Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.     

Molecular characterization of mitocalcin, a novel mitochondrial Ca2+-binding protein with EF-hand and coiled-coil domains

Here we have identified and characterized a novel mitochondrial Ca2+-binding protein, mitocalcin. Western blot analysis demonstrated that mitocalcin was widely expressed in mouse tissues. The expression in brain was increased during post-natal to adult development. Further analyses were carried out in newly established neural cell lines. The protein was expressed specifically in neurons but not in glial cells. Double-labeling studies revealed that mitocalcin was colocalized with mitochondria in neurons differentiated from 2Y-3t cells. In addition, mitocalcin was enriched in the mitochondrial fraction purified from the cells. Immunohistochemical studies on mouse cerebellum revealed that the expression pattern of mitocalcin in glomeruli of the internal granular and molecular layers was well overlapped by the distribution pattern of mitochondria. Immunogold electron microscopy showed that mitocalcin was associated with mitochondrial inner membrane. Overexpression of mitocalcin in 2Y-3t cells resulted in neurite extension. Inhibition of the expression in 2Y-3t cells caused suppression of neurite outgrowth and then cell death. These findings suggest that mitocalcin may play roles in neuronal differentiation and function through the control of mitochondrial function.     

Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion.

Monoclonal and polyclonal L1 antibodies react by indirect immunofluorescence with the cell surface of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, but not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. During cerebellar development L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after 3 days in culture. In sections of the early post-natal cerebellum, L1 antigen is found on pre-migratory neurons in the internal, but not in the external part of the external granular layer. In the adult cerebellum, L1 antigen is predominantly localized in the molecular layer and around Purkinje cells. Fibers in white matter and the granular layer are also L1 antigen-positive. Granule cell bodies and synaptic glomeruli are weakly antigen-positive. Several cell lines derived from neuroblastoma C1300 also express L1 antigen. The antigen is not detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are found in brains of rat, guinea pig, hamster, chicken, rabbit and man, but not in frog, while monoclonal antibody reacts detectably only with mouse brain. The molecular species recognized by both monoclonal and polyclonal antibodies display two prominent bands by SDS-PAGE under reducing and non-reducing conditions with apparent mol. wts. of 140 and 200 kd. L1 antigen isolated from cultured cerebellar cells consists mainly of a band in the 200-kd range and a faint one at 140 kd. L1 antigen from neuroblastoma N2A shows two bands with slightly higher apparent mol. wts. All molecular forms of L1 antigen can be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continuous cell line N2A derived from the murine neuroblastoma C1300 is inhibited by Fab fragments of the polyclonal, but not of monoclonal antibody, both of which are known to react with the surface membrane of these cells.     

CXCR3 mediates region-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis

Regional differences in inflammation during viral infections of the CNS suggest viruses differentially induce patterns of chemoattractant expression, depending on their cellular targets. Previous studies have shown that expression of the chemokine CXCL10 by West Nile virus (WNV)-infected neurons is essential for the recruitment of CD8 T cells for the purpose of viral clearance within the CNS. In the current study we used mice deficient for the CXCL10 receptor, CXCR3, to evaluate its role in leukocyte-mediated viral clearance of WNV infection within various CNS compartments. WNV-infected CXCR3-deficient mice exhibited significantly enhanced mortality compared with wild-type controls. Immunologic and virologic analyses revealed that CXCR3 was dispensable for control of viral infection in the periphery and in most CNS compartments but, surprisingly, was required for CD8 T cell-mediated antiviral responses specifically within the cerebellum. WNV-specific, CXCR3-expressing T cells preferentially migrated into the cerebellum, and WNV-infected cerebellar granule cell neurons expressed higher levels of CXCL10 compared with similarly infected cortical neurons. These results indicate that WNV differentially induces CXCL10 within neuronal populations and suggest a novel model for nonredundancy in chemokine-mediated inflammation among CNS compartments.     

Lifeguard/neuronal membrane protein 35 regulates Fas ligand-mediated apoptosis in neurons via microdomain recruitment

Fas ligand (FasL)-receptor system plays an essential role in regulating cell death in the developing nervous system, and it has been implicated in neurodegenerative and inflammatory responses in the CNS. Lifeguard (LFG) is a protein highly expressed in the hippocampus and the cerebellum, and it shows a particularly interesting regulation by being up-regulated during postnatal development and in the adult. We show that over-expression of LFG protected cortical neurons from FasL-induced apoptosis and decreased caspase-activation. Reduction of endogenous LFG expression by small interfering RNA sensitized cerebellar granular neurons to FasL-induced cell death and caspase-8 activation, and also increased sensitivity of cortical neurons. In differentiated cerebellar granular neurons, protection from FasL-induced cell death could be attributed exclusively to LFG and appears to be independent of FLICE inhibitor protein. Thus, LFG is an endogenous inhibitor of FasL-mediated neuronal death and it mediates the FasL resistance of CNS differentiated neurons. Finally, we also demonstrate that LFG is detected in lipid rafts microdomains, where it may interact with Fas receptor and regulate FasL-activated signaling pathways.     

Induction of galanin receptor-1 (GalR1) expression in external granule cell layer of post-natal mouse cerebellum

Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (- LI), [(125)I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [(125)I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80-170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p < 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3-5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10-12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.     

Altered Ontogenesis of Muscarinic Receptors in Agranular Cerebellar Cortex

Abstract: The developmental pattern, the agonist binding properties and the cellular origin(s) of muscarinic binding sites were investigated in agranular cerebellum of x-irradiated rats, of Gunn rats with hereditary hyperbilirubinemia, and of staggerer mutant mice. The density of muscarinic binding sites was found to be higher than normal in all of these cerebellar types, indicating that granular neurons do not greatly contribute to binding of acetylcholine in the rodent cerebellum. The total number of muscarinic binding sites as measured by binding of [³H]4NMPB remains unchanged in the agranular cerebellum of x-irradiated rats. However, the number of muscarinic sites is reduced by about 30% in the agranular cerebellum of homozygous Gunn rats (jj), in which fibrous astrocytes and Purkinje cells are also damaged. In the cerebellum of staggerer mice (sg/sg), where a cascade of events leads to massive damage to mossy fibers and Golgi cells in addition to granular neurons and Purkinje cells, the content of muscarinic receptors is reduced by 50%. Thus, the number of muscarinic binding sites in the rodent cerebellum seems to depend on the integrity of the additional cell types and cellular elements, damaged in these agranular models. The ontogenetic variations in the affinity of cerebellar muscarinic sites for binding of carbamylcholine in normal and Gunn rat cerebellum were compared with those observed in x-irradiated and staggerer cerebellum, where elimination of granular neurons induces the formation of ‘heterologous’ synapses. Muscarinic binding affinity increases 10-fold during postnatal development in the cerebellum of normal and Gunn rats. In the immature x-irradiated cerebellum, the affinity of muscarinic binding sites was found to be nearly as high as that detected in the adult normal cerebellum. In contrast, cerebella of 5-month-old staggerer mice display 5-fold lower affinity than their normal counterpart values, as low as that determined in normal immature cerebellum. The characteristic ontogenetic pattern of muscarink binding is therefore indicated to be related to the formation of correct circuitry, but not to the presence of granular neurons, in the developing rat cerebellum.     

An Aberrant Cerebellar Development in Mice Lacking Matrix Metalloproteinase-3

Cell–cell and cell–matrix interactions are necessary for neuronal patterning and brain wiring during development. Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of remodelling the pericellular environment and regulating signaling pathways through cleavage of a large degradome. MMPs have been suggested to affect cerebellar development, but the specific role of different MMPs in cerebellar morphogenesis remains unclear. Here, we report a role for MMP-3 in the histogenesis of the mouse cerebellar cortex. MMP-3 expression peaks during the second week of postnatal cerebellar development and is most prominently observed in Purkinje cells (PCs). In MMP-3 deficient (MMP-3^−/−) mice, a protracted granule cell (GC) tangential migration and a delayed GC radial migration results in a thicker and persistent external granular layer, a retarded arrival of GCs in the inner granular layer, and a delayed GABAergic interneuron migration. Importantly, these neuronal migration anomalies, as well as the consequent disturbed synaptogenesis on PCs, seem to be caused by an abnormal PC dendritogenesis, which results in reduced PC dendritic trees in the adult cerebellum. Of note, these developmental and adult cerebellar defects might contribute to the aberrant motor phenotype observed in MMP-3^−/− mice and suggest an involvement of MMP-3 in mouse cerebellar development.     

The interaction between cytoplasmic prion protein and the hydrophobic lipid core of membrane correlates with neurotoxicity

Prion protein (PrP), normally a cell surface protein, has been detected in the cytosol of a subset of neurons. The appearance of PrP in the cytosol could result from either retro-translocation of misfolded PrP from the endoplasmic reticulum (ER) or impaired import of PrP into the ER. Transgenic mice expressing cytoplasmic PrP (cyPrP) developed neurodegeneration in cerebellar granular neurons, although no detectable pathology was observed in other brain regions. In order to understand why granular neurons in the cerebellum were most susceptible to cyPrP-induced degeneration, we investigated the subcellular localization of cyPrP. Interestingly, we found that cyPrP is membrane-bound. In transfected cells, it binds to the ER and plasma/endocytic vesicular membranes. In transgenic mice, it is associated with synaptic and microsomal membranes. Furthermore, the cerebellar neurodegeneration in transgenic mice correlates with the interaction between cyPrP and the hydrophobic lipid core of the membrane but not with either the aggregation status or the dosage of cyPrP. These results suggest that lipid membrane perturbation could be a cellular mechanism for cyPrP-induced neurotoxicity and explain the seemingly conflicting results concerning cyPrP.     

Molecular morphology of neuronal apoptosis: Analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.     

CEREBELLAR DEVELOPMENT IN THE RAT AFTER EARLY POSTNATAL DAMAGE BY METHYLAZOXY-METHANOL: DNA, RNA AND PROTEIN DURING RECOVERY

—Total cerebellar DNA, RNA, protein and wet weight were determined for normal cerebellum and for cerebellum damaged early in postnatal development by the neurotoxic action of the drug methylazoxymethanol (MAM) acetate. Injections of a 10 mg/kg dose of the drug were given on postnatal days 1, 2, 3 and 4 as a means of inducing a transient insult to the proliferating stem cell population of the external granular layer. Cerebellar DNA and other quantitative measures were reduced to 50 per cent of normal at day 7. Growth in the MAM-treated cerebellum was relatively more rapid than in normals so that the eventual deficit in the adult was 80 per cent of normal. We concluded that early postnatal transient injury of the developing cerebellum is followed by a regenerative phase of repair but that quantitatively significant mitosis in the injured cerebellum ceases normally at about post-natal day 17. The conditions evaluated in this investigation may serve as a model for other types of injury induced by drugs, irradiation, viruses or nutritional factors.     

Monoclonal antibody (M2) to glial and neuronal cell surfaces

A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface Of all GFA protein-positive astrocytes and on more immature oligodendrocytes, that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxinpositive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.     

Neurotrophin-3 controls proliferation of granular precursors as well as survival of mature granule neurons in the developing rat cerebellum

Levels of neurotrophin-3 markedly decrease in the rat cerebellum after the first 10 days of life, suggesting an importance during early development. To further examine the effect of neurotrophin-3 on the developing cerebellum, we injected a monoclonal antibody against neurotrophin-3 into the lateral ventricle of 7.5-day-old rats. The resultant depletion of neurotrophin-3 caused a significant decrease in cerebellar wet weights noted at 7 and 23 days thereafter. Other changes noted 48 h after injection of monoclonal antibodies against neurotrophin-3 included reduced incorporation of bromode-oxyuridine into granule neurons in the external germinal layer, an elevated density of atrophic neurons that had just migrated under the Purkinje cell layer, and an increased number of apoptotic neurons in the internal granule cell layer. These changes were limited to the central lobe. The concentration of neurotrophin-3 protein in the posterior region, including the central lobe, was about four- and threefold higher than that in the anterior region of the cerebellum of 9.5- and 30-day-old rats, respectively. Immunocytochemical examination showed higher amounts of neurotrophin-3 protein in the central lobe than in the anterior lobe. Our results provide evidence that neurotrophin-3 regulates the proliferation of granule precursors and supports the survival of mature granule neurons in restricted lobules, suggesting an involvement in limited regions at a specific stage in development of the rat cerebellum.     

Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.