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Proceedings of the National Academy of... Apr 2024Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of...
Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. K differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and β-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.
Topics: Qa-SNARE Proteins; Secretagogins; Cell Membrane; Membrane Fusion; Synaptosomal-Associated Protein 25; Exocytosis; Cell Communication; Syntaxin 1; Protein Binding
PubMed: 38593081
DOI: 10.1073/pnas.2309211121 -
Frontiers in Neurology 2024Delirium represents a common terminal pathway of heterogeneous neurological conditions characterized by disturbances in consciousness and attention. Contemporary...
Delirium represents a common terminal pathway of heterogeneous neurological conditions characterized by disturbances in consciousness and attention. Contemporary theories highlight the acute impairment of synaptic function and network connectivity, driven by neuroinflammation, oxidative stress, and neurotransmitter imbalances. However, established biomarkers are still missing. Innovative diagnostic techniques, such as single-molecule array analysis, enable the detection of biomarkers in blood at picomolar concentrations. This approach paves the way for deeper insights into delirium and potentially therapeutic targets for tailored medical treatments. In a retrospective 3-year study, we investigated seven biomarkers indicative of neuroaxonal damage [neurofilament light chain (NFL), ubiquitin carboxyl-terminal hydrolase (UCHL-1), and tau protein], microglial activation [glial fibrillary acidic protein (GFAP) and soluble triggering receptor expressed on myeloid cells 2 (sTREM2)], and synaptic dysfunction [synaptosomal-associated protein 25 (SNAP-25) and neuronal pentraxin 2 (NPTX2)]. The analysis of 71 patients with delirium, Alzheimer's disease (AD), and non-AD controls revealed that serum NFL levels are higher in delirium cases compared to both AD and non-AD. This suggests that elevated NFL levels in delirium are not exclusively the result of dementia-related damage. Serum tau levels were also elevated in delirium cases compared to controls. Conversely, cerebrospinal fluid (CSF) SNAP-25 showed higher levels in AD patients compared to controls only. These findings add to the increasing body of evidence suggesting that serum NFL could be a valuable biomarker of neuroaxonal damage in delirium research. Although SNAP-25 and NPTX2 did not exhibit significant differences in delirium, the exploration of synaptic biomarkers remains promising for enhancing our understanding of this condition.
PubMed: 38566855
DOI: 10.3389/fneur.2024.1356575 -
Circulation Research May 2024Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane...
BACKGROUND
Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis.
METHODS
Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies.
RESULTS
Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V procoagulant endothelial CD62E (E-selectin) and neutrophil (Ly6G) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082.
CONCLUSIONS
Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.
Topics: Animals; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Exocytosis; Humans; Mice; von Willebrand Factor; Venous Thrombosis; Mice, Knockout; Human Umbilical Vein Endothelial Cells; Inflammation; Mice, Inbred C57BL; Neutrophils; Endothelial Cells; Cells, Cultured; Vena Cava, Inferior; Male; Neutrophil Infiltration; NF-kappa B
PubMed: 38563147
DOI: 10.1161/CIRCRESAHA.124.324214 -
GeroScience Apr 2024Down syndrome (DS) is a genetic condition where the person is born with an extra chromosome 21. DS is associated with accelerated aging; people with DS are prone to...
Neurobehavioral dysfunction in a mouse model of Down syndrome: upregulation of cystathionine β-synthase, HS overproduction, altered protein persulfidation, synaptic dysfunction, endoplasmic reticulum stress, and autophagy.
Down syndrome (DS) is a genetic condition where the person is born with an extra chromosome 21. DS is associated with accelerated aging; people with DS are prone to age-related neurological conditions including an early-onset Alzheimer's disease. Using the Dp(17)3Yey/ + mice, which overexpresses a portion of mouse chromosome 17, which encodes for the transsulfuration enzyme cystathionine β-synthase (CBS), we investigated the functional role of the CBS/hydrogen sulfide (HS) pathway in the pathogenesis of neurobehavioral dysfunction in DS. The data demonstrate that CBS is higher in the brain of the DS mice than in the brain of wild-type mice, with primary localization in astrocytes. DS mice exhibited impaired recognition memory and spatial learning, loss of synaptosomal function, endoplasmic reticulum stress, and autophagy. Treatment of mice with aminooxyacetate, a prototypical CBS inhibitor, improved neurobehavioral function, reduced the degree of reactive gliosis in the DS brain, increased the ability of the synaptosomes to generate ATP, and reduced endoplasmic reticulum stress. HS levels in the brain of DS mice were higher than in wild-type mice, but, unexpectedly, protein persulfidation was decreased. Many of the above alterations were more pronounced in the female DS mice. There was a significant dysregulation of metabolism in the brain of DS mice, which affected amino acid, carbohydrate, lipid, endocannabinoid, and nucleotide metabolites; some of these alterations were reversed by treatment of the mice with the CBS inhibitor. Thus, the CBS/HS pathway contributes to the pathogenesis of neurological dysfunction in DS in the current animal model.
PubMed: 38558215
DOI: 10.1007/s11357-024-01146-8 -
Brain, Behavior, and Immunity Jul 2024Microglia modulate synaptic refinement in the central nervous system (CNS). We have previously shown that a mouse model with innate high anxiety-related behavior (HAB)...
Microglia modulate synaptic refinement in the central nervous system (CNS). We have previously shown that a mouse model with innate high anxiety-related behavior (HAB) displays higher CD68 microglia density in the key regions of anxiety circuits compared to mice with normal anxiety-related behavior (NAB) in males, and that minocycline treatment attenuated the enhanced anxiety of HAB male. Given that a higher prevalence of anxiety is widely reported in females compared to males, little is known concerning sex differences at the cellular level. Herein, we address this by analyzing microglia heterogeneity and function in the HAB and NAB brains of both sexes. Single-cell RNA sequencing revealed ten distinct microglia clusters varied by their frequency and gene expression profile. We report striking sex differences, especially in the major microglia clusters of HABs, indicating a higher expression of genes associated with phagocytosis and synaptic engulfment in the female compared to the male. On a functional level, we show that female HAB microglia engulfed a greater amount of hippocampal vGLUT1 excitatory synapses compared to the male. We moreover show that female HAB microglia engulfed more synaptosomes compared to the male HAB in vitro. Due to previously reported effects of minocycline on microglia, we finally administered oral minocycline to HABs of both sexes and showed a significant reduction in the engulfment of synapses by female HAB microglia. In parallel to our microglia-specific findings, we further showed an anxiolytic effect of minocycline on female HABs, which is complementary to our previous findings in the male HABs. Our study, therefore, identifies the altered function of synaptic engulfment by microglia as a potential avenue to target and resolve microglia heterogeneity in mice with innate high anxiety.
Topics: Animals; Minocycline; Microglia; Female; Anxiety; Male; Mice; Sex Characteristics; Brain; Mice, Inbred C57BL; Hippocampus; Disease Models, Animal; Synapses; Phagocytosis
PubMed: 38552926
DOI: 10.1016/j.bbi.2024.03.035 -
Brain, Behavior, and Immunity Jul 2024Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple...
Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.
Topics: Animals; Humans; Mice; Brain; Cells, Cultured; Complement C3; Complement System Proteins; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Microglia; Neurons; Synapses; Thalamus
PubMed: 38552925
DOI: 10.1016/j.bbi.2024.03.041 -
International Journal of Molecular... Mar 2024Synaptic transmission is essential for nervous system function and the loss of synapses is a known major contributor to dementia. Alzheimer's disease dementia (ADD) is...
Synaptic transmission is essential for nervous system function and the loss of synapses is a known major contributor to dementia. Alzheimer's disease dementia (ADD) is characterized by synaptic loss in the mesial temporal lobe and cerebral neocortex, both of which are brain areas associated with memory and cognition. The association of synaptic loss and ADD was established in the late 1980s, and it has been estimated that 30-50% of neocortical synaptic protein is lost in ADD, but there has not yet been a quantitative profiling of different synaptic proteins in different brain regions in ADD from the same individuals. Very recently, positron emission tomography (PET) imaging of synapses is being developed, accelerating the focus on the role of synaptic loss in ADD and other conditions. In this study, we quantified the densities of two synaptic proteins, the presynaptic protein Synaptosome Associated Protein 25 (SNAP25) and the postsynaptic protein postsynaptic density protein 95 (PSD95) in the human brain, using enzyme-linked immunosorbent assays (ELISA). Protein was extracted from the cingulate gyrus, hippocampus, frontal, primary visual, and entorhinal cortex from cognitively unimpaired controls, subjects with mild cognitive impairment (MCI), and subjects with dementia that have different levels of Alzheimer's pathology. SNAP25 is significantly reduced in ADD when compared to controls in the frontal cortex, visual cortex, and cingulate, while the hippocampus showed a smaller, non-significant reduction, and entorhinal cortex concentrations were not different. In contrast, all brain areas showed lower PSD95 concentrations in ADD when compared to controls without dementia, although in the hippocampus, this failed to reach significance. Interestingly, cognitively unimpaired cases with high levels of AD pathology had higher levels of both synaptic proteins in all brain regions. SNAP25 and PSD95 concentrations significantly correlated with densities of neurofibrillary tangles, amyloid plaques, and Mini Mental State Examination (MMSE) scores. Our results suggest that synaptic transmission is affected by ADD in multiple brain regions. The differences were less marked in the entorhinal cortex and the hippocampus, most likely due to a ceiling effect imposed by the very early development of neurofibrillary tangles in older people in these brain regions.
Topics: Humans; Aged; Alzheimer Disease; Neurofibrillary Tangles; Brain; Cognitive Dysfunction; tau Proteins; Positron-Emission Tomography
PubMed: 38542104
DOI: 10.3390/ijms25063130 -
Biomedicines Feb 2024Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural...
Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural compound with neuroprotective potential, on glutamate release in isolated nerve terminals (synaptosomes) prepared from the rat cerebral cortex were examined. In this study, 4-aminopyridine (4-AP), a potassium channel blocker, was utilized to induce the release of glutamate, which was subsequently quantified via a fluorometric assay. Our findings revealed that phillygenin reduced 4-AP-induced glutamate release, and this inhibitory effect was reversed by removing extracellular Ca or inhibiting vesicular transport with bafilomycin A1. However, exposure to the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate (dl-TOBA) did not influence the inhibitory effect. Moreover, phillygenin did not change the synaptosomal membrane potential but lowered the 4-AP-triggered increase in intrasynaptosomal Ca concentration ([Ca]). Antagonizing Ca2.2 (N-type) calcium channels blocked the inhibition of glutamate release by phillygenin, whereas pretreatment with the mitochondrial Na/Ca exchanger inhibitor, CGP37157 or the ryanodine receptor inhibitor, dantrolene, both of which block intracellular Ca release, had no effect. The effect of phillygenin on glutamate release triggered by 4-AP was completely abolished when MAPK/ERK inhibitors were applied. Furthermore, phillygenin attenuated the phosphorylation of ERK1/2 and its major presynaptic target, synapsin I, a protein associated with synaptic vesicles. These data collectively suggest that phillygenin mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca through Ca2.2 calcium channels, thereby subsequently suppressing the MAPK/ERK/synapsin I signaling cascade.
PubMed: 38540109
DOI: 10.3390/biomedicines12030495 -
Membranes Mar 2024Among the most prevalent neurological disorders, epilepsy affects about 1% of the population worldwide. We previously found, using human epileptic tissues, that...
Among the most prevalent neurological disorders, epilepsy affects about 1% of the population worldwide. We previously found, using human epileptic tissues, that GABAergic neurotransmission impairment is a key mechanism that drives the pathological phenomena that ultimately lead to generation and recurrence of seizures. Using both a "microtransplantation technique" and synaptosomes preparations from drug-resistant temporal lobe epilepsies (TLEs), we used the technique of two-electrode voltage clamp to record GABA-evoked currents, focusing selectively on the synaptic "fast inhibition" mediated by low-affinity GABA receptors. Here, we report that the use-dependent GABA current desensitization (i.e., GABA rundown, which is evoked by applying to the cells consecutive pulses of GABA, at high concentration), which is a distinguishing mark of TLE, is mainly dependent on a dysfunction that affects synaptic GABA receptors. In addition, using the same approaches, we recorded a depolarized GABA reversal potential in synaptosomes samples from the human epileptic subicula of TLE patients. These results, which confirm previous experiments using total membranes, suggest an altered chloride homeostasis in the synaptic area. Finally, the lack of a Zn block of GABA-evoked currents using the synaptosomes supports the enrichment of "synaptic fast inhibitory" GABA receptors in this preparation. Altogether, our findings suggest a pathophysiological role of low-affinity GABA receptors at the synapse, especially during the fast and repetitive GABA release underlying recurrent seizures.
PubMed: 38535283
DOI: 10.3390/membranes14030064 -
BMC Medicine Mar 2024Synaptic dysfunction with reduced synaptic protein levels is a core feature of Alzheimer's disease (AD). Synaptic proteins play a central role in memory processing,...
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.
Topics: Humans; Mice; Animals; Alzheimer Disease; Synaptotagmin I; Amyloid beta-Peptides; Neurogranin; Cognitive Dysfunction; Extracellular Vesicles; Atrophy; Biomarkers
PubMed: 38528511
DOI: 10.1186/s12916-024-03359-2