Effect of Cerebellohypothalamic Glutamatergic Projections on Immune Function.

Our previous work has shown that lesions of the cerebellar interposed nuclei (IN) suppress immune cell functions. Since there is no direct structural connection between the cerebellum and immune system, we explored the pathway mediating the cerebellar immunomodulation at the profile of cerebellohypothalamic projections to understand this modulation. Anterograde tracing of nerve tracts from the cerebellar IN to the hypothalamus was conducted by injection of anterograde tracer dextran-texas red (dextran-TR) in the cerebellar IN. We observed that dextran-TR-labeled nerve fibers, which were sent by cerebellar IN neurons, traveled in the superior cerebellar peduncle (SCP), crossed in SCP decussation, and entered the hypothalamus. In the hypothalamus, the fibers mostly terminated in the lateral hypothalamic area (LHA). Retrograde tracing by injection of retrograde tracer fluoro-ruby (FR) in the LHA found that FR-labeled neurons appeared in contralateral cerebellar IN. Fluorescent immunohistochemistry for glutamate revealed that many of the FR-labeled neurons were glutamatergic. These results demonstrate a direct glutamatergic projection from the cerebellar IN to the LHA. Reduction of the cerebellohypothalamic glutamatergic projections by microinjection of 6-diazo-5-oxo- L-norleucine (DON), an inhibitor of glutaminase for glutamate synthesis, in bilateral cerebellar IN led to suppression of peripheral lymphocyte number, T lymphocyte proliferation, and serum anti-sheep red blood cell IgM level. But the DON injection in the cerebellar cortex that does not send axons to the hypothalamus did not significantly alter all the immune parameters. These findings suggest that cerebellohypothalamic glutamatergic projection modulates immune function, and that via the pathway, the cerebellum implements its immunoregulatory effect.

Antigenic compartmentation of the cerebellar cortex in an Australian marsupial, the tammar wallaby Macropus eugenii.

The mammalian cerebellar cortex is apparently uniform in composition, but a complex heterogeneous pattern can be revealed by using biochemical markers such as zebrin II/aldolase C, which is expressed by a subset of Purkinje cells that form a highly reproducible array of transverse zones and parasagittal stripes. The architecture revealed by zebrin II expression is conserved among many taxa of birds and mammals. In this report zebrin II immunohistochemistry has been used in both section and whole-mount preparations to analyze the cerebellar architecture of the Australian tammar wallaby (Macropus eugenii). The gross appearance of the wallaby cerebellum is remarkable, with unusually elaborate cerebellar lobules with multiple sublobules and fissures. However, despite the morphological complexity, the underlying zone and stripe architecture is conserved and the typical mammalian organization is present.

Familial hemophagocytic lymphohistiocytosis in a pediatric patient diagnosed by brain magnetic resonance imaging.

Familial hemophagocytic lymphohistiocytosis (fHLH) is an autosomal recessive disorder characterized by proliferation and infiltration of several organs by activated lymphocytes and macrophages. Without allogeneic stem cell transplantation, fHLH is fatal. We describe a previously healthy 11-month-old boy with a rapidly progressive encephalopathy. An older brother died at 8 months following a subacute encephalopathy diagnosed as meningoencephalitis. The family history led to the suspicion of a metabolic disease, but metabolic studies were unrevealing. MRI showed multiple inhomogeneous signal abnormalities in the cortex and white matter, most prominent in the cerebral hemispheres and around the dentate nucleus. Gadolinium-enhanced T1-weighted images showed a multitude of enhancing foci, suggestive of perivascular enhancement. Based on MRI pattern with multiple lesions, perivascular enhancement and family history, fHLH was suspected. DNA analysis showed that the patient was compound-heterozygous for the c.445 G>A (p.Gly149Ser) mutation in exon 1 and the c.757 G>A (p.Glu253Lys) mutation in exon 2 of the perforin 1 gene. The patient was treated according to the international HLH-2004 protocol (dexamethasone, etoposide, cyclosporine, intrathecal methotrexate and prednisolone) followed by allogeneic cord blood transplantation. He showed a significant neurological and radiological improvement. The reported case demonstrates that MRI pattern recognition can lead to early diagnosis of fHLH, with subsequent adequate treatment.

Clonazepam responsive opsoclonus myoclonus syndrome: additional evidence in favour of fastigial nucleus disinhibition hypothesis?

Opsoclonus myoclonus syndrome is a rare paraneoplastic syndrome seen in 50% of children with neuroblastoma. Neural generator of opsoclonus and myoclonus is not known but evidences suggest the role of fastigial nucleus disinhibition from the loss of function of inhibitory (GABAergic) Purkinje cells in the cerebellum. We present a child with paraneoplastic opsoclonus myoclonus syndrome who responded well to clonazepam. Response to clonazepam is an evidence for the involvement of GABAergic neural circuits in the genesis of opsoclonus myoclonus syndrome and is in agreement with fastigial nucleus disinhibition hypothesis.

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration.

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.

A Mab to a unique cerebellar neuron generated by immunosuppression and rapid immunization.

The cerebellar cortex is perhaps the best characterized structure in the mammalian central nervous system. Although the major cerebellar cell classes are well known, a new class of cerebellar cortical neuron has now been identified with a monoclonal antibody (Mab) generated by a procedure for rapid immunization and selective immunosuppression of antibody responses. This procedure generates a high frequency of immunoglobulin G-class antibodies of desired specificity, and has allowed the generation of two antibodies that recognize subsets of cerebellar cortical neurons. One of these antibodies defines a previously unrecognized class of cerebellar neuron. The distribution and antigenic characteristics of this neuron suggest that it has a distinct role in cerebellar circuitry.

Late Brain Involvement after Neonatal Immune Activation.

The neonatal immune system is still immature, which makes it more susceptible to the infectious agents. Neonatal immune activation is associated with increased permeability of the blood-brain barrier, causing an inflammatory cascade in the CNS and altering behavioral and neurochemical parameters. One of the hypotheses that has been studied is that neuroinflammation may be involved in neurodegenerative processes, such as Alzheimer's disease (AD). We evaluate visuospatial memory, cytokines levels, and the expression of tau and GSK-3ß proteins in hippocampus and cortex of animals exposed to neonatal endotoxemia. C57BL/6 mice aging two days received a single injection of subcutaneous lipopolysaccharide (LPS). At 60,120, and 180 days of age, visual-spatial memory was evaluated and the hippocampus and cortex were dissected to evaluate the cytokines levels and expression of tau and GSK-3ß proteins. The animals exposed to LPS in the neonatal period present with visuospatial memory impairment at 120 and 180 days of age. Here there was an increase of TNF-α and IL-1ß levels in the hippocampus and cortex only at 60 days of age. Here there was an increase in the expression of GSK-3ß in hippocampus of the animals at 60, 120, and 180 days of age. In the cortex, this increase occurred in the 120 and 180 days of age. Tau protein expression was high in hippocampus and cortex at 120 days of age and in hippocampus at 180 days of age. The data observed show that neonatal immune activation may be associated with visuospatial memory impairment, neuroinflammation, and increased expression of GSK-3ß and Tau proteins in the long term.

Preservation of ultrastructure and immunoreactivity in cryosections of brain tissue stored in a sucrose-gelatin solution at freezing temperatures.

We evaluated the preservation of ultra-structure and immunoreactivity in cryosections of central nervous system tissue mounted with and stored in a sucrose-gelatin solution for one month at -20 degrees C or -80 degrees C. The ultra-structure of synaptic structure in these sections was well preserved and comparable to that of freshly cut cryosections. Quantitative analysis of mitochondrial ultra-structure demonstrated gradually lower degrees of preservation in sections stored at -20 degrees C and -80 degrees C compared with that in freshly cut sections. We observed distinct metabotropic glutamate receptor 1 (mGluR1)-immunogold labelling at peri-synaptic sites in freshly cut sections and also in those stored at -20 degrees C and -80 degrees C. Quantitative analysis of mGluR1 immunoreactivity revealed that the total number of immunogold particles per synapse and the number of non-specifically bound particles were similar under all three conditions. However, the percentage of gold particles bound to a specific synaptic region was greatest in freshly cut sections (79.0%) and progressively lower in sections stored at -20 degrees C (76.1%), in which sections were not frozen, and in sections stored at -80 degrees C (68.0%). These data indicate that ultra-thin cryosections may be conveniently stored in a sucrose-gelatin solution at -20 degrees C for cryoultramicrotomy-immunolabelling.

Inflammation-induced reversible switch of the neuron-specific enolase promoter from Purkinje neurons to Bergmann glia.

Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs.

Targeted gene therapy to antigen-presenting cells in the central nervous system using hematopoietic stem cells.

BACKGROUND: Hematopoietic stem cells (HSC) have been previously used as vectors for gene therapy of systemic disease. The effectiveness of HSC-mediated gene therapy largely depends on efficient gene delivery into long-term repopulating progenitors and targeted transgene expression in an appropriate progeny of the transduced pluripotent HSCs. In the present study, we examined the feasibility of using HSC transduced with self-inactivating (SIN) lentiviral vectors for the delivery of gene therapy to the central nervous system (CNS). MATERIAL AND METHODS: We constructed two SIN lentiviral vectors, EF.GFP and DR.GFP, to express the green fluorescent protein (GFP) gene controlled solely by the promoter of either a housekeeping gene EF-1alpha or the human HLA-DRalpha gene, which is selectively expressed in antigen-presenting cells. RESULTS: We demonstrated that both vectors efficiently transduced human pluripotent CD34+ cells capable of engrafting NOD/SCID mice. Only the DR.GFP vector mediated transgene expression in the murine CNS containing human HLA-DR+ cells. These cells express surface markers characteristic of resident CNS microglia. Furthermore, human dendritic cells derived from transduced and engrafted human cells potently stimulated allogeneic T cell proliferation. CONCLUSIONS: The present study demonstrated successful targeting of transgene expression to CNS microglia after stable gene transduction of pluripotent HSC.

Antigenic map of the rat cerebellar cortex: the distribution of parasagittal bands as revealed by monoclonal anti-Purkinje cell antibody mabQ113.

Both anatomical and physiological mapping methods have revealed that the mammalian cerebellar cortex consists of a family of parasagittal bands of cells, each band with its own pattern of afferent and efferent axons. Monoclonal antibody mabQ113 recognizes an unknown polypeptide antigen that is confined to a subset of rat cerebellar Purkinje cells. Immunoreactive cells are arranged into parasagittal bands extending throughout the vermis and hemispheres. Expression of the Q113 epitope by individual Purkinje cells may not be all-or-nothing, since the bands tend to be more strongly stained in the vermis than the hemispheres. The band display is symmetrical about the midline and reproducible from individual to individual. Whole-mount immunocytochemistry and serial reconstruction reveal a median band of mabQ113+ Purkinje cells adjacent to the midline (P1+) and six other positive bands disposed symmetrically at either side (P2+ to P7+). Bands are distinct throughout most of the cortex but tend to fuse ventrally and caudally. There are two sources of interindividual differences. Firstly, most animals express supernumerary "satellite" bands in the vermis. Satellite bands are usually only one cell wide, are not bilaterally symmetrical, and differ in position and number from individual to individual. Secondly, the precise position of an individual band can differ, perhaps according to the variable cortical lobulation, for example, the position of P4+ in lobules VIII/IX and P6+ in lobule VII. While a scheme of parasagittal bands is a good description of the vermian organization, the distribution of mabQ113+ and mabQ113- Purkinje cells in the hemispheres may be better described as a checkerboard of antigenic patches.

Antigenic compartmentation in the mouse cerebellar cortex: zebrin and HNK-1 reveal a complex, overlapping molecular topography.

Two monoclonal antibodies--anti-zebrin I and anti-HNK-1--have been used to study the compartmentation of the mouse cerebellar cortex. As in other species, the pattern of localization of the Purkinje cell specific antigen zebrin I is confined to a subset of Purkinje cells that are organized into parasagittal bands. The basic pattern consists of two abutting paramedian bands (P1+) and up to three additional vermal bands on either side (P2(+)-P4+). This pattern is altered in the vermal regions of lobules X and VI-VII where all Purkinje cells are immunoreactive. In the hemisphere there are three additional bands present (P5(+)-P7+) plus two shorter bands in the paravermal area (P4b+ and P5a+) that extend from the paramedian lobule through the lobulus simplex. This pattern is very similar, but perhaps not identical, to that previously described for the rat. These results suggest a common mammalian plan for the expression and localization of zebrin I. By using a monoclonal antibody to an epitope associated with HNK-1, we have now identified a novel pattern of compartmentation in mouse cerebellum. The HNK-1 epitope is expressed most notably on Purkinje cells and Golgi cells. The molecular layer immunoreactivity associated with the Purkinje cell dendrites varies in intensity in a systematic and reproducible fashion. This reveals a novel cerebellar compartmentation that is sometimes complementary, sometimes overlapping, to that revealed by anti-zebrin. As a result, it is now possible to subdivide the cerebellar cortex into a still finer mosaic of antigenic patches and bands than was possible by using zebrins alone.

Destruction of meningeal cells over the medial cerebral hemisphere of newborn hamsters prevents the formation of the infrapyramidal blade of the dentate gyrus.

Meningeal cells participate in the development of the cerebellum both by stabilizing the extracellular matrix of the pial surface and by organizing the radial glial scaffold and the lamination of the cerebellar cortex. In the present study we investigated possible influences of meningeal cells on the development of the dentate gyrus, whose ontogenesis has many similarities to that of the cerebellum. Meningeal cells were selectively destroyed by injecting newborn hamsters with 25 micrograms 6-hydroxydopamine (6-OHDA) into the interhemispheric fissure. Twenty-four hours postinjection (p.i.) the meningeal cells over the medial cerebral hemispheres were completely destroyed. Thirty days p.i. the infrapyramidal blade of the dentate gyrus was almost completely missing, while the suprapyramidal blade was hypertrophied, extending with its medial tip almost up to the medial surface of the cortex. In order to ascertain that this maldevelopment was caused by the destruction of meningeal cells, another group of hamsters was pretreated with normetanephrine (NMN) which inhibits the extraneuronal uptake of 6-OHDA into meningeal cells. In this group the meningeal cells were unaffected by the treatment, and the morphology of the dentate gyrus was normal 30 days p.i. of 6-OHDA plus NMN. When the meningeal cells were destroyed in later stages of development (postnatal days 1-5), alterations of the dentate gyrus could be induced only up to the fourth postnatal day; thereafter, 6-OHDA treatment left it unchanged. This indicates a critical period of meningeal cell influence that coincides with the period of existence of the subpial dentate matrix. Analysis of the time course of the defective development revealed that in the first 5 days p.i. 1) meningeal cells over the medial cerebral hemisphere were destroyed and removed, 2) the pial basement membrane over both the dentate anlage and the diencephalon thinned and ruptured, and the adjacent brain parts fused focally, 3) many cells of the subpial dentate matrix disappeared from their subsurface position, 4) the number of "immature" cells increased in the hilus and the subgranular zone of the suprapyramidal blade, 5) the suprapyramidal blade elongated and thickened considerably, while the infrapyramidal blade did not form. Beyond 5 days p.i. those parts of the pial surface of the dentate anlage that had not fused with the diencephalon were repopulated with meningeal cells. This reappearance of meningeal cells was accompanied by 1) the restitution of the normal morphology of the basement membrane, 2) the reappearance of neuronal and glial cells below the pial surface, and 3) the formation of fragments of the infrapyramidal blade which later developed a normal appearing lamination.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-6 produces neuronal loss in developing cerebellar granule neuron cultures.

CNS levels of the cytokine interleukin-6 (IL-6) are elevated during CNS injury and disease, but it is unclear if IL-6 contributes to the pathologic process. Our studies show that in a well-characterized CNS developmental model system, primary cultures of rodent cerebellar granule neurons, chronic exposure to IL-6 during neuronal development can result in cell damage and death in a subpopulation of developing granule neurons. Chronic exposure to IL-6 also increased the susceptibility of the granule neurons to a toxic insult produced by excessive activation of NMDA receptors. These results are consistent with a role for IL-6 in the neuropathology observed in the developing CNS during injury and disease.

Increased sensitivity to N-methyl-D-aspartate receptor-induced excitotoxicity in cerebellar granule cells from interleukin-1 receptor type I-deficient mice.

The effects of chronic exposure to excitatory amino acids (EAAs) were examined in cultured cerebellar granule cells (CGCs) from wild type (WT) and interleukin-1 receptor type I (IL-1RI)-deficient mice. After 8 days in culture, the cells were exposed to 100 microM glutamate or 300 microM N-methyl-D-aspartate (NMDA) for 24 h. Analysis of cell viability, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and phase-contrast microscopy revealed that CGCs from IL-1RI-deficient mice were more vulnerable to EAAs as compared to the WT controls. The results indicate that IL-1RI signalling is important for neuronal survival. The effect of glutamate on the CGCs from IL-1RI-deficient mice was decreased by the non-competitive NMDA-receptor antagonist MK-801, supporting the involvement of NMDA receptors in the glutamate-induced excitotoxicity.

Chemokine receptor CXCR2 regulates the functional properties of AMPA-type glutamate receptor GluR1 in HEK cells.

Experiments were conducted in both HEK cells and cerebellar neurons to investigate whether CXC chemokine receptor 2 (CXCR2) is functionally coupled to GluR1. The co-expression of CXCR2 with GluR1 in HEK cells increased (i) the GluR1 "apparent" affinity for the transmitter; (ii) the GluR1 channel open probability; and (iii) GluR1 binding site cooperativity upon CXCR2 stimulation with CXC chemokine ligand 2 (CXCL2). The affinity of C-terminal-deleted GluR1 for glutamate (Glu) remained stable instead. Furthermore, CXCL2 increased the binding site cooperativity of AMPA receptors in rat cerebellar granule cells; and the amplitude of spontaneous excitatory postsynaptic current (sEPSCs) in Purkinje neurons (PNs). Our findings indicate that the coupling of CXCR2 with GluR1 may modulate glutamatergic synaptic transmission.

Differential subcellular distribution of the alpha 6 subunit versus the alpha 1 and beta 2/3 subunits of the GABAA/benzodiazepine receptor complex in granule cells of the cerebellar cortex.

The distribution of the alpha 6 subunit of the GABAA receptor has been established in rat cerebellum and compared to the distribution of the alpha 1 (cat) and the beta 2/3 (rat, cat) subunits, using immunocytochemistry. The synapses established by Golgi cell terminals on the dendrites of granule cells were immunoreactive for the alpha 6, alpha 1 and beta 2/3 subunits in virtually all glomeruli, indicating that two variants (alpha 1 and alpha 6) of the same subunit are co-localized at the same synapses. The somatic membranes of the granule cells, which receive no synapses, were immunopositive for the alpha 1 and beta 2/3 subunits, but not for the alpha 6 subunit. Thus, the alpha 1 and the beta 2/3 subunits are located at both synaptic and extrasynaptic sites, but the alpha 6 subunit is detectable only at synaptic sites.

The organization of neurofilaments accumulated in perikaryon following aluminum administration: relationship between structure and phosphorylation of neurofilaments.

Neurofilaments accumulated in perikarya and dendrites of anterior horn cells and Purkinje cells of rabbit treated by aluminum chloride were analysed with a variety of techniques. Four different monoclonal antibodies against phosphorylated and nonphosphorylated epitopes on neurofilament H subunit were used to compare phosphorylation state of these accumulated neurofilaments with that of axonal neurofilaments. Although immunoblotting revealed no significant difference in phosphorylation between control and aluminum-treated brains, accumulated neurofilaments were immunocytochemically more phosphorylated than control perikaryal or dendritic neurofilaments. With detailed analysis of cryothin-section immunogold labeling, accumulated neurofilaments were, however, significantly less phosphorylated than axonal neurofilaments. With quick-freeze deep etching, core filaments of accumulated neurofilaments are as dense as axonal neurofilaments but much less regularly aligned. Cross-bridges of accumulated neurofilaments were less frequent and more branched than those of axonal neurofilaments, and when examined with combined immunocytochemistry and deep etching, were less phosphorylated. These results suggest that there is a relationship between the phosphorylation and the structural organization of neurofilaments. The phosphorylation of neurofilament H subunit may be necessary for formation of frequent and straight cross-bridges and resulting regular alignment of core filaments.

Immunohistochemical visualization of a metabotropic glutamate receptor.

The immunocytochemical localization of the recently cloned metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was demonstrated with a C-terminus specific antibody in rat cerebellar cortex. This antibody detects a 138-140 kDa major, and a 46 kDa minor band in membrane preparations of rat cortex and cerebellum. mGluR1 alpha immunoreactivity (mGRi) was present in Purkinje and basket cells. Purkinje cell dendritic spines and their postsynaptic membranes showed selective labelling. Presynaptic membranes, parallel fibres and glial processes were devoid of mGRi. It is suggested that the selective postsynaptic localization of this receptor at the dendritic spines of Purkinje cells serves as the morphological basis for long term depression processes in the molecular layer of the cerebellar cortex.

Developmental changes of chick cerebellar cortex revealed by monoclonal antibodies.

Immunohistochemistry studies of the embryonic and newly hatched chick cerebellum were performed with 13 monoclonal antibodies (MAbs) raised against the embryonic chick optic nerve and a MAb which binds to cell nuclei. Neural MAbs differentially stained Purkinje cells, the external granular layer, molecular layer, internal granular layer, climbing fibers, basket cell axons, Bergmann glia and Ramón y Cajal's ansiform fibers. At the different developmental stages each component responded to MAbs differently. For example, staining of Purkinje cells with MAbs 23C10, 82E10 and 94C2 appeared on day 11 of incubation and disappeared sequentially after day 18. These results reveal molecular heterogeneity not only in cerebellar neurons but also at various developmental stages.