Exome sequencing and microarray identified a novel large exonic deletion in SYT2 gene in an ultra-rare case with recessive CMS type 7.

Congenital myasthenic syndromes (CMSs) are a diverse group of diseases that have an underlying defect in transmission of signals from nerve cells to muscles that lead to muscular weakness. A 13-year-old male child born of consanguineous parents with profound motor developmental delay and normal cognition was referred to us. The younger male sibling aged 9 months was similarly affected. Electromyography (EMG) and nerve conduction studies revealed CMS. Clinical exome sequencing revealed a novel large deletion including the exons 2 to 9 of SYT2 gene which confirmed the diagnosis of presynaptic CMS type 7 in the siblings. The deletion was confirmed on a chromosomal exon microarray. The parents were confirmed carriers of the same mutation and were normal on clinical and EMG studies. This is the second case of CMS type 7 described with a large deletion of SYT2 gene, a first case with SYT2 gene mutation from India and overall 10th recessive case in the world.

Molecular landscape of BoNT/B bound to a membrane-inserted synaptotagmin/ganglioside complex.

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

Expression and Neurotransmitter Association of the Synaptic Calcium Sensor Synaptotagmin in the Avian Auditory Brain Stem.

In the avian auditory brain stem, acoustic timing and intensity cues are processed in separate, parallel pathways via the two divisions of the cochlear nucleus, nucleus angularis (NA) and nucleus magnocellularis (NM). Differences in excitatory and inhibitory synaptic properties, such as release probability and short-term plasticity, contribute to differential processing of the auditory nerve inputs. We investigated the distribution of synaptotagmin, a putative calcium sensor for exocytosis, via immunohistochemistry and double immunofluorescence in the embryonic and hatchling chick brain stem (Gallus gallus). We found that the two major isoforms, synaptotagmin 1 (Syt1) and synaptotagmin 2 (Syt2), showed differential expression. In the NM, anti-Syt2 label was strong and resembled the endbulb terminals of the auditory nerve inputs, while anti-Syt1 label was weaker and more punctate. In NA, both isoforms were intensely expressed throughout the neuropil. A third isoform, synaptotagmin 7 (Syt7), was largely absent from the cochlear nuclei. In nucleus laminaris (NL, the target nucleus of NM), anti-Syt2 and anti-Syt7 strongly labeled the dendritic lamina. These patterns were established by embryonic day 18 and persisted to postnatal day 7. Double-labeling immunofluorescence showed that Syt1 and Syt2 were associated with vesicular glutamate transporter 2 (VGluT2), but not vesicular GABA transporter (VGAT), suggesting that these Syt isoforms were localized to excitatory, but not inhibitory, terminals. These results suggest that Syt2 is the major calcium binding protein underlying excitatory neurotransmission in the timing pathway comprising NM and NL, while Syt2 and Syt1 regulate excitatory transmission in the parallel intensity pathway via cochlear nucleus NA.

Knockin mouse models demonstrate differential contributions of synaptotagmin-1 and -2 as receptors for botulinum neurotoxins.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are also utilized to treat a wide range of disorders including muscle spasm, overactive bladder, and pain. BoNTs' ability to target neurons determines their specificity, potency, and therapeutic efficacy. Homologous synaptic vesicle membrane proteins synaptotagmin-1 (Syt1) and synaptotagmin-2 (Syt2) have been identified as receptors for BoNT family members including BoNT/B, DC, and G, but their contributions at physiologically relevant toxin concentrations in vivo have yet to be validated and established. Here we generated two knockin mutant mouse models containing three designed point-mutations that specifically disrupt BoNT binding in endogenous Syt1 or Syt2, respectively. Utilizing digit abduction score assay by injecting toxins into the leg muscle, we found that Syt1 mutant mice showed similar sensitivity as the wild type mice, whereas Syt2 mutant mice showed reduced sensitivity to BoNT/B, DC, and G, demonstrating that Syt2 is the dominant receptor at skeletal neuromuscular junctions. We further developed an in vivo bladder injection assay for analyzing BoNT action on bladder tissues and demonstrated that Syt1 is the dominant toxin receptor in autonomic nerves controlling bladder tissues. These findings establish the critical role of protein receptors for the potency and specificity of BoNTs in vivo and demonstrate the differential contributions of Syt1 and Syt2 in two sets of clinically relevant target tissues.

Pharmacological and optical activation of TrkB in Parvalbumin interneurons regulate intrinsic states to orchestrate cortical plasticity.

Elevated states of brain plasticity typical for critical periods of early postnatal life can be reinstated in the adult brain through interventions, such as antidepressant treatment and environmental enrichment, and induced plasticity may be critical for the antidepressant action. Parvalbumin-positive (PV) interneurons regulate the closure of developmental critical periods and can alternate between high and low plasticity states in response to experience in adulthood. We now show that PV plasticity states and cortical networks are regulated through the activation of TrkB neurotrophin receptors. Visual cortical plasticity induced by fluoxetine, a widely prescribed selective serotonin reuptake inhibitor (SSRI) antidepressant, was lost in mice with reduced expression of TrkB in PV interneurons. Conversely, optogenetic gain-of-function studies revealed that activation of an optically activatable TrkB (optoTrkB) specifically in PV interneurons switches adult cortical networks into a state of elevated plasticity within minutes by decreasing the intrinsic excitability of PV interneurons, recapitulating the effects of fluoxetine. TrkB activation shifted cortical networks towards a low PV configuration, promoting oscillatory synchrony, increased excitatory-inhibitory balance, and ocular dominance plasticity. OptoTrkB activation promotes the phosphorylation of Kv3.1 channels and reduces the expression of Kv3.2 mRNA providing a mechanism for the lower excitability. In addition, decreased expression and puncta of Synaptotagmin2 (Syt2), a presynaptic marker of PV interneurons involved in Ca2+-dependent neurotransmitter release, suggests lower inputs onto pyramidal neurons suppressing feed-forward inhibition. Together, the results provide mechanistic insights into how TrkB activation in PV interneurons orchestrates the activity of cortical networks and mediating antidepressant responses in the adult brain.

ATR regulates neuronal activity by modulating presynaptic firing.

Ataxia Telangiectasia and Rad3-related (ATR) protein, as a key DNA damage response (DDR) regulator, plays an essential function in response to replication stress and controls cell viability. Hypomorphic mutations of ATR cause the human ATR-Seckel syndrome, characterized by microcephaly and intellectual disability, which however suggests a yet unknown role for ATR in non-dividing cells. Here we show that ATR deletion in postmitotic neurons does not compromise brain development and formation; rather it enhances intrinsic neuronal activity resulting in aberrant firing and an increased epileptiform activity, which increases the susceptibility of ataxia and epilepsy in mice. ATR deleted neurons exhibit hyper-excitability, associated with changes in action potential conformation and presynaptic vesicle accumulation, independent of DDR signaling. Mechanistically, ATR interacts with synaptotagmin 2 (SYT2) and, without ATR, SYT2 is highly upregulated and aberrantly translocated to excitatory neurons in the hippocampus, thereby conferring a hyper-excitability. This study identifies a physiological function of ATR, beyond its DDR role, in regulating neuronal activity.

Dominant and recessive congenital myasthenic syndromes caused by SYT2 mutations.

INTRODUCTION/AIMS: We studied a patient with a congenital myasthenic syndrome (CMS) caused by a dominant mutation in the synaptotagmin 2 gene (SYT2) and compared the clinical features of this patient with those of a previously described patient with a recessive mutation in the same gene. METHODS: We performed electrodiagnostic (EDX) studies, genetic studies, muscle biopsy, microelectrode recordings and electron microscopy (EM). RESULTS: Both patients presented with muscle weakness and bulbar deficits, which were worse in the recessive form. EDX studies showed presynaptic failure, which was more prominent in the recessive form. Microelectrode studies in the dominant form showed a marked reduction of the quantal content, which increased linearly with higher frequencies of nerve stimulation. The MEPP frequencies were normal at rest but increased markedly with higher frequencies of nerve stimulation. The EM demonstrated overdeveloped postsynaptic folding, and abundant endosomes, multivesicular bodies and degenerative lamellar bodies inside small nerve terminals. DISCUSSION: The recessive form of CMS caused by a SYT2 mutation showed far more severe clinical manifestations than the dominant form. The pathogenesis of the dominant form likely involves a dominant-negative effect due to disruption of the dual function of synaptotagmin as a Ca2+ -sensor and modulator of synaptic vesicle exocytosis.

Late onset of Synaptotagmin 2a expression at synapses relevant to social behavior.

As they form, synapses go through various stages of maturation and refinement. These steps are linked to significant changes in synaptic function, potentially resulting in emergence and maturation of behavioral outputs. Synaptotagmins are calcium-sensing proteins of the synaptic vesicle exocytosis machinery, and changes in Synaptotagmin proteins at synapses have significant effects on vesicle release and synaptic function. Here, we examined the distribution of the synaptic vesicle protein Synaptotagmin 2a (Syt2a) during development of the zebrafish nervous system. Syt2a is widely distributed throughout the midbrain and hindbrain early during larval development but very weakly expressed in the forebrain. Later in development, Syt2a expression levels in the forebrain increase, particularly in regions associated with social behavior, and most intriguingly, around the time social behavior becomes apparent. We provide evidence that Syt2a localizes to synapses onto neurons implicated in social behavior in the ventral forebrain and show that Syt2a is colocalized with tyrosine hydroxylase, a biosynthetic enzyme in the dopamine pathway. Our results suggest a developmentally important role for Syt2a in maturing synapses in the forebrain, coinciding with the emergence of social behavior.

A new de novo SYT2 mutation presenting as distal weakness. Neuropathy or neuromuscular junction dysfunction?

We report the case of a patient with a clinical phenotype characterized by distal lower limb weakness and pes cavus. The electrophysiological study showed slightly reduced or normal amplitude of motor potentials, a decremental response to repetitive nerve stimulation and post-exercise facilitation. Muscle biopsy showed only mild neurogenic features. Genetic analysis included a clinical exome sequencing, followed by Sanger analysis. Three-dimensional (3D) models were generated with a SwissModel (https://swissmodel.expasy.org/) to explain the clinical observations and reinforce the pathogenic nature of the genetic variant identified. Genetic analysis demonstrated a new de novo heterozygous in frame deletion of the SYT2 gene (NM_177402.4: c.1082_1096del), confirmed by Sanger sequencing, which removes five aminoacids in the C2B domain of synaptotagmin-2 protein, that cause a profound effect on the structure and function of this synaptic vesicle protein. We identified a de novo genetic variant in the SYT2 gene, further supporting its association with a highly stereotyped clinical and electrophysiological phenotype. Our case showed electrophysiological features consistent with a presynaptic dysfunction in the neuromuscular junction with normal post-exercise amplitudes, not supporting the presence of predominant axonal damage. Although the analysis of SYT2 gene should be included in genetic analysis of patients presenting with this clinical phenotype that mimics motor neuropathy, clinicians have to consider the study of neuromuscular transmission to early identify this potentially treatable condition.

Identification of a Novel de Novo Variant in the SYT2 Gene Causing a Rare Type of Distal Hereditary Motor Neuropathy.

OBJECTIVE: To report the first de novo missense mutation in the SYT2 gene causing distal hereditary motor neuropathy. METHODS: Genetic testing was carried out, including clinical exome sequencing for the proband and Sanger sequencing for the proband and his parents. We described the clinical and electrophysiological features found in the patient. RESULTS: We reported a proband with a new de novo missense mutation, c.917C>T (p.Ser306Leu), in the C2B domain of SYT2. The clinical presentation was similar to that of phenotypes described in previous studies. A notable feature in our study was normal electrophysiological testing results of the patient. CONCLUSIONS: In this study we reinforced the association between SYT2 mutations and distal hereditary motor neuropathy. We also described the clinical presentation of the patient carrying this pathogenic variant and provided unusual results of electrophysiological testing. The results showed that a diagnosis of SYT2-associated neuropathy should be based on the similarity of clinical manifestations, rather than the results of electrophysiological testing.

Biallelic loss of function variants in SYT2 cause a treatable congenital onset presynaptic myasthenic syndrome.

Synaptotagmins are integral synaptic vesicle membrane proteins that function as calcium sensors and regulate neurotransmitter release at the presynaptic nerve terminal. Synaptotagmin-2 (SYT2), is the major isoform expressed at the neuromuscular junction. Recently, dominant missense variants in SYT2 have been reported as a rare cause of distal motor neuropathy and myasthenic syndrome, manifesting with stable or slowly progressive distal weakness of variable severity along with presynaptic NMJ impairment. These variants are thought to have a dominant-negative effect on synaptic vesicle exocytosis, although the precise pathomechanism remains to be elucidated. Here we report seven patients of five families, with biallelic loss of function variants in SYT2, clinically manifesting with a remarkably consistent phenotype of severe congenital onset hypotonia and weakness, with variable degrees of respiratory involvement. Electrodiagnostic findings were consistent with a presynaptic congenital myasthenic syndrome (CMS) in some. Treatment with an acetylcholinesterase inhibitor pursued in three patients showed clinical improvement with increased strength and function. This series further establishes SYT2 as a CMS-disease gene and expands its clinical and genetic spectrum to include recessive loss-of-function variants, manifesting as a severe congenital onset presynaptic CMS with potential treatment implications.

Recessive congenital myasthenic syndrome caused by a homozygous mutation in SYT2 altering a highly conserved C-terminal amino acid sequence.

Defects in the gene encoding synaptotagmin 2 (SYT2) have been linked to a presynaptic congenital myasthenic syndrome (CMS) and motor neuropathies. However, to date only dominant forms of the disease have been described. We report here a consanguineous patient with a severe recessive form of presynaptic CMS and denervation atrophy caused by the homozygous mutation c.1191delG, p.Arg397Serfs*37 in SYT2. The affected 2-year-old girl had profound weakness and areflexia with moderate bulbar deficit. Repetitive nerve stimulation revealed an extreme reduction of compound muscle action potential amplitudes at rest, with a striking facilitation followed by a progressive decline at fast stimulation rates. These findings were reminiscent, but not identical to those seen in the Lambert-Eaton myasthenic syndrome. 3,4 diaminopyridine and pyridostigmine were effective to ameliorate muscle fatigue, but albuterol was ineffective. Modeling of the mutation using the rat Syt1 C2B x-ray structure revealed that Arg397Serfs*37 disrupts a highly conserved amino acid sequence at the bottom face of the C2B domain not directly involved in calcium binding, but crucial for synaptotagmin-SNARE interaction and exocytosis. Thus, this report describes a recessive form of synaptotagmin 2-CMS and highlights the importance of the synaptotagmin C-terminal on synaptic vesicle fusion and exocytosis.

A novel mosaic 1q32.1 microduplication identified through Chromosome Microarray Analysis: narrowing the smallest critical region including KDM5B gene found associated with neurodevelopmetal disorders.

Microduplications involving 1q32.1 chromosomal region have been rarely reported in literature. Patients with these microduplications suffer from intellectual disability, developmental delay and a number of dysmorphic features, although no clear karyotype/phenotype correlation has yet been determined. In this case report we describe two monochorionic-diamniotic twins with intellectual disability, abnormality of coordination and dysmorphic features associated with a de novo 280 kb mosaic microduplication of 1q32.1 chromosomal region, identified using a Chromosome Microarray Analysis (CMA) and confirmed by quantitative PCR analysis. The duplicated region encompassed entirely three OMIM genes KDM5B (*605393), KLHL12 (*614522), RABIF (*603417) and involved partially SYT2 (*600104). This unique case report allows to redefine the critical 1q32.1 microduplicated region implicated in the ethiopathogenesis of intellectual disability and developmental delay. Furthermore, it suggests that KDM5B gene can have a pivotal role in the development of neurodevelopmental disorders through its demethylase activity.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor.

Inositol polyphosphate multikinase deficiency leads to aberrant induction of synaptotagmin-2 in the forebrain.

Inositol polyphosphate multikinase (IPMK), the key enzyme responsible for the synthesis of higher inositol polyphosphates and phosphatidylinositol 3, 4, 5-trisphosphate, is known to mediate various biological events, such as cellular growth and metabolism. Conditional deletion of IPMK in excitatory neurons of the mouse postnatal forebrain results in enhanced extinction of fear memory accompanied by activation of p85 S6 kinase 1 signaling in the amygdala; it also facilitates hippocampal long-term potentiation. However, the molecular changes triggered by IPMK deletion in the brain have not been fully elucidated. In the present study, we investigated gene expression changes in the hippocampal region of IPMK conditional knockout (cKO) mice by performing genome-wide transcriptome analyses. Here we show that expression of synaptotagmin 2 (Syt2), a synaptic vesicle protein essential for Ca2+-dependent neurotransmitter release, is robustly upregulated in the forebrain of IPMKcKO mice. Compared to wild-type mice, in which weak Syt2 expression was detected in the forebrain, IPMKcKO mice showed marked increases in both Syt2 mRNA and protein expression in the hippocampus as well as the amygdala. Collectively, our results suggest a physiological role for IPMK in regulating expression of Syt2, providing a potential underlying molecular mechanism to explain IPMK-mediated neural functions.

Structural maturation of cortical perineuronal nets and their perforating synapses revealed by superresolution imaging.

Parvalbumin-positive (PV+) interneurons play a pivotal role in orchestrating windows of experience-dependent brain plasticity during development. Critical period closure is marked by the condensation of a perineuronal net (PNN) tightly enwrapping subsets of PV+ neurons, both acting as a molecular brake on plasticity and maintaining mature PV+ cell signaling. As much of the molecular organization of PNNs exists at length scales near or below the diffraction limit of light microscopy, we developed a superresolution imaging and analysis platform to visualize the structural organization of PNNs and the synaptic inputs perforating them in primary visual cortex. We identified a structural trajectory of PNN maturation featuring a range of net structures, which was accompanied by an increase in Synaptotagmin-2 (Syt2) signals on PV+ cells suggestive of increased inhibitory input between PV+ neurons. The same structural trajectory was followed by PNNs both during normal development and under conditions of critical period delay by total sensory deprivation or critical period acceleration by deletion of MeCP2, the causative gene for Rett syndrome, despite shifted maturation levels under these perturbations. Notably, superresolution imaging further revealed a decrease in Syt2 signals alongside an increase in vesicular glutamate transporter-2 signals on PV+ cells in MeCP2-deficient animals, suggesting weaker recurrent inhibitory input between PV+ neurons and stronger thalamocortical excitatory inputs onto PV+ cells. These results imply a latent imbalanced circuit signature that might promote cortical silencing in Rett syndrome before the functional regression of vision.

Proportional loss of parvalbumin-immunoreactive synaptic boutons and granule cells from the hippocampus of sea lions with temporal lobe epilepsy.

One in 26 people develop epilepsy and in these temporal lobe epilepsy (TLE) is common. Many patients display a pattern of neuron loss called hippocampal sclerosis. Seizures usually start in the hippocampus but underlying mechanisms remain unclear. One possibility is insufficient inhibition of dentate granule cells. Normally parvalbumin-immunoreactive (PV) interneurons strongly inhibit granule cells. Humans with TLE display loss of PV interneurons in the dentate gyrus but questions persist. To address this, we evaluated PV interneuron and bouton numbers in California sea lions (Zalophus californianus) that naturally develop TLE after exposure to domoic acid, a neurotoxin that enters the marine food chain during harmful algal blooms. Sclerotic hippocampi were identified by the loss of Nissl-stained hilar neurons. Stereological methods were used to estimate the number of granule cells and PV interneurons per dentate gyrus. Sclerotic hippocampi contained fewer granule cells, fewer PV interneurons, and fewer PV synaptic boutons, and the ratio of granule cells to PV interneurons was higher than in controls. To test whether fewer boutons was attributable to loss versus reduced immunoreactivity, expression of synaptotagmin-2 (syt2) was evaluated. Syt2 is also expressed in boutons of PV interneurons. Sclerotic hippocampi displayed proportional losses of syt2-immunoreactive boutons, PV boutons, and granule cells. There was no significant difference in the average numbers of PV- or syt2-positive boutons per granule cell between control and sclerotic hippocampi. These findings do not address functionality of surviving synapses but suggest reduced granule cell inhibition in TLE is not attributable to anatomical loss of PV boutons.

Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models.

Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1MY) and E1191Q/S1199W (rBoNT/B1QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.

How to Spot Congenital Myasthenic Syndromes Resembling the Lambert-Eaton Myasthenic Syndrome? A Brief Review of Clinical, Electrophysiological, and Genetics Features.

Congenital myasthenic syndromes (CMS) are heterogeneous genetic diseases in which neuromuscular transmission is compromised. CMS resembling the Lambert-Eaton myasthenic syndrome (CMS-LEMS) are emerging as a rare group of distinct presynaptic CMS that share the same electrophysiological features. They have low compound muscular action potential amplitude that increment after brief exercise (facilitation) or high-frequency repetitive nerve stimulation. Although clinical signs similar to LEMS can be present, the main hallmark is the electrophysiological findings, which are identical to autoimmune LEMS. CMS-LEMS occurs due to deficits in acetylcholine vesicle release caused by dysfunction of different components in its pathway. To date, the genes that have been associated with CMS-LEMS are AGRN, SYT2, MUNC13-1, VAMP1, and LAMA5. Clinicians should keep in mind these newest subtypes of CMS-LEMS to achieve the correct diagnosis and therapy. We believe that CMS-LEMS must be included as an important diagnostic clue to genetic investigation in the diagnostic algorithms to CMS. We briefly review the main features of CMS-LEMS.