A novel GABRG2 mutation, p.R136*, in a family with GEFS+ and extended phenotypes.

Genetic mutations in voltage-gated and ligand-gated ion channel genes have been identified in a small number of Mendelian families with genetic generalised epilepsies (GGEs). They are commonly associated with febrile seizures (FS), childhood absence epilepsy (CAE) and particularly with generalised or genetic epilepsy with febrile seizures plus (GEFS+). In clinical practice, despite efforts to categorise epilepsy and epilepsy families into syndromic diagnoses, many generalised epilepsies remain unclassified with a presumed genetic basis. During the systematic collection of epilepsy families, we assembled a cohort of families with evidence of GEFS+ and screened for variations in the γ2 subunit of the γ-aminobutyric acid (GABA) type A receptor gene (GABRG2). We detected a novel GABRG2(p.R136*) premature translation termination codon in one index-case from a two-generation nuclear family, presenting with an unclassified GGE, a borderline GEFS+ phenotype with learning difficulties and extended behavioural presentation. The GABRG2(p.R136*) mutation segregates with the febrile seizure component of this family's GGE and is absent in 190 healthy control samples. In vitro expression assays demonstrated that γ2(p.R136*) subunits were produced, but had reduced cell-surface and total expression. When γ2(p.R136*) subunits were co-expressed with α1 and ß2 subunits in HEK 293T cells, GABA-evoked currents were reduced. Furthermore, γ2(p.R136*) subunits were highly-expressed in intracellular aggregations surrounding the nucleus and endoplasmic reticulum (ER), suggesting compromised receptor trafficking. A novel GABRG2(p.R136*) mutation extends the spectrum of GABRG2 mutations identified in GEFS+ and GGE phenotypes, causes GABAA receptor dysfunction, and represents a putative epilepsy mechanism.

CSF1R mutations in hereditary diffuse leukoencephalopathy with spheroids are loss of function.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) in humans is a rare autosomal dominant disease characterized by giant neuroaxonal swellings (spheroids) within the CNS white matter. Symptoms are variable and can include personality and behavioural changes. Patients with this disease have mutations in the protein kinase domain of the colony-stimulating factor 1 receptor (CSF1R) which is a tyrosine kinase receptor essential for microglia development. We investigated the effects of these mutations on Csf1r signalling using a factor dependent cell line. Corresponding mutant forms of murine Csf1r were expressed on the cell surface at normal levels, and bound CSF1, but were not able to sustain cell proliferation. Since Csf1r signaling requires receptor dimerization initiated by CSF1 binding, the data suggest a mechanism for phenotypic dominance of the mutant allele in HDLS.

Glucose sensor O-GlcNAcylation coordinates with phosphorylation to regulate circadian clock.

Posttranslational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3ß-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3ß. Interestingly, OGT activity is regulated by GSK3ß; hence, OGT and GSK3ß exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock.

Dynamic changes in myelin aberrations and oligodendrocyte generation in chronic amyloidosis in mice and men.

Myelin loss is frequently observed in human Alzheimer's disease (AD) and may constitute to AD-related cognitive decline. A potential source to repair myelin defects are the oligodendrocyte progenitor cells (OPCs) present in an adult brain. However, until now, little is known about the reaction of these cells toward amyloid plaque deposition neither in human AD patients nor in the appropriate mouse models. Therefore, we analyzed cells of the oligodendrocyte lineage in a mouse model with chronic plaque deposition (APPPS1 mice) and samples from human patients. In APPPS1 mice defects in myelin integrity and myelin amount were prevalent at 6 months of age but normalized to control levels in 9-month-old mice. Concomitantly, we observed an increase in the proliferation and differentiation of OPCs in the APPPS1 mice at this specific time window (6-8 months) implying that improvements in myelin aberrations may result from repair mechanisms mediated by OPCs. However, while we observed a higher number of cells of the oligodendrocyte lineage (Olig2+ cells) in APPPS1 mice, OLIG2+ cells were decreased in number in postmortem human AD cortex. Our data demonstrate that oligodendrocyte progenitors specifically react to amyloid plaque deposition in an AD-related mouse model as well as in human AD pathology, although with distinct outcomes. Strikingly, possible repair mechanisms from newly generated oligodendrocytes are evident in APPPS1 mice, whereas a similar reaction of oligodendrocyte progenitors seems to be strongly limited in final stages of human AD pathology.

Spatially coordinated kinase signaling regulates local axon degeneration.

In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3ß, we show that the ß isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3ß in axon degeneration.

Single particle tracking of alpha7 nicotinic AChR in hippocampal neurons reveals regulated confinement at glutamatergic and GABAergic perisynaptic sites.

Alpha7 neuronal nicotinic acetylcholine receptors (alpha7-nAChR) form Ca(2+)-permeable homopentameric channels modulating cortical network activity and cognitive processing. They are located pre- and postsynaptically and are highly abundant in hippocampal GABAergic interneurons. It is unclear how alpha7-nAChRs are positioned in specific membrane microdomains, particularly in cultured neurons which are devoid of cholinergic synapses. To address this issue, we monitored by single particle tracking the lateral mobility of individual alpha7-nAChRs labeled with alpha-bungarotoxin linked to quantum dots in live rat cultured hippocampal interneurons. Quantitative analysis revealed different modes of lateral diffusion of alpha7-nAChR dependent on their subcellular localization. Confined receptors were found in the immediate vicinity of glutamatergic and GABAergic postsynaptic densities, as well as in extrasynaptic clusters of alpha-bungarotoxin labeling on dendrites. alpha7-nAChRs avoided entering postsynaptic densities, but exhibited reduced mobility and long dwell times at perisynaptic locations, indicative of regulated confinement. Their diffusion coefficient was lower, on average, at glutamatergic than at GABAergic perisynaptic sites, suggesting differential, synapse-specific tethering mechanisms. Disruption of the cytoskeleton affected alpha7-nAChR mobility and cell surface expression, but not their ability to form clusters. Finally, using tetrodotoxin to silence network activity, as well as exposure to a selective alpha7-nAChR agonist or antagonist, we observed that alpha7-nAChRs cell surface dynamics is modulated by chronic changes in neuronal activity. Altogether, given their high Ca(2+)-permeability, our results suggest a possible role of alpha7-nAChR on interneurons for activating Ca(2+)-dependent signaling in the vicinity of GABAergic and glutamatergic synapses.

Localization of glycine receptors in the human forebrain, brainstem, and cervical spinal cord: an immunohistochemical review.

Inhibitory neurotransmitter receptors for glycine (GlyR) are heteropentameric chloride ion channels that are comprised of four functional subunits, alpha1-3 and beta and that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We have investigated the distribution of GlyRs in the human forebrain, brainstem, and cervical spinal cord using immunohistochemistry at light and confocal laser scanning microscopy levels. This review will summarize the present knowledge on the GlyR distribution in the human brain using our established immunohistochemical techniques. The results of our immunohistochemical labeling studies demonstrated GlyR immunoreactivity (IR) throughout the human basal ganglia, substantia nigra, various pontine regions, rostral medulla oblongata and the cervical spinal cord present an intense and abundant punctate IR along the membranes of the neuronal soma and dendrites. This work is part of a systematic study of inhibitory neurotransmitter receptor distribution in the human CNS, and provides a basis for additional detailed physiological and pharmacological studies on the inter-relationship of GlyR, GABA(A)R and gephyrin in the human brain. This basic mapping exercise, we believe, will provide important baselines for the testing of future pharmacotherapies and drug regimes that modulate neuroinhibitory systems. These findings provide new information for understanding the complexity of glycinergic functions in the human brain, which will translate into the contribution of inhibitory mechanisms in paroxysmal disorders and neurodegenerative diseases such as Epilepsy, Huntington's and Parkinson's Disease and Motor Neuron Disease.

AP2gamma regulates basal progenitor fate in a region- and layer-specific manner in the developing cortex.

An important feature of the cerebral cortex is its layered organization, which is modulated in an area-specific manner. We found that the transcription factor AP2gamma regulates laminar fate in a region-specific manner. Deletion of AP2gamma (also known as Tcfap2c) during development resulted in a specific reduction of upper layer neurons in the occipital cortex, leading to impaired function and enhanced plasticity of the adult visual cortex. AP2gamma functions in apical progenitors, and its absence resulted in mis-specification of basal progenitors in the occipital cortex at the time at which upper layer neurons were generated. AP2gamma directly regulated the basal progenitor fate determinants Math3 (also known as Neurod4) and Tbr2, and its overexpression promoted the generation of layer II/III neurons in a time- and region-specific manner. Thus, AP2gamma acts as a regulator of basal progenitor fate, linking regional and laminar specification in the mouse developing cerebral cortex.

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection.

Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed.

PICK1 interacts with alpha7 neuronal nicotinic acetylcholine receptors and controls their clustering.

Central to synaptic function are protein scaffolds associated with neurotransmitter receptors. Alpha7 neuronal nicotinic acetylcholine receptors (nAChRs) modulate network activity, neuronal survival and cognitive processes in the CNS, but protein scaffolds that interact with these receptors are unknown. Here we show that the PDZ-domain containing protein PICK1 binds to alpha7 nAChRs and plays a role in their clustering. PICK1 interacted with the alpha7 cytoplasmic loop in yeast in a PDZ-dependent way, and the interaction was confirmed in recombinant pull-down experiments and by co-precipitation of native proteins. Some alpha7 and PICK1 clusters were adjacent at the surface of SH-SY5Y cells and GABAergic interneurons in hippocampal cultures. Expression of PICK1 caused decreased alpha7 clustering on the surface of the interneurons in a PDZ-dependent way. These data show that PICK1 negatively regulates surface clustering of alpha7 nAChRs on hippocampal interneurons, which may be important in inhibitory functions of alpha7 in the hippocampus.

Glycine receptors in the striatum, globus pallidus, and substantia nigra of the human brain: an immunohistochemical study.

Glycine receptors (GlyRs) are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian spinal cord and brain. GlyRs have four functional subunits, alpha1-3 and beta, which likely exist in heteromeric alphabeta combinations. Mutations in GlyR alpha1 and beta subunits are well known for their involvement in hyperekplexia, a paroxysmal motor disorder. In this study we present the first detailed immunohistochemical investigation at the regional, cellular, and subcellular levels of GlyRs in the human basal ganglia. The results show that GlyRs are present at the regional level in low concentrations in the striatum and globus pallidus and are present in the highest concentrations in the substantia nigra. At the cellular level, GlyRs are present only in discrete populations of neurons immunoreactive for choline acetyltransferase (ChAT), parvalbumin, and calretinin in the human striatum, on a subpopulation of parvalbumin- and calretinin-positive neurons in the globus pallidus, and in the substantia nigra GlyRs are present on approximately three-fourths of all pars compacta and one-third of all pars reticulata neurons. They also form a distinct band of immunoreactive neurons in the intermedullary layers of the globus pallidus. At the subcellular level in the substantia nigra pars reticulata (SNr), GlyRs show a localized distribution on the soma and dendrites that partially complements but does not overlap with the distribution of gamma-aminobutyric acid (GABA)A receptors. Our results demonstrate the precise cellular and subcellular localization of GlyRs in the human basal ganglia and suggest that glycinergic receptors may play an important complementary role to other inhibitory receptors in modulating cholinergic, dopaminergic, and GABAergic neuronal pathways in the basal ganglia.

Sox-2 is expressed by glial and progenitor cells and Pax-6 is expressed by neuroblasts in the human subventricular zone.

Transcription factors (TFs) are responsible for the specification and fate determination of cells as they develop from progenitor cells into specific types of cells in the brain. Sox-2 and Pax-6 are TFs with key functional roles in the developing brain, although less is known about TFs in the rudimentary germinal zones in the adult human brain. In this study we have investigated the distribution and characterization of Sox-2 and Pax-6 in the human subventricular zone (SVZ). Sox-2 immunoreactivity showed a nuclear labeling pattern and colocalised on GFAP immunoreactive cells as well as on bromodeoxyuridine (BrdU)-positive cells, whereas Pax-6 immunoreactivity was detectable in the nucleus and the cytoplasm of SVZ cells and colocalised with PSA-NCAM-positive progenitor cells. Thus, our data surprisingly reveal that these TFs are differentially expressed in the adult human SVZ where Sox-2 and Pax-6 specify a glial and neuronal fate, respectively.

Differential actions of NPY on seizure modulation via Y1 and Y2 receptors: evidence from receptor knockout mice.

PURPOSE: Neuropeptide Y (NPY) has been shown to modulate seizure activities. To provide further understanding of the involvement of two of the most abundantly expressed NPY receptors, Y1 and Y2, we assessed the effect of Y1 and Y2 gene deletion on systemic kainic acid-induced seizures. We also examined the effect of rAAV-mediated hippocampal NPY overexpression on seizure susceptibility in these receptor knockout mice. METHODS: Recombinant adeno-associated viral vector overexpressing NPY (rAAV-NPY) or an empty vector control (rAAV-Empty) was injected into the hippocampus of adult C57BL/6-129/SvJ wild-type male mice and mice deficient of Y1 or Y2 receptors on the same background. Four weeks after vector injection, mice were subjected to systemic kainic acid-induced seizures, and the seizure behaviors were scored. RESULTS: The rAAV-mediated hippocampal overexpression of NPY led to a twofold reduction in seizures induced by systemic kainic acid in wild-type mice and Y1 receptor knockout mice but not in mice deficient of Y2 receptors. A differential action by the receptors was observed in the seizure-induced mortality rate, with increased fatality in Y2-/- mice. In addition, although NPY overexpression did not significantly affect the mortality rate in Y2-/- and wild-type mice, it abolished KA-induced mortality in Y1-/-mice. CONCLUSIONS: This study shows for the first time an altered susceptibility to chemically induced seizures in Y1 and Y2 knockout mice and demonstrates a differential seizure modulation mediated by these receptors via a genetic approach.

Immunohistochemical staining of post-mortem adult human brain sections.

One of the challenges for modern neuroscience is to understand the basis of coordinated neuronal function and networking in the human brain. Some of these questions can be addressed using low- and high-resolution imaging techniques on post-mortem human brain tissue. We have established a versatile protocol for fixation of post-mortem adult human brain tissue, storage of the tissue in a human brain bank, and immunohistochemical analysis in order to understand human brain functions in normal controls and in neuropathological conditions. The brains are fixed by perfusion through the internal carotid and basilar arteries to enhance the penetration of fixative throughout the brain, then blocked, postfixed, cryoprotected, snap-frozen and stored at -80 degrees C. Sections are processed for immunohistochemical single- or double-label staining and conventional-, electron- or confocal laser scanning-microscopy analysis. The results gained using this tissue and protocol are vital for determining the localization of neurochemicals throughout the human brain and to document the changes that occur in neurological diseases.

Activation of a GST-tagged AKT2/PKBbeta.

The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (DeltaPH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr309 and Ser474, and to activation of the kinase. Likewise, phosphorylation of Thr309 in vitro by recombinant PDK1 or mutation of Thr309 and Ser474 to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent Km value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr309 in the activation loop of the kinase is largely responsible for the observed reduction in Km and for a subsequent 150-fold increase in the catalytic efficiency (k(cat)/Km) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr309 or Ser474.

X-ray structure of glutathione S-transferase from Schistosoma japonicum in a new crystal form reveals flexibility of the substrate-binding site.

The crystal structure of the 26 kDa glutathione S-transferase from Schistosoma japonicum (SjGST) was determined at 3 A resolution in the new space group P2(1)2(1)2(1). The structure of orthorhombic SjGST reveals unique features of the ligand-binding site and dimer interface when compared with previously reported structures. SjGST is recognized as the major detoxification enzyme of S. japonicum, a pathogenic helminth causing schistosomiasis. As resistance against the established inhibitor of SjGST, praziquantel, has been reported these results might prove to be valuable for the development of novel drugs.

Recombinant AAV-mediated expression of galanin in rat hippocampus suppresses seizure development.

Galanin, a 29- or 30-amino acid neuropeptide, has been implicated in the modulation of seizures. In this study, we constructed a recombinant adeno-associated viral (AAV) vector to constitutively over-express galanin (AAV-GAL). The vector mediated efficient transduction of HEK 293 cells in vitro and robust galanin expression in vivo when injected into the rat dorsal hippocampus. Rats were administered kainic acid intrahippocampally 2.5 months following AAV-GAL or empty vector (AAV-Empty) injection to study the effect of vector-mediated galanin over-expression on seizures. AAV-GAL-injected rats showed a decreased number of seizure episodes and total time spent in seizures compared to AAV-Empty rats, despite similar latencies to development of the first EEG seizure and similar levels of neuronal damage in the CA3 region for both groups. These data show that recombinant AAV mediates strong and stable over-expression of galanin when injected into the rat hippocampus resulting in a significant anticonvulsive effect. The seizure suppression effect of galanin expression in the hippocampus by viral vectors may lead to novel therapeutic strategies for the treatment and management of intractable seizures with focal onset such as temporal lobe epilepsy.

Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia.

Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABAA receptors to the microtubular matrix of the neuronal postsynaptic membrane. Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia). This neuromotor disorder is associated with mutations in the GlyR alpha1 and beta subunit genes (GLRA1 and GLRB). Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN). In addition, we adopted a yeast two-hybrid screen, using the GlyR beta subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia. Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene. This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue. Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y). Functional testing demonstrated that GPHNN10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering. We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain. Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation.

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.