Reduced expression of the TrkB receptor in Huntington's disease mouse models and in human brain

Deficits of neurotrophic support caused by reduced levels of brain-derived neurotrophic factor (BDNF) have been implicated in the selective vulnerability of striatal neurones in Huntington's disease (HD). Therapeutic strategies based on BDNF administration have been proposed to slow or prevent the disease progression. However, the effectiveness of BDNF may depend on the proper expression of its receptor TrkB. In this study, we analysed the expression of TrkB in several HD models and in postmortem HD brains. We found a specific reduction of TrkB receptors in transgenic exon-1 and full-length knock-in HD mouse models and also in the motor cortex and caudate nucleus of HD brains. Our findings also demonstrated that continuous expression of mutant huntingtin is required to down-regulate TrkB levels. This was shown by findings in an inducible HD mouse model showing rescue of TrkB by turning off mutant huntingtin expression. Interestingly, the length of the polyglutamine tract in huntingtin appears to modulate the reduction of TrkB. Finally, to analyse the effect of BDNF in TrkB we compared TrkB expression in mutant huntingtin R6/1 and double mutant (R6/1 : BDNF+/-) mice. Similar TrkB expression was found in both transgenic mice suggesting that reduced TrkB is not a direct consequence of decreased BDNF. Therefore, taken together our findings identify TrkB as an additional component that potentially might contribute to the altered neurotrophic support in HD.     

Increased truncated TrkB receptor expression and decreased BDNF/TrkB signaling in the frontal cortex of reeler mouse model of schizophrenia

Heterozygous reeler mouse has been used as an animal model for schizophrenia based on several neuropathological and behavioral abnormalities homologous to schizophrenia. Since some of these abnormalities are primarily associated with altered BDNF signaling we investigated BDNF signaling in the frontal cortex of reeler mice in order to shed some light on the neuropathology and treatment of schizophrenia. BDNF, TrkB receptor isoforms (full-length and truncated), reelin, GAD67, GAD65, p75NTR, and NRH-2 levels were measured in the frontal cortex samples from reeler (B6C3Fe a/a-Reln^rl/+) and wild-type (WT) mice. BDNF protein levels were significantly higher in reeler compared to WT. The protein levels of full-length TrkB were not altered in reeler mice, but both mRNA and protein levels of truncated TrkB were significantly higher. Protein analysis showed that TrkB activity, as indicated by the levels of tyrosine-phosphorylated TrkB, was lower in reeler mice. We did not find any significant change in the levels of p75NTR and NRH-2, regulatory proteins of TrkB signaling, in the reeler mice. Furthermore, we found significant reduction in reelin and GAD67 expressions, but not GAD65 expression in reeler compared to WT mice. In summary, molecular processes associated with defective BDNF signaling in reeler mice provide new therapeutic targets for neuroprotective pharmacotherapy for schizophrenia.     

Bromocriptine and clozapine regulate dopamine 2 receptor gene expression in the mouse striatum

In a previous study, we showed that the psychoactive drug caffeine alters the expression of the dopamine 2 receptor (D2R) gene in vitro and in vivo. Here, we report that acute administration of antipsychotic and anti-parkinsonian drugs also regulate D2R gene expression in PC12 cells and in the mouse striatum. Treatment of PC12 cells with the atypical antipsychotic and specific 5-HT antagonist clozapine (60 microM) reduced D2R/luciferase reporter expression by 46% after 24 h. However, male and female mice treated with a clinical dose of clozapine (10 mg/kg) showed no changes in striatal D2R mRNA expression when assayed by quantitative RT-PCR. Treatment of PC12 cells with the specific D2R agonist anti-parkinsonian drug, bromocriptine mesylate (BCM; 5 microM) also resulted in decreased D2R/luciferase reporter activity (27%). In contrast to clozapine, a clinical dose of BCM (16 mg/kg) led to a 21% decrease and a 45% increase in striatal D2R mRNA expression in male and female mice, respectively, after 24 h. Coadministration of clozapine and BCM in PC12 cells resulted in a synergistic decrease in D2R/luciferase reporter expression (68%), and coadministration of these drugs in vivo led to decreases in striatal D2R mRNA expression in both male and female mice (45% and 22%, respectively). Collectively, these results indicate that clozapine, BCM, or a combination of these drugs have differential effects on dopamine receptor gene expression and might also affect striatal physiology in a sexually dimorphic manner.     

Expression and localization of the fibronectin receptor in the mouse nervous system

Cell surface receptors for extracellular matrix components have recently been characterized as integral membrane complexes with common features in their structural and functional properties. We have investigated the expression of the mammalian fibronectin receptor in the mouse nervous system using immunocytological and immunochemical methods. The fibronectin receptor was detectable on immature oligodendrocytes and immature and mature astrocytes in culture, while central nervous system neurons did not reveal detectable levels of fibronectin receptor at the developmental stages studied. In the peripheral nervous system both glia and neurons were found to express the fibronectin receptor. The receptor complex in both peripheral and central nervous system has an apparent molecular weight of approximately 140 kD under reducing conditions and resolves into two or three distinct protein bands under nonreducing conditions. The fibronectin receptor expresses the L2/HNK-1 epitope that is characteristic of several adhesion molecules, including L1, N-CAM, the myelin-associated glycoprotein, and J1 and thus is another member of the L2/HNK-1 family of adhesion molecules. The L2/HNK-1 carbohydrate epitope is expressed differently and independently of the fibronectin receptor protein backbone in that it is detectable in neonatal brain but not in adult brain. Our observations attribute a functional role to the fibronectin receptor and its L2/HNK-1 carbohydrate epitope during development and maintenance of cell interactions in the central and peripheral nervous systems.     

Chemokine and chemokine receptor expression in the central nervous system

A decade ago several new cytokines were described that orchestrated the activation and migration of immune cells. These newly described cytokines, of which interleukin-8 (IL-8) was a representative member, defined a novel group of molecules called chemokines (chemotactic cytokines). Chemokines are low molecular weight, 8-12 kDa, basic proteins that have been classified into four distinct families, CXC, CC, C and CX3C, based on the position of their first two conserved cysteine residues. The expression and biological function of chemokines along with their cognate receptors have been well described on various subsets of leukocytes. Only more recently have these molecules been described on various cells within the central nervous system. These pro-inflammatory proteins have been implicated in a variety of diseases within the central nervous system from Multiple Sclerosis to AIDS dementia. While chemokines are likely to enhance the evolution of central nervous system inflammatory disorders they also have other roles in normal brain function and development. This review summarizes the role of chemokines and their receptors in the normal and pathophysiological brain.     

Dynamic expression of TrkB receptor protein on proliferating and maturing cells in the adult mouse dentate gyrus

Brain-derived neurotrophic factor (BDNF) is implicated in the regulation of adult hippocampal neurogenesis, presumably via its primary receptor, TrkB. However, controversy exists about how BDNF affects neurogenesis (e.g., proliferation vs. survival/differentiation). This controversy arises, in part, due to the lack of information about whether and when TrkB is expressed on adult neural precursors in vivo. We utilized multiple methods to analyze proliferating and maturing cells in the adult mouse subgranular zone (SGZ) for TrkB protein. Using bromodeoxyuridine (BrdU) to "birthdate" cells, we found that the proportion of proliferating cells that were TrkB-immunoreactive (IR) was low and remained low for at least 1 week, but increased with further survival after BrdU labeling. Use of the nestin-GFP transgenic mouse and the immature neuron marker, doublecortin (Dcx), revealed that the likelihood of being TrkB-IR increased with presumed maturity of the cell type. Stem-like cells, which rarely divide, were likely to express TrkB protein. However, early progenitors (GFP+/Dcx-) and late progenitors (GFP+/Dcx+), both of which are still in the cell cycle, were unlikely to be TrkB-IR. Immature neuroblasts (GFP-/Dcx+) were more likely to express TrkB, especially as they presented a more mature morphology. Taken together, these findings emphasize that expression of TrkB protein is closely linked to progression toward neuronal maturity. This provides evidence that maturing, but not proliferating, cells in the adult mouse SGZ have the molecular machinery necessary to respond directly to BDNF. Furthermore, these findings lay critical groundwork for further exploration of the role of BDNF-TrkB signaling in regulation of adult hippocampal neurogenesis.     

Different expression of synemin isoforms in glia and neurons during nervous system development

The synemin gene encodes proteins belonging to the intermediate filament family. These proteins confer resistance to mechanical stress and modulate cell shape. Three synemin isoforms, of 180 (H), 150 (M) and 41 (L) kDa, are produced by alternative splicing of the pre-mRNA and are regulated differently during development. The three isoforms differ in their C-terminal tail domains, while their IF rod domains are identical. Synemins H/M occurred together with nestin and vimentin in glial progenitors during the early differentiation of the developing mouse central nervous system. They are later found in GFAP-labeled cells. In contrast, the L isoform appeared only in neurons, together with neurofilaments and betaIII-tubulin in the brain after birth. However, synemin L appeared from E13 in the peripheral nervous system, where it was confined to the neurons of spinal ganglia. In the meantime, the synemin H/M isoforms were found in both the neurons and Schwann cells of the sensorial ganglia from E11. Tissue fractionation and purification of IFs from adult mouse spinal cord revealed that the synemin L isoform binds to neurofilaments associated with the membrane compartment. This report describes the synthesis of the three synemin isoforms by selective cell types, and their temporal and spatial distributions. Mechanisms specific to neurons and glia probably control the splicing of the common synemin mRNA and the synthesis of each synemin isoform.     

mRNA expression of ephrins and Eph receptor tyrosine kinases in the neonatal and adult mouse central nervous system

Ephrins and Eph receptors are a family of molecules that have been implicated in axonal pathfinding. A unique feature of B-class ephrins and Eph receptors is their ability to transmit bidirectional signals in both ephrin- and Eph receptor-expressing cells upon cell-cell contact. These signals can lead to cytoskeletal alterations that have been attributed to regulating neuronal growth responses. Examination of gene-target knockout mice has supported this hypothesis, revealing numerous developmental defects in the nervous systems of mice mutant for both B-class ephrins and Eph receptors. To examine the potential scope of action for these genes in the nervous system, we have used in situ hybridization to study the mRNA expression of ephrins (B1, B2, and B3) and Eph receptors (B1, B2, B3, A4) in neonatal and adult mice. We found ephrins and Eph receptors to be expressed throughout the CNS. Expression was observed in the epithelium and migratory regions of the neonate and adult tissues as well as in discrete regions of high plasticity, including the adult olfactory bulb, hippocampus, and cerebellum. These studies suggest additional potential roles for these molecules in the postnatal and adult CNS and will serve as a guide in the detailed evaluation of mutant mice.     

Immunohistochemical detection of Ndrg2 in the mouse nervous system

NDRG2, a member of the N-myc downstream-regulated gene (NDRG) family, is involved in cell differentiation and development. However, the distribution and function of Ndrg2 in the central nervous system remains unclear. Here, we analyzed the expression and distribution of Ndrg2 in the mouse brain and explored the potential physiological functions of Ndrg2. Ndrg2 was expressed in different regions of the brain, including the cerebral cortex, olfactory bulb, midbrain, hippocampus, and thalamus, with high levels in the midbrain and thalamus. Immunohistochemistry assay revealed that Ndrg2-positive cells distributed widely in the adult mouse brain and some of them showed nuclear staining. Indirect immunofluorescence and confocal microscopy studies showed that Ndrg2 protein colocalized with glial fibrillary acidic protein, indicating that Ndrg2 is expressed in astrocytes. Furthermore, Ndrg2 expression increased in glioma cells that were differentiating into astrocytes. Taken together, these findings suggest that Ndrg2 is possibly associated with glial cell proliferation and differentiation based on its immunolocalization in this study.     

Site-specific and developmental expression of pannexin1 in the mouse nervous system

Until recently, members of the connexin gene family were believed to comprise the sole molecular component forming gap junction channels in vertebrates. The recent discovery of the pannexin gene family has challenged this view, as these genes may encode for a putative second class of gap junction proteins in vertebrates. The expression of pannexin genes overlaps with those cellular networks known to exhibit a high degree of gap junctional coupling. We investigated the spatio-temporal mRNA distribution of one member of this gene family, pannexin1 (Panx1), in the brain and retina of mice using quantitative real-time polymerase chain reaction and a combination of in situ hybridization and immunohistochemistry for cellular resolution. Our results demonstrate a widespread expression of Panx1 in the brain, retina and other non-neuronal tissues. In the cortex, cerebellum and eye, Panx1 is expressed at early embryonic time points and peaks around embryonic day 18 followed by a decline towards adulthood. Most notably, Panx1 is detectable in neurons of many brain nuclei, which are known to be coupled by gap junctions as well as in previously unrecognized areas. Abundant expression was found in the adult hippocampal and neocortical pyramidal cells and interneurons, neurons of the reticular thalamus, the inferior olive, magnocellular hypothalamic neurons, midbrain and brain stem motoneurons, Purkinje cells and the retina.     

Immunocytochemical localization of TrkB in the central nervous system of the adult rat

The TrkB family of transmembrane proteins serve as receptors for brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-4/5, and possibly NT-3, three members of the neurotrophin family of neurotrophic factors. In order to understand the potential roles played by these receptors, we have examined the distribution of the TrkB receptor proteins in the adult rat brain by using immunohistochemistry. Several different antisera, directed against either synthetic peptides corresponding to different regions of TrkB or a recombinant fusion protein comprising part of the extracellular domain, were generated. Each of these antisera was directed to epitopes found on all known TrkB isoforms (both the tyrosine kinase-possessing isoform and the truncated kinase-lacking isoforms). In addition, a commercially available antibody to the intracellular domain of TrkB was also used. Widespread and distinct staining was observed on the surface of neuronal cell bodies, axons, and dendrites in many structures, including the cerebral cortex, hippocampus, dentate gyrus, striatum, septal nuclei, substantia nigra, cerebellar Purkinje cells, brainstem and spinal motor neurons, and brainstem sensory nuclei. Staining was also observed in the pia matter, on a subpopulation of ependymal cells lining the cerebral ventricle wall, and other nonneuronal cells. The expression pattern of TrkB receptor protein suggests that TrkB plays a broad role in the central nervous system. In addition, the detection of TrkB immunoreactivity on cell bodies and dendrites is consistent with recent models suggesting that neurotrophins may be derived from presynaptic and/or autocrine sources in addition to the classical postsynaptic target. J Comp Neurol 378:135–157, 1997. 1997 © Wiley-Liss, Inc.     

Cyclooxygenase-2 modulates brain inflammation-related gene expression in central nervous system radiation injury

Although the contribution of cyclooxygenase-2 (COX-2) to peripheral inflammation is well documented, little is known about its role in brain inflammation. For this purpose we studied COX-2 expression in the mouse brain following ionizing radiation in vivo, as well as in murine glial cell cultures in vitro. The possible role of COX-2 in modulating brain inflammation was examined utilizing NS-398, a COX-2 selective inhibitor. Our results indicate that COX-2 is significantly induced in astrocyte and microglial cultures by radiation injury as well as in brain. Increased levels of prostaglandin E(2) in irradiated brain were reduced by NS-398. Moreover, NS-398 administration significantly attenuated levels of induction for the majority of inflammatory mediators examined, including TNFalpha, IL-1beta, IL-6, iNOS, ICAM-1, and MMP-9. In contrast, the chemokines MIP-2 and MCP-1 showed enhanced levels of induction following NS-398 administration. These results indicate that COX-2 modulates the inflammatory response in brain following radiation injury, and suggest the use of COX-2 selective inhibitors for the management of CNS inflammation.     

Demyelination and altered expression of myelin-associated glycoprotein isoforms in the central nervous system of galactolipid-deficient mice

Vertebrate myelin is enriched in the lipid galactocerebroside (GalC) and its sulfated derivated sulfatide. To understand the in vivo function of these lipids, we analyzed myelination in mice that contain a null mutation in the gene encoding UDP-galactose:ceramide galactosyltransferase, the enzyme responsible for catalyzing the final step in GalC synthesis. Galactolipid-deficient myelin is regionally unstable and progressively degenerates. At postnatal day 30, demyelination is restricted to the midbrain and hindbrain, but by postnatal day 90, it spreads throughout the central nervous system. Activated microglial cells and reactive astrocytes appear with the loss of myelin in older animals. Nonetheless, major myelin protein gene mRNA levels are normal throughout the life of these animals, suggesting that widespread oligodendrocyte death is not the primary cause of demyelination. The developmental switch in myelin-associated glycoprotein isoform expression, however, does not occur normally in these mice, suggesting an alteration in oligodendrocyte maturation. Taken together, these findings indicate that GalC and sulfatide are required for the long-term maintenance of myelin and that their absence may have subtle effects on the development of oligodendrocytes. J. Neurosci. Res. 54:613–622, 1998. © 1998 Wiley-Liss, Inc.