ABSTRACT
Recent evidence has shown support for the United Nations Development Programme (UNDP) accelerator concept, which highlights the need to identify interventions or programmatic areas that can affect multiple sustainable development goals (SDGs) at once to boost their achievement. These data have also clearly shown enhanced effects when interventions are used in combination, above and beyond the effect of single interventions. However, detailed knowledge is now required on optimum combinations and relative gain in order to derive policy guidance. Which accelerators work for which outcomes, what combinations are optimum, and how many combinations are needed to maximise effect? The current study utilised pooled data from the Young Carers (n = 1402) and Child Community Care (n = 446) studies. Data were collected at baseline (n = 1848) and at a 1 to 1.5- year follow-up (n = 1740) from children and young adolescents aged 9-13 years, living in South Africa. Measures in common between the two databases were used to generate five accelerators (caregiver praise, caregiver monitoring, food security, living in a safe community, and access to community-based organizations) and to investigate their additive effects on 14 SDG-related outcomes. Predicted probabilities and predicted probability differences were calculated for each SDG outcome under the presence of none to five accelerators to determine optimal combinations. Results show that various accelerator combinations are effective, though different combinations are needed for different outcomes. Some accelerators ramified across multiple outcomes. Overall, the presence of up to three accelerators was associated with marked improvements over multiple outcomes. The benefit of targeting access to additional accelerators, with additional costs, needs to be weighed against the relative gains to be achieved with high quality but focused interventions. In conclusion, the current data show the detailed impact of various protective factors and provides implementation guidance for policy makers in targeting and distributing interventions to maximise effect and expenditure. Future work should investigate multiplicative effects and synergistic interactions between accelerators.
Subject(s)
Policy Making , Sustainable Development , Child , Humans , Adolescent , Protective Factors , United Nations , ProbabilityABSTRACT
PURPOSE: Synaptotagmin-1 (SYT1) is a critical mediator of neurotransmitter release in the central nervous system. Previously reported missense SYT1 variants in the C2B domain are associated with severe intellectual disability, movement disorders, behavioral disturbances, and electroencephalogram abnormalities. In this study, we expand the genotypes and phenotypes and identify discriminating features of this disorder. METHODS: We describe 22 individuals with 15 de novo missense SYT1 variants. The evidence for pathogenicity is discussed, including the American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria, known structure-function relationships, and molecular dynamics simulations. Quantitative behavioral data for 14 cases were compared with other monogenic neurodevelopmental disorders. RESULTS: Four variants were located in the C2A domain with the remainder in the C2B domain. We classified 6 variants as pathogenic, 4 as likely pathogenic, and 5 as variants of uncertain significance. Prevalent clinical phenotypes included delayed developmental milestones, abnormal eye physiology, movement disorders, and sleep disturbances. Discriminating behavioral characteristics were severity of motor and communication impairment, presence of motor stereotypies, and mood instability. CONCLUSION: Neurodevelopmental disorder-associated SYT1 variants extend beyond previously reported regions, and the phenotypic spectrum encompasses a broader range of severities than initially reported. This study guides the diagnosis and molecular understanding of this rare neurodevelopmental disorder and highlights a key role for SYT1 function in emotional regulation, motor control, and emergent cognitive function.
Subject(s)
Intellectual Disability , Movement Disorders , Neurodevelopmental Disorders , Synaptotagmin I , Calcium/metabolism , Genotype , Humans , Intellectual Disability/genetics , Movement Disorders/genetics , Neurodevelopmental Disorders/genetics , Phenotype , Synaptotagmin I/geneticsABSTRACT
BACKGROUND: This study aimed to identify possible entry points for interventions that can act as development accelerators for children and adolescents in South Africa and Malawi. METHODS: This study was a secondary data analysis. Data were sourced from the Child Community Care longitudinal study which tracked child well-being outcomes among 989 children (4-13 years) and their caregivers affected by HIV and enrolled in community-based organizations in South Africa and Malawi. We examined associations between five hypothesized accelerating services/household provisions-measured as access at baseline and follow-up and 12 child outcomes that relate to indicators within the Sustainable Development Goals (SDGs) framework. We calculated the adjusted probabilities of experiencing each SDG aligned outcome conditional on receipt of single, combined or all identified accelerators. RESULTS: The results show household food security is associated with positive child education and cognitive development outcomes. Cash grants were positively associated with nutrition and cognitive development outcomes. Living in a safe community was positively associated with all mental health outcomes. Experiencing a combination of two factors was associated with higher probability of positive child outcomes. However, experiencing all three accelerators was associated with better child outcomes, compared with any of the individual factors by themselves with substantial improvements noted in child education outcomes. CONCLUSIONS: Combined delivery of specific interventions or services may yield greater improvements in child outcomes across different developmental domains. It is recommended that multiple support avenues in combination like improving food security and safe communities, as well as social protection grants, should be provided for vulnerable children to maximize the impact.
Subject(s)
HIV Infections , Sustainable Development , Adolescent , Child , Child, Preschool , HIV Infections/epidemiology , HIV Infections/prevention & control , HIV Infections/psychology , Humans , Longitudinal Studies , Malawi/epidemiology , South Africa/epidemiologyABSTRACT
Fast, high-fidelity neurotransmission and synaptic efficacy requires tightly regulated coordination of pre- and postsynaptic compartments and alignment of presynaptic release sites with postsynaptic receptor nanodomains. Neuroligin-1 (Nlgn1) is a postsynaptic cell-adhesion protein exclusively localised to excitatory synapses that is crucial for coordinating the transsynaptic alignment of presynaptic release sites with postsynaptic AMPA receptors as well as postsynaptic transmission and plasticity. However, little is understood about whether the postsynaptic machinery can mediate the molecular architecture and activity of the presynaptic nerve terminal, and thus it remains unclear whether there are presynaptic contributions to Nlgn1-dependent control of signalling and plasticity. Here, we employed a presynaptic reporter of neurotransmitter release and synaptic vesicle dynamics, synaptophysin-pHluorin (sypHy), to directly assess the presynaptic impact of loss of Nlgn1. We show that lack of Nlgn1 had no effect on the size of the readily releasable or entire recycling pool of synaptic vesicles, nor did it impact exocytosis. However, we observed significant changes in the retrieval of synaptic vesicles by compensatory endocytosis, specifically during activity. Our data extends growing evidence that synaptic adhesion molecules critical for forming transsynaptic scaffolds are also important for regulating activity-induced endocytosis at the presynapse.
ABSTRACT
The synapse is formed between a presynapse (which releases neurotransmitter) and the postsynapse (which transduces this chemical signal). Over the past decade, presynaptic dysfunction has emerged as a key mediator of a series of neurodevelopmental and neurodegenerative disorders. This special issue will highlight some of the important presynaptic molecules and mechanisms that are disrupted in these conditions and reveal potential routes for therapy.
Subject(s)
Neurodegenerative Diseases , Neurotransmitter Agents/metabolism , Presynaptic Terminals/metabolism , Synapses/metabolism , Synaptic Vesicles/metabolism , Animals , Humans , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/metabolismABSTRACT
The revolution in genetic technology has ushered in a new age for our understanding of the underlying causes of neurodevelopmental, neuromuscular and neurodegenerative disorders, revealing that the presynaptic machinery governing synaptic vesicle fusion is compromised in many of these neurological disorders. This builds upon decades of research showing that disturbance to neurotransmitter release via toxins can cause acute neurological dysfunction. In this review, we focus on disorders of synaptic vesicle fusion caused either by toxic insult to the presynapse or alterations to genes encoding the key proteins that control and regulate fusion: the SNARE proteins (synaptobrevin, syntaxin-1 and SNAP-25), Munc18, Munc13, synaptotagmin, complexin, CSPα, α-synuclein, PRRT2 and tomosyn. We discuss the roles of these proteins and the cellular and molecular mechanisms underpinning neurological deficits in these disorders.
Subject(s)
Exocytosis/physiology , Neurons/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Animals , Humans , Membrane Fusion/physiology , Synaptotagmins/metabolismABSTRACT
Huntington's disease (HD) is caused by CAG repeat expansion within the HTT gene, with the dysfunction and eventual loss of striatal medium spiny neurons a notable feature. Since medium spiny neurons receive high amounts of synaptic input, we hypothesised that this vulnerability originates from an inability to sustain presynaptic performance during intense neuronal activity. To test this hypothesis, primary cultures of either hippocampal or striatal neurons were prepared from either wild-type mice or a knock-in HD mouse model which contains 140 poly-glutamine repeats in the huntingtin protein (httQ140/Q140). We identified a striatum-specific defect in synaptic vesicle (SV) endocytosis in httQ140/Q140 neurons that was only revealed during high frequency stimulation. This dysfunction was also present in neurons that were heterozygous for the mutant HTT allele. Depletion of endogenous huntingtin using hydrophobically-modified siRNA recapitulated this activity-dependent defect in wild-type neurons, whereas depletion of mutant huntingtin did not rescue the effect in httQ140/Q140 neurons. Importantly, this SV endocytosis defect was corrected by overexpression of wild-type huntingtin in homozygous httQ140/Q140 neurons. Therefore, we have identified an activity-dependent and striatum-specific signature of presynaptic dysfunction in neurons derived from pre-symptomatic HD mice, which is due to loss of wild-type huntingtin function. This presynaptic defect may render this specific neuronal subtype unable to operate efficiently during high frequency activity patterns, potentially resulting in dysfunctional neurotransmission, synapse failure and ultimately degeneration.
Subject(s)
Corpus Striatum/metabolism , Endocytosis/physiology , Huntingtin Protein/metabolism , Huntington Disease/metabolism , Neurons/metabolism , Synaptic Vesicles/metabolism , Animals , Disease Models, Animal , Gene Knock-In Techniques , Humans , Huntingtin Protein/genetics , Huntington Disease/genetics , Mice , Mice, Inbred C57BLABSTRACT
BACKGROUND: Approximately 250 million children under the age of five in low and middle-income countries (LMICs) will not achieve their developmental potential due to poverty and stunting alone. Investments in programming to improve early childhood development (ECD) have the potential to disrupt the cycle of poverty and therefore should be prioritised. Support for ECD has increased in recent years. Nevertheless, donors and policies continue to neglect ECD, in part from lack of evidence to guide policy makers and donors about where they should focus policies and programmes. Identification and investment in research is needed to overcome these constraints and in order to achieve high quality implementation of programmes to improve ECD. METHODS: The Child Health and Nutrition Research Initiative (CHNRI) priority setting methodology was applied in order to assess research priorities for improving ECD. A group of 348 global and local experts in ECD-related research were identified and invited to generate research questions. This resulted in 406 research questions which were categorised and refined by study investigators into 54 research questions across six thematic goals which were evaluated using five criteria: answerability, effectiveness, feasibility, impact, and effect on equity. Research options were ranked by their final research priority score multiplied by 100. RESULTS: The top three research priority options from the LMIC experts came from the third thematic goal of improving the impact of interventions, whereas the top three research priority options from high-income country experts came from different goals: improving the integration of interventions, increasing the understanding of health economics and social protection strategies, and improving the impact of interventions. CONCLUSION: The results of this process highlight that priorities for future research should focus on the need for services and support to parents to provide nurturing care, and the training of health workers and non-specialists in implementation of interventions to improve ECD. Three of the six thematic goals of the present priority setting centred on interventions (ie, improving impact, implementation of interventions and improving the integration of interventions). In order to achieve higher coverage through sustainable interventions to improve ECD with equitable reach, interventions should be integrated and not be sector driven.
Subject(s)
Child Development , Global Health , Research , Child, Preschool , Humans , Infant , Sustainable DevelopmentABSTRACT
CONTEXT: Although adolescent mental health interventions are widely implemented, little consensus exists about elements comprising successful models. OBJECTIVE: We aimed to identify effective program components of interventions to promote mental health and prevent mental disorders and risk behaviors during adolescence and to match these components across these key health outcomes to inform future multicomponent intervention development. DATA SOURCES: A total of 14 600 records were identified, and 158 studies were included. STUDY SELECTION: Studies included universally delivered psychosocial interventions administered to adolescents ages 10 to 19. We included studies published between 2000 and 2018, using PubMed, Medline, PsycINFO, Scopus, Embase, and Applied Social Sciences Index Abstracts databases. We included randomized controlled, cluster randomized controlled, factorial, and crossover trials. Outcomes included positive mental health, depressive and anxious symptomatology, violence perpetration and bullying, and alcohol and other substance use. DATA EXTRACTION: Data were extracted by 3 researchers who identified core components and relevant outcomes. Interventions were separated by modality; data were analyzed by using a robust variance estimation meta-analysis model, and we estimated a series of single-predictor meta-regression models using random effects. RESULTS: Universally delivered interventions can improve adolescent mental health and reduce risk behavior. Of 7 components with consistent signals of effectiveness, 3 had significant effects over multiple outcomes (interpersonal skills, emotional regulation, and alcohol and drug education). LIMITATIONS: Most included studies were from high-income settings, limiting the applicability of these findings to low- and middle-income countries. Our sample included only trials. CONCLUSIONS: Three program components emerged as consistently effective across different outcomes, providing a basis for developing future multioutcome intervention programs.
Subject(s)
Adolescent Behavior/psychology , Mental Health , Risk Reduction Behavior , Risk-Taking , Adolescent , Humans , Program Evaluation/methods , Randomized Controlled Trials as Topic/methodsABSTRACT
The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin-II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter-pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release.
Subject(s)
Neurons/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Synaptophysin/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Animals , Cells, Cultured , Hippocampus/metabolism , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Treatment with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (l-DOPA) provides symptomatic relief arising from DA denervation in Parkinson's disease. Mounting evidence that DA autooxidation to neurotoxic quinones is involved in Parkinson's disease pathogenesis has raised concern about potentiation of oxidative stress by l-DOPA. The rate of DA quinone formation increases in the presence of excess redox-active iron (Fe), which is a pathological hallmark of Parkinson's disease. Conversely, l-DOPA has pH-dependent Fe-chelating properties, and may act to 'redox silence' Fe and partially allay DA autoxidation. We examined the effects of l-DOPA in three murine models of parkinsonian neurodegeneration: early-life Fe overexposure in wild-type mice, transgenic human (h)A53T mutant α-synuclein (α-syn) over-expression, and a combined 'multi-hit' model of Fe-overload in hA53T mice. We found that l-DOPA was neuroprotective and prevented age-related Fe accumulation in the substantia nigra pars compacta (SNc), similar to the mild-affinity Fe chelator clioquinol. Chronic l-DOPA treatment showed no evidence of increased oxidative stress in wild-type midbrain and normalized motor performance, when excess Fe was present. Similarly, l-DOPA also did not exacerbate protein oxidation levels in hA53T mice, with or without excess nigral Fe, and showed evidence of neuroprotection. The effects of l-DOPA in Fe-fed hA53T mice were somewhat muted, suggesting that Fe-chelation alone is insufficient to attenuate neuron loss in an animal model also recapitulating altered DA metabolism. In summary, we found no evidence in any of our model systems that l-DOPA treatment accentuated neurodegeneration, suggesting DA replacement therapy does not contribute to oxidative stress in the Parkinson's disease brain.
Subject(s)
Antiparkinson Agents/pharmacology , Brain/drug effects , Levodopa/pharmacology , Oxidative Stress/drug effects , Parkinson Disease/metabolism , Animals , Brain/pathology , Disease Models, Animal , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/pathology , Humans , Iron/metabolism , Iron Overload , Mice , Mice, Transgenic , Nerve Degeneration/pathology , alpha-SynucleinABSTRACT
This protocol utilizes lipophilic FM dyes to monitor membrane recycling in real time. FM dyes are virtually nonfluorescent in solution but when membrane bound are intensely fluorescent, combined with the flexibility of different emission wavelengths make these dyes an excellent choice for investigating clathrin-mediated endocytosis, among other membrane trafficking and recycling pathways.
Subject(s)
Endocytosis , Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Animals , Cells, Cultured , Clathrin/metabolism , Neurons/metabolismABSTRACT
Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals' phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.
Subject(s)
Synaptotagmin I/genetics , Synaptotagmin I/physiology , Action Potentials , Adolescent , Animals , Calcium/metabolism , Child , Child, Preschool , Electrophysiological Phenomena , Endocytosis , Female , Humans , Intellectual Disability/genetics , Male , Mice , Mice, Inbred C57BL , Movement Disorders/genetics , Mutation, Missense/genetics , Neurodevelopmental Disorders/metabolism , Neurons/metabolism , Rats , Synaptic Transmission , Synaptic Vesicles/genetics , Synaptic Vesicles/metabolism , Synaptic Vesicles/physiology , Young AdultABSTRACT
Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15-2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance.
Subject(s)
Hippocampus/cytology , Neurons/cytology , Synaptic Vesicles/metabolism , Synaptophysin/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Animals , Cells, Cultured , Endocytosis , Female , Hippocampus/metabolism , Male , Mice, Inbred C57BL , Neurons/metabolism , Synaptophysin/analysis , Vesicle-Associated Membrane Protein 2/analysisABSTRACT
The reformation of synaptic vesicles (SVs) during endocytosis is essential for the maintenance of neurotransmission in central nerve terminals. Newly formed SVs must be generated with the correct protein cargo in the correct stoichiometry to be functional for exocytosis. Classical clathrin adaptor protein complexes play a key role in sorting and clustering synaptic vesicle cargo in this regard. However it is becoming increasingly apparent that additional "fail-safe" mechanisms exist to ensure the accurate retrieval of essential cargo molecules. For example, the monomeric adaptor proteins AP180/CALM and stonin-2 are required for the efficient retrieval of synaptobrevin II (sybII) and synaptotagmin-1 respectively. Furthermore, recent studies have revealed that sybII and synaptotagmin-1 interact with other SV cargoes to ensure a high fidelity of retrieval. These cargoes are synaptophysin (for sybII) and SV2A (for synaptotagmin-1). In this review, we summarize current knowledge regarding the retrieval mechanisms for both sybII and synaptotagmin-1 during endocytosis. We also define and set criteria for a new functional group of SV molecules that facilitate the retrieval of their interaction partners. We have termed these molecules intrinsic trafficking partners (iTRAPs) and we discuss how the function of this group impacts on presynaptic performance in both health and disease.
ABSTRACT
Synaptotagmin-1 (SYT1) is a calcium-binding synaptic vesicle protein that is required for both exocytosis and endocytosis. Here, we describe a human condition associated with a rare variant in SYT1. The individual harboring this variant presented with an early onset dyskinetic movement disorder, severe motor delay, and profound cognitive impairment. Structural MRI was normal, but EEG showed extensive neurophysiological disturbances that included the unusual features of low-frequency oscillatory bursts and enhanced paired-pulse depression of visual evoked potentials. Trio analysis of whole-exome sequence identified a de novo SYT1 missense variant (I368T). Expression of rat SYT1 containing the equivalent human variant in WT mouse primary hippocampal cultures revealed that the mutant form of SYT1 correctly localizes to nerve terminals and is expressed at levels that are approximately equal to levels of endogenous WT protein. The presence of the mutant SYT1 slowed synaptic vesicle fusion kinetics, a finding that agrees with the previously demonstrated role for I368 in calcium-dependent membrane penetration. Expression of the I368T variant also altered the kinetics of synaptic vesicle endocytosis. Together, the clinical features, electrophysiological phenotype, and in vitro neuronal phenotype associated with this dominant negative SYT1 mutation highlight presynaptic mechanisms that mediate human motor control and cognitive development.
Subject(s)
Intellectual Disability/genetics , Motor Skills Disorders/genetics , Movement Disorders/genetics , Mutation, Missense , Point Mutation , Presynaptic Terminals/physiology , Synaptic Vesicles/physiology , Synaptotagmin I/physiology , Amino Acid Sequence , Animals , Calcium/metabolism , Cells, Cultured , Child , Endocytosis/genetics , Endocytosis/physiology , Evoked Potentials, Visual , Exocytosis/genetics , Exocytosis/physiology , Genes, Dominant , Hippocampus/cytology , Humans , Kinetics , Male , Membrane Fusion , Mice , Molecular Sequence Data , Rats , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Synaptotagmin I/geneticsABSTRACT
Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau-tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.
Subject(s)
Casein Kinase I/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Synaptotagmin I/metabolism , Amino Acid Sequence , Animals , Brain Chemistry/genetics , Cells, Cultured , Endocytosis/physiology , HEK293 Cells , Hippocampus/cytology , Hippocampus/metabolism , Humans , Mice , Mice, Knockout , Molecular Sequence Data , Neurons/metabolism , Phosphorylation , Synaptotagmin I/chemistry , Synaptotagmin I/isolation & purificationABSTRACT
Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.
Subject(s)
Abnormalities, Multiple/physiopathology , Cataract/congenital , Cornea/abnormalities , Cytoskeleton/physiology , Disease Models, Animal , Eye/growth & development , Hypogonadism/physiopathology , Intellectual Disability/physiopathology , Microcephaly/physiopathology , Neurons/physiology , Optic Atrophy/physiopathology , rab GTP-Binding Proteins/physiology , Animals , Cataract/physiopathology , Cornea/physiopathology , Mice , Mice, Knockout , rab GTP-Binding Proteins/geneticsABSTRACT
Synaptobrevin II (sybII) is a key fusogenic molecule on synaptic vesicles (SVs) therefore the active maintenance of both its conformation and location in sufficient numbers on this organelle is critical in both mediating and sustaining neurotransmitter release. Recently three proteins have been identified having key roles in the presentation, trafficking and retrieval of sybII during the fusion and endocytosis of SVs. The nerve terminal protein α-synuclein catalyses sybII entry into SNARE complexes, whereas the monomeric adaptor protein AP-180 is required for sybII retrieval during SV endocytosis. Overarching these events is the tetraspan SV protein synaptophysin, which is a major sybII interaction partner on the SV. This review will evaluate recent studies to propose working models for the control of sybII traffic by synaptophysin and other Sybtraps (sybII trafficking partners) and suggest how dysfunction in sybII traffic may contribute to human disease.
Subject(s)
Monomeric Clathrin Assembly Proteins/metabolism , R-SNARE Proteins/metabolism , Synaptophysin/metabolism , alpha-Synuclein/metabolism , Animals , Humans , Protein Binding , Protein TransportABSTRACT
Synaptophysin is an integral synaptic vesicle (SV) protein that accounts for â¼10% of total SV protein cargo. Deletion of synaptophysin results in the defective retrieval of synaptobrevin II (sybII) from the plasma membrane during endocytosis, coupled with a slowing in the speed of endocytosis. Synaptophysin has been implicated in X-linked intellectual disability, with a recent study identifying four separate synaptophysin gene mutations in families affected by the disorder. To determine how these mutations may affect synaptophysin function, we expressed them in cultured neurons derived from synaptophysin knock-out mice. Two distinct truncating mutants were mislocalized throughout the axon and phenocopied the arrest of sybII retrieval in synaptophysin knock-out cultures. The remaining two mutants displayed a nerve terminal localization but did not support efficient sybII retrieval. Interestingly, one mutant fully rescued SV endocytosis kinetics, suggesting that sybII retrieval and endocytosis speed are independent from each other. These studies suggest that the efficient retrieval of sybII by synaptophysin may be key to maintaining synaptic health and perturbation of this event may contribute to the pathogenesis underlying neurodevelopmental disorders such as X-linked intellectual disability.