Toshihiro Yoshizawa

Reduction of GluR2 RNA editing, a molecular change that increases calcium influx through AMPA receptors, selective in the spinal ventral gray of patients with amyotrophic lateral sclerosis

Enhancement of calcium influx through the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate receptor is a plausible mechanism underlying selective neuronal death in amyotrophic lateral sclerosis (ALS). The calcium conductance of the AMPA receptor is regulated by the GluR2 subunit that is edited at the glutamine/arginine residue site in the subunit assembly. We investigated the molecular changes of GluR2 mRNA in the spinal cord of ALS cases, those of cases with other neurological diseases, and those of normal cases using reverse transcription–polymerase chain reaction combined with restriction enzyme cleavage. We found that the editing efficiency was significantly lower only in the ventral gray of ALS cases (virtually 0% in 2 cases) than in any spinal region of the disease controls and normal controls. In addition, expression of GluR2 mRNA is lower in the ventral gray of the ALS cases and disease controls than in that of the normal controls. The above molecular changes of GluR2 mRNA in the ventral gray of ALS cases may enhance calcium influx through AMPA receptors, thereby promoting neuronal vulnerability. The decrement of GluR2 mRNA editing efficiency is unique to the ventral gray of ALS cases and may be closely linked to the etiology of ALS.

Endothelin-3 is a novel neuropeptide: Isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain

The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system.

Endothelin localizes in the dorsal horn and acts on the spinal neurones: Possible involvement of dihydropyridine-sensitive calcium channels and substance P release

The neural effect of endothelin, a vasoconstrictor peptide from vascular endothelium, was investigated in the in vitro spinal cord preparation of the newborn rat. In addition, an immunohistochemical study of endothelin was performed in the porcine spinal cord. Endothelin produced ventral root depolarization in a dose-dependent manner in the newborn rat spinal cord. Endothelin (5 x 10(-8) M)-induced depolarization was depressed by the dihydropyridine-sensitive Ca2+ channel blocker, nicardipine (10(-7) M), or the substance P antagonist, spantide (5 x 10(-6) M). These results suggest that endothelin may cause substance P release and that dihydropyridine-sensitive Ca2+ channels in the spinal cord may be involved in this process. Furthermore, endothelin-like immunoreactivity was localized in dot- and fibre-like structures and neurones in the dorsal horn of the porcine spinal cord. Therefore, it is suggested that endothelin or endothelin-related peptide(s) have a neuromodulatory function in the spinal cord.

Clinical, neuropathological, and molecular study in two families with spinocerebellar ataxia type 6 (SCA6)

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.

Presence of endothelin-1 in porcine spinal cord: Isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.

Chemical Chaperones Reduce Aggregate Formation and Cell Death Caused by the Truncated Machado–Joseph Disease Gene Product with an Expanded Polyglutamine Stretch

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 are 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.

Ectoine alters subcellular localization of inclusions and reduces apoptotic cell death induced by the truncated Machado-Joseph disease gene product with an expanded polyglutamine stretch.

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.

Screening of the mis-sense mutation producing the 717Val → Ile substitution in the amyloid precursor protein in Japanese familial and sporadic Alzheimer's disease

We investigated a C to T transition at base pair 2149 in the amyloid precursor protein gene in 41 Japanese cases of early-onset familial Alzheimers disease (FAD), late-onset FAD and sporadic Alzheimers disease (AD) by polymerase chain reaction and restriction enzyme polymorphism with BclI. Among 9 early-onset FAD patients derived from independent families, only one patient had the mis-sense mutation. Neither 5 patients with late-onset FAD nor 27 patients with sporadic AD had the mutation. Our result and the previous reports from Japan indicate that this type of mis-sense mutation is present in several cases of Japanese early-onset FAD. On the other hand, our data suggest that this mutation is not a common cause of Japanese early-onset FAD. Moreover, this mutation could be absent in late-onset FAD and sporadic AD in Japan. Because the mutation has been reported to be rare in Caucasian early-onset FAD and to be absent in Caucasian late-onset FAD and sporadic AD, the situation of this mutation in Alzheimers disease may be common beyond the ethnic background.

Magnetic resonance imaging demonstrates differential atrophy of pontine base and tegmentum in Machado–Joseph disease

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.

Endothelin-1 depolarizes a ventral root potential in the newborn rat spinal cord.

Summary The effect of endothelin-1 (ET-1) was investigated in the isolated spinal cord of the newborn rat. Bath-applied ET-1 produced a depolarization of a lumbar ventral root, which was suppressed by nicardipine or a substance P antagonist (spantide). In addition, the slow ventral root depolarization from L4 segment evoked by electrical stimulation of the contralateral sciatic nerve was also suppressed by spantide or nicardipine. However, nicardipine did not affect the ventral root depolarization induced by substance P, itself. These results indicate that the dihydropyridine-sensitive Ca2+ channels are associated with substance P release and that the effect of endothelin is probably mediated by substance P.