Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses.

Antibody (Ab)-based imaging techniques rely on reagents whose performance may be application specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here, we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique.

High-volume hybridoma sequencing on the NeuroMabSeq platform enables efficient generation of recombinant monoclonal antibodies and scFvs for neuroscience research.

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses.

Antibody-based imaging techniques rely on reagents whose performance may be application-specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique.

NeuroMabSeq: high volume acquisition, processing, and curation of hybridoma sequences and their use in generating recombinant monoclonal antibodies and scFvs for neuroscience research.

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as searchable DNA sequence database ( neuromabseq.ucdavis.edu ) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

NPC1-dependent alterations in KV2.1-CaV1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease.

Lysosomes communicate through cholesterol transfer at endoplasmic reticulum (ER) contact sites. At these sites, the Niemann Pick C1 cholesterol transporter (NPC1) facilitates the removal of cholesterol from lysosomes, which is then transferred to the ER for distribution to other cell membranes. Mutations in NPC1 result in cholesterol buildup within lysosomes, leading to Niemann-Pick Type C (NPC) disease, a progressive and fatal neurodegenerative disorder. The molecular mechanisms connecting NPC1 loss to NPC-associated neuropathology remain unknown. Here we show both in vitro and in an animal model of NPC disease that the loss of NPC1 function alters the distribution and activity of voltage-gated calcium channels (CaV). Underlying alterations in calcium channel localization and function are KV2.1 channels whose interactions drive calcium channel clustering to enhance calcium entry and fuel neurotoxic elevations in mitochondrial calcium. Targeted disruption of KV2-CaV interactions rescues aberrant CaV1.2 clustering, elevated mitochondrial calcium, and neurotoxicity in vitro. Our findings provide evidence that NPC is a nanostructural ion channel clustering disease, characterized by altered distribution and activity of ion channels at membrane contacts, which contribute to neurodegeneration.

Global metabolic profiles in a non-human primate model of maternal immune activation: implications for neurodevelopmental disorders.

Epidemiological evidence implicates severe maternal infections as risk factors for neurodevelopmental disorders, such as ASD and schizophrenia. Accordingly, animal models mimicking infection during pregnancy, including the maternal immune activation (MIA) model, result in offspring with neurobiological, behavioral, and metabolic phenotypes relevant to human neurodevelopmental disorders. Most of these studies have been performed in rodents. We sought to better understand the molecular signatures characterizing the MIA model in an organism more closely related to humans, rhesus monkeys (Macaca mulatta), by evaluating changes in global metabolic profiles in MIA-exposed offspring. Herein, we present the global metabolome in six peripheral tissues (plasma, cerebrospinal fluid, three regions of intestinal mucosa scrapings, and feces) from 13 MIA and 10 control offspring that were confirmed to display atypical neurodevelopment, elevated immune profiles, and neuropathology. Differences in lipid, amino acid, and nucleotide metabolism discriminated these MIA and control samples, with correlations of specific metabolites to behavior scores as well as to cytokine levels in plasma, intestinal, and brain tissues. We also observed modest changes in fecal and intestinal microbial profiles, and identify differential metabolomic profiles within males and females. These findings support a connection between maternal immune activation and the metabolism, microbiota, and behavioral traits of offspring, and may further the translational applications of the MIA model and the advancement of biomarkers for neurodevelopmental disorders such as ASD or schizophrenia.

Age-Related Changes in Synaptic Plasticity Associated with Mossy Fiber Terminal Integration during Adult Neurogenesis.

Mouse hippocampus retains the capacity for neurogenesis throughout lifetime, but such plasticity decreases with age. Adult hippocampal neurogenesis (AHN) involves the birth, maturation, and synaptic integration of newborn granule cells (GCs) into preexisting hippocampal circuitry. While functional integration onto adult-born GCs has been extensively studied, maturation of efferent projections onto CA3 pyramidal cells is less understood, particularly in aged brain. Here, using combined light and reconstructive electron microscopy (EM), we describe the maturation of mossy fiber bouton (MFB) connectivity with CA3 pyramidal cells in young adult and aged mouse brain. We found mature synaptic contacts of newborn GCs were formed in both young and aged brains. However, the dynamics of their spatiotemporal development and the cellular process by which these cells functionally integrated over time were different. In young brain newborn GCs either formed independent nascent MFB synaptic contacts or replaced preexisting MFBs, but these contacts were pruned over time to a mature state. In aged brain only replacement of preexisting MFBs was observed and new contacts were without evidence of pruning. These data illustrate that functional synaptic integration of AHN occurs in young adult and aged brain, but with distinct dynamics. They suggest elimination of preexisting connectivity is required for the integration of adult-born GCs in aged brain.

A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

Astroglial-targeted expression of the fragile X CGG repeat premutation in mice yields RAN translation, motor deficits and possible evidence for cell-to-cell propagation of FXTAS pathology.

The fragile X premutation is a CGG trinucleotide repeat expansion between 55 and 200 repeats in the 5'-untranslated region of the fragile X mental retardation 1 (FMR1) gene. Human carriers of the premutation allele are at risk of developing the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Characteristic neuropathology associated with FXTAS includes intranuclear inclusions in neurons and astroglia. Previous studies recapitulated these histopathological features in neurons in a knock-in mouse model, but without significant astroglial pathology. To determine the role of astroglia in FXTAS, we generated a transgenic mouse line (Gfa2-CGG99-eGFP) that selectively expresses a 99-CGG repeat expansion linked to an enhanced green fluorescent protein (eGFP) reporter in astroglia throughout the brain, including cerebellar Bergmann glia. Behaviorally these mice displayed impaired motor performance on the ladder-rung test, but paradoxically better performance on the rotarod. Immunocytochemical analysis revealed that CGG99-eGFP co-localized with GFAP and S-100ß, but not with NeuN, Iba1, or MBP, indicating that CGG99-eGFP expression is specific to astroglia. Ubiquitin-positive intranuclear inclusions were found in eGFP-expressing glia throughout the brain. In addition, intracytoplasmic ubiquitin-positive inclusions were found outside the nucleus in distal astrocyte processes. Intriguingly, intranuclear inclusions, in the absence of eGFP mRNA and eGFP fluorescence, were present in neurons of the hypothalamus and neocortex. Furthermore, intranuclear inclusions in both neurons and astrocytes displayed immunofluorescent labeling for the polyglycine peptide FMRpolyG, implicating FMRpolyG in the pathology found in Gfa2-CGG99 mice. Considered together, these results show that Gfa2-CGG99 expression in mice is sufficient to induce key features of FXTAS pathology, including formation of intranuclear inclusions, translation of FMRpolyG, and deficits in motor function.

A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain.

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.

Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons.

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

Heterogeneity in Kv2 Channel Expression Shapes Action Potential Characteristics and Firing Patterns in CA1 versus CA2 Hippocampal Pyramidal Neurons.

The CA1 region of the hippocampus plays a critical role in spatial and contextual memory, and has well-established circuitry, function and plasticity. In contrast, the properties of the flanking CA2 pyramidal neurons (PNs), important for social memory, and lacking CA1-like plasticity, remain relatively understudied. In particular, little is known regarding the expression of voltage-gated K+ (Kv) channels and the contribution of these channels to the distinct properties of intrinsic excitability, action potential (AP) waveform, firing patterns and neurotransmission between CA1 and CA2 PNs. In the present study, we used multiplex fluorescence immunolabeling of mouse brain sections, and whole-cell recordings in acute mouse brain slices, to define the role of heterogeneous expression of Kv2 family Kv channels in CA1 versus CA2 pyramidal cell excitability. Our results show that the somatodendritic delayed rectifier Kv channel subunits Kv2.1, Kv2.2, and their auxiliary subunit AMIGO-1 have region-specific differences in expression in PNs, with the highest expression levels in CA1, a sharp decrease at the CA1-CA2 boundary, and significantly reduced levels in CA2 neurons. PNs in CA1 exhibit a robust contribution of Guangxitoxin-1E-sensitive Kv2-based delayed rectifier current to AP shape and after-hyperpolarization potential (AHP) relative to that seen in CA2 PNs. Our results indicate that robust Kv2 channel expression confers a distinct pattern of intrinsic excitability to CA1 PNs, potentially contributing to their different roles in hippocampal network function.

Developing high-quality mouse monoclonal antibodies for neuroscience research - approaches, perspectives and opportunities.

High-quality antibodies (Abs) are critical to neuroscience research, as they remain the primary affinity proteomics reagent used to label and capture endogenously expressed protein targets in the nervous system. As in other fields, neuroscientists are frequently confronted with inaccurate and irreproducible Ab-based results and/or reporting. The UC Davis/NIH NeuroMab Facility was created with the mission of addressing the unmet need for high-quality Abs in neuroscience research by applying a unique approach to generate and validate mouse monoclonal antibodies (mAbs) optimized for use against mammalian brain (i.e., NeuroMabs). Here we describe our methodology of multi-step mAb screening focused on identifying mAbs exhibiting efficacy and specificity in labeling mammalian brain samples. We provide examples from NeuroMab screens, and from the subsequent specialized validation of those selected as NeuroMabs. We highlight the particular challenges and considerations of determining specificity for brain immunolabeling. We also describe why our emphasis on extensive validation of large numbers of candidates by immunoblotting and immunohistochemistry against brain samples is essential for identifying those that exhibit efficacy and specificity in those applications to become NeuroMabs. We describe the special attention given to candidates with less common non-IgG1 IgG subclasses that can facilitate simultaneous multiplex labeling with subclass-specific secondary antibodies. We detail our recent use of recombinant cloning of NeuroMabs as a method to archive all NeuroMabs, to unambiguously define NeuroMabs at the DNA sequence level, and to re-engineer IgG1 NeuroMabs to less common IgG subclasses to facilitate their use in multiplex labeling. Finally, we provide suggestions to facilitate Ab development and use, as to design, execution and interpretation of Ab-based neuroscience experiments. Reproducibility in neuroscience research will improve with enhanced Ab validation, unambiguous identification of Abs used in published experiments, and end user proficiency in Ab-based assays.

Distinct Cell- and Layer-Specific Expression Patterns and Independent Regulation of Kv2 Channel Subtypes in Cortical Pyramidal Neurons.

The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K(+) current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT: Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.

Mitochondrial complex I deficiency leads to inflammation and retinal ganglion cell death in the Ndufs4 mouse.

Mitochondrial complex I (NADH dehydrogenase) is a major contributor to neuronal energetics, and mutations in complex I lead to vision loss. Functional, neuroanatomical and transcriptional consequences of complex I deficiency were investigated in retinas of the Ndufs4 knockout mouse. Whole-eye ERGs and multielectrode arrays confirmed a major retinal ganglion cell functional loss at P32, and retinal ganglion cell loss at P42. RNAseq demonstrated a mild and then sharp increase in innate immune and inflammatory retinal transcripts at P22 and P33, respectively, which were confirmed with QRT-PCR. Intraperitoneal injection of the inflammogen lipopolysaccharide further reduced retinal ganglion cell function in Ndufs4 KO, supporting the connection between inflammatory activation and functional loss. Complex I deficiency in the retina clearly caused innate immune and inflammatory markers to increase coincident with loss of vision, and RGC functional loss. How complex I incites inflammation and functional loss is not clear, but could be the result of misfolded complex I generating a 'non-self' response, and induction of innate immune response transcripts was observed before functional loss at P22, including ß-2 microglobulin and Cx3cr1, and during vision loss at P31 (B2m, Tlr 2, 3, 4, C1qa, Cx3cr1 and Fas). These data support the hypothesis that mitochondrial complex I dysfunction in the retina triggers an innate immune and inflammatory response that results in loss of retinal ganglion cell function and death, as in Leber's hereditary Optic Neuropathy and suggests novel therapeutic routes to counter mitochondrial defects that contribute to vision loss.

Neuronal excitability and calcium/calmodulin-dependent protein kinase type II: location, location, location.

Calcium/calmodulin-dependent protein kinase type II (CaMKII) is a highly abundant serine/threonine kinase comprising a significant fraction of total protein in mammalian forebrain and forming a major component of the postsynaptic density. CaMKII is essential for certain forms of synaptic plasticity and memory consolidation and this is mediated through substrate binding and intramolecular phosphorylation of holoenzyme subunits. CaMKII is multifunctional; it targets a variety of cellular substrates, and this diversity depends on holoenzyme subunit composition. CaMKII comprises homooligomeric and heterooligomeric complexes generated from four subunits (α, ß, δ, and γ) encoded by separate genes that are further expanded by extensive alternative splicing to more than 30 different isoforms. Much attention has been paid to understanding the regulation of CaMKII function through its structural diversity and/or substrate specificity. However, given the importance of subunit composition to holoenzyme activity, it is likely that specificity of cellular expression of CaMKII isoforms also plays a major role in regulation of enzyme function. Herein we review the cellular colocalization of CaMKII isoforms with special regard to the cell-type specificity of isoform expression in brain. In addition, we highlight the remarkable specificity of subcellular localization by the CaMKIIα isoform. In addition, we discuss the role that this cellular specificity of expression might play in propagating the type of recurrent neuronal activity associated with disorders such as temporal lobe epilepsy.

Abnormal dendrite and spine morphology in primary visual cortex in the CGG knock-in mouse model of the fragile X premutation.

The fragile X mental retardation 1 gene (Fmr1) is polymorphic for CGG trinucleotide repeat number in the 5'-untranslated region, with repeat lengths <45 associated with typical development and repeat lengths >200 resulting in hypermethylation and transcriptional silencing of the gene and mental retardation in the fragile X Syndrome (FXS). Individuals with CGG repeat expansions between 55 and 200 are carriers of the fragile X premutation (PM). PM carriers show a phenotype that can include anxiety, depression, social phobia, and memory deficits. They are also at risk for developing fragile X-associated tremor/ataxia syndrome (FXTAS), a late onset neurodegenerative disorder characterized by tremor, ataxia, cognitive impairment, and neuropathologic features including intranuclear inclusions in neurons and astrocytes, loss of Purkinje cells, and white matter disease. However, very little is known about dendritic morphology in PM or in FXTAS. Therefore, we carried out a Golgi study of dendritic complexity and dendritic spine morphology in layer II/III pyramidal neurons in primary visual cortex in a knock-in (KI) mouse model of the PM. These CGG KI mice carry an expanded CGG trinucleotide repeat on Fmr1, and model many features of the PM and FXTAS. Compared to wild-type (WT) mice, CGG KI mice showed fewer dendritic branches proximal to the soma, reduced total dendritic length, and a higher frequency of longer dendritic spines. The distribution of morphologic spine types (e.g., stubby, mushroom, filopodial) did not differ between WT and KI mice. These findings demonstrate that synaptic circuitry is abnormal in visual cortex of mice used to model the PM, and suggest that such changes may underlie neurologic features found in individuals carrying the PM as well as in individuals with FXTAS.

Sema3E/plexin-D1 mediated epithelial-to-mesenchymal transition in ovarian endometrioid cancer.

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration.

Low-threshold calcium channel subunit Ca(v) 3.3 is specifically localized in GABAergic neurons of rodent thalamus and cerebral cortex.

Relatively little is known about the subcellular localization of low threshold Ca²+ channels (T-channels) in the brain. Using immunocytochemical labeling and preembedding immunoperoxidase and silver-enhanced immunogold electron microscopy, we localized T-channel subunit Ca(v) 3.3 in rodent cerebral cortex and thalamus. Double immunofluorescent staining demonstrated that Ca(v) 3.3-labeled neurons in cerebral cortex are a subgroup of GABAergic interneurons that coexpress calbindin and in half of the cases parvalbumin. In the thalamus, virtually all reticular nucleus (RTN) neurons were immunopositive for Ca(v) 3.3, while neurons in dorsal thalamic nuclei were nonimmunoreactive. At the electron microscopic (EM) level, in cortical layers IV-V and RTN neurons, Ca(v) 3.3 immunoreactivity was mainly associated with membranes of dendrites but with some localization in cytoplasm. None was found in axon terminals. In cortex, ≈73% of immunogold particles were present in close proximity to synaptic contacts (<0.5 µm from the postsynaptic density), while 27% were distributed along membranes at extrasynaptic sites (>0.5 µm from the postsynaptic density). In RTN, ≈57% particles were evenly distributed along perisynaptic membranes and the remaining 43% of particles were diffusely localized at extrasynaptic membranes. The density of particles along the dendritic membranes of cortical neurons was 40% higher than in RTN neurons. These results suggest that Ca(v) 3.3 plays a role in regulating GABAergic neurons whose actions underlie thalamocortical rhythmicity.

Molecular correlates of laminar differences in the macaque dorsal lateral geniculate nucleus.

In anthropoid primates, cells in the magnocellular and parvocellular layers of the dorsal lateral geniculate nucleus (dLGN) are distinguished by unique retinal inputs, receptive field properties, and laminar terminations of their axons in visual cortex. To identify genes underlying these phenotypic differences, we screened RNA from magnocellular and parvocellular layers of adult macaque dLGN for layer-specific differences in gene expression. Real-time quantitative reverse transcription-PCR and in situ hybridization were used to confirm gene expression in adult and fetal macaque. Cellular localization of gene expression revealed 11 new layer-specific markers, of which 10 were enriched in magnocellular layers (BRD4, CAV1, EEF1A2, FAM108A1, INalpha, KCNA1, NEFH, NEFL, PPP2R2C, and SFRP2) and one was enriched in parvocellular and koniocellular layers (TCF7L2). These markers relate to functions involved in development, transcription, and cell signaling, with Wnt/beta-catenin and neurofilament pathways figuring prominently. A subset of markers was differentially expressed in the fetal dLGN during a developmental epoch critical for magnocellular and parvocellular pathway formation. These results provide new evidence for the molecular differentiation of magnocellular and parvocellular streams through the primate dLGN.