalance specifically disrupt Ca 2+ -dependent vesicle fusion.Cholesterol has no effect on the membrane binding or insertion of synaptotagmin-1, a   Ca 2+ sensor for vesicle fusion 23 .Once synaptotagmin-1 is inserted into the membrane, cholesterol stabilizes and strengthens the local membrane bending and deformation induced by synaptotagmin-1 23 .The membrane is highly flexible, so it can reform into its original shape due to membrane elasticity 24 .Because cholesterol reduces membrane fluidity and increases membrane rigidity 25 , cholesterol can strengthen local membrane deformation and bending, thus lowering the energy barrier for Ca 2+ -dependent fusion 23 (Fig. 1).Membrane bending and curvature play crucial roles in the process of vesicle fusion by lowering the energy barrier 26 .The energy stored in the curvature of the membrane can be released to facilitate the merging of two separate lipid bilayers [26][27][28] .For instance, smaller vesicles, which have a greater curvature, have greater bending energy per unit surface area, leading to a more efficient fusion process 26 .The insertion of proteins such as the C2AB domain of synaptotagmin-1 into the plasma membrane contributes to this curvature 29,30 , creating a high-energy state that can drive vesicle fusion.
Cholesterol enhances membrane curvature and thus lowers the energy barrier for fusion 18 .It also strengthens local bending and deformation, particularly in the presence of Ca 2+ and synaptotagmin-1, thereby driving Ca 2+dependent vesicle fusion 23 .Mechanical forces of membrane bending are critical for the dynamic process of vesicle fusion 31 , and cholesterol is an essential lipid for synaptic transmission because it strengthens membrane bending 23 .
Synaptic transmission involves the release of neurotransmitters from synaptic vesicles into synapses, where neurotransmitters bind to receptors on postsynaptic neurons, thereby transmitting signals across the neural network 32 .When synaptic transmission is impaired due to disruptions in Ca 2+ -dependent vesicle fusion, neural network formation becomes impaired 33 .Disruption of Ca 2+ -dependent vesicle fusion and synaptic transmission via a cholesterol imbalance in the plasma membrane leads to reduced neural network activity and synaptic dysfunction and ultimately contributes to neurodegeneration.The loss of the neural network caused by the dysregulation of cholesterol homeostasis can result in declines in cognitive and motor functions associated with neurodegenerative diseases.2. Membrane structure and protein clustering: cholesterol regulates membrane structure, fluidity, and curvature 25 .The plasma membrane is enriched in cholesterol 15 , which stabilizes membrane curvature and promotes vesicle fusion 17,25,34,35 .The membrane curvature and deformation stabilized by cholesterol bring the two membranes close together and enable fusion.Cholesterol also contributes to vesicle fusion by stabilizing fusion pores 25,[36][37][38] .Therefore, cholesterol deficiency in neurons causes defects in membrane structure, resulting in neurodegeneration.
Cholesterol also mediates the protein clustering involved in vesicle fusion.Exocytosis of neurotransmitter release is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins 39,40 .Neuronal SNARE proteins consist of Q-SNARE in the plasma membrane (syntaxin-1 and SNAP-25) and R-SNARE in the vesicle membrane (synaptobrevin-2 or vesicle-associated membrane protein-2 (VAMP-2)) 39 .Specialized microdomains within the plasma membrane, e.g., lipid rafts, detergentresistant membranes, or liquid-ordered membrane microdomains, are enriched in cholesterol and concentrate signaling molecules [41][42][43] .Cholesterol plays an important role in the function and organization of SNARE proteins; syntaxin-1A, a neuronal SNARE protein, is concentrated in cholesterol-enriched domains in the plasma membrane 44,45 .
The cholesterol-enriched membrane microdomains provide a specialized environment where syntaxin-1A interacts with its binding partners.In the context of synaptic transmission, the clustering of syntaxin-1A in cholesterol-enriched microdomains may enhance its interactions with other SNARE proteins, such as SNAP-25 and VAMP-2, to form the core SNARE complex necessary for vesicle fusion.This organization may influence the overall stability and efficiency of neurotransmitter release at the synapse, suggesting that cholesterol imbalance leads to defects in the clustering of the vesicle fusion machinery.3. Oligomers of amyloid beta (Aβ) protein: cholesterol imbalance influences the aggregation and misfolding of proteins involved in neurodegenerative diseases, such as amyloid precursor protein (APP) 46 .The accumulation of Aβ plaques in the brain is a hallmark pathology of AD 47,48 .Aβ is derived from APP through enzymatic cleavage by the β-secretase Bace1 49 , and cholesterol modulates the processing of APP and the generation of Aβ 50,51 .High cholesterol levels can promote and accelerate the cleavage of APP by Bace1, resulting in increased Aβ production and aggregation, which contributes to the formation of toxic plaques [50][51][52] .Aβ40/Aβ42 peptides are the primary constituents of Aβ oligomers and plaques, which can be stabilized by biomolecules, including carbohydrates, nucleic acids, and lipids, e.g., cholesterol 53 .Extracellular cholesterol strengthens Aβ fibrils and oligomers against degradation by directly binding to Aβ 53,54 .Cholesterol has been implicated in the aggregation of Aβ peptides, particularly in the formation of Aβ oligomers [55][56][57] .Free cholesterol interacts with specific residues in Aβ peptides, particularly Phe19 55 .Cholesterol binds to the aromatic side chains of the Aβ peptide, thus increasing β-sheet formation in Aβ peptide oligomers 55 .A stable interaction between cholesterol and Phe19 leads to the formation of Aβ oligomers 55 , suggesting that the interaction of cholesterol with Aβ contributes to the formation of toxic Aβ oligomers, which play a critical role in AD pathology 57 .
Cholesterol dramatically enhances and accelerates the onset of Aβ42 aggregation through a heterogeneous nucleation pathway 58 .Cholesterol imbalance and elevated extracellular levels of cholesterol can promote the production and accumulation of Aβ peptides, which induce the formation of Aβ oligomers in the brain, thus contributing to neuronal damage and cognitive decline 57 (Fig. 2).Aβ monomers misfold and form β-sheet-rich oligomers that eventually impair synaptic plasticity and neuronal survival 59 .The direct interaction of cholesterol with Aβ can stimulate and activate toxic Aβ oligomerization, which is a critical factor in AD pathogenesis.
The membrane curvature induced by cholesterol might contribute to Aβ aggregation 60 .High membrane curvature promotes Aβ nucleation, accelerating amyloid fibril formation 61 .Aβ aggregates can readily form on membranes with high curvature 62 .High membrane curvature, such as that associated with lipid rafts and membrane budding processes, might provide favorable conditions for the nucleation of Aβ peptides [61][62][63] .Given that cholesterol stabilizes high membrane curvature, cholesterol-mediated curvature of membranes may lead to altered lipid packing that engages Aβ hydrophobic groups and promotes Aβ fibrillar structures 64 .Lipid packing defects that occur in curved membranes may induce conformational changes in Aβ peptides, thus promoting Aβ fibrils and aggregation 64 .This aggregation is a hallmark of AD, and understanding the underlying mechanisms of membrane curvature is crucial for developing potential therapeutic strategies.The interplay between membrane curvature and Aβ aggregation is complex, and ongoing research continues to unveil the molecular details involved.4. Tau aggregation: while Aβ oligomers and aggregation are primarily associated with AD, another hallmark of AD is the aggregation of hyperphosphorylated tau proteins into neurofibrillary tangles (NFTs) 65 .Tau is a cytoskeletal protein that stabilizes microtubules in neurons, but it is hyperphosphorylated in AD 66 .The interaction between tau proteins and cell membranes, particularly in cholesterol-rich regions, is a critical factor in the tau aggregation process 67,68 .The membrane binding of tau can induce conformational changes and aggregation with β-sheet-rich structures 69 .NFTs in AD brains contain cholesterol 54,70 , which could modulate tau-membrane interactions and affect tau aggregation 68,71 .
As cholesterol can influence membrane curvature, high membrane curvature caused by cholesterol can induce changes in the conformation of tau proteins and promote tau aggregation 72 .Membranes with high curvature that contain cholesterol induce tau fibril formation, whereas cholesterol depletion abolishes tau fibril formation 72 .Cholesterol-free membranes fail to induce the formation of tau fibrils, suggesting that cholesterol-mediated tau aggregation is essential for the pathology of tauopathies 72 .Membrane morphologies result in different hydrophobic interactions that lead to the β-sheet structures of tau proteins 72 .The association of tau with highly curved membranes is initiated by electrostatic attraction between the Lys sidechains of tau and the lipid headgroups; cholesterol might further strengthen this electrostatic attraction for tau fibril formation 72 .However, how cholesterol facilitates tau nucleation remains a topic of further study, and understanding the underlying molecular mechanisms is crucial for developing therapeutic strategies involving the disruption of tau aggregation.5. α-Synuclein (α-syn) aggregation: a characteristic feature of PD is the accumulation of misfolded α-syn proteins in Lewy bodies (LBs) 73 .The interaction between α-syn and lipids is important for fibril formation, and the aggregation of α-syn is induced by binding to membrane lipids 74 .Lipids dramatically enhance the primary nucleation of α-syn to form aggregates associated with neurodegeneration 74 .
Together with α-syn, cholesterol, which is a component of LBs 75 , accelerates α-syn aggregation and LB formation 76 .Cholesterol interacts with α-syn, and high cholesterol levels can promote αsyn aggregation 76,77 , leading to the formation of toxic LBs, which contributes to neurodegeneration in PD.
α-Syn binds to membranes through electrostatic interactions and hydrogen bonding, but cholesterol reduces the coulomb interactions and hydrophobic interactions between α-syn and membranes 78 .Cholesterol decreases lipid packing defects and lipid fluidity, thereby dysregulating the membrane binding of αsyn 78 ; membrane-bound α-syn can have a β-sheet structure that induces the formation of toxic α-syn oligomers and fibrils.Together, the imbalance and dysregulated distribution of cholesterol in neurons cause neurodegeneration by accelerating α-syn aggregation and LB formation.

Possible therapeutic approaches for cholesterol imbalance
The apolipoprotein E (ApoE) gene is involved in the metabolism and transport of cholesterol 79,80 .There are three main variants or alleles of the APOE gene, namely, ApoE2, ApoE3, and ApoE4 81 .The ApoE4 protein is the most important risk factor for late-onset AD 82 , i.e., the most common form of the disease that occurs after age 65.The molecular mechanisms by which ApoE4 contributes to AD are complex and not fully understood, but ApoE4 likely promotes Aβ aggregation by transporting cholesterol 79,83 .ApoE4 can induce cholesterol imbalance by transporting cholesterol from the plasma membrane in neurons to protein aggregates (Fig. 2).Given that cholesterol imbalance causes neurodegeneration, ApoE4 may be a possible target for mitigating Aβ aggregation and treating cholesterol imbalance.
Overall, the maintenance of cholesterol homeostasis is important for preventing or slowing the pathogenesis of neurodegenerative diseases.Strategies aimed at regulating cholesterol levels or targeting cholesterol-mediated pathways involved in protein aggregation might be potential therapeutic approaches to treating neurodegenerative diseases, although neurodegenerative diseases are complex and cholesterol imbalance is one of many contributing factors 11,84 .

CONCLUSION
Cholesterol is an essential component of the body that helps maintain the integrity of cell membranes.The role of cholesterol dysregulation in neurodegeneration is an active area of research, and the precise mechanisms involved may vary depending on the specific condition.The balance between the beneficial and detrimental effects of cholesterol remains complex.An imbalance in cholesterol regulation is a common feature of neurodegenerative conditions such as AD and PD.Therapeutic approaches for modulating cholesterol metabolism or targeting specific cholesterol-related pathways could be potential strategies for mitigating neurodegeneration.While cholesterol-lowering drugs, e.g., statins, have shown some potential in reducing the risk of certain neurodegenerative diseases, further research is required to fully understand the role of cholesterol and develop targeted therapeutic interventions.

Fig. 1
Fig.1Schematic illustration of the roles of cholesterol in Ca 2+ -dependent vesicle fusion.Cholesterol is essential for Ca 2+ -dependent vesicle fusion.Synaptotagmin-1, a Ca 2+ sensor that triggers fusion, induces local deformation of the plasma membrane.The plasma membrane is normally flexible and can return to its original shape due to membrane elasticity.However, cholesterol makes the membrane less fluid and more rigid, which helps to strengthen the membrane curvature and deformation, thus lowering the energy barrier for fusion.This image was created with BioRender.com.

Fig. 2
Fig. 2 Schematic overview of cholesterol transport to promote Aβ aggregation for neurodegeneration.Cholesterol enhances and accelerates APP cleavage by Bace1, leading to increased Aβ oligomer and plaque formation.Cholesterol binds to Aβ and increases the resistance of Aβ fibrils and oligomers to degradation.Cholesterol imbalance and high extracellular cholesterol levels can stimulate the production and accumulation of Aβ peptides, which cause Aβ oligomer formation and aggregation in the brain, resulting in neuronal damage.This image was created with BioRender.com.

Table 1 .
Cholesterol and neurodegenerative diseases.