Moritz J. Rossner

A TRANSGENIC RAT MODEL OF CHARCOT-MARIE-TOOTH DISEASE

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy in humans and has been associated with a partial duplication of chromosome 17 (CMT type 1A). We have generated a transgenic rat model of this disease and provide experimental evidence that CMT1A is caused by increased expression of the gene for peripheral myelin protein-22 (PMP22, gas-3). PMP22-transgenic rats develop gait abnormalities caused by a peripheral hypomyelination, Schwann cell hypertrophy (onion bulb formation), and muscle weakness. Reduced nerve conduction velocities closely resemble recordings in human patients with CMT1A. When bred to homozygosity, transgenic animals completely fail to elaborate myelin. We anticipate that the CMT rat model will facilitate the identification of a cellular disease mechanism and serve in the evaluation of potential treatment strategies.

The Transcriptional Repressor DEC2 Regulates Sleep Length in Mammals

Reducing Sleep Length Humans, like most animals, need their beauty sleep. But the preferred amount and quality of sleep varies between individuals—and some individuals exhibit a heritable, lifetime tendency to sleep less then 6 hours per night. He et al. (p. 866; see the Perspective by Hor and Tafti) identified a mutation in humans associated with people who regularly require shorter than usual sleep duration. The mutation is found in the gene encoding a transcriptional repressor, DEC2, already implicated in regulation of circadian rhythms. Related mutations introduced into mice and flies similarly resulted in shortened sleep phases. Some people are genetically determined to get by with less sleep than other people. Sleep deprivation can impair human health and performance. Habitual total sleep time and homeostatic sleep response to sleep deprivation are quantitative traits in humans. Genetic loci for these traits have been identified in model organisms, but none of these potential animal models have a corresponding human genotype and phenotype. We have identified a mutation in a transcriptional repressor (hDEC2-P385R) that is associated with a human short sleep phenotype. Activity profiles and sleep recordings of transgenic mice carrying this mutation showed increased vigilance time and less sleep time than control mice in a zeitgeber time– and sleep deprivation–dependent manner. These mice represent a model of human sleep homeostasis that provides an opportunity to probe the effect of sleep on human physical and mental health.

Elevated phosphatidylinositol 3,4,5-trisphosphate in glia triggers cell-autonomous membrane wrapping and myelination.

In the developing nervous system, constitutive activation of the AKT/mTOR (mammalian target of rapamycin) pathway in myelinating glial cells is associated with hypermyelination of the brain, but is reportedly insufficient to drive myelination by Schwann cells. We have hypothesized that it requires additional mechanisms downstream of NRG1/ErbB signaling to trigger myelination in the peripheral nervous system. Here, we demonstrate that elevated levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) have developmental effects on both oligodendrocytes and Schwann cells. By generating conditional mouse mutants, we found that Pten-deficient Schwann cells are enhanced in number and can sort and myelinate axons with calibers well below 1 μm. Unexpectedly, mutant glial cells also spirally enwrap C-fiber axons within Remak bundles and even collagen fibrils, which lack any membrane surface. Importantly, PIP3-dependent hypermyelination of central axons, which is observed when targeting Pten in oligodendrocytes, can also be induced after tamoxifen-mediated Cre recombination in adult mice. We conclude that it requires distinct PIP3 effector mechanisms to trigger axonal wrapping. That myelin synthesis is not restricted to early development but can occur later in life is relevant to developmental disorders and myelin disease.

Cell type- and brain region-resolved mouse brain proteome

Brain transcriptome and connectome maps are being generated, but an equivalent effort on the proteome is currently lacking. We performed high-resolution mass spectrometry–based proteomics for in-depth analysis of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type–specific proteins defined as tenfold more abundant than average expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.

Common Variants at VRK2 and TCF4 Conferring Risk of Schizophrenia

Common sequence variants have recently joined rare structural polymorphisms as genetic factors with strong evidence for association with schizophrenia. Here we extend our previous genome-wide association study and meta-analysis (totalling 7 946 cases and 19 036 controls) by examining an expanded set of variants using an enlarged follow-up sample (up to 10 260 cases and 23 500 controls). In addition to previously reported alleles in the major histocompatibility complex region, near neurogranin (NRGN) and in an intron of transcription factor 4 (TCF4), we find two novel variants showing genome-wide significant association: rs2312147[C], upstream of vaccinia-related kinase 2 (VRK2) [odds ratio (OR) = 1.09, P = 1.9 × 10(-9)] and rs4309482[A], between coiled-coiled domain containing 68 (CCDC68) and TCF4, about 400 kb from the previously described risk allele, but not accounted for by its association (OR = 1.09, P = 7.8 × 10(-9)).

P2X1 and P2X5 subunits form the functional P2X receptor in mouse cortical astrocytes.

ATP plays an important role in signal transduction between neuronal and glial circuits and within glial networks. Here we describe currents activated by ATP in astrocytes acutely isolated from cortical brain slices by non-enzymatic mechanical dissociation. Brain slices were prepared from transgenic mice that express enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp. Exogenous ATP evoked inward currents in 75 of 81 astrocytes. In the majority (∼65%) of cells, ATP-induced responses comprising a fast and delayed component; in the remaining subpopulation of astrocytes, ATP triggered a smoother response with rapid peak and slowly decaying plateau phase. The fast component of the response was sensitive to low concentrations of ATP (with EC50 of ∼40 nm). All ATP-induced currents were blocked by pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS); they were insensitive to ivermectin. Quantitative real-time PCR demonstrated strong expression of P2X1 and P2X5 receptor subunits and some expression of P2X2 subunit mRNAs. The main properties of the ATP-induced response in cortical astrocytes (high sensitivity to ATP, biphasic kinetics, and sensitivity to PPADS) were very similar to those reported for P2X1/5 heteromeric receptors studied previously in heterologous expression systems.

Tumor necrosis factor-like weak inducer of apoptosis-induced neurodegeneration

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) family of cytokines. It has proangiogenic and proinflammatory properties in vivo and induces cell death in tumor cell lines. TWEAK effects are mediated by the membrane receptor Fn14. In a systematic search for genes regulated in a murine stroke model with the tag-sequencing technique massively parallel signature sequencing, we have identified TWEAK as an induced gene. After 24 hr of focal cerebral ischemia in vivo or oxygen glucose deprivation in primary cortical neurons, both TWEAK and its receptor Fn14 were significantly upregulated. TWEAK induced cell death in primary neurons. Transfection of a nuclear factor (NF)-κB-luciferase fusion gene demonstrated that TWEAK stimulated transcriptional activity of NF-κB through Fn14 and the IκB kinase. Inhibition of NF-κB reduced TWEAK-stimulated neuronal cell death, suggesting that NF-κB mediates TWEAK-induced neurodegeneration at least in part. Intraperitoneal injection of a neutralizing anti-TWEAK antibody significantly reduced the infarct size after 48 hr of permanent cerebral ischemia. In summary, our data show that TWEAK induces neuronal cell death and is involved in neurodegeneration in vivo.

Cognitive and Sensorimotor Gating Impairments in Transgenic Mice Overexpressing the Schizophrenia Susceptibility Gene Tcf4 in the Brain

BACKGROUND The combined analysis of several large genome-wide association studies identified the basic helix-loop-helix (bHLH) transcription factor TCF4 as one of the most significant schizophrenia susceptibility genes. Its function in the adult brain, however, is not known. TCF4 belongs to the E-protein subfamily known to be involved in neurodevelopment. The messenger RNA expression of Tcf4 is sustained in the adult mouse brain, suggesting a function in the adult nervous system. Tcf4 null mutant mice die perinatally, and haploinsufficiency of TCF4 in humans causes severe mental retardation. METHODS To investigate the possible function of TCF4 in the adult central nervous system, we generated transgenic mice that moderately overexpress TCF4 postnatally in the brain to reduce the risk of developmental effects possibly interfering with adult brain functions. Tcf4 transgenic mice were characterized with molecular, histological, and behavioral methods. RESULTS Tcf4 transgenic mice display profound deficits in contextual and cued fear conditioning and sensorimotor gating. Furthermore, we show that TCF4 interacts with the neurogenic bHLH factors NEUROD and NDRF in vivo. Molecular analyses revealed the dynamic circadian deregulation of neuronal bHLH factors in the adult hippocampus. CONCLUSIONS We conclude that TCF4 likely acts in concert with other neuronal bHLH transcription factors contributing to higher-order cognitive processing. Moderate transcriptional deregulation of Tcf4 in the brain interferes with cognitive functions and might alter circadian processes in mice. These observations provide insight for the first time into the physiological function of TCF4 in the adult brain and its possible contributions to neuropsychiatric disease conditions.

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting.

Global Transcriptome Analysis of Genetically Identified Neurons in the Adult Cortex

The enormous cellular complexity of the brain is a major obstacle for gene expression profiling of neurological disease models, because physiologically relevant changes of transcription in a specific neuronal subset are likely to be lost in the presence of other neurons and glia. We solved this problem in transgenic mice by labeling genetically defined cells with a nuclear variant of GFP. When combined with laser-directed microdissection, intact RNA from unfixed, freeze-dried sections can be isolated, which is a prerequisite for high-quality global transcriptome analysis. Here, we compared gene expression profiles between pyramidal motor neurons and pyramidal somatosensory neurons captured from layer V of the adult neocortex. One striking feature of motor neurons is the elevated expression of ribosomal genes and genes involved in ATP synthesis. This suggests a molecular adaptation of the upper motor neurons to longer axonal projections and higher electrical activity. These molecular signatures were not detected when cortical layers and microareas were analyzed in toto. Additionally, we used microarrays to determine the global mRNA expression profiles of microdissected Purkinje cells and cellularly complex cerebellar cortex microregions. In summary, our analysis shows that cellularly complex targets lead to averaged gene expression profiles that lack substantial amounts of cell type-specific information. Thus, cell type-restricted sampling strategies are mandatory in the CNS. The combined use of a genetic label with laser-microdissection offers an unbiased approach to map patterns of gene expression onto practically any cell type of the brain.