Identification of the C-terminal activation domain of the NeuroD-related factor (NDRF)

NeuroD-related factor (NDRF) is a basic helix–loop–helix (bHLH) protein whose expression is restricted to the central nervous system, and is considered to be responsible for maintenance of differentiated neurons as well as neurogenesis. NDRF structurally resembles NeuroD in the bHLH region and can induce neurogenesis ectopically in ectodermal cells of the Xenopus embryo. In this study, we delineated the functional domains of NDRF. Using GAL4/NDRF fusion proteins, we identified the C-terminal activation domain (C-AD) in NDRF between amino acid positions 294 and 383. This region was highly homologous to one part of the activation domain of NeuroD. We further investigated the transactivational function of C-AD in the mouse type 1 inositol 1,4,5-trisphosphate receptor promoter, which has an NDRF site. Truncation of C-AD resulted in reduction of the activation function, whereas the DNA-binding specificity was not affected. These results suggest that C-AD has a stimulatory function in the mammalian nervous system.     

Expression pattern of ACK1 tyrosine kinase during brain development in the mouse

Ack1 is a non-receptor tyrosine kinase that is highly expressed in the adult central nervous system (CNS). Here, we studied the distribution of Ack1 mRNA throughout the development of mouse CNS. Expression was detected in all areas of the brain but especially high levels were observed in the neocortex, hippocampus, and cerebellum. Interestingly, expression levels were prominent in areas of proliferation such as the subventricular zone and areas that originate other structures such the pontine nucleus and the ganglionic eminence. During development, several areas showed an increase in Ack1 expression, especially the dentate gyrus and CA3 in the hippocampus, layer V in the neocortex, and the Purkinje cell layer in the cerebellum. These results demonstrate that this kinase is up-regulated during development and that it is expressed in proliferative areas and in migratory pathways in the developing brain.     

Computational analysis of calcium signaling and membrane electrophysiology in cerebellar Purkinje neurons associated with ataxia

ABSTRACT: BACKGROUND: Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1delta18/delta18 mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins. RESULTS: We create a computational model of pathological alterations in calcium signaling in cerebellar Purkinje neurons to investigate several forms of spinocerebellar ataxia associated with changes in the abundance, sensitivity, or activity of the calcium channel IP3R1. We find that increasing IP3R1 sensitivity to IP3 in computational models of SCA15/16 can restore normal calcium response if IP3R1 abundance is not too low. The studied range in IP3R1 levels reflects variability found in human and mouse ataxic models. Further, the required fold increases in sensitivity are within experimental ranges from experiments that use IP3R1 phosphorylation status to adjust its sensitivity to IP3. Results from our simulations of polyglutamine SCAs suggest that downregulation of some calcium signaling proteins may be partially compensatory. However, the downregulation of calcium buffer proteins observed in the SCA1 mice may contribute to pathology. Finally, our model suggests that the calcium-activated voltage-gated potassium channels may provide an important link between calcium metabolism and membrane potential in Purkinje cell function. CONCLUSION: Thus, we have established an initial platform for computational evaluation and prediction of ataxia pathophysiology. Specifically, the model has been used to investigate SCA15/16, SCA1, SCA2, and SCA3. Results suggest that experimental studies treating mouse models of any of these ataxias with appropriately chosen peptides resembling the C-terminal of IP3R1 could adjust receptor sensitivity, and thereby modulate calcium release and normalize IP3 response. In addition, the model supports the hypothesis of IP3R1 supersensitivity in SCA1.     

Sonic hedgehog--regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system

During development, basic helix-loop-helix (bHLH) proteins regulate formation of neurons from multipotent progenitor cells. However, bHLH factors linked to gliogenesis have not been described. We have isolated a pair of oligodendrocyte lineage genes (Olg-1 and Olg-2) that encode bHLH proteins and are tightly associated with development of oligodendrocytes in the vertebrate central nervous system (CNS). Ectopic expression of Olg-1 in rat cortical progenitor cell cultures promotes formation of oligodendrocyte precursors. In developing mouse embryos, Olg gene expression overlaps but precedes the earliest known markers of the oligodendrocyte lineage. Olg genes are expressed at the telencephalon-diencephalon border and adjacent to the floor plate, a source of the secreted signaling molecule Sonic hedgehog (Shh). Gain- and loss-of-function analyses in transgenic mice demonstrate that Shh is both necessary and sufficient for Olg gene expression in vivo.     

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y₁R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y₁R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09792-9.     

Diverse Expression of β1,4-N-Acetylgalactosaminyltransferase Gene in the Adult Mouse Brain

Abstract: Among various tissues of mouse, β1,4- N -acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.     

Diverse human aldolase C gene promoter regions are required to direct specific LacZ expression in the hippocampus and Purkinje cells of transgenic mice

Aldolase C is selectively expressed in the hippocampus and Purkinje cells in adult mammalian brain. The gene promoter regions governing cell-specific aldolase C expression are obscure. We show that aldolase C messenger expression in the hippocampus is restricted to CA3 neurons. The human distal promoter region (-200/-1200 bp) is essential for beta-galactosidase (beta-gal) expression in CA3 neurons and drives high stripe-like beta-gal expression in Purkinje cells. The 200 bp proximal promoter region is sufficient to drive low brain-specific and stripe-like beta-gal expression in Purkinje cells. Thus, the human aldolase C gene sequences studied drive endogenous-like expression in the brain.     

Glucagon-like peptide (GLP)-2 action in the murine central nervous system is enhanced by elimination of GLP-1 receptor signaling

Glucagon-like peptide-2 (GLP-2) regulates energy homeostasis via effects on nutrient absorption and maintenance of gut mucosal epithelial integrity. The biological actions of GLP-2 in the central nervous system (CNS) remain poorly understood. We studied the sites of endogenous GLP-2 receptor (GLP-2R) expression, the localization of transgenic LacZ expression under the control of the mouse GLP-2R promoter, and the actions of GLP-2 in the murine CNS. GLP-2R expression was detected in multiple extrahypothalamic regions of the mouse and rat CNS, including cell groups in the cerebellum, medulla, amygdala, hippocampus, dentate gyrus, pons, cerebral cortex, and pituitary. A 1.5-kilobase fragment of the mouse GLP-2R promoter directed LacZ expression to the gastrointestinal tract and CNS regions in the mouse that exhibited endogenous GLP-2R expression, including the cerebellum, amygdala, hippocampus, and dentate gyrus. Intracerebroventricular injection of GLP-2 significantly inhibited food intake during dark-phase feeding in wild-type mice. Disruption of glucagon-like peptide-1 receptor (GLP-1R) signaling with the antagonist exendin-(9-39) in wild-type mice or genetically in GLP-1R(-)/- mice significantly potentiated the anorectic actions of GLP-2. These findings illustrate that CNS GLP-2R expression is not restricted to hypothalamic nuclei and demonstrate that the anorectic effects of GLP-2 are transient and modulated by the presence or absence of GLP-1R signaling in vivo.