The proteomic landscape of synaptic diversity across brain regions and cell types.

Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered ∼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

Delivering synaptic protein mRNAs via extracellular vesicles ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

BACKGROUND: Synaptic dysfunction with reduced synaptic protein levels is a core feature of Alzheimer's disease (AD). Synaptic proteins play a central role in memory processing, learning, and AD pathogenesis. Evidence suggests that synaptic proteins in plasma neuronal-derived extracellular vesicles (EVs) are reduced in patients with AD. However, it remains unclear whether levels of synaptic proteins in EVs are associated with hippocampal atrophy of AD and whether upregulating the expression of these synaptic proteins has a beneficial effect on AD. METHODS: In this study, we included 57 patients with AD and 56 healthy controls. We evaluated their brain atrophy through magnetic resonance imaging using the medial temporal lobe atrophy score. We measured the levels of four synaptic proteins, including synaptosome-associated protein 25 (SNAP25), growth-associated protein 43 (GAP43), neurogranin, and synaptotagmin 1 in both plasma neuronal-derived EVs and cerebrospinal fluid (CSF). We further examined the association of synaptic protein levels with brain atrophy. We also evaluated the levels of these synaptic proteins in the brains of 5×FAD mice. Then, we loaded rabies virus glycoprotein-engineered EVs with messenger RNAs (mRNAs) encoding GAP43 and SNAP25 and administered these EVs to 5×FAD mice. After treatment, synaptic proteins, dendritic density, and cognitive function were evaluated. RESULTS: The results showed that GAP43, SNAP25, neurogranin, and synaptotagmin 1 were decreased in neuronal-derived EVs but increased in CSF in patients with AD, and the changes corresponded to the severity of brain atrophy. GAP43 and SNAP25 were decreased in the brains of 5×FAD mice. The engineered EVs efficiently and stably delivered these synaptic proteins to the brain, where synaptic protein levels were markedly upregulated. Upregulation of synaptic protein expression could ameliorate cognitive impairment in AD by promoting dendritic density. This marks the first successful delivery of synaptic protein mRNAs via EVs in AD mice, yielding remarkable therapeutic effects. CONCLUSIONS: Synaptic proteins are closely related to AD processes. Delivery of synaptic protein mRNAs via EVs stands as a promising effective precision treatment strategy for AD, which significantly advances the current understanding of therapeutic approaches for the disease.

Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.

Central nervous system specific high molecular weight ALS2/alsin homophilic complex is enriched in mouse brain synaptosomes.

ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function.

Alterations in the Proteome of Developing Neocortical Synaptosomes in the Absence of MET Signaling Revealed by Comparative Proteomics.

Alterations in the expression of genes encoding proteins involved in synapse formation, maturation, and function are a hallmark of many neurodevelopmental and psychiatric disorders. For example, there is reduced neocortical expression of the MET receptor tyrosine kinase (MET) transcript and protein in Autism Spectrum Disorder (ASD) and Rett syndrome. Preclinical in vivo and in vitro models manipulating MET signaling reveal that the receptor modulates excitatory synapse development and maturation in select forebrain circuits. The molecular adaptations underlying the altered synaptic development remain unknown. We performed a comparative mass spectrometry analysis of synaptosomes generated from the neocortex of wild type and Met null mice during the peak of synaptogenesis (postnatal day 14; data are available from ProteomeXchange with identifier PXD033204). The analyses revealed broad disruption of the developing synaptic proteome in the absence of MET, consistent with the localization of MET protein in pre- and postsynaptic compartments, including proteins associated with the neocortical synaptic MET interactome and those encoded by syndromic and ASD risk genes. In addition to an overrepresentation of altered proteins associated with the SNARE complex, multiple proteins in the ubiquitin-proteasome system and associated with the synaptic vesicle, as well as proteins that regulate actin filament organization and synaptic vesicle exocytosis/endocytosis, were disrupted. Taken together, the proteomic changes are consistent with structural and functional changes observed following alterations in MET signaling. We hypothesize that the molecular adaptations following Met deletion may reflect a general mechanism that produces circuit-specific molecular changes due to loss or reduction of synaptic signaling proteins.

Localised Cdr1as activity is required for fear extinction memory.

Circular RNAs (circRNAs) comprise a novel class of regulatory RNAs that are abundant in the brain, particularly within synapses. They are highly stable, dynamically regulated, and display a range of functions, including serving as decoys for microRNAs and proteins and, in some cases, circRNAs also undergo translation. Early work in animal models revealed an association between circRNAs and neurodegenerative and neuropsychiatric disorders; however, little is known about the link between circRNA function and memory. To address this, we examined circRNA in synaptosomes derived from the medial prefrontal cortex of fear extinction-trained male C57BL/6J mice and found 12,837 circRNAs that were enriched at the synapse, including cerebellar degeneration-related protein 1 antisense RNA (Cdr1as). Targeted knockdown of Cdr1as in the neural processes of the infralimbic cortex led to impaired fear extinction memory. These findings highlight the involvement of localised circRNA activity at the synapse in memory formation.

Intrathecal Injection of Botulinum Toxin Type A has an Analgesic Effect in Male Rats CCI Model by Inhibiting the Activation of Spinal P2X4R.

Purinergic receptor P2X4 (P2X4R) plays an essential role in neuropathic pain. However, the specific mechanism needs to be clarified. Botulinum toxin type A is a neurotoxin produced by Clostridium botulinum type A. This study found that intrathecal injection of botulinum toxin type A produced an excellent analgesic effect in a rat model of chronic constriction sciatic nerve injury and inhibited the activation of P2X4R, microglia, and astrocytes. The administration of a P2X4R activator can up-regulate the expression of P2X4R and eliminate the analgesic effect of intrathecal injection of botulinum toxin type A. In addition, we found that microglia and astrocytes in the spinal cord of rats injected with botulinum toxin type A were reactivated after administration of the P2X4R activator. Our results suggest that intrathecal injection of botulinum toxin type A has an analgesic effect in a rat model of chronic constriction sciatic nerve injury by inhibiting the activation of P2X4R in the spinal cord.

Synaptophysin controls synaptobrevin-II retrieval via a cryptic C-terminal interaction site.

The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures.

Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

Neural cell adhesion molecules 1 (NCAM1) and 2 (NCAM2) belong to the cell adhesion molecules of the immunoglobulin superfamily and have been shown to regulate formation, maturation, and maintenance of synapses. NCAM1 and NCAM2 undergo proteolysis, but the identity of all the proteases involved and how proteolysis is used to regulate their functions are not known. We report here that NCAM1 and NCAM2 are BACE1 substrates in vivo. NCAM1 and NCAM2 overexpressed in HEK cells were both cleaved by metalloproteinases or BACE1, and NCAM2 was also processed by γ-secretase. We identified the BACE1 cleavage site of NCAM1 (at Glu 671) and NCAM2 (at Glu 663) using mass spectrometry and site-directed mutagenesis. Next, we assessed BACE1-mediated processing of NCAM1 and NCAM2 in the mouse brain during aging. NCAM1 and NCAM2 were cleaved in the olfactory bulb of BACE1+/+ but not BACE1-/- mice at postnatal day 10 (P10), 4 and 12 months of age. In the hippocampus, a BACE1-specific soluble fragment of NCAM1 (sNCAM1ß) was only detected at P10. However, we observed an accumulation of full-length NCAM1 in hippocampal synaptosomes in 4-month-old BACE1-/- mice. We also found that polysialylated NCAM1 (PSA-NCAM1) levels were increased in BACE1-/- mice at P10 and demonstrated that BACE1 cleaves both NCAM1 and PSA-NCAM1 in vitro. In contrast, we did not find evidence for BACE1-dependent NCAM2 processing in the hippocampus at any age analyzed. In summary, our data demonstrate that BACE1 differentially processes NCAM1 and NCAM2 depending on the region of brain, subcellular localization, and age in vivo.

SNAP25 is a potential prognostic biomarker for prostate cancer.

BACKGROUND: Prostate cancer (PCa) is one of the most lethal cancers in male individuals. The synaptosome associated protein 25 (SNAP25) gene is a key mediator of multiple biological functions in tumors. However, its significant impact on the prognosis in PCa remains to be elucidated. METHODS: We performed a comprehensive analysis of the Cancer Genome Atlas dataset (TCGA) to identify the differentially expressed genes between PCa and normal prostate tissue. We subjected the differentially expressed genes to gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes functional analysis, and constructed a protein-protein interaction network. We then screened for pivotal genes to identify the hub genes of prognostic significance by performing Cox regression analysis. We identified SNAP25 as one such gene and analyzed the relationship between its expression in PCa to poor prognosis using GEPIA interactive web server. RESULTS: TCGA database demonstrated that SNAP25 was significantly downregulated in PCa. The progressive decrease in SNAP25 expression with the increase in the clinical staging and grading of PCa demonstrates that reduced SNAP25 expression considerably exacerbates the clinical presentation. Our findings confirm that SNAP25 expression strongly correlates with overall survival, which was determined using the Gleason score. We also validated the role of SNAP25 expression in the prognosis of patients with PCa. We used Gene Set Enrichment and Gene Ontology analyses to evaluate the function of SNAP25 and further explored the association between SNAP25 expression and tumor-infiltrating immune cells using the Tumor Immune Assessment Resource database. We found for the first time that SNAP25 is involved in the activation, differentiation, and migration of immune cells in PCa. Its expression was positively correlated with immune cell infiltration, including B cells, CD8+ T cells, CD4+ T cells, neutrophils, dendritic cells, macrophages, and natural killer cells. SNAP25 expression also positively correlated with chemokines/chemokine receptors, suggesting that SNAP25 may regulate the migration of immune cells. In addition, our experimental results verified the low expression of SNAP25 in PCa cells. CONCLUSION: Our findings indicate a relationship between SNAP25 expression and PCa, demonstrating that SNAP25 is a potential prognostic biomarker due to its vital role in immune infiltration.

Proteomic Profiling of Cerebrum Mitochondria, Myelin Sheath, and Synaptosome Revealed Mitochondrial Damage and Synaptic Impairments in Association with 3 × Tg-AD Mice Model.

Alzheimer's disease (AD) is the most common age-associated dementia with complex pathological hallmarks. Mitochondrion, synaptosome, and myelin sheath appear to be vulnerable and play a key role in the pathogenesis of AD. To clarify the early mechanism associated with AD, followed by subcellular components separation, we performed iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics analysis to simultaneously investigate the differentially expressed proteins (DEPs) within the mitochondria, synaptosome, and myelin sheath in the cerebrum of the 6-month-old triple transgenic AD (3 × Tg-AD) and 6-month-old wild-type (WT) mice. A large number of DEPs between the AD and WT mice were identified. Most of them are related to mitochondria and synaptic dysfunction and cytoskeletal protein change. Differential expressions of Lrpprc, Nefl, and Sirpa were verified by Western blot analysis. The results suggest that decreased energy metabolism, impaired amino acid metabolism and neurotransmitter synthesis, increase compensatory fatty acid metabolism, up-regulated cytoskeletal protein expression, and oxidative stress are the early events of AD. Among these, mitochondrial damage, synaptic dysfunction, decreased energy metabolism, and abnormal amino acid metabolism are the most significant events. The results indicate that it is feasible to separate and simultaneously perform proteomics analysis on the three subcellular components.

Coupling of GABA Metabolism to Mitochondrial Glucose Phosphorylation.

Glucose and oxygen (O2) are vital to the brain. Glucose metabolism and mitochondria play a pivotal role in this process, culminating in the increase of reactive O2 species. Hexokinase (HK) is a key enzyme on glucose metabolism and is coupled to the brain mitochondrial redox modulation by recycling ADP for oxidative phosphorylation (OXPHOS). GABA shunt is an alternative pathway to GABA metabolism that increases succinate levels, a Krebs cycle intermediate. Although glucose and GABA metabolisms are intrinsically connected, their interplay coordinating mitochondrial function is poorly understood. Here, we hypothesize that the HK and the GABA shunt interact to control mitochondrial metabolism differently in the cortex and the hypothalamus. The GABA shunt stimulated mitochondrial O2 consumption and H2O2 production higher in hypothalamic synaptosomes (HSy) than cortical synaptosomes (CSy). The GABA shunt increased the HK coupled to OXPHOS activity in both population of synaptosomes, but the rate of activation was higher in HSy than CSy. Significantly, malonate and vigabatrin blocked the effects of the GABA shunt in the HK activity coupled to OXPHOS. It indicates that the glucose phosphorylation is linked to GABA and Krebs cycle reactions. Together, these data shed light on the HK and SDH role on the metabolism of each region fed by GABA turnover, which depends on the neurons' metabolic route.

Inhibition of Synaptic Glutamate Exocytosis and Prevention of Glutamate Neurotoxicity by Eupatilin from Artemisia argyi in the Rat Cortex.

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca2+ elevation, inhibition of P/Q-type Ca2+ channels, decreased synapsin I Ca2+-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca2+ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.

The Acute Antiallodynic Effect of Tolperisone in Rat Neuropathic Pain and Evaluation of Its Mechanism of Action.

Current treatment approaches to manage neuropathic pain have a slow onset and their use is largely hampered by side-effects, thus there is a significant need for finding new medications. Tolperisone, a centrally acting muscle relaxant with a favorable side effect profile, has been reported to affect ion channels, which are targets for current first-line medications in neuropathic pain. Our aim was to explore its antinociceptive potency in rats developing neuropathic pain evoked by partial sciatic nerve ligation and the mechanisms involved. Acute oral tolperisone restores both the decreased paw pressure threshold and the elevated glutamate level in cerebrospinal fluid in neuropathic rats. These effects were comparable to those of pregabalin, a first-line medication in neuropathy. Tolperisone also inhibits release of glutamate from rat brain synaptosomes primarily by blockade of voltage-dependent sodium channels, although inhibition of calcium channels may also be involved at higher concentrations. However, pregabalin fails to affect glutamate release under our present conditions, indicating a different mechanism of action. These results lay the foundation of the avenue for repurposing tolperisone as an analgesic drug to relieve neuropathic pain.

Differential compartmental processing and phosphorylation of pathogenic human tau and native mouse tau in the line 66 model of frontotemporal dementia.

Synapse loss is associated with motor and cognitive decline in multiple neurodegenerative disorders, and the cellular redistribution of tau is related to synaptic impairment in tauopathies, such as Alzheimer's disease and frontotemporal dementia. Here, we examined the cellular distribution of tau protein species in human tau overexpressing line 66 mice, a transgenic mouse model akin to genetic variants of frontotemporal dementia. Line 66 mice express intracellular tau aggregates in multiple brain regions and exhibit sensorimotor and motor learning deficiencies. Using a series of anti-tau antibodies, we observed, histologically, that nonphosphorylated transgenic human tau is enriched in synapses, whereas phosphorylated tau accumulates predominantly in cell bodies and axons. Subcellular fractionation confirmed that human tau is highly enriched in insoluble cytosolic and synaptosomal fractions, whereas endogenous mouse tau is virtually absent from synapses. Cytosolic tau was resistant to solubilization with urea and Triton X-100, indicating the formation of larger tau aggregates. By contrast, synaptic tau was partially soluble after Triton X-100 treatment and most likely represents aggregates of smaller size. MS corroborated that synaptosomal tau is nonphosphorylated. Tau enriched in the synapse of line 66 mice, therefore, appears to be in an oligomeric and nonphosphorylated state, and one that could have a direct impact on cognitive function.

Mass-tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events.

Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes. Up to 20 individual samples, each with a unique combinatorial barcode, were pooled for labeling with an antibody cocktail. Our synaptosome protocol used six palladium-based barcoding reagents, and in combination with sequential gating increased the identification of presynaptic events approximately fourfold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.

Synaptic mitochondrial dysfunction and septin accumulation are linked to complement-mediated synapse loss in an Alzheimer's disease animal model.

Synaptic functional disturbances with concomitant synapse loss represent central pathological hallmarks of Alzheimer's disease. Excessive accumulation of cytotoxic amyloid oligomers is widely recognized as a key event that underlies neurodegeneration. Certain complement components are crucial instruments of widespread synapse loss because they can tag synapses with functional impairments leading to their engulfment by microglia. However, an exact understanding of the affected synaptic functions that predispose to complement-mediated synapse elimination is lacking. Therefore, we conducted systematic proteomic examinations on synaptosomes prepared from an amyloidogenic mouse model of Alzheimer's disease (APP/PS1). Synaptic fractions were separated according to the presence of the C1q-tag using fluorescence-activated synaptosome sorting and subjected to proteomic comparisons. The results raised the decline of mitochondrial functions in the C1q-tagged synapses of APP/PS1 mice based on enrichment analyses, which was verified using flow cytometry. Additionally, proteomics results revealed extensive alterations in the level of septin protein family members, which are known to dynamically form highly organized pre- and postsynaptic supramolecular structures, thereby affecting synaptic transmission. High-resolution microscopy investigations demonstrated that synapses with considerable amounts of septin-3 and septin-5 show increased accumulation of C1q in APP/PS1 mice compared to the wild-type ones. Moreover, a strong positive correlation was apparent between synaptic septin-3 levels and C1q deposition as revealed via flow cytometry and confocal microscopy examinations. In sum, our results imply that deterioration of synaptic mitochondrial functions and alterations in the organization of synaptic septins are associated with complement-dependent synapse loss in Alzheimer's disease.

Local apoptotic-like mechanisms underlie complement-mediated synaptic pruning.

C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. However, the molecular mechanisms underlying C1q-controlled synaptic pruning are mostly unknown. This study addresses distortions in the synaptic proteome leading to C1q-tagged synapses. Our data demonstrated the preferential localization of C1q to the presynapse. Proteomic investigation and pathway analysis of C1q-tagged synaptosomes revealed the presence of apoptotic-like processes in C1q-tagged synapses, which was confirmed experimentally with apoptosis markers. Moreover, the induction of synaptic apoptotic-like mechanisms in a model of sensory deprivation-induced synaptic depression led to elevated C1q levels. Our results unveiled that C1q label-based synaptic pruning is triggered by and directly linked to apoptotic-like processes in the synaptic compartment.

Chlorogenic Acid Decreases Glutamate Release from Rat Cortical Nerve Terminals by P/Q-Type Ca2+ Channel Suppression: A Possible Neuroprotective Mechanism.

The glutamatergic neurotransmitter system has received substantial attention in research on the pathophysiology and treatment of neurological disorders. The study investigated the effect of the polyphenolic compound chlorogenic acid (CGA) on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). CGA inhibited 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes. This inhibition was prevented in the absence of extracellular Ca2+ and was associated with the inhibition of 4-AP-induced elevation of Ca2+ but was not attributed to changes in synaptosomal membrane potential. In line with evidence observed through molecular docking, CGA did not inhibit glutamate release in the presence of P/Q-type Ca2+ channel inhibitors; therefore, CGA-induced inhibition of glutamate release may be mediated by P/Q-type Ca2+ channels. CGA-induced inhibition of glutamate release was also diminished by the calmodulin and Ca2+/calmodilin-dependent kinase II (CaMKII) inhibitors, and CGA reduced the phosphorylation of CaMKII and its substrate, synapsin I. Furthermore, pretreatment with intraperitoneal CGA injection attenuated the glutamate increment and neuronal damage in the rat cortex that were induced by kainic acid administration. These results indicate that CGA inhibits glutamate release from cortical synaptosomes by suppressing P/Q-type Ca2+ channels and CaMKII/synapsin I pathways, thereby preventing excitotoxic damage to cortical neurons.

Quantitative proteomics of synaptosome S-nitrosylation in Alzheimer's disease.

Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2-/- mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies.