Integrity of neural extracellular matrix is required for microglia-mediated synaptic remodeling.

Microglia continuously remodel synapses, which are embedded in the extracellular matrix (ECM). However, the mechanisms, which govern this process remain elusive. To investigate the influence of the neural ECM in synaptic remodeling by microglia, we disrupted ECM integrity by injection of chondroitinase ABC (ChABC) into the retrosplenial cortex of healthy adult mice. Using in vivo two-photon microscopy we found that ChABC treatment increased microglial branching complexity and ECM phagocytic capacity and decreased spine elimination rate under basal conditions. Moreover, ECM attenuation largely prevented synaptic remodeling following synaptic stress induced by photodamage of single synaptic elements. These changes were associated with less stable and smaller microglial contacts at the synaptic damage sites, diminished deposition of calreticulin and complement proteins C1q and C3 at synapses and impaired expression of microglial CR3 receptor. Thus, our findings provide novel insights into the function of the neural ECM in deposition of complement proteins and synaptic remodeling by microglia.

Focal clusters of peri-synaptic matrix contribute to activity-dependent plasticity and memory in mice.

Recent findings show that effective integration of novel information in the brain requires coordinated processes of homo- and heterosynaptic plasticity. In this work, we hypothesize that activity-dependent remodeling of the peri-synaptic extracellular matrix (ECM) contributes to these processes. We show that clusters of the peri-synaptic ECM, recognized by CS56 antibody, emerge in response to sensory stimuli, showing temporal and spatial coincidence with dendritic spine plasticity. Using CS56 co-immunoprecipitation of synaptosomal proteins, we identify several molecules involved in Ca2+ signaling, vesicle cycling, and AMPA-receptor exocytosis, thus suggesting a role in long-term potentiation (LTP). Finally, we show that, in the CA1 hippocampal region, the attenuation of CS56 glycoepitopes, through the depletion of versican as one of its main carriers, impairs LTP and object location memory in mice. These findings show that activity-dependent remodeling of the peri-synaptic ECM regulates the induction and consolidation of LTP, contributing to hippocampal-dependent memory.

Microvascular damage, neuroinflammation and extracellular matrix remodeling in Col18a1 knockout mice as a model for early cerebral small vessel disease.

Collagen type XVIII (COL18) is an abundant heparan sulfate proteoglycan in vascular basement membranes. Here, we asked (i) if the loss of COL18 would result in blood-brain barrier (BBB) breakdown, pathological alterations of small arteries and capillaries and neuroinflammation as found in cerebral small vessel disease (CSVD) and (ii) if such changes may be associated with remodeling of synapses and neural extracellular matrix (ECM). We found that 5-month-old Col18a1-/- mice had elevated BBB permeability for mouse IgG in the deep gray matter, and intravascular erythrocyte accumulations were observed brain-wide in capillaries and arterioles. BBB permeability increased with age and affected cortical regions and the hippocampus in 12-month-old Col18a1-/- mice. None of the Col18a1-/- mice displayed hallmarks of advanced CSVD, such as hemorrhages, and did not show perivascular space enlargement. Col18a1 deficiency-induced BBB leakage was accompanied by activation of microglia and astrocytes, a loss of aggrecan in the ECM of perineuronal nets associated with fast-spiking inhibitory interneurons and accumulation of the perisynaptic ECM proteoglycan brevican and the microglial complement protein C1q at excitatory synapses. As the pathway underlying these regulations, we found increased signaling through the TGF-ß1/Smad3/TIMP-3 cascade. We verified the pivotal role of COL18 for small vessel wall structure in CSVD by demonstrating the protein's involvement in vascular remodeling in autopsy brains from patients with cerebral hypertensive arteriopathy. Our study highlights an association between the alterations of perivascular ECM, extracellular proteolysis, and perineuronal/perisynaptic ECM, as a possible substrate of synaptic and cognitive alterations in CSVD.

Spatio-temporal dynamics of microglia phenotype in human and murine cSVD: impact of acute and chronic hypertensive states.

Vascular risk factors such as chronic hypertension are well-established major modifiable factors for the development of cerebral small vessel disease (cSVD). In the present study, our focus was the investigation of cSVD-related phenotypic changes in microglia in human disease and in the spontaneously hypertensive stroke-prone rat (SHRSP) model of cSVD. Our examination of cortical microglia in human post-mortem cSVD cortical tissue revealed distinct morphological microglial features specific to cSVD. We identified enlarged somata, an increase in the territory occupied by thickened microglial processes, and an expansion in the number of vascular-associated microglia. In parallel, we characterized microglia in a rodent model of hypertensive cSVD along different durations of arterial hypertension, i.e., early chronic and late chronic hypertension. Microglial somata were already enlarged in early hypertension. In contrast, at late-stage chronic hypertension, they further exhibited elongated branches, thickened processes, and a reduced ramification index, mirroring the findings in human cSVD. An unbiased multidimensional flow cytometric analysis revealed phenotypic heterogeneity among microglia cells within the hippocampus and cortex. At early-stage hypertension, hippocampal microglia exhibited upregulated CD11b/c, P2Y12R, CD200R, and CD86 surface expression. Detailed analysis of cell subpopulations revealed a unique microglial subset expressing CD11b/c, CD163, and CD86 exclusively in early hypertension. Notably, even at early-stage hypertension, microglia displayed a higher association with cerebral blood vessels. We identified several profound clusters of microglia expressing distinct marker profiles at late chronic hypertensive states. In summary, our findings demonstrate a higher vulnerability of the hippocampus, stage-specific microglial signatures based on morphological features, and cell surface protein expression in response to chronic arterial hypertension. These results indicate the diversity within microglia sub-populations and implicate the subtle involvement of microglia in cSVD pathogenesis.

Prenatal Cannabinoid Exposure Elicits Memory Deficits Associated with Reduced PSA-NCAM Expression, Altered Glutamatergic Signaling, and Adaptations in Hippocampal Synaptic Plasticity.

Cannabis is now one of the most commonly used illicit substances among pregnant women. This is particularly concerning since developmental exposure to cannabinoids can elicit enduring neurofunctional and cognitive alterations. This study investigates the mechanisms of learning and memory deficits resulting from prenatal cannabinoid exposure (PCE) in adolescent offspring. The synthetic cannabinoid agonist WIN55,212-2 was administered to pregnant rats, and a series of behavioral, electrophysiological, and immunochemical studies were performed to identify potential mechanisms of memory deficits in the adolescent offspring. Hippocampal-dependent memory deficits in adolescent PCE animals were associated with decreased long-term potentiation (LTP) and enhanced long-term depression (LTD) at hippocampal Schaffer collateral-CA1 synapses, as well as an imbalance between GluN2A- and GluN2B-mediated signaling. Moreover, PCE reduced gene and protein expression of neural cell adhesion molecule (NCAM) and polysialylated-NCAM (PSA-NCAM), which are critical for GluN2A and GluN2B signaling balance. Administration of exogenous PSA abrogated the LTP deficits observed in PCE animals, suggesting PSA mediated alterations in GluN2A- and GluN2B- signaling pathways may be responsible for the impaired hippocampal synaptic plasticity resulting from PCE. These findings enhance our current understanding of how PCE affects memory and how this process can be manipulated for future therapeutic purposes.

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy.

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

Golgi satellites are essential for polysialylation of NCAM and expression of LTP at distal synapses.

The complex cytoarchitecture of neurons poses significant challenges for the maturation of synaptic membrane proteins. It is currently unclear whether locally secreted synaptic proteins bypass the Golgi or whether they traffic through Golgi satellites (GSs). Here, we create a transgenic GS reporter mouse line and show that GSs are widely distributed along dendrites and are capable of mature glycosylation, in particular sialylation. We find that polysialylation of locally secreted NCAM takes place at GSs. Accordingly, in mice lacking a component of trans-Golgi network-to-plasma membrane trafficking, we find fewer GSs and significantly reduced PSA-NCAM levels in distal dendrites of CA1 neurons that receive input from the temporoammonic pathway. Induction of long-term potentiation at those, but not more proximal, synapses is severely impaired. We conclude that GSs serve the need for local mature glycosylation of synaptic membrane proteins in distal dendrites and thereby contribute to rapid changes in synaptic strength.

Brain microvascular disease and functional network connectivity-a call for a stage-based approach.

This scientific commentary refers to 'Functional connectivity in older adults-the effect of cerebral small vessel disease' by Drenth et al. (https://doi.org/10.1093/braincomms/fcad126).

Amisulpride as a potential disease-modifying drug in the treatment of tauopathies.

INTRODUCTION: Hyperphosphorylation and aggregation of the microtubule-associated protein tau cause the development of tauopathies, such as Alzheimer's disease and frontotemporal dementia (FTD). We recently uncovered a causal link between constitutive serotonin receptor 7 (5-HT7R) activity and pathological tau aggregation. Here, we evaluated 5-HT7R inverse agonists as novel drugs in the treatment of tauopathies. METHODS: Based on structural homology, we screened multiple approved drugs for their inverse agonism toward 5-HT7R. Therapeutic potential was validated using biochemical, pharmacological, microscopic, and behavioral approaches in different cellular models including tau aggregation cell line HEK293 tau bimolecular fluorescence complementation, primary mouse neurons, and human induced pluripotent stem cell-derived neurons carrying an FTD-associated tau mutation as well as in two mouse models of tauopathy. RESULTS: Antipsychotic drug amisulpride is a potent 5-HT7R inverse agonist. Amisulpride ameliorated tau hyperphosphorylation and aggregation in vitro. It further reduced tau pathology and abrogated memory impairment in mice. DISCUSSION: Amisulpride may be a disease-modifying drug for tauopathies.

A shift in the mechanisms controlling hippocampal engram formation during brain maturation.

The ability to form precise, episodic memories develops with age, with young children only able to form gist-like memories that lack precision. The cellular and molecular events in the developing hippocampus that underlie the emergence of precise, episodic-like memory are unclear. In mice, the absence of a competitive neuronal engram allocation process in the immature hippocampus precluded the formation of sparse engrams and precise memories until the fourth postnatal week, when inhibitory circuits in the hippocampus mature. This age-dependent shift in precision of episodic-like memories involved the functional maturation of parvalbumin-expressing interneurons in subfield CA1 through assembly of extracellular perineuronal nets, which is necessary and sufficient for the onset of competitive neuronal allocation, sparse engram formation, and memory precision.

TRPC4 Channel Knockdown in the Hippocampal CA1 Region Impairs Modulation of Beta Oscillations in Novel Context.

Hippocampal local field potentials (LFP) are highly related to behavior and memory functions. It has been shown that beta band LFP oscillations are correlated with contextual novelty and mnemonic performance. Evidence suggests that changes in neuromodulators, such as acetylcholine and dopamine, during exploration in a novel environment underlie changes in LFP. However, potential downstream mechanisms through which neuromodulators may alter the beta band oscillation in vivo remain to be fully understood. In this paper, we study the role of the membrane cationic channel TRPC4, which is modulated by various neuromodulators through G-protein-coupled receptors, by combining shRNA-mediated TRPC4 knockdown (KD) with LFP measurements in the CA1 region of the hippocampus in behaving mice. We demonstrate that the increased beta oscillation power seen in the control group mice in a novel environment is absent in the TRPC4 KD group. A similar loss of modulation was also seen in the low-gamma band oscillations in the TRPC4 KD group. These results demonstrate that TRPC4 channels are involved in the novelty-induced modulation of beta and low-gamma oscillations in the CA1 region.

Brain Vascular Health in ALS Is Mediated through Motor Cortex Microvascular Integrity.

Brain vascular health appears to be critical for preventing the development of amyotrophic lateral sclerosis (ALS) and slowing its progression. ALS patients often demonstrate cardiovascular risk factors and commonly suffer from cerebrovascular disease, with evidence of pathological alterations in their small cerebral blood vessels. Impaired vascular brain health has detrimental effects on motor neurons: vascular endothelial growth factor levels are lowered in ALS, which can compromise endothelial cell formation and the integrity of the blood-brain barrier. Increased turnover of neurovascular unit cells precedes their senescence, which, together with pericyte alterations, further fosters the failure of toxic metabolite removal. We here provide a comprehensive overview of the pathogenesis of impaired brain vascular health in ALS and how novel magnetic resonance imaging techniques can aid its detection. In particular, we discuss vascular patterns of blood supply to the motor cortex with the number of branches from the anterior and middle cerebral arteries acting as a novel marker of resistance and resilience against downstream effects of vascular risk and events in ALS. We outline how certain interventions adapted to patient needs and capabilities have the potential to mechanistically target the brain microvasculature towards favorable motor cortex blood supply patterns. Through this strategy, we aim to guide novel approaches to ALS management and a better understanding of ALS pathophysiology.

Rescue of synaptic and cognitive functions in polysialic acid-deficient mice and dementia models by short polysialic acid fragments.

Dysregulated cortical expression of the neural cell adhesion molecule (NCAM) and deficits of its associated polysialic acid (polySia) have been found in Alzheimer's disease and schizophrenia. However, the functional role of polySia in cortical synaptic plasticity remains poorly understood. Here, we show that acute enzymatic removal of polySia in medial prefrontal cortex (mPFC) slices leads to increased transmission mediated by the GluN1/GluN2B subtype of N-methyl-d-aspartate receptors (NMDARs), increased NMDAR-mediated extrasynaptic tonic currents, and impaired long-term potentiation (LTP). The latter could be fully rescued by pharmacological suppression of GluN1/GluN2B receptors, or by application of short soluble polySia fragments that inhibited opening of GluN1/GluN2B channels. These treatments and augmentation of synaptic NMDARs with the glycine transporter type 1 (GlyT1) inhibitor sarcosine also restored LTP in mice deficient in polysialyltransferase ST8SIA4. Furthermore, the impaired performance of polySia-deficient mice and two models of Alzheimer's disease in the mPFC-dependent cognitive tasks could be rescued by intranasal administration of polySia fragments. Our data demonstrate the essential role of polySia-NCAM in the balancing of signaling through synaptic/extrasynaptic NMDARs in mPFC and highlight the therapeutic potential of short polySia fragments to restrain GluN1/GluN2B-mediated signaling.

Mice deficient in synaptic protease neurotrypsin show impaired spaced long-term potentiation and blunted learning-induced modulation of dendritic spines.

Neurotrypsin (NT) is a neuronal trypsin-like serine protease whose mutations cause severe mental retardation in humans. NT is activated in vitro by Hebbian-like conjunction of pre- and postsynaptic activities, which promotes the formation of dendritic filopodia via proteolytic cleavage of the proteoglycan agrin. Here, we investigated the functional importance of this mechanism for synaptic plasticity, learning, and extinction of memory. We report that juvenile neurotrypsin-deficient (NT-/-) mice exhibit impaired long-term potentiation induced by a spaced stimulation protocol designed to probe the generation of new filopodia and their conversion into functional synapses. Behaviorally, juvenile NT-/- mice show impaired contextual fear memory and have a sociability deficit. The latter persists in aged NT-/- mice, which, unlike juvenile mice, show normal recall but impaired extinction of contextual fear memories. Structurally, juvenile mutants exhibit reduced spine density in the CA1 region, fewer thin spines, and no modulation in the density of dendritic spines following fear conditioning and extinction in contrast to wild-type littermates. The head width of thin spines is reduced in both juvenile and aged NT-/- mice. In vivo delivery of adeno-associated virus expressing an NT-generated fragment of agrin, agrin-22, but not a shorter agrin-15, elevates the spine density in NT-/- mice. Moreover, agrin-22 co-aggregates with pre- and postsynaptic markers and increases the density and size of presynaptic boutons and presynaptic puncta, corroborating the view that agrin-22 supports the synaptic growth.

Heparan Sulfates Regulate Axonal Excitability and Context Generalization through Ca2+/Calmodulin-Dependent Protein Kinase II.

Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of ankyrin G at the axon initial segments in the CA1 region of the hippocampus ex vivo, impaired context discrimination in vivo, and increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in vitro. Here, we show that in vivo delivery of heparinase 1 in the CA1 region of the hippocampus elevated autophosphorylation of CaMKII 24 h after injection in mice. Patch clamp recording in CA1 neurons revealed no significant heparinase effects on the amplitude or frequency of miniature excitatory and inhibitory postsynaptic currents, while the threshold for action potential generation was increased and fewer spikes were generated in response to current injection. Delivery of heparinase on the next day after contextual fear conditioning induced context overgeneralization 24 h after injection. Co-administration of heparinase with the CaMKII inhibitor (autocamtide-2-related inhibitory peptide) rescued neuronal excitability and expression of ankyrin G at the axon initial segment. It also restored context discrimination, suggesting the key role of CaMKII in neuronal signaling downstream of heparan sulfate proteoglycans and highlighting a link between impaired CA1 pyramidal cell excitability and context generalization during recall of contextual memories.

Real-time mechanisms of exacerbated synaptic remodeling by microglia in acute models of systemic inflammation and tauopathy.

Remodeling of synapses by microglia is essential for synaptic plasticity in the brain. However, during neuroinflammation and neurodegenerative diseases, microglia can induce excessive synaptic loss, although the precise underlying mechanisms are unknown. To directly observe microglia-synapse interactions under inflammatory conditions, we performed in vivo two-photon time-lapse imaging of microglia-synapse interactions after bacterial lipopolysaccharide administration to model systemic inflammation, or after inoculation of Alzheimer's disease (AD) brain extracts to model disease-associated neuroinflammatory microglial response. Both treatments prolonged microglia-neuron contacts, decreased basal surveillance of synapses and promoted synaptic remodeling in response to synaptic stress induced by focal single-synapse photodamage. Spine elimination correlated with the expression of microglial complement system/phagocytic proteins and the occurrence of synaptic filopodia. Microglia were observed contacting spines, then stretching and phagocytosing spine head filopodia. Thus, in response to inflammatory stimuli microglia exacerbated spine remodeling through prolonged microglial contact and elimination of spines 'tagged' by synaptic filopodia.

Aging-Associated Changes in Cognition, Expression and Epigenetic Regulation of Chondroitin 6-Sulfotransferase Chst3.

Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight into aging-associated decline in synaptic and cognitive functions. Hence, in this study, we compared the expression levels of ECM-related genes in the hippocampus of young, aged and very aged mice. ECM gene expression was downregulated, despite the accumulation of ECM proteoglycans during aging. The most robustly downregulated gene was carbohydrate sulfotransferase 3 (Chst3), the enzyme responsible for the chondroitin 6-sulfation (C6S) of proteoglycans. Further analysis of epigenetic mechanisms revealed a decrease in H3K4me3, three methyl groups at the lysine 4 on the histone H3 proteins, associated with the promoter region of the Chst3 gene, resulting in the downregulation of Chst3 expression in non-neuronal cells. Cluster analysis revealed that the expression of lecticans-substrates of CHST3-is tightly co-regulated with this enzyme. These changes in ECM-related genes were accompanied by an age-confounded decline in cognitive performance. Despite the co-directional impairment in cognitive function and average Chst3 expression in the studied age groups, at the individual level we found a negative correlation between mRNA levels of Chst3 and cognitive performance within the very aged group. An analysis of correlations between the expression of ECM-related genes and cognitive performance in novel object versus novel location recognition tasks revealed an apparent trade-off in the positive gene effects in one task at the expense of another. Further analysis revealed that, despite the reduction in the Chst3 mRNA, the expression of CHST3 protein is increased in glial cells but not in neurons, which, however, does not lead to changes in the absolute level of C6S and even results in the decrease in C6S in perineuronal, perisynaptic and periaxonal ECM relative to the elevated expression of its protein carrier versican.

Neddylation-dependent protein degradation is a nexus between synaptic insulin resistance, neuroinflammation and Alzheimer's disease.

BACKGROUND: The metabolic syndrome is a consequence of modern lifestyle that causes synaptic insulin resistance and cognitive deficits and that in interaction with a high amyloid load is an important risk factor for Alzheimer's disease. It has been proposed that neuroinflammation might be an intervening variable, but the underlying mechanisms are currently unknown. METHODS: We utilized primary neurons to induce synaptic insulin resistance as well as a mouse model of high-risk aging that includes a high amyloid load, neuroinflammation, and diet-induced obesity to test hypotheses on underlying mechanisms. RESULTS: We found that neddylation and subsequent activation of cullin-RING ligase complexes induced synaptic insulin resistance through ubiquitylation and degradation of the insulin-receptor substrate IRS1 that organizes synaptic insulin signaling. Accordingly, inhibition of neddylation preserved synaptic insulin signaling and rescued memory deficits in mice with a high amyloid load, which were fed with a 'western diet'. CONCLUSIONS: Collectively, the data suggest that neddylation and degradation of the insulin-receptor substrate is a nodal point that links high amyloid load, neuroinflammation, and synaptic insulin resistance to cognitive decline and impaired synaptic plasticity in high-risk aging.

Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R.

The brain extracellular matrix (ECM) consists of extremely long-lived proteins that assemble around neurons and synapses, to stabilize them. The ECM is thought to change only rarely, in relation to neuronal plasticity, through ECM proteolysis and renewed protein synthesis. We report here an alternative ECM remodeling mechanism, based on the recycling of ECM molecules. Using multiple ECM labeling and imaging assays, from super-resolution optical imaging to nanoscale secondary ion mass spectrometry, both in culture and in brain slices, we find that a key ECM protein, Tenascin-R, is frequently endocytosed, and later resurfaces, preferentially near synapses. The TNR molecules complete this cycle within ~3 days, in an activity-dependent fashion. Interfering with the recycling process perturbs severely neuronal function, strongly reducing synaptic vesicle exo- and endocytosis. We conclude that the neuronal ECM can be remodeled frequently through mechanisms that involve endocytosis and recycling of ECM proteins.

Context value updating and multidimensional neuronal encoding in the retrosplenial cortex.

The retrosplenial cortex (RSC) has diverse functional inputs and is engaged by various sensory, spatial, and associative learning tasks. We examine how multiple functional aspects are integrated on the single-cell level in the RSC and how the encoding of task-related parameters changes across learning. Using a visuospatial context discrimination paradigm and two-photon calcium imaging in behaving mice, a large proportion of dysgranular RSC neurons was found to encode multiple task-related dimensions while forming context-value associations across learning. During reversal learning requiring increased cognitive flexibility, we revealed an increased proportion of multidimensional encoding neurons that showed higher decoding accuracy for behaviorally relevant context-value associations. Chemogenetic inactivation of RSC led to decreased behavioral context discrimination during learning phases in which context-value associations were formed, while recall of previously formed associations remained intact. RSC inactivation resulted in a persistent positive behavioral bias in valuing contexts, indicating a role for the RSC in context-value updating.