Emergence of non-canonical parvalbumin-containing interneurons in hippocampus of a murine model of type I lissencephaly.

Type I lissencephaly is a neuronal migration disorder caused by haploinsuffiency of the PAFAH1B1 (mouse: Pafah1b1) gene and is characterized by brain malformation, developmental delays, and epilepsy. Here, we investigate the impact of Pafah1b1 mutation on the cellular migration, morphophysiology, microcircuitry, and transcriptomics of mouse hippocampal CA1 parvalbumin-containing inhibitory interneurons (PV+INTs). We find that WT PV+INTs consist of two physiological subtypes (80% fast-spiking (FS), 20% non-fast-spiking (NFS)) and four morphological subtypes. We find that cell-autonomous mutations within interneurons disrupts morphophysiological development of PV+INTs and results in the emergence of a non-canonical 'intermediate spiking (IS)' subset of PV+INTs. We also find that now dominant IS/NFS cells are prone to entering depolarization block, causing them to temporarily lose the ability to initiate action potentials and control network excitation, potentially promoting seizures. Finally, single-cell nuclear RNAsequencing of PV+INTs revealed several misregulated genes related to morphogenesis, cellular excitability, and synapse formation.

BRAFV600E expression in neural progenitors results in a hyperexcitable phenotype in neocortical pyramidal neurons.

Somatic mutations have emerged as the likely cause of focal epilepsies associated with developmental malformations and epilepsy-associated glioneuronal tumors (GNT). Somatic BRAFV600E mutations in particular have been detected in the majority of low-grade neuroepithelial tumors (LNETS) and in neurons in focal cortical dysplasias adjacent to epilepsy-associated tumors. Furthermore, conditional expression of an activating BRAF mutation in neocortex causes seizures in mice. In this study we characterized the cellular electrophysiology of layer 2/3 neocortical pyramidal neurons induced to express BRAFV600E from neural progenitor stages. In utero electroporation of a piggyBac transposase plasmid system was used to introduce transgenes expressing BRAF wild type (BRAFwt), BRAFV600E, and/or enhanced green fluorescent protein (eGFP) and monomeric red fluorescent protein (mRFP) into radial glia progenitors in mouse embryonic cortex. Whole cell patch-clamp recordings of pyramidal neurons in slices prepared from both juvenile and adult mice showed that BRAFV600E resulted in neurons with a distinct hyperexcitable phenotype characterized by depolarized resting membrane potentials, increased input resistances, lowered action potential (AP) thresholds, and increased AP firing frequencies. Some of the BRAFV600E-expressing neurons normally destined for upper cortical layers by their birthdate were stalled in their migration and occupied lower cortical layers. BRAFV600E-expressing neurons also displayed increased hyperpolarization-induced inward currents (Ih) and decreased sustained potassium currents. Neurons adjacent to BRAFV600E transgene-expressing neurons, and neurons with TSC1 genetically deleted by CRISPR or those induced to carry PIK3CAE545K transgenes, did not show an excitability phenotype similar to that of BRAFV600E-expressing neurons. Together, these results indicate that BRAFV600E leads to a distinct hyperexcitable neuronal phenotype.NEW & NOTEWORTHY This study is the first to report the cell autonomous effects of BRAFV600E mutations on the intrinsic neuronal excitability. We show that BRAFV600E alters multiple electrophysiological parameters in neocortical neurons. Similar excitability changes did not occur in cells neighboring BRAFV600E-expressing neurons, after overexpression of wild-type BRAF transgenes, or after introduction of mutations affecting the mammalian target of rapamycin (mTOR) or the catalytic subunit of phosphoinositide 3-kinase (PIK3CA). We conclude that BRAFV600E causes a distinct, cell autonomous, highly excitable neuronal phenotype when introduced somatically into neocortical neuronal progenitors.

AMPA receptor deletion in developing MGE-derived hippocampal interneurons causes a redistribution of excitatory synapses and attenuates postnatal network oscillatory activity.

Inhibitory interneurons derived from the medial ganglionic eminence represent the largest cohort of GABAergic neurons in the hippocampus. In the CA1 hippocampus excitatory synapses onto these cells comprise GluA2-lacking, calcium-permeable AMPARs. Although synaptic transmission is not established until early in their postnatal life, AMPARs are expressed early in development, however their role is enigmatic. Using the Nkx2.1-cre mouse line we genetically deleted GluA1, GluA2, GluA3 selectively from MGE derived interneurons early in development. We observed that the number of MGE-derived interneurons was preserved in mature hippocampus despite early elimination of AMPARs, which resulted in >90% decrease in spontaneous excitatory synaptic activity. Of particular interest, excitatory synaptic sites were shifted from dendritic to somatic locations while maintaining a normal NMDAR content. The developmental switch of NMDARs from GluN2B-containing early in development to GluN2A-containing on maturation was similarly unperturbed despite the loss of AMPARs. Early network giant depolarizing potential oscillatory activity was compromised in early postnatal days as was both feedforward and feedback inhibition onto pyramidal neurons underscoring the importance of glutamatergic drive onto MGE-derived interneurons for hippocampal circuit function.

Diverse roles for ionotropic glutamate receptors on inhibitory interneurons in developing and adult brain.

Glutamate receptor-mediated recruitment of GABAergic inhibitory interneurons is a critical determinant of network processing. Early studies observed that many, but not all, interneuron glutamatergic synapses contain AMPA receptors that are GluA2-subunit lacking and Ca(2+) permeable, making them distinct from AMPA receptors at most principal cell synapses. Subsequent studies demonstrated considerable alignment of synaptic AMPA and NMDA receptor subunit composition within specific subtypes of interneurons, suggesting that both receptor expression profiles are developmentally and functionally linked. Indeed glutamate receptor expression profiles are largely predicted by the embryonic origins of cortical interneurons within the medial and caudal ganglionic eminences of the developing telencephalon. Distinct complements of AMPA and NMDA receptors within different interneuron subpopulations contribute to the differential recruitment of functionally divergent interneuron subtypes by common afferent inputs for appropriate feed-forward and feedback inhibitory drive and network entrainment. In contrast, the lesser-studied kainate receptors, which are often present at both pre- and postsynaptic sites, appear to follow an independent developmental expression profile. Loss of specific ionotropic glutamate receptor (iGluR) subunits during interneuron development has dramatic consequences for both cellular and network function, often precipitating circuit inhibition-excitation imbalances and in some cases lethality. Here we briefly review recent findings highlighting the roles of iGluRs in interneuron development.

Synapse-associated protein 97 regulates the membrane properties of fast-spiking parvalbumin interneurons in the visual cortex.

Fast-spiking parvalbumin (PV)-positive interneurons in layers 2/3 of the visual cortex regulate gain control and tuning of visual processing. Synapse-associated protein 97 (SAP97) belongs to a family of proteins that have been implicated in regulating glutamatergic synaptic transmission at pyramidal-to-pyramidal connections in the nervous system. For PV interneurons in mouse visual cortex, the expression of SAP97 is developmentally regulated, being expressed in almost all juvenile but only a fraction, ~40%, of adult PV interneurons. Using whole-cell patch-clamping, single-cell RT-PCR to assay endogenous expression of SAP97 and exogenous expression of SAP97, we investigated the functional significance of SAP97 in PV interneurons in layers 2/3 of the visual cortex. PV interneurons expressing SAP97, either endogenously or via exogenous expression, showed distinct membrane properties from those not expressing SAP97. This included an overall decrease in membrane excitability, as indexed by a decrease in membrane resistance and an increase in the stimulus threshold for the first action potential firing. Additionally, SAP97-expressing PV interneurons fired action potentials more frequently and, at moderate stimulus intensities, showed irregular or stuttering firing patterns. Furthermore, SAP97-expressing PV interneurons showed increased glutamatergic input and more extensive dendritic branching when compared with non-expressing PV interneurons. These differences in membrane and synaptic properties would significantly alter how PV interneurons expressing SAP97 compared with those not expressing SAP97 would function in local networks. Thus, our results indicate that the scaffolding protein SAP97 is a critical molecular factor regulating the input-output relationships of cortical PV interneurons.

Microglia actively regulate the number of functional synapses.

Microglia are the immunocompetent cells of the central nervous system. In the physiological setting, their highly motile processes continually survey the local brain parenchyma and transiently contact synaptic elements. Although recent work has shown that the interaction of microglia with synapses contributes to synaptic remodeling during development, the role of microglia in synaptic physiology is just starting to get explored. To assess this question, we employed an electrophysiological approach using two methods to manipulate microglia in culture: organotypic hippocampal brain slices in which microglia were depleted using clodronate liposomes, and cultured hippocampal neurons to which microglia were added. We show here that the frequency of excitatory postsynaptic current increases in microglia-depleted brain slices, consistent with a higher synaptic density, and that this enhancement ensures from the loss of microglia since it is reversed when the microglia are replenished. Conversely, the addition of microglia to neuronal cultures decreases synaptic activity and reduces the density of synapses, spine numbers, surface expression of AMPA receptor (GluA1), and levels of synaptic adhesion molecules. Taken together, our findings demonstrate that non-activated microglia acutely modulate synaptic activity by regulating the number of functional synapses in the central nervous system.

Subgroups of parvalbumin-expressing interneurons in layers 2/3 of the visual cortex.

The input, processing, and output characteristics of inhibitory interneurons help shape information flow through layers 2/3 of the visual cortex. Parvalbumin (PV)-positive interneurons modulate and synchronize the gain and dynamic responsiveness of pyramidal neurons. To define the diversity of PV interneurons in layers 2/3 of the developing visual cortex, we characterized their passive and active membrane properties. Using Ward's and k-means multidimensional clustering, we identified four PV interneuron subgroups. The most notable difference between the subgroups was their firing patterns in response to moderate stimuli just above rheobase. Two subgroups showed regular and continuous firing at all stimulus intensities above rheobase. The difference between these two continuously firing subgroups was that one fired at much higher frequencies and transitioned into this high-frequency firing rate at or near rheobase. The two other subgroups showed irregular, stuttering firing patterns just above rheobase. Both of these subgroups typically transitioned to regular and continuous firing at intense stimulations, but one of these subgroups, the strongly stuttering subgroup, showed irregular firing across a wider range of stimulus intensities and firing frequencies. The four subgroups also differed in excitatory synaptic input, providing independent support for the classification of subgroups. The subgroups of PV interneurons identified here would respond differently to inputs of varying intensity and frequency, generating diverse patterns of PV inhibition in the developing neural circuit.

Interaction of the M4 segment with other transmembrane segments is required for surface expression of mammalian α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

Ionotropic glutamate receptors (GluRs) are ligand-gated ion channels with a modular structure. The ion channel itself shares structural similarity, albeit an inverted membrane topology, with P-loop channels. Like P-loop channels, prokaryotic GluR subunits (e.g. GluR0) have two transmembrane segments. In contrast, eukaryotic GluRs have an additional transmembrane segment (M4), located C-terminal to the ion channel core. However, the structural/functional significance of this additional transmembrane segment is poorly defined. Although topologically similar to GluR0, mammalian AMPA receptor (GluA1) subunits lacking the M4 segment do not display surface expression. This lack of expression is not due to the M4 segment serving as an anchor to the ligand-binding domain because insertion of an artificial polyleucine transmembrane segment does not rescue surface expression. Specific interactions between M4 and the ligand-binding domain are also unlikely because insertion of polyglycines into the linker connecting them has no deleterious effects on function or surface expression. However, tryptophan and cysteine scanning mutagenesis of the M4 segment, as well as recovery of function in the polyleucine background, defined a unique face of the M4 helix that is required for GluR surface expression. In the AMPA receptor structure, this face forms intersubunit contacts with the transmembrane helices of the ion channel core (M1 and M3) from another subunit within the homotetramer. Thus, our experiments show that a highly specific interaction of the M4 segment with an adjacent subunit is required for surface expression of AMPA receptors. This interaction may represent a mechanism for regulating AMPA receptor biogenesis.

Expression pattern of membrane-associated guanylate kinases in interneurons of the visual cortex.

GABAergic interneurons are key elements regulating the activity of local circuits, and abnormal inhibitory circuits are implicated in certain psychiatric and neurodevelopmental diseases. The glutamatergic input that interneurons receive is a key determinant of their activity, yet its molecular structure and development, which are often distinct from those of glutamatergic input to pyramidal cells, are poorly defined. The membrane-associated guanylate kinase (MAGUK) homologs PSD-95/SAP90, PSD-93/chapsyn110, SAP97, and SAP102 are central organizers of the postsynaptic density at excitatory synapses on pyramidal neurons. We therefore studied the cell-type-specific and developmental expression of MAGUKs in the nonoverlapping parvalbumin (PV)- and somatostatin (SOM)-positive interneurons in the visual cortex. These interneuron subtypes account for the vast majority of interneurons in the cortex and have different functional properties and postsynaptic structures, being either axodendritic (PV(+)) or axospinous (SOM(+)). To study cell-type-specific MAGUK expression, we used DIG-labeled riboprobes against each MAGUK along with antibodies against either PV or SOM and examined tissue from juvenile (P15) and adult mice. Both PV(+) and SOM(+) interneurons express mRNA for PSD-95, PSD-93, and SAP102 in P15 and adult tissue. In contrast, these interneuron subtypes express SAP97 at P15, but for adult visual cortex we found that most PV(+) and SOM(+) interneurons show low or no expression of SAP97. Given the importance of SAP97 in regulating AMPA receptor GluA1 subunit and NMDA receptor subunits at glutamatergic synapses, these results suggest a developmental shift in glutamate receptor subunit composition and regulation of glutamatergic synapses on PV(+) and SOM(+) interneurons.

PI3K-AKT-mTOR pathway is dominant over androgen receptor signaling in prostate cancer cells.

BACKGROUND: Androgen receptor (AR) and the phosphatidylinositol-3 kinase (PI3K) signaling are two of the most important pathways implicated in prostate cancer. Previous work has shown that there is crosstalk between these two pathways; however, there are conflicting findings and the molecular mechanisms are not clear. Here we studied the AR-PI3K pathway crosstalk in prostate cancer cells in vitro as well as in vivo. METHODS: Quantitative PCR, Western analysis, reporter assays, and proliferation analyses in vitro and in vivo were used to evaluate the effect of PI3K pathway inhibition on AR signaling and cell growth. RESULTS: Transcriptional activity of AR was increased when the PI3K pathway was inhibited at different levels. In the androgen responsive prostate cancer cell line LNCaP, androgen and the mTOR inhibitor rapamycin synergistically activated androgen target genes. Despite increased androgen signaling, rapamycin treatment reduced LNCaP cell growth; the AR antagonist bicalutamide potentiated this effect. Furthermore, the rapamycin derivative CCI-779 reduced the growth of CWR22 prostate cancer xenografts while increasing AR target gene expression. CONCLUSION: These findings suggest that inhibition of the PI3K pathway activates AR signaling. Despite the increase in AR signaling which has proliferative effects, the result of PI3K pathway inhibition is antiproliferative. These findings suggest that the PI3K pathway is dominant over AR signaling in prostate cancer cells which should be considered in developing novel therapeutic strategies for prostate cancer.

Role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells.

The goal of this study was to determine the role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells. Strain 2951 and its P(k) mutant strain 2951 galE were used in this study. This study suggests that the P(k) epitope of LOS is not an adhesin for M. catarrhalis, but plays a crucial role by its surface charge in the initial stage of attachment.