Regulatory role of sorting nexin 5 in protein stability and vesicular targeting of vesicular acetylcholine transporter to synaptic vesicle-like vesicles in PC12 cells

Accurate targeting of vesicular acetylcholine transporter(VAChT)to synaptic vesicles(SVs)is indispensable for efficient cholinergic transmission.Previous studies have suggested that the dileucine motif within the C-terminus of the transporter is sufficient for its targeting to SVs.However,the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear.Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5(SNX5)as its novel interacting protein.SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells.Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5.Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway.More importantly,we found that,in PC12 cells,the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles(SVLVs)localization of VAChT.Therefore,the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT.

Synaptic dysfunction in Alzheimer's disease:the effects of amyloid beta on synaptic vesicle dynamics as a novel target for therapeutic intervention

The most prevalent form of dementia in the elderly is Alzheimer＇s disease.A significant contributing factor to the progression of the disease appears to be the progressive accumulation of amyloid-β42（Aβ42）,a small hydrophobic peptide.Unfortunately,attempts to develop therapies targeting the accumulation of Aβ42 have not been successful to treat or even slow down the disease.It is possible that this failure is an indication that targeting downstream effects rather than the accumulation of the peptide itself might be a more effective approach.The accumulation of Aβ42 seems to affect various aspects of physiological cell functions.In this review,we provide an overview of the evidence that implicates Aβ42 in synaptic dysfunction,with a focus on how it contributes to defects in synaptic vesicle dynamics and neurotransmitter release.We discuss data that provide new insights on the Aβ42 induced pathology of Alzheimer＇s disease and a more detailed understanding of its contribution to the synaptic deficiencies that are associated with the early stages of the disease.Although the precise mechanisms that trigger synaptic dysfunction are still under investigation,the available data so far has enabled us to put forward a model that could be used as a guide to generate new therapeutic targets for pharmaceutical intervention.

Designing the lipid raft marker protein for synaptic vesicles

Lipid rafts are cholesterol-enriched microdomains and implicated in many essential physiological ac-tivities such as the neurotransmitter release.Many studies have been carried out on the function of rafts inthe plasma membranes,whereas little is known about the information of such microdomains in subcellularcompartments especially synaptic vesicles(SVs).In the well-studied plasma membranes,several proteinshave been recognized as raft markers,which are used to label or trace rafts.But the raft marker proteinon SVs has not been identified yet.Although some SV proteins,including VAMP and CPE,have beenfound in raft fractions,they cannot be used as markers due to their low abundance in rafts.In this work,we designed several chimera proteins and tested their characteristics for using as SV raft makers.First,we detected whether they located in SVs,and then the chimeras exhibiting the better localization in SVswere further examined for their enrichment in raft using detergent treatment and gradient density floatationanalysis.Our results indicate that one of the chimeric proteins is primarily located in SVs and distributedin raft microdomains,which strongly suggests that it could be served as a raft marker for SVs.

SV2B defines a subpopulation of synaptic vesicles

Synaptic vesicles can undergo several modes of exocytosis,endocytosis,and trafficking within individual synapses,and their fates may be linked to different vesicular protein compositions.Here,we mapped the intrasynaptic distribution of the synaptic vesicle proteins SV2B and SV2A in glutamatergic synapses of the hippocampus using three-dimensional electron microscopy.SV2B was almost completely absent from docked vesicles and a distinct cluster of vesicles found near the active zone.In contrast,SV2A was found in all domains of the synapse and was slightly enriched near the active zone.SV2B and SV2A were found on the membrane in the peri-active zone,suggesting the recycling from both clusters of vesicles.SV2B knockout mice displayed an increased seizure induction threshold only in a model employing high-frequency stimulation.Our data show that glutamatergic synapses generate molecularly distinct populations of synaptic vesicles and are able to maintain them at steep spatial gradients.The almost complete absence of SV2B from vesicles at the active zone of wildtype mice may explain why SV2A has been found more important for vesicle release.

Dysfunction of synaptic endocytic trafficking in Parkinson's disease

Parkinson's disease is characterized by the selective degeneration of dopamine neurons in the nigrostriatal pathway and dopamine deficiency in the striatum.The precise reasons behind the specific degeneration of these dopamine neurons remain largely elusive.Genetic investigations have identified over 20 causative PARK genes and 90 genomic risk loci associated with both familial and sporadic Parkinson's disease.Notably,several of these genes are linked to the synaptic vesicle recycling process,particularly the clathrinmediated endocytosis pathway.This suggests that impaired synaptic vesicle recycling might represent an early feature of Parkinson's disease,followed by axonal degeneration and the eventual loss of dopamine cell bodies in the midbrain via a"dying back"mechanism.Recently,several new animal and cellular models with Parkinson's disease-linked mutations affecting the endocytic pathway have been created and extensively characterized.These models faithfully recapitulate certain Parkinson's disease-like features at the animal,circuit,and cellular levels,and exhibit defects in synaptic membrane trafficking,further supporting the findings from human genetics and clinical studies.In this review,we will first summarize the cellular and molecular findings from the models of two Parkinson's disease-linked clathrin uncoating proteins:auxilin(DNAJC6/PARK19)and synaptojanin 1(SYNJ1/PARK20).The mouse models carrying these two PARK gene mutations phenocopy each other with specific dopamine terminal pathology and display a potent synergistic effect.Subsequently,we will delve into the involvement of several clathrin-mediated endocytosis-related proteins(GAK,endophilin A1,SAC2/INPP5 F,synaptotagmin-11),identified as Parkinson's disease risk factors through genome-wide association studies,in Parkinson's disease pathogenesis.We will also explore the direct or indirect roles of some common Parkinson's disease-linked proteins(alpha-synuclein(PARK1/4),Parkin(PARK2),and LRRK2(PARK8))in synaptic endocytic trafficking.Additionally,we will discuss the emerging novel functions of these endocytic proteins in downstream membrane traffic pathways,particularly autophagy.Given that synaptic dysfunction is considered as an early event in Parkinson's disease,a deeper understanding of the cellular mechanisms underlying synaptic vesicle endocytic trafficking may unveil novel to rgets for early diagnosis and the development of interventional therapies for Parkinson's disease.Future research should aim to elucidate why generalized synaptic endocytic dysfunction leads to the selective degeneration of nigrostriatal dopamine neurons in Parkinson's disease.

Lipid Rafts Identified on Synaptic Vesicles from Rat Brain

For a long time, lipid rafts have been thought to participate in regulating neurotransmitter release. However, the existence of lipid rafts on synaptic vesicles （SVs） and the mechanism by which exocytosisrelative proteins distribute on this structure have not been fully investigated. There is also much controversial data concerning rafts on SVs and synaptic vesicle proteins which makes the results difficult to interpret. This study systematically analyzed the existence and properties of lipid rafts on purified SVs by sucrose density gradient centrifugation, cholesterol depletion, and temperature variation. The data reveals that typical lipid rafts on SVs are both cholesterol dependent and temperature sensitive. Previous confusing results may have been caused by improper treatment or side effects of particular reagent. We also screened the lateral distribution of major exocytosis-related SV proteins and found that only the synaptobrevin （syb） and synaptotagmin （syt） produce detectable association with lipid rafts in 1% Triton X-100.

Real-time imaging of single synaptic vesicles in live neurons

Recent advances in fluorescence microscopy have provided researchers with powerful new tools to visualize cellular processes occurring in real time, giving researchers an unprecedented opportunity to address many biological questions that were previously inaccessible. With respect to neurobiology, these real-time imaging techniques have deepened our understanding of molecular and cellular processes, including the movement and dynamics of single proteins and organelles in living cells. In this review, we summarize recent advances in the field of real-time imaging of single synaptic vesicles in live neurons.

Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C

Huntington＇s disease （HD） is a neurodegenera- tive disease caused by a polyglutamine expansion in the huntingtin （Htt） protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 （SV2s）, a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic （TG） mice and Neuro2a mouse neuroblastoma cells （N2a cells） expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not signifi- cantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progres- sively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt- mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

Promoting neuronal regeneration using extracellular vesicles loaded with superparamagnetic iron oxide nanoparticles

Intercellular communication between neurons and glial cells via extracellular vesicles（EVs）as a novel mechanism of information transfer has been shown to be involved in regeneration processes within the central nervous system（CNS）（Rajendran et al.,2014）.Hence,to take advantage of EV signaling for therapeutic applications appears to be a completely new approach to promote regeneration.

Selective deletion of zinc transporter 3 in amacrine cells promotes retinal ganglion cell survival and optic nerve regeneration after injury

Vision depends on accurate signal conduction from the retina to the brain through the optic nerve,an important part of the central nervous system that consists of bundles of axons originating from retinal ganglion cells.The mammalian optic nerve,an important part of the central nervous system,cannot regenerate once it is injured,leading to permanent vision loss.To date,there is no clinical treatment that can regenerate the optic nerve and restore vision.Our previous study found that the mobile zinc(Zn^(2+))level increased rapidly after optic nerve injury in the retina,specifically in the vesicles of the inner plexiform layer.Furthermore,chelating Zn^(2+)significantly promoted axonal regeneration with a long-term effect.In this study,we conditionally knocked out zinc transporter 3(ZnT3)in amacrine cells or retinal ganglion cells to construct two transgenic mouse lines(VGAT^(Cre)ZnT3^(fl/fl)and VGLUT2^(Cre)ZnT3^(fl/fl),respectively).We obtained direct evidence that the rapidly increased mobile Zn^(2+)in response to injury was from amacrine cells.We also found that selective deletion of ZnT3 in amacrine cells promoted retinal ganglion cell survival and axonal regeneration after optic nerve crush injury,improved retinal ganglion cell function,and promoted vision recovery.Sequencing analysis of reginal ganglion cells revealed that inhibiting the release of presynaptic Zn^(2+)affected the transcription of key genes related to the survival of retinal ganglion cells in postsynaptic neurons,regulated the synaptic connection between amacrine cells and retinal ganglion cells,and affected the fate of retinal ganglion cells.These results suggest that amacrine cells release Zn^(2+)to trigger transcriptomic changes related to neuronal growth and survival in reginal ganglion cells,thereby influencing the synaptic plasticity of retinal networks.These results make the theory of zinc-dependent retinal ganglion cell death more accurate and complete and provide new insights into the complex interactions between retinal cell networks.

Therapeutic target for nephrotic syndrome: Identification of novel slit diaphragm associated molecules

The slit diaphragm bridging the neighboring foot pro-cesses functions as a fnal barrier of glomerular capil-lary wall for preventing the leak of plasma proteins into primary urine. It is now accepted that the dysfunction of the sit diaphragm contributes to the development of proteinuria in several glomerular diseases. Neph-rin, a gene product of NPHS1, a gene for a congenital nephrotic syndrome of Finnish type, constitutes an ex-tracellular domain of the slit diaphragm. Podocin was identified as a gene product of NPHS2 , a gene for a familial steroid-resistant nephrotic syndrome of French. Podocin binds the cytoplasmic domain of nephrin. After then, CD2 associated protein, NEPH1 and transient re-ceptor potential-6 were also found as crucial molecules of the slit diaphragm. In order to explore other novel molecules contributing to the development of protein-uria, we performed a subtraction hybridization assay with a normal rat glomerular RNA and a glomerular RNA of rats with a puromycin aminonucleoside ne-phropathy, a mimic of a human minimal change type nephrotic syndrome. Then we have found that synaptic vesicle protein 2B, ephrin-B1 and neurexin were already downregulated at the early stage of puromycin aminonucleoside nephropathy, and that these molecules were localized close to nephrin. It is conceivable that these molecules are the slit diaphragm associated molecules, which participate in the regulation of the barrier func-tion. These molecules could be targets to establish a novel therapy for nephrotic syndrome.

Acrylamide-induced Subacute Neurotoxic Effects on the Cerebral Cortex and Cerebellum at the Synapse Level in Rats

Objective To investigate acrylamide （ACR）-induced subacute neurotoxic effects on the central nervous system （CNS） at the synapse level in rats. Methods Thirty-six Sprague Dawley （SD） rats were randomized into three groups, （1） a 30 mg/kg ACR-treated group, （2） a 50 mg/kg ACR-treated group, and （3） a normal saline （NS）-treated control group. Body weight and neurological changes were recorded each day. At the end of the test, cerebral cortex and cerebellum tissues were harvested and viewed using light and electron microscopy. Additionally, the expression of Synapsin I and P-Synapsin I in the cerebral cortex and cerebellum were investigated. Results The 50 mg/kg ACR-treated rats showed a significant reduction in body weight compared with untreated individuals （P 〈 0.05）. Rats exposed to ACR showed a significant increase in gait scores compared with the NS control group （P 〈 0.05）. Histological examination indicated neuronal structural damage in the 50 mg/kg ACR treatment group. The active zone distance （AZD） and the nearest neighbor distance （NND） of synaptic vesicles in the cerebral cortex and cerebellum were increased in both the 30 mg/kg and 50 mg/kg ACR treatment groups. The ratio of the distribution of synaptic vesicles in the readily releasable pool （RRP） was decreased. Furthermore, the expression levels of Synapsin I and P-Synapsin I in the cerebral cortex and cerebellum were decreased in both the 30 mg/kg and 50 mg/kg ACR treatment groups. Conclusion Subacute ACR exposure contributes to neuropathy in the rat CNS. Functional damage of synaptic proteins and vesicles may be a mechanism of ACR neurotoxicity.

Physiological Roles of β-amyloid in Regulating Synaptic Function:Implications for AD Pathophysiology

The physiological functions of endogenous amyloid-β(Aβ),which plays important role in the pathology of Alzheimer's disease(AD),have not been paid enough attention.Here,we review the multiple physiological effects of Aβ,particularly in regulating synaptic transmission,and the possible mechanisms,in order to decipher the real characters of Aβunder both physiological and pathological conditions.Some worthy studies have shown that the deprivation of endogenous Aβgives rise to synaptic dysfunction and cognitive deficiency,while the moderate elevation of this peptide enhances long term potentiation and leads to neuronal hyperexcitability.In this review,we provide a new view for understanding the role of Aβin AD pathophysiology from the perspective of physiological meaning.

Development of SV2A Ligands for Epilepsy Treatment:A Review of Levetiracetam,Brivaracetam,and Padsevonil

Epilepsy is a common neurological disorder that is primarily treated with antiseizure medications(ASMs).Although dozens of ASMs are available in the clinic,approximately 30%of epileptic patients have medically refractory seizures;other limitations in most traditional ASMs include poor tolerability and drug-drug interactions.Therefore,there is an urgent need to develop alternative ASMs.Levetiracetam(LEV)is a first-line ASM that is well tolerated,has promising efficacy,and has little drug-drug interaction.Although it is widely accepted that LEV acts through a unique therapeutic target synaptic vesicle protein(SV)2A,the molecular basis of its action remains unknown.Even so,the next-generation SV2A ligands against epilepsy based on the structure of LEV have achieved clinical success.This review highlights the research and development(R&D)process of LEV and its analogs,brivaracetam and padsevonil,to provide ideas and experience for the R&D of novel ASMs.

STED Imaging of Vesicular Endocytosis in the Synapse

Endocytosis is a fundamental biological process that couples exocytosis to maintain the homeostasis of the plasma membrane and sustained neurotransmission.Super-resolution microscopy enables optical imaging of exocytosis and endocytosis in live cells and makes an essential contribution to understanding molecular mechanisms of endocytosis in neuronal somata and other types of cells.However,visualization of exo-endocytic events at the single vesicular level in a synapse with optical imaging remains a great challenge to reveal mechanisms governing the synaptic exo-endocytotic coupling.In this protocol,we describe the technical details of stimulated emission depletion(STED)imaging of synaptic endocytosis at the single-vesicle level,from sample preparation and microscopy calibration to data acquisition and analysis.

Bulk endocytosis at neuronal synapses

Neurotransmitter-containing synaptic vesicle(SV)fusion with the nerve terminal plasma membrane initiates neurotransmission in response to neuronal excitation.Under mild stimulation,the fused vesicular membrane is retrieved via kiss-and-run and/or clathrin-mediated endocytosis,which is sufficient to maintain recycling of SVs.When neurons are challenged with very high stimulation,the number of fused SVs can be extremely high,resulting in significant plasma membrane addition.Under such conditions,a higher capacity retrieval pathway,bulk endocytosis,is activated to redress this large membrane imbalance.Despite first being described more than 40 years ago,the molecular mechanisms underpinning this important process have yet to be clearly defined.In this review,we highlight the current evidence for bulk endocytosis and its prevalence in various neuronal models,as well as discuss the underlying molecular components.