Acitretin reverses early functional network degradation in a mouse model of familial Alzheimer’s disease

Aberrant activity of local functional networks underlies memory and cognition deficits in Alzheimer’s disease (AD). Hyperactivity was observed in microcircuits of mice AD-models showing plaques, and also recently in early stage AD mutants prior to amyloid deposition. However, early functional effects of AD on cortical microcircuits remain unresolved. Using two-photon calcium imaging, we found altered temporal distributions (burstiness) in the spontaneous activity of layer II/III visual cortex neurons, in a mouse model of familial Alzheimer’s disease (5xFAD), before plaque formation. Graph theory (GT) measures revealed a distinct network topology of 5xFAD microcircuits, as compared to healthy controls, suggesting degradation of parameters related to network robustness. After treatment with acitretin, we observed a re-balancing of those network measures in 5xFAD mice; particularly in the mean degree distribution, related to network development and resilience, and post-treatment values resembled those of age-matched controls. Further, behavioral deficits, and the increase of excitatory synapse numbers in layer II/III were reversed after treatment. GT is widely applied for whole-brain network analysis in human neuroimaging, we here demonstrate the translational value of GT as a multi-level tool, to probe networks at different levels in order to assess treatments, explore mechanisms, and contribute to early diagnosis.

Immunohistochemistry. For each genotype/condition 3 slices of 3 animals were analyzed. An additional slice was used as a control and only treated with secondary antibodies (Supplementary Fig. 4). Layers II/III, layer IV and layers V/VI were detected in each slice using DAPI staining. Slices were washed 3 times for 5 min in 1 × PBS before they were incubated in blocking solution (20% BSA, 0.2% Triton in 1 × PBS) for 2 h at RT. The solution was exchanged with fresh blocking solution (10% BSA, = 0.2% Triton in 1 × PBS) including primary antibodies for 48 h. Refer to SI for details on antibodies and concentrations used (Table S4).
Significance statement. Targeting early network dysregulations prior to irrevocable neurodegeneration might represent a promising therapeutic approach in Alzheimer's disease (AD). Here, we applied graph theoretical approaches to identify these dysregulations in a mouse model of AD, prior to neurodegeneration and plaque formation. We find a distinct network pathology characterized by a significant degradation of parameters related to the network's robustness and resilience in cortical microcircuits. Notably, upon treatment with the clinically available retinoid acitretin, deterioration of network topology measures, and structural synapse numbers was reversed, alongside an amelioration of behavioral dysfunction. This study may lay the foundation for neurophysiologically-informed, network-centered therapeutic interventions, aiming for stabilizing network function and delaying disease progression.

Results
Aβ deposition and behavioural deficits in early-stage 5xFAD mice. In 5xFAD mice, it has been shown that Aβ depositions first appear in deep layers of the cortex and the subiculum, to later spread towards other cortical regions and hippocampus 37 . We compared Aβ deposition in cortex and hippocampus of 5xFAD mice and wild type littermates (WT) at 4 months of age (Fig. 1A). In cortical tissue, soluble peptides only represented a fractional amount and plaque-associated peptides still did not reach high levels (1.9 ± 0.7 vs 422.6 ± 118.1 ng/g; Fig. 1B, top row). Whereas in hippocampus, soluble and insoluble Aβ 42 peptides both were detected at higher, similar levels (Fig. 1B, bottom row; p = 0.0113 for comparison of soluble A-beta in both tissue specimen; p = 0.0077 for comparison of Formic Acid (FA) extracted A-beta in both tissue specimen).
These results are consistent with the fact that first signs of memory loss are found at 4 months of age in 5xFAD mice (e.g., 43 ). However, these animals are still unimpaired in sensory-motor abilities, and it known that neuronal loss occurs much later 44 . These findings led us to choose 4 months as a model of early-stage AD in 5xFAD mice for analyzing early changes in visual cortex network dynamics and their potential restoration by acitretin treatment. 5xFAD mice display aberrant temporal distribution of calcium transients in a small portion of visual cortex. To investigate microcircuit network dynamics with single-cell resolution, we performed two-photon microscopy in layer II/III of the visual cortex in 5xFAD and WT mice ( Fig. 2A). Upon bolus loading with the synthetic calcium indicator Oregon Green BAPTA-1AM (OGB-1; 45 ), we labeled neuronal somata in WT ( Fig. 2B; left panel) and 5xFAD animals (Fig. 2 B; right panel; note that cortical plaques would be clearly visible, as they are stained by OGB-1 14 ). We recorded ongoing activity in the lightly anesthetized mouse 20,21 and deconvolved the calcium transients (using Online Active Set method to Infer Spikes OASIS 46 , for details see SI Materials and Methods and Supplementary Fig. 1) for WT controls (Fig. 3A,B) and 5xFAD animals (Fig. 3C,D). We observed bursting patterns in a small fraction of neurons in the 5xFAD animals (Fig. 3C,F). Those bursts of activity were notably distinctive from the sparse appearance of calcium transients observed in the WT controls ( Fig. 3E (WT), 3 F (5xFAD)). We quantified this phenomenon by analyzing Inter Event Intervals (IEI) of calcium transients, where bursting neurons should be represented by shorter intervals. Indeed, the IEI histogram showed an enhanced proportion of shorter IEIs in the histogram for 5xFAD as compared with WT controls (Fig. 3G, two-sample Kolmogorov-Smirnov = 0.0795, p = 4.47xE-140). We further analyzed the amplitudes of those calcium transients, and found lower amplitudes for 5xFAD mice than for WT controls (Chi-sq(2) = 563.32, p = 4.7589*E-123, mean ranks WT = 17,629, 5xFAD = 14,438, Supplementary Fig. 2).
Neuronal network dynamics de-organize and lose robustness in cortical microcircuits of 5xFAD mice. We next assessed network dynamics of cortical microcircuits with graph theoretic measures ( Fig. 4A,B). Indeed, whereas visual capability of 5xFAD mice does not seem to be impaired at this stage (measured by visual discrimination test, data not shown), complex network measures of the imaged assemblies already differed between 5xFAD and WT. Networks are described in terms of nodes and links 29 . We considered neuronal somata as network nodes, and their functional association (in the form of correlations) as network links. We assessed these functional profiles, derived from their calcium transients' functional activity, in terms of different network parameters, which are   Fig. 4C). We focused on degree distribution as an important marker of network development and resilience 29,48 and thus a crucial differential marker between complex neural networks in health and disease. We observed a lower mean degree distribution in the 5xFAD group compared with WT control group ( Acitretin rescues network patterns and restores early behavioral deficits in 5xFAD mice. Acitretin is a synthetic retinoid used in humans for treatment of psoriasis 50 . Re-purposing approaches for acitretin as a candidate AD drug revealed promising results in in vitro studies 38 . Its potential as an inducer of the non-amyloidogenic alpha-secretase ADAM10 has been subsequently demonstrated in primary cells, AD model mice, and a pilot study in human patients ( 38,39,51 ; Fig. 5A), indicating efficacy of the drug in mild to moderately affected patients 51 . A probable positive impact of acitretin during early stages of AD prior to plaque deposition and neuronal loss has not been investigated so far. We explored the effect of short-term acitretin treatment ( Fig. 5B) on behavioral performance as well as on cortical microcircuit network dynamics. Acitretin treatment significantly improved behavioral impairments assessed by the water maze in 5xFAD mice, almost reaching wild type levels for delta latency (5xFAD + Ac = − 36. Observation of the cortical activity pattern in acitretin-treated mice reveals regular and sparse patterns of calcium transients (Fig. 5E,F), resembling calcium transient patterns of WT controls (cp. Fig. 3A,B), and absence of the aberrant firing patterns observed in 5xFAD (Fig. 3C,F). Furthermore, the distributions of IEIs for the treatment group differed significantly from 5xFAD and were undistinguishable from WT controls (Kruskal-Wallis H test, Chi-sq(2) = 57.48, p = 3.2938xE-13, mean ranks 5xFAD + Ac = 83553.28, WT = 84099.84, 5xFAD = 86016.23). The calcium transients' rate, however, was downregulated for the group treated with acitretin ( Supplementary  Fig. 3C).
Regarding network analysis measures, acitretin-treated 5xFAD mice diverged significantly from untreated 5xFAD animals, and showed values closer to WT controls, in the parameter mean degree distribution, critical for network resilience and development. For the networks recorded in the group treated with acitretin, mean degree distribution was significantly higher from the 5xFAD, and approached control values (Kruskal-Wallis H test, Chi-sq(2) = 18.42, p = 0.0001, mean ranks 5xFAD + Act = 32.75, WT = 39.45, 5xFAD = 17.22; Fig. 5G,H).
To decipher how acitretin might achieve stabilized network activity, we subsequently analyzed parameters indicating neuronal signaling, synaptic activity, and immune reaction within visual cortex (Fig. 6). Interestingly, nitric oxid production was elevated by acitretin administration (Fig. 6A) even if the synthesizing enzyme nNOS was not increased on protein level. At this comparably young age, no significant astrogliosis was detectable in the visual cortex, as indicated by GFAP quantification (Fig. 6B). Also IbaI and NFкb p65 levels were not altered; GluR1 as a representative of neurotransmitter receptors was also not affected. Lastly, we also assessed the amount Excitatory synaptic density in 5xFAD is increased in layer II/III only, restored by acitretin treatment. The altered firing patterns could be underpinned by changes in synaptic densities, and said changes may, or not, be layer specific. To contrast this hypothesis, we conducted immunohistochemical stainings of V1  www.nature.com/scientificreports/ coronal sections with antibodies for all experimental groups (Fig. 7A-H), demarcating excitatory (anti-VGlut1, anti-Homer1) and inhibitory synapses (anti-VGat, anti-Gephyrin). Importantly, we found a selective increase in the density of excitatory synapses (Fig. 7I,K) in layer II/III only. Excitatory synapses in other layers (Fig. 7K, Supplementary Fig. 5) and inhibitory synapses in all layers analyzed were not affected, neither by the genotype, nor by acitretin treatment (Fig. 7J,L). Moreover, the increase in excitatory synapse levels in layer II/III was completely restored upon acitretin treatment. This finding suggests, that indeed, the dysregulation of the functional network in layer II/III is accompanied by changes in excitatory synapse numbers. Notably, layer II/III integrating intracortical connections appears to be considerably plastic in this context, compared to the other cortical layers.

Discussion
For microcircuit data, graph theoretical approaches have not been applied, mostly due to restrictions on the generalization of certain network parameters across individuals. Arguably, some measures of local integration have different interpretations at microcircuit level, as for whole brain analysis to which graph theory has classically been applied. However, it is possible to characterize network parameters for each local network imaged, and subsequently compare indexes among groups in order to establish differences in network integration. In this study, we explored ongoing neuronal interaction dynamics in layer II/III of V1 in a mouse model resembling amyloid pathology of familial AD, both on the single-cell and the topological level. Since we investigated animals prior to the onset of pronounced cortical plaque deposition, neuronal network qualities should not reveal changes due to mere loss of neurons but instead due to early functional changes. We therefore applied graph theory to probe network vulnerability and impairment in an early stage of AD.

Complex network measures capture early functional disruption in AD.
In human AD patients, apparent cognitive deterioration begins when a large number of neurons has already been lost 52 , leaving clinicians little time to attempt treatment. A plausible explanation for this notably initial robustness of the system to sustained neuronal loss is the capability of the neuronal network to supply the function of lost neurons by distribution among other nodes, i.e., unaffected neuronal ensembles present in the network 29 . Following this rationale, any behavioural impairment must be preceded by subtle network disruptions which, if detected, could carry diagnostic value. Thus, it is relevant to characterize network dynamics in search of signs of early disruptions, explore the networks' robustness in regards to vulnerability to damaged nodes, and further assess whether they correlate with pre-clinical symptoms. In our study, we found only one of the network indexes to be altered in 5xFAD mice V1. This suggests that at this early stage, only subtle changes of neuronal communication modes become apparent, in accordance with the finding that loss of pyramidal neurons in 5XFAD models does not commence until 9-12 months of age 44,53 . It is plausible that mean degree distribution would have been more sensitive than other markers (i.e. assortativity coefficient). Considering that distributions of values can constitute a more global description of the network, with their central measurements acting as a more stable marker of a network feature, in this case, network resilience 29,54 . Few neurons exhibited bursting calcium-transient patterns in the AD model, captured by their IEI distributions. The minimal proportion of cells with burst-like aberrant activity might play a key role in the distortion of neuronal communication, possibly by enhancing noise beyond functional levels, thereby impacting higher level network dynamics. Graph theory predicts scale-free topologies including hubs (i.e., highly interconnected neurons) to be an effective design to organize network communication 55 . With our approach we can only image the present state of the recorded neurons' functional network as their connection degrees are defined dynamically as correlation. The current state of a neuron would not inform us about its past functional role, given that the correlation with other neurons eventually has changed and a neuron that was a highly linked hub neuron before neurodegeneration, might cease to be a hub after functional collapse. Aberrant firing patterns have more detrimental effects on network communication if the abnormal cell is a hub neuron with a high number of connections (degree). Arguably, if a fraction of those neurons would exhibit the aberrant patterns we observed in our data, their impact on network dynamics would be amplified by their central role in network communication. Proposed action-mechanism of acitretin. Acitretin itself was reported not to act as a direct ligand to retinoic acid receptors. However, it displaces retinoic acid from its cytoplasmic binding proteins (CRABP) and thereby increases intracellular availability of this bioactive ligand. Retinoic acid has been shown to exert different cellular mechanisms with potential benefit for AD. (B) Schematic of acitretin treatment (5 days of daily injection, 2 days of rest, 2 days of daily injections, imaging/behavioural testing). (C) Swim paths of representative animals of the 5xFAD group treated with acitretin, on day 1 and 4 of Morris water maze show improvement in finding the hidden platform after learning. (D) Differences between distance (left) and latencies to reach the hidden platform (right) for WT and 5xFAD animals in Morris water maze for WT, 5xFAD, and 5xFAD + Acitretin (Ac) groups. Treated 5xFAD mice do not differ significantly from WT animals (n = 5 animals per group). (E) Representative traces of 10 neurons from 5xFAD group treated with acitretin; ongoing activity in layer II/III neuronal assemblies in V1 cortex. (F) Raster plot from entire cell population from which selection in (E) was drawn shows calcium transient pattern across whole imaging session. (G) Network internal connectivity in the 5xFAD group treated with acitretin (n = 5 mice). Connections with higher than 50% of connection strength are depicted. (H) Modular organization of same network as in (G) in the acitretin-treated group. Neuronal correlations sorted by modular organization with overlapping modules (purple). Note that both correlation distributions and approximate size and number of modular organization resemble those observed in WT group (Fig. 4A). Relevance of ongoing activity to study neuronal network dynamics. Keeping neuronal ensembles in a constant and stable state is a fundamental condition for the interpretation of network analysis parameters to be meaningful. We chose the lightly sedated state to reduce physiological noise and keep the network's intrinsic state constant as during awake periods neural networks undergo constant state fluctuations [56][57][58] . These changes occur randomly during wakefulness as patterns of activity vary on slow and rapid time scales, shaping the ongoing signal 58,59 . Such moment-to-moment fluctuations during waking 58,60 would not necessarily influence the aberrant firing of neurons, but the rest of the network, adding multi-dimensional activity 56 . As recently, findings of early hyperactivity in a mouse model of Huntington´s disease in this controlled state 20 have been recapitulated in awake animals 61 , we opted for the lightly sedated condition as the basis for graph theoretical measures.
Acitretin reverses network measures, protein expression and synapse densities in 5xFAD mice. Subchronic treatment of 5xFAD mice with acitretin stabilized abnormal network activity with regard to firing patterns and distributions of mean IEI, and reversed behavioural performances in MWM. Aβ increases spontaneous neuronal activity early during disease progression in the absence of amyloid plaques or neurofibrillary tangles 62 . We previously reported that single stereotactic application of acitretin in another AD mouse model (APP/PS1) was sufficient to re-balance APP processing with a strong reduction of Aβ 38 . By activating ADAM10 transcriptional activity, the alpha-secretase that prevents Aβ synthesis 63 , acitretin is able to lower A-beta production 43 . The amount of soluble Aβ was comparably low in the cortex of 5xFAD mice at the investigated time point. Small reductions in Aβ synthesis might still be beneficial in an early state, while in a pronounced pathological state, efficacy of ADAM10-enhancement by such subtle changes might be not sufficient to have beneficial effects. Another possible mechanism for the reversal effects in the treated 5xFAD animals might be due to elevation of IL-1 64 or IL-6 65 evoked by acitretin. The activation of a cytokine network in the brain might play an important role in plasticity and can be relevant for LTP maintenance, for example an overexpression of IL-6 might be able to trigger the outgrowth of new fibers 66 . Acitretin itself is not a ligand to retinoic acid receptors. It has been shown to replace retinoic acid from intracellular binding proteins and thereby increase the available pool of this bioactive compound 67 . Several studies found an increase of neuroprotective gene expression by retinoic acid receptors: e.g., elevated expression of neurotrophic tyrosine kinases 1 and 2 in human neuroblastoma cells 43 . Administration of β-carotene, the precursor of retinoic acid, in an autism mouse model increased BDNF concentration 68 . More recently, homeostatic synaptic plasticity at inhibitory synapses in murine visual cortical circuits has been shown to depend on retinoic acid signaling indicating a potential inhibitory mode of action 69 . We did not observe increased amounts of GluR1 in the cortex. However, nitric oxide levels were increased by acitretin treatment. NO/cGMP signaling participates in synaptic plasticity in the visual cortex 70 and. another synthetic retinoid receptor ligand -AM-80 -linked retinoid signaling via NO to ERK-dependent BDNF synthesis in midbrain dopaminergic neurons 71 . We observed an increase of this neurotrophic factor in visual cortex tissue. BDNF suppresses responses to exogenously added glutamate in slices from rat visual cortex 72 and promotes development of dendrites and synapses of cultivated rodent V1 GABAergic neurons 73 . These factors, taken together might explain the restoration of homeostatic network conditions we observed for acitretin-treated animals.
Notably, acitretin was capable of rebalancing the increased density of excitatory synapses specifically in layer II/III of visual cortex in 5xFAD mice. Previous work suggested maladaptive synaptic plasticity changes to be related to network dysregulations in mouse models of relapse-remitting MS 20 . While the pathophysiological mechanisms of relapse-remitting MS and early stage AD are distinct, it is plausible that the early dysregulations in cortical regions we observed, represent a plasticity driven phenomenon. This notion is supported by the fact that acitretin treatment restored excitatory synapse counts, alongside with rebalancing network parameters and ameliorating behavior. Hence, acitretin seems to be a valuable therapeutic approach in the early stage of AD.

Network-based analyses of cortical microcircuits indicate treatment-related functional restoration in early-stage neurodegenerative disorders.
Increasing evidence points to the particular importance of studying cortical network dynamics in early stages of neurodegenerative and neuroimmunological disorders as diverse as Huntington's disease 20 , AD 18 , and relapse-remitting MS 21 . Importantly, the first studies of cortical neuronal assemblies in a mouse model of AD described hyperactivity in the late stage of the disease, dependent on the spatial proximity to plaques 12 . These individual hyperactive neurons most likely change their activity state due to the cytotoxic environment. Here, we describe a distinctively different, early, type of network dysregulation, which might be closely related to (mal-) adaptive changes in the network. Indeed, the selective increase in excitatory synapse densities in layer II/II which could be rebalanced by acitretin treatment points to these early, subtle changes to be of different origin than the later changes. While the cortex is not the first brain region affected by the molecular pathology in neither of these disorders, it seems to react in a maladaptive fashion prior to the onset of neuronal loss and apparent protein aggregates or T-cell infiltrations. Our choice to image neuronal ensembles in V1 was driven by the evidence reported by other groups showing that visual cortex represents an area which is compromised in AD progression directly after medial entorhinal cortex, so relatively early on during the disease 74 www.nature.com/scientificreports/ target of preventive therapies. Our study suggests that using computational methods dedicated to the analysis of cortical network interactions, in combination with the application of therapeutic targets, might provide new ways of understanding the local cortical network as a pathophysiological entity. Network analyses as used here, rely on information related to response amplitude of calcium transients, which are highly non-linear in GCaMP-based measurements. For synthetic calcium indicators as OGB-1 fluorescence changes from successive action potentials combine nearly linearly 76,77 and can be inferred by deconvolution (e.g. 78,79 ). The same inferences remain challenging for imaging data based on genetically encoded calcium indicators as the associated responses are complex, nonlinear and variable over neurons [80][81][82] . This nonlinearity and variability can be explained by GCaMP's four calmodulin-derived binding sites, dependence of fluorescence amplitude and shape of transients on total GCaMP concentration depending on expression, as well as by its lower dynamic range compared to OGB-1 81 . The neuronal networks in our case are functional, as opposed to structural networks. In a functional network, the edges or connections between the (neuronal) nodes are defined by a measure of functional association, commonly the Pearson's product-moment correlation coefficient, which captures the strength and direction of a monotonical relationship between two variables, in this case neuronal activity levels.
Graph theory is commonly used for gathering information about interactions of whole-brain networks by treating brain regions as nodes, and highly interconnected areas as hubs. Employing the same analytic approach on the microcircuit level allows to monitor the influence of disturbances in the temporal distribution of single-cell activity on the topology of the local network. In contrast to studies in late stage AD 12 , we did not find a "hyperactivity phenotype" close to plaques, but a rather complex dysregulation of local neuronal networks, potentially underpinned by synaptic density. In contrast to human brain network modelling, a node in the local neuronal network index the activity of an individual neuron. Abnormal activity of individual cells eventually leads to a breakdown of the higher-order interactions in the local network which might serve as the seed of brain-wide functional degradation. Further investigation could shed light on the "tipping-point" where local networks break down of too many components exhibit aberrant activity. A neurodegenerative disease is a complex collection of events at multiple levels. Combining graph theoretical analysis, promising drugs such as acitretin, and synaptic density counting, holds diagnostic and therapeutic value. Early intervention yields the opportunity to restore cortical network dynamics while the disease is still in its prodromal stage, thereby granting valuable time to attempt further treatment.