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Alzheimer's disease (AD) is characterized by the extensive deposition of amyloid beta protein (Abeta) in the brain cortex. Abeta is produced from beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. beta-Secretase has been identified as beta-site APP cleaving enzyme1 (BACE1). We produced rabbit polyclonal antibodies against the amino and the carboxyl terminals of BACE1. Using these antibodies, BACE1 was characterized in temporal lobe cortices by Western blotting and immunohistochemistry. Immunohistochemical studies employing anti-GFAP and anti-MAP2 antibodies as well as anti-BACE1 antibodies showed that BACE1 was expressed exclusively in neurons but not in glial cells. Brain samples were directly extracted by 0.5% SDS and analyzed by Western blotting and densitometer. Although the mean level of BACE1/mg protein in AD brains was not increased, the ratio of BACE1 to MAP2 or to NSE was significantly increased compared with that in control brains. Taken together, these findings suggest that those neurons that survive in AD brains might generate more BACE1 than normal neurons in control brains, indicating that increased BACE1 activity could be one of the causes of AD. This could justify the development of anti-BACE1 drugs for AD treatment.

Protein misfolding is considered a key event in the pathogenesis of polyglutamine disease such as Machado-Joseph disease (MJD). Overexpression of chaperone proteins and the application of chemical chaperones are reported to suppress polyglutamine induced cytotoxicity in vitro and in vivo. The effects of compatible solutes, which are osmoprotectants in bacteria and possess the action in stabilizing proteins under stress, have not, to our knowledge, been studied. We explored the protective effects of the compatible solutes ectoine, hydroxyectoine, and betaine on apoptotic cell death produced by the truncated MJD gene product with an expanded polyglutamine tract in cultured neuro2a cells. Ectoine, but not hydroxyectoine or betaine, decreased large cytoplasmic inclusions and increased the frequency of nuclear inclusions. Immunoblot analysis showed that ectoine reduced the total amount of aggregates. Despite the presence of nuclear inclusions, apoptotic features were less frequently observed after ectoine application. Our findings suggest that ectoine, a natural osmoprotectant in bacteria, may function as a novel molecule protecting cells from polyglutamine-induced toxicity.

Coordinated development of excitatory and inhibitory synapses is crucial for normal function of neuronal circuits. Using homo- and heterochronic cultures of hippocampal neurons, we compared the formation of glutamatergic and GABAergic synapses at different stages and asked whether the age of dendrites affects their ability to accept new glutamatergic and GABAergic synapses. Neurons were transfected with either CFP-actin as a dendritic marker or GFP-synaptophysin as a presynaptic marker. We found that GFP-synaptophysin clusters formed on CFP-actin-labeled dendrites at similar density regardless of pre- and postsynaptic cell type or the age of dendrites (0-2 weeks) upon co-culturing. Therefore, the age of mature dendrites does not affect their ability to accept new synapses. Because GABAergic transmission switches from depolarizing to hyperpolarizing during 1-2 weeks in these cultures, our observations also suggest that this developmental switch does not alter the formation of GABAergic synapses.

In order to clarify the clinical and genetic features of SCA6, we retrospectively analyzed 140 patients. We observed an inverse correlation between the age of onset and the length of the expanded allele, and also between the age of onset and the sum of CAG repeats in the normal and the expanded alleles. The ages of onset of four homozygous patients correlated better with the sum of CAG repeats in both alleles rather than with the expanded allele calculated from heterozygous SCA6 subjects. Clinically, unsteadiness of gait was the main initial symptom, followed by vertigo and oscillopsia, and cerebellar signs were detected in nearly 100% of the patients. In contrast, extracerebellar signs were relatively mild and infrequent. The results of neuro-otological examination performed in 22 patients suggested the purely cerebellar abnormalities of ocular movements in nature. There was a close relationship between downbeat positioning nystagmus (DPN) and positioning vertigo, which became more common in the later stage. We conclude that total number of CAG repeat-units in both alleles is a good parameter for assessment of age of onset in SCA6 including homozygous patients. In addition, clinical and neuro-otological examinations suggested that SCA6 is a disease with predominantly cerebellar dysfunction.

The pons is one of the brain areas demonstrating selective degeneration in Machado–Joseph disease (MJD), which is caused by the expansion of a polyglutamine stretch in the protein called ataxin-3. Although the resultant pontine atrophy is readily recognized by magnetic resonance imaging (MRI), the features and natural process of atrophy are not fully understood. To characterize them, we analyzed the midsagittal images of the pons obtained by MRI. We found a difference in atrophy between the pontine base and tegmentum. The reduced size of the pontine tegmentum was prominent early after the onset of clinical symptoms. No overlap was seen in the range of the area of pontine tegmentum between MJD and controls. The quotient of atrophy of the pontine tegmentum divided by age correlated well with the CAG repeat number. In contrast, the area of the pontine base correlated negatively with disease duration. Particularly, the size of the pontine base remained in the range of controls for a relatively long time after the onset of symptoms. These results suggest that the atrophic process is not uniform in the pons in MJD and that the different patterns of atrophy may be derived from the differential vulnerability in pontine structures.

The neurotoxicity of amyloid beta (Abeta) is widely believed to play a seminal role in neurodegeneration in Alzheimer's disease. We examined the effect of Chrysamine G (CG) on such neurotoxicity using the specific measurement of surviving neurons. CG was found to reduce the neurodegeneration induced by both the active short fragment of Abeta(25-35) and full-sized Abeta(1-40). In this study, we synthesized a new chemical compound from a monovalent structure of CG (hCG), with a lower affinity for Abeta, and compared its activity with that of CG. Both CG and hCG were found to be equally efficacious in reducing Abeta-induced neuronal death at a concentration of 0.1-1 microM, indicating that the mechanism of action for CG was not due to its chelating activity, but rather due to its anti-oxidant activity.

Machado–Joseph disease/spinocerebellar ataxia-3 (MJD/SCA-3) is an inherited neurodegenerative disorder caused by expansion of the polyglutamine stretch in the MJD gene-encoded protein ataxin-3. The truncated form of mutated ataxin-3 causes aggregation and cell death in vitro and in vivo . Abnormal conformation and misfolding of the pathological protein are assumed critical to pathogenesis. To test this hypothesis, we transfected BHK-21 and Neuro2a cells transiently with N-terminal truncated ataxin-3 with an expanded polyglutamine stretch. We then studied the effects of organic solvent dimethyl sulfoxide (DMSO), cellular osmolytes glycerol, and trimethylamine N -oxide (TMAO) on aggregate formation and cell death. These reagents stabilize proteins in their native conformation and called chemical chaperones based on their influence on protein folding. Aggregate formation and cytotoxicity induced by truncated expanded ataxin-3 were reduced by exposing cells to these chemical chaperones. Our results indicate the potentially useful therapeutic strategy of the chemical chaperones in preventing cell death in MJD.

Little information is available on molecular defects involved in adult Sandhoff disease presenting as motor neuron disease phenotype. We studied enzyme activities of beta-hexosaminidase (Hex) and the HEXB gene encoding the beta-subunit of Hex in a family of the Japanese case. Enzyme assay with 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside revealed a reduction in Hex A and B activity in proband's leukocytes. Although the activity of both in the mother were intermediate between those of controls and the proband, only Hex B reduction determined with heat inactivation was found in the father. Analysis of HEXB gene demonstrated two novel point mutations. The first mutation, IVS2-1G>A, was located at the 3'-splice acceptor site of intron 2 derived from the mother, causing exon 3 skipping. The resultant mRNA encoded a shorter beta-chain, which may not form an active enzyme. The second mutation was a G-to-A transition in exon 13 (c.1598G>A) derived from the father and resulted in arginine-to-histidine substitution at amino acid position 533 (R533H). Expression of R533H mutation in COS-1 cells demonstrated a lack of normal Hex activity, indicating that this mutation is pathological. Compound heterozygosity of these two mutations may trigger the development of adult Sandhoff disease with a motor neuron disease phenotype.

We investigated the expression of neuronal nitric oxide synthase (nNOS) and dystrophin in the regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis by immunohistochemical studies and western blot analysis. In normal muscles, nNOS was moderately immunostained on type 2B fibers, but was faintly immunostained on type 2A or type 1 fibers. In immunohistochemical studies of regenerating muscles, nNOS was first observed at the sarcolemma of type 2B fibers on day 10, when the type discrimination between types 2A and 2B was first detected by ATP reactions. Subsequently, the immunostaining of nNOS grew progressively stronger in type 2B fibers, with faint staining in type 2A and type 1 fibers until day 28. Meanwhile, the immunostaining of dystrophin grew stronger equally in all three fibers until day 21. In western blot analysis of regenerating muscles, nNOS regenerated more slowly than dystrophin. The present data suggest that the expression of nNOS is related to the muscle fiber type differentiation, and that the role of nNOS is related to the function of the type 2B fibers of the muscle.

The expression of α-dystrobrevin and dystrophin in rat tibialis anterior muscles was chronologically evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. In immunohistochemical studies, α-dystrobrevin and dystrophin were first stained weakly at the sarcolemma of some regenerating muscle fibers on day 5. On day 7, α-dystrobrevin was still stained weakly, whereas dystrophin was stained conspicuously. After day 10, α-dystrobrevin and dystrophin were both stained conspicuously on almost all regenerating muscle fibers. In the Western blot analysis, α-dystrobrevin and dystrophin were first detected as visible bands on days 5 and 7, respectively. The bands of α-dystrobrevin and dystrophin both darkened sequentially up to day 10. The protein levels based on the densitometrical analysis of the bands on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. The sequential line based on these data showed that α-dystrobrevin and dystrophin reached 50% of the protein level on day 28 by 6.6 and 5.3 days, respectively. These data provide evidence that α-dystrobrevin regenerates more slowly than dystrophin in skeletal muscle.

We evaluated re-expression of dystrophin, alpha-sarcoglycan and beta-dystroglycan in regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis in order to understand the dynamic behaviour of these proteins during the regeneration process. Immunohistochemical staining of these proteins almost disappeared in the sarcolemma of necrotic fibers on the 1st day, and was obscured due to non-specific staining on the 3rd day. Dystrophin was labeled faintly at the sarcolemma of regenerating muscle fibers on the 5th day. From the 5th day to the 10th day, levels of immunostaining of dystrophin increased. After the 14th day, dystrophin was stained conspicuously. alpha-Sarcoglycan was labeled weakly at the sarcolemma of small regenerating muscle fibers on the 5th day and was labeled conspicuously after the 7th day. beta-Dystroglycan was labeled moderately at the sarcolemma of regenerating muscle fibers on the 5th day and was labeled conspicuously after the 7th day. In western blot analysis, beta-dystroglycan persisted throughout the entire cycle of myonecrosis and regeneration, and re-expression of alpha-sarcoglycan progressed faster than that of dystrophin. We speculate that regeneration advances from the basement membrane side to the subsarcolemmal side, and that proteins at the basement membrane side resist disruption and have a high capacity for regeneration.

Ataxin-3, a protein coded by the Machado-Joseph disease gene, possesses a polyglutamine stretch whose expansion is known to produce neuronal intranuclear inclusion and neurodegeneration. Although previous studies describe the aggregate formation and toxic effect of the expanded polyglutamine tract in vitro and in vivo, differences in the susceptibility of different cultured cell lines has not been reported. Using the plasmid expressing N-terminal truncated ataxin-3 with an expanded polyglutamine stretch, we evaluated the aggregate formation and cytotoxicity in eight cultured cell lines-HeLa, Swiss/3T3, P19, C2C12, COS-1, BHK-21, PC12, and Neuro2a-that demonstrated a diverse range of aggregate formation and cell death. Although aggregate frequency did not appear to be correlated with cell death, Neuro2a demonstrated a high frequency of both. Our data indicates that susceptibility to cell death produced by mutant truncated ataxin-3 differs significantly among different cell lines and provides useful information when using a cultured cell line as an in vitro cellular model of polyglutamine disease.

Both APP and PS-1 are causal genes for early-onset familial Alzheimer's disease (AD) and their mutation effects on cerebral Abeta deposition in the senile plaques were examined in human brains of 29 familial AD (23 PS-1, 6 APP) cases and 14 sporadic AD cases in terms of Abeta40 and Abeta42. Abeta isoform data were evaluated using repeated measures analysis of variance which adjusted for within-subject measurement variation and confounding effects of individual APP and PS-1 mutations, age at onset, duration of illness and APOE genotype. We observed that mutations in both APP and PS-1 were associated with a significant increase of Abeta42 in plaques as been documented previously. In comparison to sporadic AD cases, both APP717 and PS-1 mutation cases had an increased density (measured as the number of plaques/mm(2)) and area (%) of Abeta42 plaques. However, we found an unexpected differential effect of PS-1 but not APP717 mutation cases. At least some of PS-1 but not APP717 mutation cases had the significant increase of density and area of Abeta40-plaques as compared to sporadic AD independently of APOE genotype. Our results suggest that PS-1 mutations affect cerebral accumulation of Abeta burden in a different fashion from APP717 mutations in their familial AD brains.

The expression of dystrophin and alpha1-syntrophin in rat tibialis anterior muscles were evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. Immunohistochemical studies were performed in cryosections of muscles on days 1, 3, 5, 7, 10, 14, 21 and 28 after injection of cardiotoxin. Western blot analysis was also examined in muscle on days 1, 3, 5, 7, 10, 14, 21 and 28. In immunohistochemical studies, dystrophin was stained weakly at the sarcolemma of some regenerating muscle fibers on day 3, and by day 10 it was stained strongly on almost all regenerating muscle fibers. Alpha1-syntrophin was stained weakly at the sarcolemma of some regenerating fibers on day 5, and by day 14 it was detected on all regenerating muscle fibers. In Western blot analysis, dystrophin (DYS1) and alpha1-syntrophin (alpha1S) were completely absent on day 1. Re-expression of DYS1 and alpha1S was visible by day 5 and accelerated thereafter. The Western blots of DYS1 and alpha1S were densitometrically analyzed on each day. The protein levels on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. From the sequential line based on these data, the following results were obtained on the chronological course of DYS1 and alpha1S. DYS1: 25% of the protein level on day 28 was reached by 3.5 days, 50% was reached by 5.3 days, and 90% was reached by 6.9 days. Alpha1S: 25% of the protein level on day 28 was reached by 4.6 days, 50% was reached by 6.0 days, and 90% was reached by 12.5 days. In this study, DYS1 regenerated earlier than alpha1S at the sarcolemma of regenerating muscle fibers.

A 49-year-old man was admitted to our hospital complaining of dysarthria and involuntary movements of his neck and extremities. He had first begun to experience involuntary neck movements at the age of 40 and his symptoms gradually progressed thereafter. There was no family history of neurological disorders. On admission he showed memory disturbance, dysarthria, and choreic movements. The involuntary movements affected his face, neck, trunk, and extremities. MRI of the brain revealed atrophy of both the cerebral cortex and the head of the caudate nucleus. DNA samples for molecular analysis were obtained from the patient and both of his parents. In this pedigree, the father carried a premutated allele of 35 CAG repeats and transmitted an expanded allele of 43 CAG repeats to his son. Paternity and maternity were analyzed using a microsatellite marker located in a different chromosome. To our knowledge, this is the first report of a sporadic case of Huntington's disease in a non-caucasian population in which the disease prevalence is much lower than that in the caucasian population. A new mutation in the current Japanese population which shares the same mechanism as de novo mutation in Caucasians may have contributed to the frequency of HD in Japan at the present time.

We reported a 64-year-old male with an eight-month history of gait disturbance and sensory impairment. The patient initially noticed unsteadiness of gait and numbness in his feet, and these symptoms progressed until he was unable to walk without assistance five months later. Vibratory sensation and position sense were markedly diminished, and deep tendon reflexes were absent in all extremities. Motor conduction velocities were slow with prolonged distal latencies, and sensory nerve action potentials (SNAP) were not elicited. Sural nerve biopsy revealed a mild loss of myelinated fibres and segmental demyelination. Cerebrospinal fluid showed normal cell count with protein 526 mg/dL. Anti-GM1, anti-GM2 and anti-GA1 antibodies in serum were positive. We diagnosed chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) presenting ataxia. Steroid therapy provided immediate improvement of symptoms and signs. This case suggests that CIDP should be considered as one of the potential causes of ataxic neuropathy.

In Down syndrome (DS) brain an early, selective accumulation of amyloid β (Aβ) peptides ending at residue 42 (Aβ42) occurs. Whether this event depends on an altered processing of amyloid beta precursor protein (APP) or on defective clearance is uncertain. To investigate this issue, we measured Aβ species 40 and 42 in plasma from 61 patients with DS, 77 age-matched normal controls, and 55 mentally retarded subjects without chromosomal abnormalities. The Aβ 40 and 42 plasma levels were then correlated with apolipoprotein E (apoE) genotypes in all groups of cases, and with I.Q. and Mini Mental Status Examination values in DS subjects. Both Aβ species were significantly elevated in DS compared to control groups, and the extent of their increase reflects that expected from APP gene overexpression. Plasma levels of Aβ 40 and 42 did not correlate with apoE genotypes in DS and control cases, and with the extent of mental retardation in DS subjects. The results indicate that accumulation and clearance of plasma and cerebral Aβ are regulated by different and independent factors. Am. J. Med. Genet. 95:224–228, 2000. © 2000 Wiley-Liss, Inc.

Ischemia-reperfusion is known to induce generation of oxygen free radicals (OFRs), thereby resulting in tissue damage. To clarify the pathogenesis of diabetic neuropathy, we examined whether hyperglycemia exacerbates the effects of ischemia-reperfusion on peripheral nerves. Using light microscopy, characteristic pathological changes seen in myelin and axons of nerve fibers were classified into two types: "loose myelin" and "dark axon," respectively. After a 2-hour ischemia and 24-hour reperfusion of sciatic nerves of rats, the concentrations of lipid peroxide and "loose myelin" in the nerves were significantly increased in the diabetic rats, but not in the nondiabetic rats. Thereafter, "dark axon" was significantly increased in both diabetic and nondiabetic rats. "Loose myelin" may be increased as a result of the effects of OFRs produced by ischemia-reperfusion, which are exacerbated by hyperglycemia.

To clarify the clinical, neuropathological, and molecular characteristics of spinocerebellar ataxia type 6 (SCA6), two unrelated Japanese families with SCA6 were studied. A clinical feature of the two families was late onset "pure" cerebellar ataxia. Pathologically, three SCA6 brains consistently showed Purkinje cell dominant cortical cerebellar degeneration. Morphometric analysis showed that loss of the cerebellar granule cells and inferior olivary neurons were very mild compared with the severity of Purkinje cell loss. There was no obvious ubiquitin immunoreactive nuclear inclusions. All affected patients had identical expanded alleles, and the expansion was also homogeneously distributed throughout the brain without mosaicism. The present study showed that SCA6 is characterised by Purkinje cell dominant cortical cerebellar degeneration, highly stable transmission of the CAG repeat expansion, and lack of ubiquitin immunoreactive nuclear inclusions.

Spinocerebellar ataxia type 6 (SCA6) is one of the eight neurodegenerative diseases caused by a tri-nucleotide (CAG) repeat expansion coding polyglutamine (CAG repeat/polyglutamine diseases) and is characterized by late onset autosomal dominant cerebellar ataxia and predominant loss of cerebellar Purkinje cells. Although the causative, small and stable CAG repeat expansion for this disease has been identified in the [alpha]1A voltage-dependent calcium channel gene (CACNA1A), the mechanism which leads to predominant Purkinje cell degeneration is totally unknown. In this study, we show that the calcium channel mRNA/protein containing the CAG repeat/polyglutamine tract is most intensely expressed in Purkinje cells of human brains. In SCA6 brains, numerous oval or rod-shaped aggregates were seen exclusively in the cytoplasm of Purkinje cells. These cytoplasmic inclusions were not ubiquitinated, which contrasts with the neuronal intra-nuclear inclusions of other CAG repeat/polyglutamine diseases. In cultured cells, formation of perinuclear aggregates of the channel protein and apoptotic cell death were seen when transfected with full-length CACNA1A coding an expanded polyglutamine tract. The present study indicates that the mechanism of neurodegeneration in SCA6 is associated with cytoplasmic aggregations of the [alpha]1A calcium channel protein caused by a small CAG repeat/polyglutamine expansion in CACNA1A.

We established an efficient method for obtaining expression of a foreign marker gene transferred in vitro into myoblasts and in vivo into adult mouse skeletal muscles using adenovirus vector. After infection of the C2 myoblasts with the adenovirus vector containing the beta-actin promoter with cytomegalovirus (CMV) enhancer (CAG promoter) AxCALacZ, significantly greater number of cells express beta-galactosidase when compared with the adenovirus vector expressing the lacZ gene under the control of the SR alpha viral terminal repeat promoter (AxSRLacZL) or the myosin heavy chain (MHC) IIB promoter (AxMHCLacZ). We also injected AxCALacZ into the skeletal muscles of 5- to 6-week-old C57BL/10 mice and determined that more than 60% of their muscle fibers expressed the lacZ gene 7 days after injection. The CAG promoter may have application in the development of gene therapy for Duchenne muscular dystrophy (DMD) using adenovirus vector.

We evaluated the effect of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) on the response of blood pressure and vasoactive factors during head-up tilt in patients with multiple system atrophy (MSA). After the daily oral administration of 300 mg L-threo-DOPS for 2 weeks we observed a significant reduction in the decline of systolic blood pressure during 60 degrees head-up-tilt. In concordance with this result, we detected significant increases in postural changes in plasma renin activity and angiotensin II following L-threo-DOPS treatment. The enhanced responses of these vasoactive mediators by L-threo-DOPS during head-up tilt may contribute to the improvement in orthostatic hypotension in patients with MSA.

Cerebral presenilin-1 protein (PS-1) is normally composed of the amino-terminal fragment (NTF) with Mr 28 kDa and the carboxy-terminal fragment (CTF) with 18 kDa. We analyzed human PS-1 in brains with early-onset familial Alzheimer's disease (FAD) with and without PS-1 mutations to study whether mutated PS-1 was abnormally metabolized. Cerebral PS-1 were found to be cleaved into two fragments of NTF and CTF independently of the occurrence of PS-1 mutation in human brains. A small portion of PS-1 was recently found to suffer another processing by caspase-3, an apoptosis-related cysteine protease. In contrast to the recent finding that the Volga-German mutation on presenilin-2 (PS-2) affects the increasing caspase-3 PS-2 fragment, the PS-1 mutation did not cause a significant change in PS-1 fragmentation. We conclude that PS-1 fragmentation and other (probably caspase-3-mediated) digestion following apoptosis occur independently of PS-1 mutations.

Mice with the Otx2+/- mutation often die during the postneonatal period. Before death these animals, generated from TT2 ES cells and crossed with CBA mice, develop a dome-shaped head, weakness of the limbs, kyphosis, lethargy, drowsiness, and emaciation. Autopsy of these mice revealed eminent dilatation of lateral ventricles and a ballooned cerebrum. Histological analysis shows edematous change of the periventricular white matter. These results suggest that Otx2 functions as a head organizer, and a mutation of this gene is a likely cause of hydrocephalus in mammals. Additionally, craniobasal skeletal anomaly in half of the heterozygotes and dwarfism in some of the female heterozygotes are described.

To clarify the immunohistochemical features of canine senile plaques (SPs) and cerebral amyloid angiopathy (CAA), the distribution of the amyloid beta protein (A beta) subtypes A beta 40 and A beta 42(43), A beta precursor protein (APP), and glial cell reaction were examined in the brains of seven aged dogs (12-18 years). A beta 42(43) was found to be deposited in all types of SPs, whereas A beta 40 was deposited only in mature (classical and primitive) plaques. CAA, which was located along parenchymal and meningeal arterioles and capillaries, consisted of both subtypes of A beta. APP was exhibited in normal and degenerative neurons and swollen neurites of mature plaques. It was, therefore, considered that A beta 42(43) in diffuse plaques might be derived from APP in neurons, while A beta 40 and A beta 42(43) in mature plaques might be generated from APP in swollen neurites in the plaque. In contrast to the case in humans, in whom deposition of A beta 40 and A beta 42(43) in the mature plaques is predominantly associated with microglial reaction, in dogs we found that it was closely associated with astroglial reaction. The present findings showed characteristics of canine SPs which are different from those of humans.

To determine localization of nitric oxide synthase in diseased muscle, we performed immunohistochemical analyses of neuronal-type nitric oxide synthase (nNOS) and endothelial-type nitric oxide synthase (ec-NOS) in biopsied muscles from five patients with mitochondrial encephalomyopathies. Immunostaining of nNOS was prominent in the sarcolemmal region of the ragged-red fibers, and weak in the sarcolemmal region of normal fibers. Immunostaining of ec-NOS was strongly positive in the myofibrils of ragged-red fibers. Ec-NOS immunoreactivity corresponded to fibers positive for SDH by histochemistry. Histochemical methods revealed prominent staining for NADPH-diaphorase on surface membranes of the ragged-red fibers. Findings suggest that nitric oxide is important in the muscles of patients with mitochondrial encephalomyopathies.

To investigate the pathomechanism of amyloid beta protein (A beta) deposition in brains with Alzheimer's disease (AD), cerebrospinal fluid (CSF) levels of A beta species (CSF-A beta) with different carboxy termini, i.e. A betaX-40 and A betaX-42(43) as well as A beta1-40 and A beta1-42(43), were measured in patients with AD and age-matched controls without dementia (CTR) using sandwich enzyme-linked immunosorbent assays (ELISAs). The present study revealed that both CSF-A betaX-42(43) and A beta1-42(43) levels were significantly lower in the AD patients (P

The combination of ataxia with peripheral neuropathy (ataxic neuropathy) is rare. Six elderly patients with peripheral neuropathy who developed ataxia were studied. Of the peripheral neuropathies, ataxic neuropathy was significantly more frequent in patients aged more than 65 years compared with younger patients. Ataxic neuropathy was associated with carcinoma (2 cases), Sjögren's syndrome (1 case), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP, 1 case) and chronic idiopathic ataxic neuropathy (2 cases). The two cases of carcinomatous neuropathy initially showed ataxia, which preceded detection of the carcinomatous lesions in the lung by approximately 1 year. The study cases had many clinical features in common. In the nerve conduction study, sural nerve action potential could not be measured in five of the cases; sural nerve biopsy revealed a decreased density of myelinated fibers in all cases. In particular, the large myelinated fibers were markedly decreased. These findings were common, regardless of the underlying disease, except in the case of CIDP in which there was only a slight decrease in the number of large myelinated fibers. Differential diagnosis based on the clinicopathological features was difficult. Therefore, in cases of ataxic neuropathy, systemic evaluation is necessary to rule out the possibility of carcinoma or various systemic diseases, especially in elderly patients.

Fibrillar amyloid beta protein (A beta) deposition is increased in the brains of patients with Alzheimer's disease (AD), and is manifested as senile plaques (SPs) and congophilic angiopathy (CA). A beta 40 and A beta 42(43), two chief species of A beta, are documented in SPs and CA, as well as in cerebrospinal fluid (CSF) and cell culture media. A beta 42(43) is the major component of diffuse plaques, the earliest form of SPs. Thus, we hypothesized that determination of the amount of A beta 42(43) in CSF or plasma might provide a diagnostic laboratory test for AD. We measured amounts of different A beta species in plasma from 28 patients with sporadic probable AD, 40 age-matched neurologic patients without dementia and 25 age-matched normal controls using enzyme-linked immunosorbent assays (ELISAs). Plasma concentrations of A beta 1-40 and A beta 1-42(43) did not significantly differ among these groups. These findings suggest the unlikelihood that plasma A beta assays would be useful as a diagnostic tool for AD.

In the present study, we characterized the epitope of a monoclonal antibody against purified amyloid plaque cores (Am-3). By immunocytochemical experiments, Am-3 stained cerebrovascular and senile plaque amyloid in brain sections of patients with Alzheimer's disease (AD) in a similar manner to that of antibodies against amyloid beta-protein (A beta). By Western blotting experiments, Am-3 recognized only a 35 kDa protein, which was revealed to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and not A beta or beta amyloid precursor protein (beta PP). However, Am-3 recognized both GAPDH and purified native A beta in a dot-binding assay. Therefore, we concluded that Am-3 recognized both GAPDH and native A beta. Other monoclonal antibodies (6C6 and AmT-1) against the synthetic peptide corresponding to residues 1-28 of A beta also recognized these proteins. Because the amino acid sequences of these two proteins are not homologous, we propose that the crossreactivity between A beta and GAPDH is a consequence of their similar conformational epitopes. The possibility of crossreactions would complicate immunochemical and immunocytochemical studies of brain aging, AD and Down's syndrome. The implications of crossreactivity in developing immunological assays and in investigating the amyloid deposits of AD are discussed.

The selective vulnerability of distinct neuronal structures is a major feature of Machado-Joseph disease (MJD), also known as spinocerebellar ataxia 3 (SCA3). Vestibular dysfunction is known to be a symptom of MJD, but little is known about precisely when the vestibular system becomes impaired. Using a caloric test, we evaluated vestibular function in 2 MJD patients. One developed the initial symptom 1 year before evaluation, and the other 3 years before evaluation. Neither demonstrated a bilateral response in electronystagmography using ice-cold water irrigation, indicating severe vestibular disturbance. These results suggest that vestibular dysfunction is a symptom that develops very early in MJD and may contribute to unsteady gait as the initial symptom. The vestibular system thus appears to be one of the structures most vulnerable to MJD.

Immunohistochemical analyses were made of the superoxide dismutases (Mn-SOD and Cu/Zn-SOD) in biopsied muscles from 7 patients with mitochondrial encephalomyopathies that included mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS), and chronic progressive external ophthalmoplegia (CPEO). Mn-SOD mainly was present in the subsarcolemmal region, but it also was found in a coarsely granular, reticular, or diffuse pattern of staining within the muscle fibers. These Mn-SOD-positive fibers corresponded almost completely to the ragged-red fibers. The immunoreaction for Cu/Zn-SOD was weakly positive in some of the muscle fibers positive for Mn-SOD. In CPEO, Mn-SOD-positive fibers predominantly showed decreased cytochrome c oxidase (COX) activity. In MELAS, Mn-SOD-positive fibers tended to be stained deeply for COX although a few were COX-negative. These findings suggest that Mn-SOD-positive fibers can be used to make a differential diagnosis between CPEO and MELAS and that in mitochondrial encephalomyopathies Mn-SOD in the ragged-red fibers may protect against oxidative stress.

Diffuse plaques are immature and amorphous senile plaques and believed to be in the initial phase of plaque formation. In contrast to amyloid angiopathy and the plaque core amyloid, diffuse plaques failed to be purified in preserved forms from the brain. Here, we studied the diffuse plaques in the cerebellar region of the Alzheimer's disease brain based on immunocytochemistry and ELISA using two different monoclonal antibodies specifically recognizing the carboxyl termini of A beta molecules (BA27 for A beta 1-40 and BC05 for A beta 1-42/43). We found that the amount of A beta 1-40 was in proportion to the staining degree on amyloid angiopathy by immunohistochemistry. We found that A beta 1-42/43 comprised diffuse plaques as the major component in the cerebella of AD brains. Taking these findings into consideration, diffuse plaques, the earliest pathological change in the brain with AD, are concluded to be composed mainly of A beta 1-42/43, implicating the critical importance of this kind of A beta species deposition in the pathogenesis of AD.

Glial cytoplasmic inclusions (GCIs) have been observed in oligodendroglia-like cells, specifically in the brains of patients with multiple system atrophy (striatonigral degeneration, olivopontocerebellar atrophy and Shy-Drager syndrome). We have investigated GCIs from brains of patients with multiple system atrophy biochemically and immunochemically. While most GCIs have been reported positive for both ubiquitin and alpha B-crystallin in immunocytochemical studies, the components of GCIs have not been identified biochemically. GCI-bearing cells were partially purified from the OPCA brain by sieving with nylon meshes and discontinuous sucrose density gradient centrifugation. The fraction containing GCI-bearing cells was also found to contain a 32 kDa and a 40 kDa protein, both of which were specifically recognized by anti-ubiquitin and anti-alpha B-crystallin antibodies, neither of which was found in the same fraction derived from control brain. These immunochemical results suggest that ubiquitinated alpha B-crystallin is present in GCIs from the brains of patients with multiple system atrophy.

We investigated the clinical usefulness of N-isopropyl-p-[123I]iodoamphetamine single photon emission computed tomography (SPECT) for diagnosis of spinocerebellar degeneration. The uptake in the cerebellar hemispheres and vermis was decreased in sporadic cerebelloolivary atrophy (Holmes, n = 9) and sporadic olivopontocerebellar atrophy (OPCA, n = 6). The uptake in the pons was decreased in OPCA. The decrease of the uptake in the cerebellum and the pons was associated with clinical severity in OPCA. Our results show that semiquantitative SPECT analysis differentiates between the Holmes type and OPCA.

We present a 17-year-old boy with Becker muscular dystrophy (BMD) who developed hyperthermia and heart failure after general anesthesia. He presented clinical features of malignant hyperthermia (MH), and had masseter spasm and elevated body temperature (38.7 degrees C) with very high serum CK activity (107,000 IUl-1). Dystrophin tests confirmed a clinical diagnosis of BMD in the patient, i.e. faint and patchy immunostaining pattern of skeletal muscle, truncated dystrophin protein and a deletion of exons 3 and 4 of the dystrophin gene. To inquire into the mechanism of MH associated in the patient, we tested caffeine contracture reaction by the skinned fiber method. We found an increased sensitivity to caffeine only in type 1 muscle fibers. The rate of Ca(2+)-induced Ca2+ release (CICR) was normal, suggesting that the mechanism of "MH" observed in our patient with BMD is not the same as that of classical MH. A possible mechanism might be related to derangements of the sarcoplasmic reticulum membrane in BMD, which sensitize the membrane to caffeine or other agents.

Background: Donepezil hydrochloride (Aricept) is a selective acetylcholinesterase inhibitor for the treatment of Alzheimer’s disease (AD). However, the memory domains for which the agent is effective, especially in very early phase of the disease, have not been clarified.We observed patients with amnestic mild cognitive impairment (aMCI) for seven years to determine the efficacy and limitations of donepezil. Methods: Ten patients with aMCI were diagnosed on the basis of intact daily activity, normal Mini-Mental State Examination (MMSE) score, and deviated memory reduction (less than 85) on at least one index of the Wechsler Memory Scale Revised (WMS-R). Single photon emission tomography (SPECT) showed AD patterns in the Three-Dimensional Stereotactic Surface Projection (3DSSP) analysis. All patients received Aricept, 5 mg/day, with informed consent. The WMS-R and a set of spatial memory tests (SMT) we devised were administered at baseline and at months 6, 12, 36, 60, and 84. The SMT comprised two subtests: (a) a spatial source memory test, assessing the memory of the agent of an action (who acted) and the reality of an action (acted or imagined) using line drawings of furniture on small pieces of paper and a virtual room and (b) a line drawing furniture completion test, assessing the ability to organize spatial representations, in which patients were requested to insert an important part of a piece of furniture in the appropriate position. Both subtests assess cognitive ability to manipulate three-dimensional ideations underlying memory of a space. Results: At baseline, all patients showed one or moreWMS-R index scores below 85 along with errors on SMT. At month 6, all patients scored WMS-R index scores higher than 85, and this improvement was preserved till month 84. However, the error on SMT remained in all patients throughout the study.Conclusions:Donepezil hydrochloride, prescribed early in the course of AD, improves general (verbal and visual) memory function, as assessed by a standardized memory test, and preserves it for a considerable period of time; however, it does not affect spatial memory function, which requires three-dimensional ideation. The memory impairment related to manipulation of spatial representations might be germane to AD.

We examined the apolipoprotein E (apo E) genotypes in 47 patients with late-onset sporadic Alzheimer's disease (mean age at onset +/- standard deviation, 72.2 +/- 6.4 years), 8 with late-onset familial Alzheimer's disease (75.5 +/- 5.1 years), 18 with early-onset sporadic Alzheimer's disease (52.8 +/- 4.7 years), and 10 with early-onset familial Alzheimer's disease (52.0 +/- 6.8 years) in Japan and compared them with genotypes in control subjects. In late-onset sporadic Alzheimer's disease, apo E-epsilon 4 frequency increased significantly (epsilon 4 frequency: 0.34 vs 0.095 in controls, p < 0.0001), and the odds ratio, which represents the strength of association between Alzheimer's disease and apo E-epsilon 4, markedly increased with increasing dose of apo E-epsilon 4 gene (3 [95% confidence interval, 2-6] in one dose; 43 [95% confidence interval, 12-154] in two doses). This study also suggested that apo E-epsilon 4 is associated with both late-onset (epsilon 4: 0.31) and early-onset familial Alzheimer's disease (epsilon 4: 0.35). In contrast, we found no association between apo E-epsilon 4 and early-onset sporadic Alzheimer's disease (epsilon 4: 0.08). These results indicate that the risk of developing late-onset sporadic Alzheimer's disease is markedly dependent on the dose of apo E-epsilon 4, while apo E-epsilon 4 does not appear to be a major risk factor for early-onset sporadic Alzheimer's disease.

A case of primary ocular and central nervous system malignant lymphoma was reported which was first manifested as bilateral uveitis ten months before the neurological symptoms appeared. The lesion of lymphoma in the central nervous system was clearly demonstrated as a heavy accumulation of N-isopropyl-123I-p-iodoamphetamine by single photon emission computed tomography. Since most brain tumors reportedly show a low uptake of N-isopropyl-123I-p-iodoamphetamine, single photon emission computed tomography with N-isopropyl-123I-p-iodoamphetamine could be helpful in making the diagnosis, as well as in visualizing primary central nervous system malignant lymphoma.

症例は51歳,女性.主訴は繰り返す運動時の左上下肢のふるえ(limb shaking).入院時,神経学的に異常なく,頭部MRIで右大脳白質のラクナ梗塞,過換気時の脳SPECTで右の前頭・頭頂・側頭部および大脳基底核部の血流低下,脳血管造影で右内頸動脈閉塞像を認めた. limb shakingは基底核部の一過性脳虚血発作(TIA)に基づく不随意運動とされ,本例のように内頸動脈閉塞によるTIA症状である可能性に十分留意しなければならない.

Article abstract Thiobarbituric acid–reactive substances (TBARS), an index of lipid peroxidation, were assayed in postmortem brain. Basal TBARS levels were increased and oxidative stimulation produced more TBARS in AD relative to control brains. In addition, apolipoprotein E isoforms showed differing antioxidant activities, with E2 > E3 > E4, suggesting that the lowest antioxidant activity of E4 could contribute to its association with AD.

重症筋無力症の患者が急に筋力低下が増強し呼吸困難を訴えることを,クリーゼと呼び,救急処置が必要である.呼吸を確保し,テンシロン・テストで明らかな筋力低下に効果があれば,抗コリンエステラーゼ薬の増量を,明らかな効果が認められない場合や重症でテストを行えない場合は,抗コリンエステラーゼ薬を切る.原則に従って,テンシロン・テストを繰り返し抗コリンエステラーゼ薬の適量を決める.

We report two siblings with spinocerebellar ataxia type 6 (SCA 6), both showing downbeat nystagmus (DBN) as a predominant clinical feature. Familial hemiplegic migraine (FHM), episodic ataxia type 2 (EA-2) and SCA 6 are allelic disorders, and interestingly, the occasional presence of DBN in EA-2 was reported. Our observations suggest that common molecular mechanisms might underlie DBN in FHM, EA-2 and SCA 6. Then, these disorders should be kept in mind in diagnosing patients with DBN.

To investigate the knowledge, attitudes and practice of Japanese students regarding brain death and organ transplantation.A 22-item questionnaire was handed out among 383 Japanese students during the 2002 academic year. The data was finally analyzed using a statistical package for social sciences, SPSS.Most students knew that organ transplantation can save a life 97%, while only 38% of the students were aware that there is no treatment for brain dead patients. Overall, 60% of the respondents believed brain death is equal to human death and 63% believed that organ removal from brain dead donors is mutilation of the body. There was a negative association between the students who reported positive attitudes toward the idea that removing an organ is mutilation of the body with their willingness to be a donor. Of the students 27% were opposed to the necessity of family agreement for organ donation. Although 66% expressed their wish to be a donor, less than half of them, 31% had a donor card; and 40% did not even know how to get a donor card.Granting a veto power to the family as well as the mistaken idea that there is recovery from brain death, can be considered as obstacles to organ procurement from the brain dead in Japan. The high value of life among Japanese society and the willingness to be a donor, bring hope that giving correct medical information and proper public education can increase social acceptance of organ transplantation from the brain dead in Japan.