Ring-like Oligomers of Synaptotagmins and Related C2 Domain Proteins

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx

Synaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain

Synaptotagmin interaction with anionic lipid (phosphatidylserine/phosphatidylinositol) containing membranes, both in the absence and presence of calcium ions (Ca2+), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca2+-binding aliphatic loops on both C2A and C2B domains, are known. Here we use surface force apparatus combined with systematic mutational analysis of the functional surfaces to directly measure Syt1-membrane interaction and fully map the site-binding energetics of Syt1 both in the absence and presence of Ca2+. By correlating energetics data with the molecular rearrangements measured during confinement, we find that both C2 domains cooperate in membrane binding, with the C2B domain functioning as the main energetic driver, and the C2A domain acting as a facilitator

Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+ Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery

Rearrangements under confinement lead to increased binding energy of Synaptotagmin-1 with anionic membranes in Mg2+ and Ca2+

Synaptotagmin‐1 (Syt1) is the primary calcium sensor (Ca2+) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca2+‐dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane‐anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+ and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca2+‐activation process in synaptic transmission

Mutations in NKX6-2 Cause Progressive Spastic Ataxia and Hypomyelination

Progressive limb spasticity and cerebellar ataxia are frequently found together in clinical practice and form a heterogeneous group of degenerative disorders that are classified either as pure spastic ataxia or as complex spastic ataxia with additional neurological signs. Inheritance is either autosomal dominant or autosomal recessive. Hypomyelinating features on MRI are sometimes seen with spastic ataxia, but this is usually mild in adults and severe and life limiting in children. We report seven individuals with an early-onset spastic-ataxia phenotype. The individuals come from three families of different ethnic backgrounds. Affected members of two families had childhood onset disease with very slow progression. They are still alive in their 30s and 40s and show predominant ataxia and cerebellar atrophy features on imaging. Affected members of the third family had a similar but earlier-onset presentation associated with brain hypomyelination. Using a combination of homozygozity mapping and exome sequencing, we mapped this phenotype to deleterious nonsense or homeobox domain missense mutations in NKX6-2. NKX6-2 encodes a transcriptional repressor with early high general and late focused CNS expression. Deficiency of its mouse ortholog results in widespread hypomyelination in the brain and optic nerve, as well as in poor motor coordination in a pattern consistent with the observed human phenotype. In-silico analysis of human brain expression and network data provides evidence that NKX6-2 is involved in oligodendrocyte maturation and might act within the same pathways of genes already associated with central hypomyelination. Our results support a non-redundant developmental role of NKX6-2 in humans and imply that NKX6-2 mutations should be considered in the differential diagnosis of spastic ataxia and hypomyelination

Multimodality Imaging of Abnormal Vascular Perfusion and Morphology in Preclinical 9L Gliosarcoma Model

This study demonstrates that a dynamic susceptibility contrast-magnetic resonance imaging (DSC-MRI) perfusion parameter may indicate vascular abnormality in a brain tumor model and reflects an effect of dexamethasone treatment. In addition, X-ray computed tomography (CT) measurements of vascular tortuosity and tissue markers of vascular morphology were performed to investigate the underpinnings of tumor response to dexamethasone.One cohort of Fisher 344 rats (N = 13), inoculated intracerebrally with 9L gliosarcoma cells, was treated with dexamethasone (i.p. 3 mg/kg/day) for five consecutive days, and another cohort (N = 11) was treated with equal volume of saline. Longitudinal DSC-MRI studies were performed at the first (baseline), third and fifth day of treatments. Relative cerebral blood volume (rCBV) was significantly reduced on the third day of dexamethasone treatment (0.65 ± .13) as compared to the fifth day during treatment (1.26 ±.19, p < 0.05). In saline treated rats, relative CBV gradually increased during treatment (0.89 ±.13, 1.00 ± .21, 1.13 ± .23) with no significant difference on the third day of treatment (p>0.05). In separate serial studies, microfocal X-ray CT of ex vivo brain specimens (N = 9) and immunohistochemistry for endothelial cell marker anti-CD31 (N = 8) were performed. Vascular morphology of ex vivo rat brains from micro-CT analysis showed hypervascular characteristics in tumors, and both vessel density (41.32 ± 2.34 branches/mm(3), p<0.001) and vessel tortuosity (p<0.05) were significantly reduced in tumors of rats treated with dexamethasone compared to saline (74.29 ± 3.51 branches/mm(3)). The vascular architecture of rat brain tissue was examined with anti-CD31 antibody, and dexamethasone treated tumor regions showed reduced vessel area (16.45 ± 1.36 µm(2)) as compared to saline treated tumor regions (30.83 ± 4.31 µm(2), p<0.001) and non-tumor regions (22.80 ± 1.11 µm(2), p<0.01).Increased vascular density and tortuosity are culprit to abnormal perfusion, which is transiently reduced during dexamethasone treatment

The role of pneumolysin in mediating lung damage in a lethal pneumococcal pneumonia murine model

BACKGROUND: Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia. METHODS: To examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice. RESULTS: In bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues. CONCLUSION: The data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival