Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.

To elucidate the molecular mechanisms whereby expanded polyglutamine stretches elicit a gain of toxic function, we expressed full-length and truncated DRPLA (dentatorubral-pallidoluysian atrophy) cDNAs with or without expanded CAG repeats in COS-7 cells. We found that truncated DRPLA proteins containing an expanded polyglutamine stretch form filamentous peri- and intranuclear aggregates and undergo apoptosis. The apoptotic cell death was partially suppressed by the transglutaminase inhibitors cystamine and monodansyl cadaverine (but not putrescine), suggesting involvement of a transglutaminase reaction and providing a potential basis for the development of therapeutic measures for CAG-repeat expansion diseases.

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

A novel calpain inhibitor, ((1S)-1((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl) carbamic acid 5-methoxy-3-oxapentyl ester, protects neuronal cells from cerebral ischemia-induced damage in mice.

Cerebral ischemia induces Ca(2+) influx into neuronal cells, and activates several proteases including calpains. Since calpains play important roles in neuronal cell death, calpain inhibitors may have potential as drugs for cerebral infarction. ((1S)-1((((1S)-1-Benzyl-3- cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl) carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945) is a novel calpain inhibitor that has good membrane permeability and water solubility. We evaluated the effect of SNJ-1945 on the focal brain ischemia induced by middle cerebral artery occlusion (MCAO) in mice. Brain damage was evaluated by assessing neurological deficits at 24 h or 72 h after MCAO and also by examining 2,3,5-triphenyltetrazolium chloride (TTC) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of brain sections. When injected at 1 h after MCAO, SNJ-1945 at 30 and 100 mg/kg, i.p. decreased the infarction volume and improved the neurological deficits each assessed at 24 h. SNJ-1945 at 100 mg/kg, i.p. also showed neuroprotective effects at 72 h and reduced the number of TUNEL-positive cells at 24 h. SNJ-1945 was able to prevent neuronal cell death even when it was injected at up to 6 h, but not at 8 h, after MCAO. In addition, SNJ-1945 decreased cleaved alpha-spectrin at 6 h and 12 h, and active caspase-3 at 12 h and 24 h in ischemic brain hemisphere. These findings indicate that SNJ-1945 inhibits the activation of calpain, and offers neuroprotection against the effects of acute cerebral ischemia in mice even when given up to 6 h after MCAO. SNJ-1945 may therefore be a potential drug for stroke.

Neuroprotective effect of erythropoietin, and role of metallothionein-1 and -2, in permanent focal cerebral ischemia.

Metallothioneins (MTs) are small cysteine-rich proteins found widely throughout the mammalian body, including the CNS. MT-1 and -2 protect against reactive oxygen species and free radicals. We investigated the role of MT-1 and -2 using MT-1,-2 knockout (KO) mice. MT-1,-2 KO mice exhibited greater neuronal damage after permanent middle cerebral artery occlusion (MCAO) than wild-type mice. MT-2 mRNA was significantly increased at 6, 12, and 24 h after MCAO in the wild-type mouse brain [as detected by real-time reverse-transcription polymerase chain reaction (RT-PCR)], while MT-1 and MT-3 were decreased at 12 and 24 h. In an immunohistochemical study, MT expression displayed colocalization with glial fibrillary acidic protein (GFAP)-positive cells (astrocytes) in the penumbra area in wild-type mice. Since erythropoietin (EPO) has been reported to induce MT-1 and -2 gene expression in vitro, we examined its effect after permanent MCAO, and explored the possible underlying mechanism by examining MT-1 and -2 induction in vivo. In wild-type mice, EPO significantly reduced both infarct area and volume at 24 h after the ischemic insult. However, in MT-1,-2 KO mice EPO-treatment did not alter infarct volume (vs. vehicle-treatment). In wild-type mice at 6 h after EPO administration, real-time RT-PCR revealed increased MT-1 and -2 mRNA expression in the cerebral cortex (without MCAO). Further, MT-1 and -2 immunoreactivity was increased in the cortex of EPO-treated mice. These findings indicate that MTs are induced, and may be neuroprotective against neuronal damage, after MCAO. Furthermore, EPO is neuroprotective in vivo during permanent MCAO, and this may be at least partly mediated by MTs.

Autoantibodies against glutamate receptor epsilon2-subunit detected in a subgroup of patients with reversible autoimmune limbic encephalitis.

We investigated the presence of autoantibodies against glutamate receptor (GluR) epsilon2 in serum and cerebrospinal fluid (CSF) samples from 12 consecutive patients with acute encephalitis/encephalopathy by immunoblotting using recombinant GluR epsilon2 as antigen. In 4 patients, IgM autoantibodies against GluR epsilon2 were detected in CSF in the early phase of the disease but were not detectable after several months. Seizures and psychiatric symptoms were noted during the acute phase of the disease in these 4 patients, who showed various degrees of residual amnesia. Immunotherapy was performed on 3 patients (patients 1, 3 and 4), and they showed marked improvements. Immunohistochemistry using these patients' sera showed that immunoreactivity is specifically detected in the cytoplasm of rat hippocampal and cortical neurons. The clinical features and neuroimaging findings of patients with IgM autoantibodies against GluR epsilon2 in CSF resemble those of patients with reversible autoimmune limbic encephalitis.

Cilostazol reduces ischemic brain damage partly by inducing metallothionein-1 and -2.

The neuroprotective effect of cilostazol, an antiplatelet drug, was examined after 24 h permanent middle cerebral artery (MCA) occlusion in mice, and explored the possible underlying mechanism by examining metallothionein (MT)-1 and -2 induction in vivo. Cilostazol (30 mg/kg) was intraperitoneally administered at 12 h before, 1 h before, and just after MCA occlusion. Mice were euthanized at 24 h after the occlusion, and the neuronal damage was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cilostazol significantly reduced the infarct area and volume, especially in the cortex. Real-time RT-PCR revealed increased mRNA expressions for MT-1 and -2 in the cortex of normal brains at 6 h after cilostazol treatment without MCA occlusion. MT-1 and -2 immunoreactivity was also increased in the cortex of such mice, and this immunoreactivity was observed in the ischemic hemisphere at 24 h after MCA occlusion (without cilostazol treatment). The strongest MT-1 and -2 immunoreactivity was detected in MCA-occlused mice treated with cilostazol [in the peri-infarct zone of the cortex (penumbral zone)]. These findings indicate that cilostazol has neuroprotective effects in vivo against permanent focal cerebral ischemia, especially in the penumbral zone in the cortex, and that MT-1 and -2 may be partly responsible for these neuroprotective effects.

Linkage mapping of the gene for Charcot-Marie-Tooth disease type 2 to chromosome 1p (CMT2A) and the clinical features of CMT2A.

Charcot-Marie-Tooth disease type 2 (CMT2) is characterized by a motor conduction velocity of the median nerve of > 38 m/sec and is a genetically heterogeneous disorder with at least three loci identified: CMT2A (1p35-36), CMT2B (3q13-22), CMT2C (not linked to any known loci), and CMT2D (7p14). In this study, we performed linkage analysis of two Japanese CMT2 families using markers flanking the CMT2A, CMT2B, and CMT2D loci. The highest cumulative multipoint lod score of 3.69 was obtained at D1S244. The CMT2B and CMT2D loci were excluded by the results of linkage analysis performed using markers D3S1551, D3S1290, and D7S484. The clinical features of the CMT2A affecting the two families include similar levels of muscle weakness of the posterior and anterior tibial muscles, tendon reflexes preserved in upper extremities but reduced or absent in lower extremities, no enlargement of the peripheral nerves, and mild sensory disturbance in only 20% of affected individuals.

Uniform tissue distribution of tRNA(Lys) mutation in mitochondrial DNA in MERRF patients.

We documented the presence of a point mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA) in various postmortem tissues from two patients with myoclonus epilepsy associated with ragged-red fibers (MERRF). The percentages of the mutant mtDNA were similar (93 to 99%) in both clinically affected and unaffected tissues, suggesting that preferential clinical involvement of certain tissues in MERRF is based not only on the variation of distribution of the mutant mtDNA, but also on other factors such as differences in the threshold in various CNS regions and organs.

Shared carbohydrate antigenic determinant between the myelin-associated glycoprotein (MAG) and lung cancers. An immunohistochemical study by an anti-MAG IgM monoclonal antibody.

An immunochemical study has shown that monoclonal anti-myelin-associated glycoprotein (MAG) antibodies identify some membrane glycoproteins from cell lines of small cell lung cancer. We investigated immunohistochemically 85 specimens of lung cancer from resection and autopsy using one of the monoclonal antibodies against MAG. One adenocarcinoma was highly reactive with the anti-MAG antibody, and in three small cell carcinomas immunostained tumor cells were distributed either singly or in occasional small clusters. These results show that a shared antigenic determinant with MAG appears on the tumor cells. MAG is believed to play the role of antigen in the development of carcinomatous polyneuropathy, and contributes to cancer development by its reduction of natural killer (NK) cell activity.

Expression of growth inhibitory factor (GIF) in normal and injured rat brains.

Immunohistochemical study on growth inhibitory factor (GIF) in rat brain has revealed that a glial cell layer on the surface of cerebral cortex and the cells surrounding Purkinje cells has been reported. In addition, neurons in gray matter were weakly immunostained for GIF. In situ hybridization using digoxigenin-labeled single-strand RNA probes also demonstrated that most of the neurons and small round cells, which were presumably astrocytes, expressed GIF mRNA in the cerebral cortex of rat brain. These findings indicate that GIF is produced in neurons as well as in astrocytes. The most prominent findings in this study are, a very strong reaction of GIF and GIF mRNA in the reactive astrocytes around the site of injury induced by stab wound or kainic acid injection. These results raised the possibility that GIF may act as an acute-phase protein in reactive astrocytes and have a role in tissue repair.

Biochemical and immunocytochemical changes in glial fibrillary acidic protein after stab wounds.

Changes of glial fibrillary acidic protein (GFAP) in the forebrain of rats with stab wounds were determined by quantitative immunoblots and by immunohistochemistry. Bilateral stab wounds were made stereotaxically in the cortex and hippocampus. In control rats, the scalp was retracted and depressions were etched on the intact skull. At various times up to 21 days postoperation, one cerebral hemisphere was homogenized, proteins were separated by polyacrylamide gel electrophoresis and immunoblots were quantitated by densitometry. The contralateral hemisphere was immunostained for GFAP. Three hours postoperation GFAP+ cells were detected around the wound but there was no increase of total GFAP. At 6 h postoperation total GFAP in the forebrain decreased to 80% of the sham-operated control value and the number of GFAP+ cells was lower, compared to the controls, in layer 1 of the cortex, corpus callosum, cingulum, external capsule, internal capsule, hippocampus, optic tracts and around blood vessels. This early relative decrease in GFAP levels was actually due to an increase in GFAP in the sham-operated controls, which mounted a stronger gliotic response during the first 24 h. In neither group of animals did the GFAP levels drops below those of intact unoperated animals. At 24 h total GFAP began to increase. The number and intensity of reactive glia in the vicinity of the wound increased steadily, appearing to reach a maximum at about 7 days, then declining significantly by 21 days. The glial reaction was most pronounced in the hippocampus. Total GFAP reached 180% of the control value by 7 days and then declined to 117% by 21 days.(ABSTRACT TRUNCATED AT 250 WORDS)

GFAP mRNA levels following stab wounds in rat brain.

We previously reported that glial fibrillary acidic protein (GFAP) levels increased significantly at 3 days after stab wounds, relative to sham-operated controls, reaching a maximum of 200% of control value at 5-7 days. They then fell to near-normal values by 21 days. To determine whether these protein changes correlated with changes in GFAP mRNA we performed Northern blot analyses. Total RNA, isolated from lesioned, sham-operated and intact rat forebrains, was hybridized with 32P-labeled mouse GFAP cDNA and quantified by densitometry. The maximum increase in total RNA content in lesioned animals was only 20% over controls at 12 h. GFAP mRNA levels increased to 2-fold control values at 6 h and reached 5-fold at 12 h. Thereafter they remained at 3.5- to 6-fold until 5 days and then declined to 1.5-fold by 21 days. The rapid increase of GFAP message at 12 h preceded a significant increase in GFAP by 2 days and the decrease of message after 5 days was more precipitate than the slow decrease in GFAP content. Sham-operated animals showed no significant changes in GFAP mRNA, compared to intact controls, during the period 3 h to 14 days postoperation. GFAP mRNA and GFAP in the stab-wound model reached levels similar to those found in the experimental autoimmune encephalomyelitis (EAE) model, but returned to normal much more rapidly.

Patterns of growth inhibitory factor (GIF) and glial fibrillary acidic protein relative level changes differ following left middle cerebral artery occlusion in rats.

Growth inhibitory factor (GIF) has been identified as a new metallothionein-like protein, the level of which is decreased in the Alzheimer's disease brain. GIF and glial fibrillary acidic protein (GFAP) have been reported to be expressed in reactive astrocytes in the rat brain following stab wounds. Moreover, strong expression of GIF mRNA in reactive astrocytes after ventricular injection of kainic acid has been demonstrated. To clarify the biological functions of GIF and GFAP in repair of the CNS, we examined changes in their relative levels to sham control using a Western blotting technique in the rat left hemisphere following occlusion of the left middle cerebral artery, for 28 days after surgery. The GIF relative level declined to 56% of the sham-operated control value on day 7. Thereafter the GIF relative level increased and returned to the normal relative level by days 21-28. The GFAP relative level increased from day 3 and reached a maximum of 120% of the sham-operated control value on days 14-21. While GIF and GFAP were both detected in reactive astrocytes, an increase in the GFAP relative level occurred prior to an increase in GIF relative level following the ischemia. The patterns of changes in relative expression levels of GIF and GFAP were quite similar to those in our previous studies on effects of cerebral stab wounds in rats, although the changes were more rapid in the previous studies. GIF and GFAP appear to play different roles in the repair of the CNS. The present results also indicated that GIF could play an important role in CNS repair after cerebral ischemia and provide new insights into the mechanism of gliosis investigated mainly from the viewpoint of GFAP.

Subcellular localization of growth inhibitory factor in rat brain: light and electron microscopic immunohistochemical studies.

The subcellular localization of growth inhibitory factor (GIF), a brain-specific member of the metallothionein family, was determined in the rat brain by electron microscopic immunohistochemistry using a rabbit antiserum against a synthetic polypeptide specific for rat GIF. The major cell type that expressed a high level of GIF immunoreactivity was the astrocytes. In these cells, dense labelling was observed throughout the soma and the fine processes, in association with the free ribosomes, rough endoplasmic reticulum, small vesicles, the outer membrane of the mitochondria and part of the plasma membrane. Astrocytic end-feet around blood vessels exhibited intense immunoreactivity. Another cell type exhibiting GIF immunolabelling was the neurons. However, this immunoreactivity was restricted to a subset of the neuronal population, and in contrast to the astrocytic pattern, the labelling was localized predominantly in the processes including axons and dendrites, in association with microtubules, ribosomes, the outer membrane of the mitochondria and the plasmalemma. Synaptic elements, including dendritic spines, also showed definite immunoreactivity in association with synaptic vesicles and post-synaptic densities. No labelling was observed in the oligodendrocytes or microglia. The present data suggest that GIF is expressed in both astrocytes and neurons, and plays rather specific roles in each phenotype.

Immunoreactivity of growth inhibitory factor in normal rat brain and after stab wounds--an immunocytochemical study using confocal laser scan microscope.

The growth inhibitor factor (GIF) is a new member of the metallothionein family that is downregulated in Alzheimer's disease brain. Using a confocal laser scan microscope with polyclonal and monoclonal antibodies to GIF, and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and MAP-2, we demonstrated that GIF immunoreactivity was expressed primarily in astrocytes and much less in neurons. In astrocytes of normal rat brain GIF immunoreactivity was detected mainly in the cell bodies, while GFAP immunoreactivity was detected mainly in the processes. GIF immunoreactivity was more strongly expressed in reactive astrocytes. These findings were confirmed with both polyclonal and monoclonal antibodies. Following stab wounds, a number of GIF-positive reactive astrocytes were detected around the wounds at 3 days postoperation. After 7 days GIF immunoreactivity was detected in cell bodies and processes of reactive astrocytes. The number of GIF-positive astrocytes and the intensity of the immunoreactivity remained elevated over the control levels at least through 28 days. These immunocytochemical findings correlated well with changes in GIF protein and mRNA levels. Not only changes in GIF protein and mRNA levels but also intracellular localization of GIF in normal rat brain and after stab wounds in rat brain were different from those of GFAP. These results support the concept that GIF plays an important role in the processing of reconstruction after brain damage.

Changes of growth inhibitory factor after stab wounds in rat brain.

The growth inhibitory factor (GIF) is a new metallothionein (MT)-like protein that is downregulated in Alzheimer's disease (AD) brain. The biological function of GIF has not been fully clarified yet. We have raised an antibody to the synthetic polypeptide that is specific for rat GIF. The purified antibody reacted to recombinant GIF and native rat GIF but not to MT or maltose-binding protein. Using the antibody and GIF cDNA probe, we investigated changes of GIF and GIF mRNA by Western and Northern blotting techniques in rat brains after stab wounds. The levels of GIF and GIF mRNA began to increase 4 days postoperation, reached a maximum at 14-21 days and sustained the increased level at least through 28 days. While both glial fibrillary acidic protein (GFAP) and GIF were recognized in astrocytes, the increases of these 2 proteins after stab wounds showed different patterns. The results indicated that GIF could play an important role in the repair after brain damage and also produce new insights into the mechanism of gliosis investigated mainly from the viewpoint of GFAP.

Administration of prosaposin ameliorates spatial learning disturbance and reduces cavity formation following stab wounds in rat brain.

The effectiveness of prosaposin as a neurotrophic factor was investigated using rats with bilateral stab wounds, injecting 240 ng per day of prosaposin for 3 days. In Morris water maze task, after 3 weeks postoperation, the stab-wounds rats show significant impairment in acquisition compared with the sham-operated rats. In the transfer test the mean number of crossings of the platform place in stab-wounds was significantly lower than that in sham-operated rats (P < 0.01). The stab-wounds rats treated with prosaposin showed significant improvement (P < 0.05). The cavities following stab wounds in the rats treated with prosaposin were significantly smaller than those in the rats treated with (P < 0.05). Our data support that prosaposin is likely to be a new agent for brain injury.

An immunologic abnormality common to Bickerstaff's brain stem encephalitis and Fisher's syndrome.

The nosological position of Bickerstaff's brain stem encephalitis (BBE) has yet to be established, and its etiology is not clear. Because anti-GQ1b antibody frequently occurs in patients with Fisher's syndrome (FS) and there are clinical similarities between FS and BBE, we investigated anti-ganglioside antibodies in sera from 3 BBE patients who had transient long tract signs in addition to acute ophthalmoplegia and cerebellar-like ataxia in order to clarify the etiology and nosological position of BBE. High IgG anti-GQ1b antibody titers were present in all 3 sera samples but decreased with the clinical course of the illness. In contrast, no anti-GQ1b antibody was found in sera from patients with other neurologic diseases which were able to produce transient brain stem disturbance: multiple sclerosis, neuro-Behçet's disease, brain stem infarction, herpes simplex virus encephalitis, and Wernicke's encephalopathy. The finding that BBE and FS shared common autoantibody suggests that autoimmune mechanism common to FS is likely in BBE, and that both conditions represent a distinct disease with a wide spectrum of symptoms that include ophthalmoplegia and ataxia.

Accumulation of glycosphingolipids in spinal and sympathetic ganglia of a symptomatic heterozygote of Fabry's disease.

Fabry's disease is an X-linked disorder of glycolipid catabolism. We have found a symptomatic heterozygous female with cardiomyopathy and severe pain in the extremities. We studied histochemically and biochemically the accumulated glycolipids in spinal and sympathetic ganglia of the patient. Histochemical examination demonstrated the marked glycolipid deposits that have been observed in heterozygous males in these ganglia. Gas-liquid chromatography (GLC) revealed that these accumulated glycolipids were characterized as globotriaosylceramide (Gb3cer) and galabiosylceramide (Ga2cer). In the heterozygous female, the accumulations of Gb3cer in spinal and sympathetic ganglia were, respectively, 34 and 48 times the amount in normal controls. This is the first report on quantitative and qualitative analyses of the accumulated glycolipids in spinal and sympathetic ganglia of a heterozygous carrier female.

A novel monoclonal antibody which reacts with a high molecular weight neuronal cytoplasmic protein and myelin basic protein (MBP) in a patient with macroglobulinemia.

We report on the case of a 70-year-old man with primary macroglobulinemia who showed cranial polyneuropathy and extensive radiculoneuropathy. His serum contained an IgM lambda monoclonal antibody which reacted with both a high molecular weight protein in grey matter and purified myelin basic protein (MBP) on immunoblotting. In an immunohistochemical study, strong immunoreactivity was detected only in the cytoplasm of neurons and weak immunoreactivity was detected in myelin. These findings suggest that this antibody may be related to the pathogenesis of neuronal damage in patients with macroglobulinemia.