Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice.

Huntington disease (HD), an autosomal dominant, progressive neurodegenerative disorder, is caused by an expanded CAG repeat sequence leading to an increase in the number of glutamine residues in the encoded protein. The normal CAG repeat range is 5-36, whereas 38 or more repeats are found in the diseased state; the severity of disease is roughly proportional to the number of CAG repeats. HD shows anticipation, in which subsequent generations display earlier disease onsets due to intergenerational repeat expansion. For longer repeat lengths, somatic instability of the repeat size has been observed both in human cases at autopsy and in transgenic mouse models containing either a genomic fragment of human HD exon 1 (ref. 9) or an expanded repeat inserted into the endogenous mouse gene Hdh (ref. 10). With increasing repeat number, the protein changes conformation and becomes increasingly prone to aggregation, suggesting important functional correlations between repeat length and pathology. Because dinucleotide repeat instability is known to increase when the mismatch repair enzyme MSH2 is missing, we examined instability of the HD CAG repeat by crossing transgenic mice carrying exon 1 of human HD (ref. 16) with Msh2-/- mice. Our results show that Msh2 is required for somatic instability of the CAG repeat.

The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8.

The neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of progressive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in various tissues. Progressive epilepsy with mental retardation (EPMR, MIM 600143) was recently recognized as a new NCL subtype (CLN8). It is an autosomal recessive disorder characterized by onset of generalized seizures between 5 and 10 years, and subsequent progressive mental retardation. Here we report the positional cloning of a novel gene, CLN8, which is mutated in EPMR. It encodes a putative transmembrane protein. EPMR patients were homozygous for a missense mutation (70C-->G, R24G) that was not found in homozygosity in 433 controls. We also cloned the mouse Cln8 sequence. It displays 82% nucleotide identity with CLN8, conservation of the codon harbouring the human mutation and is localized to the same region as the motor neuron degeneration mouse, mnd, a naturally occurring mouse NCL (ref. 4). In mnd/mnd mice, we identified a homozygous 1-bp insertion (267-268insC, codon 90) predicting a frameshift and a truncated protein. Our data demonstrate that mutations in these orthologous genes underlie NCL phenotypes in human and mouse, and represent the first description of the molecular basis of a naturally occurring animal model for NCL.

Cystamine and intrabody co-treatment confers additional benefits in a fly model of Huntington's disease.

Huntington's disease (HD) is a lethal, neurodegenerative disorder caused by expansion of the polyglutamine repeat in the Huntingtin gene (HTT), leading to mutant protein misfolding, aggregation, and neuronal death. Feeding a Drosophila HD model cystamine, or expressing a transgene encoding the anti-htt intracellular antibody (intrabody) C4-scFv in the nervous system, demonstrated therapeutic potential, but suppression of pathology was incomplete. We hypothesized that a combinatorial approach entailing drug and intrabody administration could enhance rescue of HD pathology in flies and that timing of treatment would affect outcomes. Feeding cystamine to adult HD flies expressing the intrabody resulted in a significant, additional rescue of photoreceptor neurodegeneration, but no additional benefit in longevity. Feeding cystamine during both larval and adult stages produced the converse result: longevity was significantly improved, but increased photoreceptor survival was not. We conclude that cystamine-intrabody combination therapies can be effective, reducing neurodegeneration and prolonging survival, depending on administration protocols.

Combinational approach of intrabody with enhanced Hsp70 expression addresses multiple pathologies in a fly model of Huntington's disease.

Intracellular antibodies (intrabodies) and the chaperone, heat shock protein 70 (Hsp70), have each shown potential as therapeutics for neurodegenerative diseases in vitro and in vivo. Investigating combinational therapy in an established Drosophila model of Huntington's disease (HD), we show that Hsp70 and intrabody actually affect different aspects of the disease. Overexpression of human Hsp70 resulted in improved survival of HD flies to eclosion and prolonged adult life compared with intrabody treatment alone. An additive effect on adult survival was observed when the two therapies were combined. Intrabody was more successful at suppressing neurodegeneration in photoreceptors than was Hsp70. Furthermore, Hsp70 treatment alone did not block aggregation of mutant huntingtin, a process slowed by intrabody. Expression of each is restricted to the nervous system, which implies different neuronal populations respond distinctly to these treatments. Importantly, a role for endogenous Hsp70 in suppression of mutant huntingtin pathology was confirmed by a separate set of genetic studies in which HD flies deficient for Hsp70 showed significantly increased pathology. We conclude that a combinational approach of intrabody with enhanced Hsp70 expression is beneficial in addressing multiple pathologies associated with HD and has potential application for other neurodegenerative disorders.

Dystonin Is Essential for Maintaining Neuronal Cytoskeleton Organization.

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons-cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology. Copyright 1998 Academic Press.

Identification and characterization of Magmas, a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction.

OBJECTIVE: The aim of this study was to identify granulocyte-macrophage colony-stimulating factor (GM-CSF) responsive genes. MATERIALS AND METHODS: Potential GM-CSF responsive genes were identified by comparing the mRNA expression pattern of the murine myeloid cell line PGMD1 grown in either interleukin-3 (IL-3) or GM-CSF by differential display. Human and murine cDNA clones of one of the bands having increased expression in GM-CSF were isolated. mRNA expression of the gene was examined by Northern blot. Immunohistochemistry and studies with a green fluorescent fusion protein were used to determine its intracellular location. Growth factor-stimulated proliferation of PGMD1 cells transfected with constitutively expressed sense and anti-sense cDNA constructs of the gene was measured by 3H-thymidine incorporation. RESULTS: A gene, named Magmas (mitochondria-associated granulocyte macrophage CSF signaling molecule), was shown to be rapidly induced when cells were switched from IL-3 to GM-CSF. Analysis of the amino acid sequence of Magmas showed it contained a mitochondrial signal peptide, but not any other known functional domains. The human and murine clones encode nearly identical 13-kDa proteins that localized to the mitochondria. Magmas mRNA expression was observed in all tissues examined. PGMD1 cells that overexpressed Magmas proliferated similarly to untransfected cells when cultured in IL-3 or GM-CSF. In contrast, cells with reduced protein levels grew normally in IL-3, but had impaired proliferation in GM-CSF. CONCLUSION: Magmas is a mitochondrial protein involved in GM-CSF signal transduction.

Localization of phosphorylase kinase subunits at the sarcoplasmic reticulum of rabbit skeletal muscle by monoclonal and polyclonal antibodies.

Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.

Neurofilament distribution is altered in the Mnd (motor neuron degeneration) mouse.

Motor neuron degeneration (Mnd) is a genetic neurodegenerative disease of the mouse that is characterized by a progressive increase in motor dysfunction, moving from hind to fore limbs, leading to paralysis. An immunocytochemical analysis of the neurofilament distribution in spinal motor neurons in Mnd mice from all stages of the disease, including the presymptomatic, was performed using antibodies to different neurofilament subunits with different degrees of phosphorylation. Perikarya that stained with antibodies to phosphorylated neurofilaments were present in Mnd and control spinal cords, but the number of stained perikarya in Mnd was not significantly different from controls. There was a marked redistribution of neurofilaments within the cytoplasm of some motor neurons in Mnd cords. In Mnd but not controls, the immunoreaction product appeared marginated, leaving areas in the cytoplasm absent of immunostaining. These areas were observed in all stages of the disease, but less predictably in presymptomatics. Both the size of the areas and the number of motoneurons containing these areas appeared to increase with the severity of the disease. The number of anterior horn neurons in the hind limb region of lamina IX in spinal segment L4 of Mnd was lower than in controls, suggesting there is a loss of neurons in Mnd.

Novel developmental boundary in the cerebellum revealed by zebrin expression in the lurcher (Lc/+) mutant mouse.

The cerebellar cortex contains at least two classes of Purkinje cells, which are organized into alternating arrays of parasagittal bands. The clearest demonstration of this compartmentation is the pattern of expression of a family of polypeptide antigens, the zebrins, which are expressed selectively by Purkinje cell subsets. Furthermore, anterograde tracing experiments show that the zebrin compartments are closely correlated with both afferent and efferent projection maps. The further subdivision of long parasagittal bands into smaller modules may occur through several different mechanisms, including the intrinsic cerebellar lobulation and the selective distribution of afferent terminal fields. However, while the longitudinal subdivisions are straightforwardly shown, the mediolateral boundaries are more subtle. In this report we describe a novel mediolateral and anteroposterior compartmentation boundary in mice, running across lobule VIII, that is revealed by the consequences of the lurcher (Lc/+) allele for zebrin expression. In normal mice zebrin compartmentation develops in several discrete stages: until postnatal day 5 (PD5) there is no zebrin expression; from PD5-PD7 zebrin is found only in the posterior lobe vermis, with immunoreactive Purkinje cells in lobules X, IX, and VIII but not elsewhere; from PD7-PD12 most Purkinje cells in the vermis become zebrin+; from PD12-PD15 immunoreactivity also appears in the hemispheres so that almost all Purkinje cells now are zebrin+; and finally, from PD15-PD25 zebrin is gradually suppressed in those Purkinje cells that are zebrin- in the adult until the mature pattern of parasagittal compartments is revealed. In the Lc/+ mutant the normal developmental progression is interrupted at around PD7. As a result, the pattern of zebrin expression becomes frozen at that stage when immunoreactive Purkinje cells are confined exclusively to the posterior lobe vermis. A reproducible boundary between expressing and nonexpressing zones runs mediolaterally across the dorsal surface of lobule VIII. Apart from zebrin expression itself, there are no obvious structural correlates of this transition. This mediolateral boundary identifies a developmental unit in the posterior lobe vermis of the cerebellum, and provides further evidence that the cerebellum is a highly heterogeneous structure.

Accumulating autofluorescent material as a marker for early changes in the spinal cord of the Mnd mouse.

The mouse mutant Motor neuron degeneration (Mnd) displays an adult-onset progressive degeneration of upper and lower motor neurons, with mild symptoms recognizable at 6 months, leading to spastic paralysis and premature death at 10-12 months on the C57B1/6 background. Despite this late onset, abnormally-accumulating autofluorescent material can be seen in both the spinal cord and other regions as early as the first month. This pigmented material is present in both increasing numbers of cells, and in increasing amounts within individual cells, as the animals age. Motor neurons then go on to degenerate, while most other cell types stabilize. The level of pathological involvement, well before the onset of clear clinical symptoms, suggests that the full degenerative process is an extremely gradual and protracted one with some selectivity for motor neurons.

Genetics of primary and timing effects in the mnd mouse.

The mnd mouse shows a spontaneous adult-onset hereditary neurological disease, with motor abnormality by 6 months of age, progressing to severe spastic paralysis and premature death. The disease is autosomal recessive, with heterozygote effects seen under stress. It maps to mouse chromosome (chr) 8. Histopathology with Nissl stains documents substantial abnormalities of upper and lower motor neurons, and there is retinal degeneration beginning in the first month, even without light exposure. Increasing levels of autofluorescent lipopigment are found in both neuronal and non-neuronal tissues as the mnd mice age. Recently, NCL-like inclusions and accumulating subunit c have also been described. When mnd is outcrossed to the AKR/J genetic background, ca. 40% of the mnd/mnd F2 progeny show early onset (onset by 4.5-5 months and death by 7 months.) This accelerated timing effect seems to be strain-specific, and unlinked to the mnd gene itself. Our current working hypothesis is that the timing effect is due to 2 or 3 unlinked dominant genes with incomplete penetrance at any single locus. In a combined RFLP/PCR fragment genetic analysis, the strongest deviation from the expected ratio of AKR vs B6 alleles occurs with markers on proximal half of chr 1. Additional loci on chrs 5 and 10 may also be involved. The mechanism of interaction of these modifying genes with the primary mnd gene may offer new therapeutic avenues.

Altered gene expression for calpain/calpastatin system in motor neuron degeneration (Mnd) mutant mouse brain and spinal cord.

The calcium-activated neutral proteases (CANP, calpains) have been implicated in both acute and chronic neurodegenerative processes. In the present study, we analyzed the in situ mRNA expression of calpain I and II and their endogenous inhibitor, calpastatin, in the motor neuron degeneration (Mnd) mutant mouse, which exhibits progressive dysfunction of the spinal cord and brain. As the disease progresses, the mutants show increasingly pronounced motor abnormalities which coincide with swelling of the spinal motor neurons, neocortex, hippocampal CA regions and cerebellar Purkinje cells. In situ hybridization studies show that the Mnd mice have a significantly higher level of calpain I, calpain II and calpastatin than the congenic controls in the following brain regions and cell types: hippocampal CA3 region, pyramidal cells, cerebellar Purkinje cells and spinal cord motor neurons. However, no differences in calpain or calpastatin mRNA levels are observed in glial and cerebellar granule cells of Mnd and control mice. Western blots and competitive RT-PCR analyses of brain and spinal cord homogenates are confirmative. Such altered gene expression in specific cell types of brain and spinal cord suggests the involvement of the calpain/calpastatin system.

The maintenance and identification of mouse cerebellar granule cells in monolayer culture.

Methods are described for maintaining postnatal mouse cerebellar cells in monolayer culture, and for identifying granule cells in such cultures. Cells from cerebella of 7-day-old mice are dissociated with trypsin and DNAse, then plated at 1-1.5 X 10(6) cells/35 mm dish in a high-potassium modification of Hams F12 medium plus 10% fetal calf serum. Under these conditions, cells grow either singly or in small clumps, and develop complex meshes of single fibers and fiber bundles over a period of several days. Granule cells are identified by a combination of several criteria including their size, shape and relative proportion of the total cell population as determined by phase contrast and scanning electron microscopy; nuclear morphology, demonstrated by transmission electron microscopy, and failure to take up [3H]gamma-aminobutyric acid (GABA) in the presence of several other cell types which do, shown by autoradiography.

In vitro behavior of granule cells from Staggerer and Weaver mutants of mice.

The survival, in monolayer culture, of cerebellar granule cells from the cerebellar mutants Staggerer and Weaver was studied to examine whether the observed in vivo granule cell degeneration is intrinsic or environmentally induced. Granule cells in vitro can be identified by means of a combination of criteria including their size, shape, nuclear morphology, relative proportion of the total cell population, and failure to take up gamma-aminobutyric acid (GABA) in the presence of several other cell types which do. Cultures from both Staggerer and Weaver cerebella had surviving granule cells for at least 3 weeks in vitro. Therefore, since the mice used as the source of the cells were 7 days of age, the degeneration observed in vivo cannot be a case of irreversibly programmed cell death determined before postnatal day 7. While the behavior of Weaver granule cells is essentially the same as littermate controls, cells from Staggerer cerebella both clump less and survive considerably longer than those from wild-type. The role of intrinsic granule cell differences vs. primary changes in some other cell type is discussed.

Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington's disease transgenic mice.

Huntington's Disease transgenic mice were used for an exploration into the stability of a trinucleotide repeat. The brain shows heterogeneous somatic instability that increases quantitatively with age. To test somatic CAG-repeat alterations during long-term culture, DNA was extracted from transgenic tissue, primary fibroblasts, and SV40-immortalized fibroblasts at intervals of approximately 100 cell doublings. In fibroblasts derived from an adult mouse, there was an initial short truncation of the repeat, followed by an emerging population of cells showing continuous slow expansion. After 15 months in continuous culture (approximately 600 cell doublings following transformation) the major CAG peak has increased from 155 to approximately 170 triplets. This in vitro system can now be used to assay factors that affect instability.

Control of transcription in the RORa-staggerer mutant mouse cerebellum: glutamate receptor delta2 mRNA.

The staggerer (transcription factor RORa-deleted) mutation blocks cerebellar Purkinje cell development shortly after birth. In double mutants, the homozygous staggerer mutation can 'rescue' Purkinje cells carrying a channel-opening mutation in the Glutamate receptor delta2 (Lurcher) from apoptotic death during the third and fourth postnatal weeks. Transcript levels for the glutamate receptor delta2, a channel subunit that is found at both climbing fiber and parallel fiber synapses on cerebellar Purkinje cells, are higher in the staggerer mutant cerebellum than in the wild-type cerebellum at age 14 days. By 21 days, the wild-type level is higher, having increased tremendously while the staggerer increase is modest. The results imply that the mechanism protecting Purkinje cells in staggerer-Lurcher double mutants operates by blocking mutant receptor protein localization, rather than mRNA transcription. Between the ages 10 and 14 days, the climbing fiber innervation of Purkinje cells is known to switch from multiple to single in wild-type, but not in the staggerer mutant. Therefore, the results also suggest that the multiple innervation and the level of the receptor message are coordinated, either directly or indirectly.

Effects of triiodothyronine (T3) on the development of rat cerebellar cells in culture.

To determine whether or not the effects of thyroxine on the cells of the external granular layer of rat cerebellum are direct or indirect, Purkinje cell-free dissociated cell cultures from 5-day-old rat cerebellum in serum-free medium were treated with triiodothyronine (T3) at concentrations of 20-3000 ng/ml. Cultures were assayed for uptake and synthesis of γ-aminobutyric acid (GABA) after 3 weeks, and for thymidine incorporation at 1, 2, 3 and 4 days. Specific (per mg protein) activity of glutamic acid decarboxylase and of GABA uptake into neurons was reduced by the more physiologic (50 or 500 ng/ml) concentrations of T3, probably due to a non-neuronal increase in overall protein synthesis, since the total per culture values seem unaffected by hormone. Thymidine uptake was significantly increased only at the highest (unphysiologic) concentration of T3 on the second day after treatment. None of the label appeared to be in the nuclei of the neuronal cells at any of the times and concentrations tested. These results are consistent with the hypothesis that T3 does not have a direct effect on the proliferation of neurons derived from the external granular layer, although it does seem to affect some non-neuronal cells. T3 also does not seem to enhance differentiated GABA functions (uptake or synthesis) beyond the levels achieved using insulin, progesterone, putrescine, selenium and transferrin.

Timecourse of effects of triiodothyronine on mouse cerebellar cells cultured by two different methods.

Dissociated cells from week-old mouse cerebellum were grown on either polylysine coated coverslips or on uncoated coverslips. Polylysine coated coverslips give rise to cultures containing all of the cerebellar cell types except Purkinje cells. Use of uncoated coverslips gives rise to cultures which are depleted of granule cells because the granule cells are unable to adhere to glass without a substrate present. The uncoated coverslip cultures are therefore enriched in glial and other non-neuronal cells. Effects of triiodothyronine on each type of culture were then examined as a function of time. On coated coverslips hormone treatment caused a noticeable increase in cell clumping at 1 week, and seemed responsible for a leveling off of the decline in total high-affinity uptake of γ-aminobutyric acid, as well as for a small increase in ß-alanine inhibited uptake between 2 and 3 weeks. There was no effect on the overall uptake of thymidine. On uncoated coverslips triiodothyronine treatment significantly increased the thymidine uptake at days 2 and 3, and increased the proportion of Bergmann-like to velate astrocytes at 1 week. There were, however, no significant differences in GABA uptake at any of the time points examined. We conclude that in cerebellar cultures lacking Purkinje cells, triiodothyronine affects both the rate of acquisition and the timecourse of morphological changes (possibly reflecting transformation to more differentiated states) of glial cells but not of neurons. These results are consistent with the hypothesis that, in vivo, thyroxine acts indirectly via Purkinje cells to give developmental signals to neuroblasts and/ or neurons.

Abnormal staggerer cerebellar cell interactions and survival in vitro.

Staggerer cerebellar cells in primary monolayer cell culture exhibit less initial clumping and unexpectedly longer overall survival than their wild-type counterparts when grown in medium containing fetal calf serum. Although polylysine coating of substratum both reduces the amount of initial clumping and increases the survival of normal cells, it does not completely eliminate the difference between mutant and control. Such cultures exhibit the same behavior only when culture conditions are changed to include supplementation with horse serum instead of fetal calf serum in addition to the substrate coating. These results are discussed in terms of the site of action of the mutation.

Tonic/clonic seizures in a mouse mutant carrying the weaver gene.

A seizure disorder, characterized by sporadic tonic/clonic seizures has been identified in a strain of mice carrying the weaver (wv) gene. Breeding studies and a review of the literature indicate that this is either a property of the wv gene or linked to it tightly.