Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat which codes for glutamine in the protein ataxin-1. We have investigated the effect of this expansion on ataxin-1 by immunoblot analysis. The wild-type protein is detected in both normal and affected individuals; however, a mutant protein which varies in its migration properties according to the size of the CAG repeat is detected in cultured cells and tissues from SCA1 individuals. The protein has a nuclear localization in all normal and SCA1 brain regions examined but a cytoplasmic localization of ataxin-1 was also observed in cerebellar Purkinje cells. Our data show that in SCA1, the expanded alleles are faithfully translated into proteins of apparently normal stability and distribution.

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.

Polyglutamine expansion down-regulates specific neuronal genes before pathologic changes in SCA1.

The expansion of an unstable CAG repeat causes spinocerebellar ataxia type 1 (SCA1) and several other neurodegenerative diseases. How polyglutamine expansions render the resulting proteins toxic to neurons, however, remains elusive. Hypothesizing that long polyglutamine tracts alter gene expression, we found certain neuronal genes involved in signal transduction and calcium homeostasis sequentially downregulated in SCA1 mice. These genes were abundant in Purkinje cells, the primary site of SCA1 pathogenesis; moreover, their downregulation was mediated by expanded ataxin-1 and occurred before detectable pathology. Similar downregulation occurred in SCA1 human tissues. Altered gene expression may be the earliest mediator of polyglutamine toxicity.

Decreased dendritic branching in frontal, motor and limbic cortex in Rett syndrome compared with trisomy 21.

The branching of dendrites of pyramidal neurons in premotor frontal, motor and limbic cortex have been identified by us using Golgi technique to be less in Rett Syndrome (RS) brains than in non-Rett control brains. Decreased dendritic branching per se is not pathognomonic of a particular condition and has been reported in numerous disorders associated with mental retardation. This study was designed to test whether the dendritic alterations in Rett Syndrome are the same or different from the alterations present in Down Syndrome (DS), 1 specific form of mental retardation. Sections from Brodmann's areas 6, 4, 20, 43, 28, and 17 of premotor frontal, motor cortex, inferior temporal gyrus, hippocampal formation and the striate cortex from 16 Rett brains, 9 non-Rett brains and 9 Down's brains were prepared for dendrite analysis using the rapid Golgi technique. Drawings of apical and basilar dendrites of pyramidal neurons from 2 cortical layers and Cal were submitted to Sholl analysis. The analyses of Rett brains were compared with the analyses of the Trisomy 21 brains using the repeated measures analysis of covariance, with age as a covariate. The studies demonstrate in our sample that basal dendrites of layer III and V of frontal, layer IV of subiculum, and layer V of motor cortex and apical dendrites of layer III of frontal cortex have a significantly reduced dendritic arborization in RS compared with Trisomy 21. This study suggests that the cortical distribution of the dendritic alterations is specific for Rett Syndrome, and that the premotor frontal, motor and subicular cortex are preferentially involved in the, as yet, undefined process which affects brain growth and function in RS.

Selective dendritic alterations in the cortex of Rett syndrome.

Rett syndrome, the commonest condition associated with severe mental retardation in girls, is diagnosed only by its clinical phenotype, because, to date, there is no consistent characteristic alteration in genetic, biochemical, neurotransmitter or morphologic marker. The clinical features at various ages suggest involvement of most parts of the nervous system, however, the brain in Rett syndrome is reduced in weight, without other obvious morphologic alterations. Because of the relative microcephaly, hypotheses regarding failure of development have been suggested. Supporting such hypotheses are the quantitative studies by Jellinger, Seitelberger and Kitt defining a decrease in the amount of melanin in the substantia nigra and by Bauman defining a global decrease in the size of the neurons. In this study the cerebral cortex has been examined using the rapid Golgi technique with the purpose of investigating dendrites of pyramidal neurons in six cortical regions of Rett girls from ages 2.9-35 years. Camera lucida drawings of apical and basal dendrites of two cortical layers and CA1 were prepared. These were submitted to the Sholl analysis. The Sholl analyses were tested for significance using the repeated measures analysis of covariance, with age as a covariate. The studies demonstrate that from our samples there is no evidence that the pyramidal neurons in Rett syndrome degenerate progressively with increasing age but that the basal dendrites of layers three and five pyramidal neurons in the motor and frontal cortex, the apical dendrites of layer five of the motor cortex, and the basal dendrites of layer four of the subiculum are significantly shorter than in non-Rett brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Predominant localization of the LIS family of gene products to Cajal-Retzius cells and ventricular neuroepithelium in the developing human cortex.

Mutations that perturb neuronal migration provide important biological clues that can lead to an understanding of the role of specific cells and molecules in the formation of the cortex. The human neuronal migration disorder, Miller-Dieker Lissencephaly, results from a hemideletion of LIS-1, which encodes a subunit of a brain platelet-activating factor acetylhydrolase. The cellular localization of the LIS-1 gene product in human fetal brain and its normal role in neuronal migration have yet to be determined. LIS-1 belongs to a family of genes that have identical coding sequences (LIS-1 [chromosome 17] and LIS-2 [chromosome 2]). In the brain, LIS-1 is the more abundant gene as determined by Northern blot analysis. Using antibodies raised against 2 epitopes of the LIS-1/LIS-2 protein sequence, we have localized the LIS family of gene products in the developing human brain to the Cajal-Retzius cells, some subplate neurons, thalamic neurons, the ventricular neuroepithelium, and at later gestational ages, to the ependyma. Therefore, LIS-1 bears some resemblance to reelin, the gene product involved in the cortical mouse mutant reeler, in that Cajal-Retzius cells demonstrate immunolocalization. However, unlike reelin, LIS proteins are expressed not only in the Cajal Retzius cells, but also in the ventricular neuroepithelium, suggesting a potential role for this structure in neuronal migration.

Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy.

Studies were undertaken to examine the effects recurrent early-life seizures have on the ability of rats to acquire spatial memories in adulthood. A minute quantity of tetanus toxin was injected unilaterally into the hippocampus on postnatal day 10. Within 48 h, rats developed recurrent seizures that persisted for 1 week. Between postnatal days 57 and 61, rats were trained in a Morris water maze. Toxin-injected rats were markedly deficient in learning this task. While these rats showed gradual improvement in escape latencies over 20 trials, their performance always lagged behind that of controls. Poor performance could not be explained by motor impairments or motivational difficulties since swimming speed was similar for the groups. Only eight of 16 toxin-injected animals showed focal interictal spikes in the hippocampus during electroencephalographic recordings. This suggests that learning deficiencies and chronic epilepsy may be independent products of recurrent early-life seizures. A quantitative analysis of hippocampus revealed a significant decrease in neuronal density in stratum pyramidale of experimental rats. However, the differences were largely explained by a concomitant increase in the area of stratum pyramidale. Studies of glial fibrillary acidic protein expression and spread of horseradish peroxidase-conjugated tetanus toxin in the hippocampus suggest that the dispersion of cell bodies in stratum pyramidale can neither be explained by a reactive gliosis nor the direct action of the toxin itself. Taken together, we suggest that recurrent seizures beginning in early life can lead to a significant deficiency in spatial learning without ongoing hippocampal synchronized network discharging or a substantial loss of hippocampal pyramidal cells.

A chronic focal epilepsy with mossy fiber sprouting follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infant rats.

Studies were conducted to characterize a chronic epileptic condition that follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infancy. Wistar rat pups received a single injection of tetanus toxin in the right CA3 region on postnatal day 10. Animals were monitored for epileptiform activity by video electroencephalographic or visual observation during the following three to five days. Repeat evaluation six months later demonstrated interictal discharges in 79% (11 of 14) and electrographic seizures in 42% (six of 14) of adult rats with tetanus toxin-induced seizures in infancy. Five of the animals had interictal activity which occurred focally in either the left (n = 2) or right (n = 3) hippocampus. One animal had focal interictal activity independently in these regions and in the left and right cortical regions. The remaining five animals had interictal activity in the hippocampus and synchronously in the ipsilateral cortex or the contralateral hippocampus. Electrographic seizures were focal (nine of 14) or bilateral (five of 14) in onset. The behaviors that accompanied these seizures were quite variable. Clonic face and forelimb movements were observed in some animals. However, a significant portion of rats had electrographic seizures with no associated behavioral change. Timm staining was performed on hippocampal sections from experimental and control animals. There was a significantly greater Timm score (aberrant Timm granules) in the inner molecular layer of the dentate gyrus in tetanus toxin-treated rats than in control rats. Our findings suggest that intrahippocampal tetanus toxin injection in infant rats results in a chronic focal epilepsy that persists for at least six months and is associated with aberrant mossy fiber sprouting in the dentate gyrus. The model described here contributes significantly to the evidence for chronic effects of recurrent seizures in early life, and provides a model for investigation of the molecular and cellular events that contribute to the development of chronic epilepsy.

Intracoccygeal and pericoccygeal glomus bodies and their relationship to coccygodynia.

BACKGROUND: Coccygodynia is an uncommon condition of diverse causes. A few cases were attributed to so-called pericoccygeal glomus tumors. However, the pericoccygeal soft tissues normally contain numerous small glomus bodies and a larger one known as the glomus coccygeum, which can reach several millimeters in diameter. Most reported cases of alleged pericoccygeal glomus tumors represent normal, incidentally discovered coccygeal glomus bodies. Recently, an intracoccygeal glomus tumor was reported as a cause of coccygodynia. However, we suspected that glomera can also occur normally within the coccyx itself. METHODS: Twenty coccyges from fetuses, newborns, infants, and adults were obtained at autopsy, embedded in toto, and examined histologically in step sections. RESULTS: Intracoccygeal glomera were present in six of the nine pediatric specimens and all 11 adult specimens. All were microscopic structures, and none appeared to have caused bony destruction or erosion. They did not differ from the structures previously reported as alleged intracoccygeal "glomus tumors." CONCLUSIONS: Pericoccygeal and intracoccygeal glomus bodies are normal findings in humans at all ages. They should not be mistaken for tumors, and their role in the pathogenesis of coccygodynia is questionable.

Substance P immunoreactivity in the enteric nervous system in Rett syndrome.

Rett syndrome is associated with profound mental retardation and motor disability in girls. It has a characteristic clinical phenotype which includes abnormalities of the autonomic nervous system. Feeding impairment and severe constipation are two symptoms of this autonomic dysfunction. Substance P, an important peptide in the autonomic nervous system, is decreased in the cerebrospinal fluid of Rett syndrome. We have demonstrated that substance P immunoreactivity is significantly decreased in Rett syndrome brain-stem and may be related to the autonomic dysfunction. In this study, we have continued the investigation of substance P in the enteric nervous system. We immunohistochemically examined the normal developing bowel in 22 controls (ages, 14 gestational weeks to 31 years) using formalin fixed tissue, with antibodies to substance P, tyrosine hydroxylase and vasoactive intestinal peptide. We compared the immunoreactivity of normal controls with 14 cases of Rett syndrome (ages, 5-41 years) and observed that the expression of substance P, tyrosine hydroxylase and vasoactive intestinal peptide immunoreactivity in the bowel in Rett syndrome was not significantly different from that of controls. This suggests that the feeding impairment and constipation in Rett syndrome relate to dysfunction of the autonomic nervous system originating outside of the bowel, in the brain-stem, as suggested by our previous study.

Substance P immunoreactivity in Rett syndrome.

Severe autonomic dysfunction occurs in Rett syndrome (RS). Substance P, a tachykinin peptide that localizes to several brain regions, including the autonomic nervous system, is reduced in the cerebrospinal fluid of patients with RS. The anatomic localization and intensity of substance P immunoreactivity and glial fibrillary acidic protein-positive astrocytes in the brains of 14 patients with RS were compared with those in the brains of 10 age-matched normal patients. Substance P immunoreactivity expression was significantly decreased in RS tissue compared with control tissue in the following regions: dorsal horns, intermediolateral column of the spinal cord, spinal trigeminal tract, solitary tract and nucleus, parvocellular and pontine reticular nuclei, and locus ceruleus. A less significant decrease of substance P immunoreactivity occurred in the substantia nigra, central gray of the midbrain, frontal cortex, caudate, putamen, globus pallidus, and thalamus. Antiglial fibrillary acidic protein-positive astrocytes were increased in the areas in which substance P immunoreactivity was decreased and in other brain regions. Because many of the brain regions with the greatest decrease in substance P immunoreactivity are involved in the control of the autonomic nervous system, especially the solitary tracts and reticular formation, reduced substance P may contribute to the autonomic dysfunction in RS.

Differential expression of survivin splice isoforms in medulloblastomas.

Survivin, a member of the inhibitor of apoptosis protein family, is implicated in the dysregulation of apoptosis in human cancers. Survivin and survivin-deltaEx3, one of its two alternatively spliced isoforms, confer anti-apoptotic activities in human tumours, while survivin-2B antagonizes such anti-apoptotic properties. The current study was undertaken to examine the mRNA expression of survivin isoforms and their correlation with clinical staging and outcome in 20 medulloblastoma (MB) tumours, three MB cell lines and normal brain tissues (a foetal and an adult cerebellum) by densitometry scanning of 32p-dCTP incorporated reverse transcription polymerase chain reaction (RT-PCR) products and quantitative real-time PCR. Our results showed that the normal adult brain only expressed low levels of survivin-deltaEx3 mRNA, while the foetal brain expressed all three isoforms, with wild-type survivin as the dominant transcript. All three survivin isoforms were detected in all the MB cell lines and tumours analysed. Immunohistochemical staining also demonstrated survivin protein expressions in all five paraffin-embedded MBs, with predominant nuclear localization. Although overexpressions of survivin were not associated with the presence of metastatic MB or tumour histological subtypes, elevated expressions of survivin-deltaEx3 were significantly associated with progressive/recurrent tumours (P-value = 0.024). Our data demonstrated that overexpression of survivin mRNA is a common feature in MBs, may contribute to their anti-apoptosis properties and clinical behaviours, and predicts a poor clinical outcome, independent of clinical staging or tumour histology.

Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.

Many neurodegenerative diseases are caused by gain-of-function mechanisms in which the disease-causing protein is altered, becomes toxic to the cell, and aggregates. Among these 'proteinopathies' are Alzheimer's and Parkinson's disease, prion disorders and polyglutamine diseases. Members of this latter group, also known as triplet repeat diseases, are caused by the expansion of unstable CAG repeats coding for glutamine within the respective proteins. Spinocerebellar ataxia type 1 (SCA1) is one such disease, characterized by loss of motor coordination due to the degeneration of cerebellar Purkinje cells and brain stem neurons. In SCA1 and several other polyglutamine diseases, the expanded protein aggregates into nuclear inclusions (NIs). Because these NIs accumulate molecular chaperones, ubiquitin and proteasomal subunits--all components of the cellular protein re-folding and degradation machinery--we hypothesized that protein misfolding and impaired protein clearance might underlie the pathogenesis of polyglutamine diseases. Over-expressing specific chaperones reduces protein aggregation in transfected cells and suppresses neurodegeneration in invertebrate animal models of polyglutamine disorders. To determine whether enhancing chaperone activity could mitigate the phenotype in a mammalian model, we crossbred SCA1 mice with mice over-expressing a molecular chaperone (inducible HSP70 or iHSP70). We found that high levels of HSP70 did indeed afford protection against neurodegeneration.