Hirohide Asai

DNA single-strand break repair is impaired in aprataxin-related ataxia

Early‐onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive form of cerebellar ataxia. The causative protein for EAOH/AOA1, aprataxin (APTX), interacts with X‐ray repair cross‐complementing 1 (XRCC1), a scaffold DNA repair protein for single‐strand breaks (SSBs). The goal of this study was to prove the functional involvement of APTX in SSB repair (SSBR).

Sympathetic disturbances increase risk of sudden cardiac arrest in sporadic ALS

BACKGROUND ALS exclusively involves motor neurons, however, accumulating evidence suggests involvement of sympathetic neurons, as in other diseases including Parkinsons disease and multiple system atrophy. In these diseases increased risk of sudden cardiac arrest is established, while that in ALS remains uncertain. METHODS The authors retrospectively studied 12 pathologically confirmed sporadic ALS patients who received no assisted ventilation. Among them, two patients died of sudden cardiac arrest. Changes in QTc interval and dispersion, indices of sympathetic activities obtainable by routine electrocardiograms, were evaluated at the early stage and the terminal stage. Pathologically, intermediolateral nucleus (IML) sympathetic neurons in the upper thoracic cord were examined. RESULTS The QTc intervals and dispersion were significantly increased at the terminal stage compared with that at the early stage (p<0.01). The numbers of IML neurons were significantly lower in ALS patients than in controls (p=0.017), and had linear inverse correlation with the rate of increases in maximum QTc interval and QTc dispersion (p=0.01, r=-0.915 and p=0.02, r=-0.884). Notably, two patients with sudden cardiac arrest showed longer QTc interval, larger QTc dispersion, and lower number of IML neurons than most of others. CONCLUSIONS Patients with ALS had reduced sympathetic activities at the terminal stage of disease, presumably due to neuronal loss in IML, which may increase risk of sudden cardiac arrest. Thus, prolonged QTc intervals and increased QTc dispersion may suggest an increased risk of sudden death in ALS, as in other neurodegenerative diseases.

Restoration of nuclear-import failure caused by triple A syndrome and oxidative stress.

Triple A syndrome is an autosomal recessive neurological disease, mimicking motor neuron disease, and is caused by mutant ALADIN, a nuclear-pore complex component. We recently discovered that the pathogenesis involved impaired nuclear import of DNA repair proteins, including DNA ligase I and the cerebellar ataxia causative protein aprataxin. Such impairment was overcome by fusing classical nuclear localization signal (NLS) and 137-aa downstream sequence of XRCC1, designated stretched NLS (stNLS). We report here that the minimum essential sequence of stNLS (mstNLS) is residues 239-276, downsized by more than 100 aa. mstNLS enabled efficient nuclear import of DNA repair proteins in patient fibroblasts, functioned under oxidative stress, and reduced oxidative-stress-induced cell death, more effectively than stNLS. The stress-tolerability of mstNLS was also exerted in control fibroblasts and neuroblastoma cells. These findings may help develop treatments for currently intractable triple A syndrome and other oxidative-stress-related neurological diseases, and contribute to nuclear compartmentalization study.

Cerebrospinal fluid-orexin levels and sleep attacks in four patients with Parkinson's disease

OBJECTIVES Sleep attacks (SAs) in Parkinsons disease (PD) are rare, but clinically important because they significantly impair the daily lives of patients. Causes of SAs include long-term activation of dopaminergic (especially D3) receptors. Recent studies suggest that SAs in PD may be related to impairment of hypothalamic orexin neurons, similar to narcolepsy. Whether orexin is associated with long-term activation of dopaminergic receptors remains uncertain. PATIENTS AND METHODS We measured levels of orexin in samples of spinal cerebrospinal fluid (CSF) from 25 patients with PD, including 9 with excessive daytime sleepiness and 4 with SAs. Furthermore, in the four patients with SAs, the selective dopamine D1/D2 agonist pergolide was substituted for the causative drugs with D3 stimulatory activity, and CSF-orexin levels were measured before and after switching treatment. RESULTS In the 25 patients with PD, including the 4 patients with SAs, lower CSF-orexin levels were associated with a longer disease duration, which has been linked to a higher incidence of SAs. Switching treatment to pergolide significantly increased CSF-orexin levels and completely resolved SAs in the four patients with PD. CONCLUSION Despite the small number of patients studied, our results suggest that orexin transmission is most likely involved in SAs in PD and that abrogation of D3 receptor stimulation may increase orexin and thereby inhibit SAs.

Protein kinase Cγ, a protein causative for dominant ataxia, negatively regulates nuclear import of recessive-ataxia-related aprataxin

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant disease caused by mutations in the gene encoding protein kinase C gamma (PKC gamma). We report an SCA14 family with a novel deletion of a termination-codon-containing region, resulting in a missense change and a C-terminal 13-amino-acid extension with increased kinase activity. Notably, one patient with a severe phenotype is the first homozygote for the mutation causing SCA14. We show the novel molecular consequences of increased kinase activities of mutants: aprataxin (APTX), a DNA repair protein causative for autosomal recessive ataxia, was found to be a preferential substrate of mutant PKC gamma, and phosphorylation inhibited its nuclear entry. The phosphorylated residue was Thr111, located adjacent to the nuclear localization signal, and disturbed interactions with importin alpha, a nuclear import adaptor. Decreased nuclear APTX increased oxidative stress-induced DNA damage and cell death. Phosphorylation-resistant APTX, kinase inhibitors, and antioxidants may be therapeutic options for SCA14.

Odor abnormalities caused by bilateral thalamic infarction.

Odor is the only sensation thought to be unrelated to the thalamus. However, accumulating evidence suggests that the dorsomedial nucleus (DM) of the thalamus is associated with odor. Although the thalamus is prone to ischemia, only a single patient with bilateral DM infarctions was reported to have odor abnormalities. We describe a second such patient with infarctions involving the left DM and the right ventral posterior nucleus and ventral lateral nucleus, nuclei adjacent to the DM, associated with transient edema. In contrast to the previous case, our patient had transient odor abnormality. These observations suggested that direct and/or indirect bilateral involvement of the DM might be associated with odor abnormalities in patients with thalamic infarction.

Cytoprotective effect of novel histone deacetylase inhibitors against polyglutamine toxicity

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurological disorder caused by a CAG repeat expansion in the DRPLA gene encoding polyglutamine (polyQ). Although previous experimental studies have demonstrated that histone deacetylase (HDAC) inhibitors are therapeutically active, known HDAC inhibitors have considerable adverse effects clinically. To identify new HDAC inhibitors for the treatment of DRPLA, we evaluated a new series of HDAC inhibitors, N-hydroxycarboxamides, with our drug screening system, which uses neuronal PC12 cells stably transfected with a part of the DRPLA gene. We found that two of four N-hydroxycarboxamides significantly reduced polyQ-induced cell death. The essential structure of these compounds is a hydroxamic acid residue, which is shared with trichostatin A, a known HDAC inhibitor. Although our study showed mild neuroprotective effects, further structural modification of compounds that retain this residue may decrease cytotoxicity and increase protective activity against polyQ toxicity.

Short half-lives of ataxia-associated aprataxin proteins in neuronal cells

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is caused by mutations in the gene encoding aprataxin (APTX). Although several in vitro findings proposed that impaired enzymatic activities of APTX are responsible for EAOH/AOA1, potential instability of mutant proteins has also been suggested as the pathogenesis based on in vivo finding that mutant proteins are almost undetectable in EAOH/AOA1 tissues or cells. The present study aimed to experimentally prove instability of mutant proteins in neuronal cells, the cell type preferentially affected by this disease. Results of pulse-chase experiments demonstrated that all of the disease-associated mutants had extremely shorter half-lives than the WT. We further found that mutants were targeted for rapid proteasome-mediated degradation. These results help establish pathogenic and physiological protein characteristics of APTX in neuronal cells.

Treatable fluctuating mental impairment in a patient with Bardet–Biedl syndrome

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by rod-cone dystrophy, polydactyly, central obesity, mental retardation, and hypogonadism. Although many organs are involved in BBS, hyperammonemia caused by portal hypertension has been reported previously in only a single patient. We describe the second such patient with BBS and hyperammonemia, associated with fluctuating mental impairment. The patient was a 17-year-old boy with BBS. Esophageal, gastric, and rectal varices and mild hepatic dysfunction started to develop at 5 years of age. A liver biopsy showed dilated portal veins with mild fibrosis in portal tract. From the age of 17 years, he often had forced laughter with apparently normal consciousness. Laboratory examinations revealed hyperammonemia (112.2mg/ml). Oral medication lowered the blood ammonia level to 69.9 mg/ml, reduced the frequency of forced laughter, and improved his IQ. Patients with BBS may have additional diseases or conditions that affect mental status, such as hyperammonemia. Physicians should explore the underlying causes of these conditions and treat such patients, who already have a compromised quality of life.

Naturally- and experimentally-designed restorations of the Parkin gene deficit in autosomal recessive juvenile parkinsonism.

Intranuclear events due to mutations in the Parkin gene remain elusive in autosomal recessive juvenile parkinsonism (ARJP). We identified a mutant PARKIN protein in fibroblast cultures from a pair of siblings with ARJP who were homozygous for the exon 4-deleted Parkin gene. Disease was mild in one patient and debilitating in the other. The detected mutant, encoded by a transcript lacking exon 3 as well as exon 4, is an in-frame deletion that removes 121 aa, resulting in a 344-aa protein (PaDel3,4). Cell culture and transfection studies revealed negative correlations between expression levels of PaDel3,4 and those of cell cycle proteins, including cyclin E, CDK2, ppRb, and E2F-1, and demonstrated that GFP-PaDel3,4 entered nucleus and ubiquitinated cyclin E as a part of SCF(hSel-10) ligase complex in the patient cells. In addition, nuclear localization signal-tagged PaDel3,4 expressed in the transfected patient cells most effectively ubiquitinated cyclin E and reduced DNA damage, protecting cells from oxidative stress. Antisense-oligonucleotide treatment promoted skipping of exon 3 and thus generated PaDel3,4, increasing cell survival. Collectively, we propose that naturally- and experimentally-induced exon skipping at least partly restores the mutant Parkin gene deficit, providing a molecular basis for the development of therapeutic exon skipping.