Outside-in regulation of the readily releasable pool of synaptic vesicles by α2δ-1.

The readily releasable pool (RRP) of synaptic vesicles is a key determinant of phasic neurotransmission. Although the size of the RRP is tightly regulated by intracellular factors, there is little evidence for its modification by extracellular signals. By studying the homogeneous population of synapses present in autaptic microcultures, we show that pregabalin, a prototypical gabapentinoid, decreases the effective RRP size. Simultaneous imaging of presynaptic calcium influx and recording of postsynaptic responses shows that the effect is not related to a reduction of calcium entry. The main cause is the impairment of the functional coupling among N-type calcium channels and the RRP, resembling an increase of intracellular mobile calcium buffers. The ectodomain of neurexin-1α shows a similar action to pregabalin, acting as an endogenous ligand of α2δ-1 that reduces the RRP size without affecting presynaptic calcium influx. The regulatory actions described for pregabalin and the ectodomain of neurexin-1α are mutually exclusive. The overexpression of α2δ-1 enhances the effect of pregabalin and the ectodomain of neurexin-1α on neurotransmission by decreasing their effective concentration. In contrast, knockdown of α2δ-1 causes a profound inhibition of synaptic transmission. These observations prompt to consider α2δ-1 as an outside-in signaling platform that binds exogenous and endogenous cues for regulating the coupling of voltage-gated calcium channels to synaptic vesicles.

Endothelial Progenitor Cell Secretome and Oligovascular Repair in a Mouse Model of Prolonged Cerebral Hypoperfusion.

BACKGROUND AND PURPOSE: Endothelial progenitor cells (EPCs) have been extensively investigated as a therapeutic approach for repairing the vascular system in cerebrovascular diseases. Beyond vascular regeneration per se, EPCs may also release factors that affect the entire neurovascular unit. Here, we aim to study the effects of the EPC secretome on oligovascular remodeling in a mouse model of white matter injury after prolonged cerebral hypoperfusion. METHODS: The secretome of mouse EPCs was analyzed with a proteome array. In vitro, the effects of the EPC secretome and its factor angiogenin were assessed on primary oligodendrocyte precursor cells and mature human cerebral microvascular endothelial cells (hCMED/D3). In vivo, mice were subjected to permanent bilateral common carotid artery stenosis, then treated with EPC secretome at 24 hours and at 1 week, and cognitive outcome was evaluated with the Y maze test together with oligodendrocyte precursor cell proliferation/differentiation and vascular density in white matter at 4 weeks. RESULTS: Multiple growth factors, cytokines, and proteases were identified in the EPC secretome, including angiogenin. In vitro, the EPC secretome significantly enhanced endothelial and oligodendrocyte precursor cell proliferation and potentiated oligodendrocyte precursor cell maturation. Angiogenin was proved to be a key factor since pharmacological blockade of angiogenin signaling negated the positive effects of the EPC secretome. In vivo, treatment with the EPC secretome increased vascular density, myelin, and mature oligodendrocytes in white matter and rescued cognitive function in the mouse hypoperfusion model. CONCLUSIONS: Factors secreted by EPCs may ameliorate white matter damage in the brain by boosting oligovascular remodeling.

Matrix metalloproteinase-13 participates in neuroprotection and neurorepair after cerebral ischemia in mice.

New neuroreparative and neuroprotective therapies are being sought to treat stroke patients. One approach is the remodeling of extracellular matrix, which participates in both brain injury and neurovascular repair when matrix metalloproteinases (MMPs) are thought to be key players. Our aim was to investigate the role of MMP-13 (collagenase-3) in the acute (24h and 3days) and delayed (2weeks) phases of stroke. Permanent and transient cerebral ischemia models involving the cortex were induced in MMP-13 knock-out (KO) and wild-type (WT) mice. In the transient model, MMP-13 deficiency reduced the amount of TTC-stained infarct tissue, reduced hemorrhagic events and improved functional outcomes (p<0.01). At two weeks, normal neuroblast (DCX+) migration from the subventricular zone toward the peri-infarct area was observed. However, MMP-13 deficiency significantly reduced the number of newborn neuroblasts (DCX+/BrdU+) in the cortical peri-infarct area (p<0.01). This result occurred in parallel with aberrant cortical vascular remodeling: post-stroke peri-infarct vessel density increased in the WT mice (p<0.01) but this increase was blocked in the MMP-13 KO mice. Prior to these vascular alterations, the levels of pro-angiogenic factors, including G-CSF, VEGF-A and angiopoietin-2, were lower in the ischemic cortex of MMP-13 KO mice than in WT mice (p<0.05). In vitro, gene-silencing of MMP-13 in endothelial progenitor cells (EPCs) confirmed the reduced ability of these cells to build tubulogenic networks in Matrigel™ substrate. Together, our results indicate that MMP-13 is a central protease in infarct development and cortical remodeling during post-stroke neurorepair, which is critical for optimal angiogenic and neurogenic responses.

Protocol to correlate electron microscopy with electrophysiology in single-cell autaptic microcultures.

Single-cell microcultures (SCMs) form a monosynaptic circuit that allows stimulation and recording of postsynaptic responses using a single electrode. Here, we present a protocol to establish autaptic cultures from rat superior cervical ganglion neurons. We describe the steps for preparing SCMs, recording synaptic currents, and identifying and processing the recorded neurons for electron microscopy. We then detail procedures for visualizing synapses. This protocol is illustrated by correlating evoked and spontaneous neurotransmitter release with the ultrastructural features of synapses recorded. For complete details on the use and execution of this protocol, please refer to Velasco et al.1.

Multivesicular release favors short term synaptic depression in hippocampal autapses.

Presynaptic terminals of the central nervous system can support univesicular and multivesicular synchronous release of neurotransmitters, however, the functional implications of the prevalence of one mechanism over the other are yet unresolved. Here, we took advantage of the expression of SF-iGluSnFR.S72A in the astrocytic feeder layer of autaptic hippocampal neuronal cultures to associate the liberation of glutamate to excitatory postsynaptic currents. The presence of the glutamate sensor in glial cells avoided any interference with the function of endogenous postsynaptic receptors. It was possible to optically detect changes in neurotransmitter release probability, which was heterogeneous among synaptic boutons studied. For each neuron investigated, the liberation of neurotransmitters occurred through a predominant mechanism. The prevalence of multivesicular over univesicular release increased synaptic strength and enhanced short-term synaptic depression. These results show that the preference of hippocampal boutons to synchronously release one or more vesicles determines the strength and low pass filtering properties of the synapses established.

Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice.

Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG-binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763.