The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.

BackgroundPresenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.Methodology/Principal FindingsTo test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ42 production are associated with a consistent conformational change in PS1.Conclusions/SignificanceThese results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ42 generation, which could be exploited therapeutically.

We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4 -7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is ␥-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with ␥-aminobutyric acid, type A, receptor (GABA A R) clusters than with ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA A R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.

We have found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the b subunits of the c-aminobutyric acid type-A receptor (GABA A R). BIG2 is a Sec7 domain-containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C-terminal fragment of BIG2 and the large intracellular loop of the GABA A R b subunits was revealed with a yeast two-hybrid assay. The native BIG2 and GABA A Rs interact in the brain since both coprecipitated from detergent extracts with either anti-GABA A R or anti-BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABA A Rs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans-Golgi network. BIG2 is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABA A Rs. BIG2 is present in both inhibitory GABAergic synapses that contain GABA A Rs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABA A R b subunits plays a role in the exocytosis and trafficking of assembled GABA A R to the cell surface.

Rat forebrain synaptosomes were extracted with Triton X-100 at 4°C and the insoluble material, which is enriched in postsynaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble c-aminobutyric acid type-A receptors (GABA A Rs) were identified: (i) a higher-density pool (q = 1.10-1.15 mg/ mL) of GABA A Rs that contains the c2 subunit (plus a and b subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (q = 1.06-1.09 mg/mL) of GABA A Rs associated to detergent-resistant membranes (DRMs) that contain a and b subunits but not the c2 subunit. Some of these GABA A Rs contain the d subunit. Two pools of GABA A Rs insoluble in Triton X-100 at 4°C were also identified in cultured hippocampal neurons: (i) a GABA A R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA A R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA A R clusters by capping with an anti-a1 GABA A R subunit antibody and that becomes solubilized in Triton X-100 at 4°C after cholesterol depletion. Thus, there is a pool of GABA A Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA A Rs. Some of the lipid raft-associated GABA A Rs might be involved in tonic inhibition.

BackgroundSynaptic loss strongly correlates with memory deterioration. Local accumulation of amyloid β (Aβ) peptide, and neurotoxic Aβ42 in particular, due to abnormal neuronal activity may underlie synaptic dysfunction, neurodegeneration, and memory impairments. To gain an insight into molecular events underlying neuronal activity-regulated Aβ production at the synapse, we explored functional outcomes of the newly discovered calcium-dependent interaction between Alzheimer’s disease-associated presenilin 1 (PS1)/γ-secretase and synaptic vesicle proteins.ResultsMass spectrometry screen of mouse brain lysates identified synaptotagmin 1 (Syt1) as a novel synapse-specific PS1-binding partner that shows Ca2+-dependent PS1 binding profiles in vitro and in vivo. We found that Aβ level, and more critically, conformation of the PS1 and the Aβ42/40 ratio, are affected by Syt1 overexpression or knockdown, indicating that Syt1 and its interaction with PS1 might regulate Aβ production at the synapse. Moreover, β-secretase 1 (BACE1) stability, β- and γ-secretase activity, as well as intracellular compartmentalization of PS1 and BACE1, but not of amyloid precursor protein (APP), nicastrin (Nct), presenilin enhancer 2 (Pen-2), or synaptophysin (Syp) were altered in the absence of Syt1, suggesting a selective effect of Syt1 on PS1 and BACE1 trafficking.ConclusionsOur findings identify Syt1 as a novel Ca2+-sensitive PS1 modulator that could regulate synaptic Aβ, opening avenues for novel and selective synapse targeting therapeutic strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0248-3) contains supplementary material, which is available to authorized users.

Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 ؎ 4% of the GABAergic synapses and 25 ؎ 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.We have recently developed a method for the preparation of a brain fraction enriched in GABAergic postsynaptic complex (1). This fraction, insoluble in Triton X-100, was enriched in Gray's type-II postsynaptic densities (type-II PSDs) 2 and in the postsynaptic GABAergic markers GABA A receptors (GABA A Rs) and gephyrin. Here we report that septin 11 is a major component of the type-II PSD fraction. Septins are a family of proteins with GTPase activity that form heterooligomeric filaments and ringlike structures that act as diffusion barriers and scaffolds. Septins are involved in cytokinesis, positioning of the mitotic spindle, cellular morphology, vesicle trafficking, apoptosis, neurodegeneration, and neoplasia (2-5). In mammals, 14 septin genes have been identified. Each septin gene is expressed in several spliced forms. Although most septins are highly expressed in the brain (6), only recently is their role in neuronal function (7-9) and in neuropathology (10 -14) is beginning to be addressed for some septins.Septin 11 is expressed in various tissues, including the brain (15), but little is known about the role of septin 11 in the brain. Septins 3, 5, 6, and 7 are localized in the presynaptic terminals, frequently associated with synaptic vesicles (6,16,17). In neurons, septin 11 forms heterooligomeric complexes with septin 7 and septin 5 (9, 18). Nevertheless, the regional and developmental distribution of septin 11 in the brain and in hippocampal cultures is not identical to that of septin 7 or septin 5 (8). These result...

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