MAB1596 Sigma-AldrichAnti-Post Synaptic Density Protein 95 Antibody, clone 6G6-1C9

STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca(2+) entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER-plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.

In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations.

The formation of synapses, the specialized points of chemical communication between neurons, is a highly regulated developmental process fundamental to establishing normal brain circuitry. Perturbations of synapse formation and function causally contribute to human developmental and degenerative neuropsychiatric disorders, such as Alzheimer's disease, intellectual disability, and autism spectrum disorders. Many genes controlling synaptogenesis have been identified, but lack of facile experimental systems has made systematic discovery of regulators of synaptogenesis challenging. Thus, we created a high-throughput platform to study excitatory and inhibitory synapse development in primary neuronal cultures and used a lentiviral RNA interference library to identify novel regulators of synapse formation. This methodology is broadly applicable for high-throughput screening of genes and drugs that may rescue or improve synaptic dysfunction associated with cognitive function and neurological disorders.

At glutamatergic synapses, local endocytic recycling of AMPA receptors (AMPARs) is important for the supply of a mobile pool of AMPARs required for synaptic potentiation. This local recycling of AMPARs critically relies on the presence of an endocytic zone (EZ) near the postsynaptic density (PSD). The precise mechanisms that couple the EZ to the PSD still remain largely elusive, with the large GTPase Dynamin-3 and the multimeric PSD adaptor protein Homer1 as the two main players identified. Here, we demonstrate that a physical interaction between the X-linked mental retardation protein oligophrenin-1 (OPHN1) and Homer1b/c is crucial for the positioning of the EZ adjacent to the PSD, and present evidence that this interaction is important for OPHN1's role in controlling activity-dependent strengthening of excitatory synapses in the rat hippocampus. Disruption of the OPHN1-Homer1b/c interaction causes a displacement of EZs from the PSD, along with impaired AMPAR recycling and reduced AMPAR accumulation at synapses, in both basal conditions and conditions that can induce synaptic potentiation. Together, our findings unveil a novel role for OPHN1 as an interaction partner of Homer1b/c in spine EZ positioning, and provide new mechanistic insight into how genetic deficits in OPHN1 can lead to impaired synapse maturation and plasticity.

Spiral ganglion neurons (SGNs) receive input from cochlear hair cells and project from the cochlea to the cochlear nucleus. After destruction of hair cells with aminoglycoside antibiotics or noise, SGNs gradually die. It has been assumed that SGN death is attributable to loss of neurotrophic factors (NTFs) derived from hair cells or supporting cells in the organ of Corti (OC). We used quantitative PCR (qPCR) to assay NTF expression-neurotrophin-3 (NT-3), BDNF, GDNF, neurturin, artemin, and CNTF-in the OC and cochlear nucleus at various ages from postnatal day 0 (P0) to P90 in control hearing and neonatally deafened rats. NT-3, neurturin, and CNTF were most abundant in the postnatal hearing OC; CNTF and neurturin most abundant in the cochlear nucleus. In the OC, NT-3 and CNTF showed a postnatal increase in expression approximately concomitant with hearing onset. In rats deafened by daily kanamycin injections (from P8 to P16), surviving inner hair cells were evident at P16 but absent by P19, with most postsynaptic boutons lost before P16. NT-3 and CNTF, which normally increase postnatally, had significantly reduced expression in the OC of deafened rats, although CNTF was expressed throughout the time that SGNs were dying. In contrast, neurturin expression was constant, unaffected by deafening or by age. CNTF and neurturin expression in the cochlear nucleus was unaffected by deafening or age. Thus, NTFs other than NT-3 are available to SGNs even as they are dying after deafening, apparently conflicting with the hypothesis that SGN death is attributable to lack of NTFs.

In recent decades, there has been increased interest in the physiological roles of the endocannabinoid (eCB) system and its receptors, the cannabinoid receptor types 1 (CB1R) and 2 (CB2R). Exposure to cannabinoids during development results in neurofunctional alterations, which implies that the eCB system is involved in the developmental processes of the brain. Because of their lipophilic nature, eCBs are synthesized on demand and are not stored in vesicles. Consequently, the enzymes responsible for their synthesis and degradation are key regulators of their physiological actions. Therefore, knowing the localization of these enzymes during development is crucial for a better understanding of the role played by eCBs during the formation of the central nervous system. In this study, we investigated the developmental protein localization of the synthesizing and catabolic enzymes of the principal eCB, 2-arachidonoylglycerol (2-AG) in the retinas of young and adult rats. The distribution of the enzymes responsible for the synthesis (DAGLα) and the degradation (MAGL) of 2-AG was determined for every retinal cell type from birth to adulthood. Our results indicate that DAGLα is present early in postnatal development. It is highly expressed in photoreceptor, horizontal, amacrine, and ganglion cells. MAGL appears later during the development of the retina and its presence is limited to amacrine and Müller cells. Overall, these results suggest that 2-AG is strongly present in early retinal development and might be involved in the regulation of the structural and functional maturation of the retina.

In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.

The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.

High-voltage activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α(1) subunit and auxiliary α(2)δ and β subunits. Although there are reports of α(2)δ(4) subunit mRNA in the mouse retina and localization of the α(2)δ(4) subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of its expression and localization in the retina. α(2)δ(4) subunit expression and distribution in the mouse and rat retina were evaluated by using reverse transcriptase polymerase chain reaction, western blot, and immunohistochemistry with specific primers and a well-characterized antibody to the α(2)δ(4) subunit. α(2)δ(4) subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α(2)δ(4) subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals, and photoreceptor outer segments. This subunit is also expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α(2)δ(4) subunit participates in the modulation of L-type Ca(2+) current regulating neurotransmitter release from photoreceptor terminals and Ca(2+)-dependent signaling pathways in bipolar and Müller cells.

MET, a replicated autism risk gene, encodes a pleiotropic receptor tyrosine kinase implicated in multiple cellular processes during development and following injury. Previous studies suggest that Met modulates excitatory synapse development in the neocortex and hippocampus, although the underlying mechanism is unknown. The peak of Met expression corresponds to the period of process outgrowth and synaptogenesis, with robust expression in hippocampal and neocortical neuropil. Resolving whether neuropil expression represents presynaptic, postsynaptic or glial localization provides insight into potential mechanisms of Met action. The subcellular distribution of Met was characterized using complementary ultrastructural, in situ proximity ligation assay (PLA), and biochemical approaches. At postnatal day (P) 7, immunoelectron microscopy revealed near-equivalent proportions of Met-immunoreactive pre- (axons and terminals) and postsynaptic (dendritic shafts and spines) profiles in the stratum radiatum in the hippocampal CA1 region. Staining was typically in elements in which the corresponding pre- or postsynaptic apposition was unlabeled. By P21, Met-immunoreactive presynaptic profiles predominated and ~20% of Met-expressing profiles were glial. A different distribution of Met-immunoreactive profiles was observed in layer V of somatosensory cortex: Met-labeled spines were rare and a smaller proportion of glial profiles expressed Met. Strikingly, Met-immunoreactive presynaptic profiles predominated over postsynaptic profiles as early as P7. PLA analysis of neurons in vitro and biochemical analysis of tissue subsynaptic fractions confirmed the localization of Met in specific synaptic subcompartments. The study demonstrates that Met is enriched at synapses during development and its activation may modulate synapse formation and stability through both pre- and postsynaptic mechanisms.