Akihiro Koumura

Patterns of levels of biological metals in CSF differ among neurodegenerative diseases

We measured the levels of some biological metals: copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn) in the cerebrospinal fluid (CSF) in patients with neurodegenerative diseases (52 patients with amyotrophic lateral sclerosis (ALS)), 21 patients with Alzheimers disease (AD), and 20 patients with Parkinsons disease (PD) by inductively coupled plasma mass spectrometry (ICP-MS). The diagnoses were additionally supported by neuroimaging techniques for AD and PD. In ALS, the levels of Mg (p<0.01 significant difference), Fe, Cu (p<0.05), and Zn (p<0.10) in CSF were higher than those in controls. Some patients showed very high levels of Cu and Zn before the critical deterioration of the disease. In AD, the levels of Cu and Zn in CSF were significantly higher in patients with late-onset AD (p<0.01). In PD, we found significantly increased levels of especially Cu and Zn in particular (p<0.01) and Mn (p<0.05) in CSF. A multiple comparison test suggested that the increased level of Mg in ALS and that of Mn in PD were the pathognomonic features. These findings suggest that Cu and Zn in particular play important roles in the onset and/or progression of ALS, AD, and PD. Therefore, Cu-chelating agents and modulators of Cu and Zn such as metallothionein (MT) can be new candidates for the treatment of ALS, AD, and PD.

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.

Combination Treatment with Normobaric Hyperoxia and Cilostazol Protects Mice against Focal Cerebral Ischemia-Induced Neuronal Damage Better Than Each Treatment Alone

Normobaric hyperoxia (NBO) and cilostazol (6-[4-(1-cyclohexy-1H-tetrazol-5-yl)butoxyl]-3,4-dihydro-2-(1H)-quinolinone) (a selective inhibitor of phosphodiesterase 3) have each been reported to exert neuroprotective effects against acute brain injury after cerebral ischemia in rodents. Here, we evaluated the potential neuroprotective effects of combination treatment with NBO and cilostazol against acute and subacute brain injuries after simulated stroke. Mice subjected to 2-h filamental middle cerebral artery (MCA) occlusion were treated with NBO (95% O2, during the ischemia) alone, with cilostazol (3 mg/kg i.p. after the ischemia) alone, with both of these treatments (combination), or with vehicle. The histological and neurobehavioral outcomes were assessed at acute (1 day) or subacute (7 days) stages after reperfusion. We measured regional cerebral blood flow (rCBF) during and after ischemia by laser-Doppler flowmetry and recovery (versus vehicle) in the combination therapy group just after reperfusion. Mean acute and subacute lesion volumes were significantly reduced in the combination group but not in the two monotherapy groups. The combination therapy increased endothelial nitric-oxide synthase (eNOS) activity in the lesion area after ischemia versus vehicle. Combination therapy with NBO plus cilostazol protected mice subjected to focal cerebral ischemia by improvement of rCBF after reperfusion, in part in association with eNOS activity.

Fasudil and ozagrel in combination show neuroprotective effects on cerebral infarction after murine middle cerebral artery occlusion

Rho kinase (ROCK), one of the serine/threonine kinases, is involved in pathologic conditions, and its activation causes neuronal cell death. Fasudil, a selective ROCK inhibitor, has been reported to cause increased cerebral blood flow (CBF) in the ischemic brain and protect against neuronal cell death by inhibiting ROCK. Ozagrel, a thromboxane A2 synthase inhibitor, inhibits platelet aggregation and causes vasodilatation, thereby increasing CBF in cerebral thrombosis. The present study evaluates the combination therapy of fasudil and ozagrel on focal brain ischemia induced by middle cerebral artery occlusion (MCAO) in mice. Each monotherapy of fasudil at 10 mg/kg i.p. and ozagrel at 30 mg/kg i.p. significantly reduced cerebral infarction. The combination therapy of fasudil (3 mg/kg i.p.) and ozagrel (10 mg/kg i.p.), which are noneffective doses, resulted in reduction of cerebral infarction, and the protective effect was observed up to 5 min, but not 3 h, after reperfusion. Regional CBF after MCAO and phosphorylation of endothelial nitric-oxide synthase (NOS) significantly increased in response to the combination therapy, whereas these effects were not observed with monotherapy of either drug. The protective effect of combination treatment was antagonized by the treatment of a NOS inhibitor, nitro-l-arginine methyl ester hydrochloride. These findings indicate that the combination treatment of fasudil and ozagrel exhibits additive effects for neuroprotection after MCAO. These findings indicate that the combination treatment of fasudil and ozagrel may be useful as a potential therapeutic strategy for the treatment of stroke.

Metallothionein-3 deficient mice exhibit abnormalities of psychological behaviors

Metallothioneins (MTs) are metal binding proteins and have four isoforms. MT-3, known as growth inhibitory factor (GIF), exists mainly in the central nervous system. It regulates zinc levels and exhibits a neuroprotective effect in the various types of brain diseases. However, the reports demonstrate that the relation between MT-3 and psychiatric disorder is still unknown. In the present study, the authors carried out behavioral tests on MT-3 knock-out (KO) mice. The duration of the MT-3 KO mices social interactions were significantly shorter than that of the wild-type (WT) mice. The acoustic startle response of the MT-3 KO mice showed diminished prepulse inhibition (PPI) at all prepulse intensities. However, the locomotor activity tests of the MT-3 KO mice displayed normal circadian rhythm, activity, and habituation to a novel environment. In the novel object recognition test, the MT-3 KO mice exhibited normal memory. These findings indicate that abnormalities of psychological behavior were observed in the MT-3 KO mice. Further experiments will be needed to clarify the involvement of MT-3 in higher brain function.

Progressive multifocal leukoencephalopathy and CD4+ T-lymphocytopenia in a patient with Sjögren syndrome

We report progressive multifocal leukoencephalopathy (PML) and CD4+ T-lymphocytopenia in a 71-year-old man with Sjögren syndrome (SjS). The patient was admitted to our hospital because of progressive dementia and gait disturbance. T2-weighted MR images showed high-intensity lesions in his left frontal white matter thalamus, cerebellum and brainstem. A pathological diagnosis of PML was made by brain biopsy. SjS is frequently accompanied with immunological complications; however, there are few reports on PML in patients with SjS. Recently, isolated CD4+ T-lymphocytopenia is reported to be one of the based immunological conditions associated with the development of PML. In the present case, CD4+ T-lymphocytopenia was also observed on admission, which is also associated with SjS.

Antibodies in patients with neuropsychiatric systemic lupus erythematosus

Objective: To investigate a target for antibodies in patients with neuropsychiatric systemic lupus erythematosus (NPSLE). Background: Pathogenesis of NPSLE may be related to autoantibody-mediated neural dysfunction, vasculopathy, and coagulopathy. However, very few autoantibodies are sensitive and specific to NPSLE because the neuropsychiatric syndromes associated with SLE are diverse in cause and presentation. Methods: We identified antibodies against brain antigens in the sera of 7 patients with NPSLE and 12 healthy controls by 2-dimensional electrophoresis, followed by Western blotting and liquid chromatography–tandem mass spectrometry (LC-MS/MS), using rat brain proteins as the antigen source. Results: Six antibodies were detected in patients with NPSLE. One of these 6 antibodies was found in antibodies against Rab guanosine diphosphate dissociation inhibitor α (αGDI) (which is specifically abundant in neurons and regulates synaptic vesicle exocytosis) in patients with NPSLE with psychosis. We tested more samples by 1-dimensional immunoblotting of human recombinant αGDI. Positivity of the anti-αGDI antibody was significantly higher in patients with NPSLE with psychosis (80%, 4 of 5) than in patients with NPSLE without psychosis (0%, 0 of 13), patients with systemic lupus erythematosus without neuropsychiatric symptoms (5.3%, 1 of 19), patients with multiple sclerosis (0%, 0 of 12), patients with infectious meningoencephalitis (0%, 0 of 13), patients with polyneuropathy (0%, 0 of 10), patients with psychotic syndromes (0%, 0 of 10), and healthy controls (0%, 0 of 12). Conclusions: We propose that the anti–Rab guanosine diphosphate dissociation inhibitor α antibody is a candidate for further exploration as diagnostic marker of psychosis associated with neuropsychiatric systemic lupus erythematosus.

Cardiac sympathetic function in the patients with amyotrophic lateral sclerosis: analysis using cardiac [123I] MIBG scintigraphy

Amyotrophic lateral sclerosis (ALS), which is the most serious form of degenerative motor neuron disease in adults, is characterized by upper and lower motor neuron degeneration, skeletal muscle atrophy, paralysis, and death. Some patients with respiratory-dependent ALS die of sudden cardiac arrest or anoxic encephalopathy following circulatory collapse, which may be associated with sympathetic hyperactivity. Cardiac [123I] MIBG scintigraphy is a diagnostic method of cardiac sympathetic function. However, few reports have addressed cardiac sympathetic function in ALS patients using this technique. We investigated cardiac sympathetic function in 63 ALS patients and 10 healthy volunteers using cardiac [123I] metaiodobenzylguanidine (MIBG) scintigraphy [heart/mediastinum ratio (H/M ratio) in the early phase and washout ratio (WR)] at the time of diagnosis. The WR of cardiac [123I] MIBG scintigraphy, which indicates cardiac sympathetic activity, was significantly increased in ALS patients compared with controls. ALS patients with an increased WR exhibited a significantly higher progression rate compared with those with normal WR. Moreover, the survival of ALS patients with increased WR was significantly decreased compared with those with normal WR. These results suggested that some patients with ALS have sympathetic hyperactivity at the time of diagnosis. ALS patients may suffer from chronic cardiac sympathetic hyperactivity, which is associated with sudden cardiac death and stress induced cardiomyopathy. Increased WR in cardiac [123I] MIBG scintigraphy may be a predictive factor in ALS patients.

High prevalence of autoantibodies against phosphoglycerate mutase 1 in patients with autoimmune central nervous system diseases.

We identified the autoantibody against phosphoglycerate mutase 1 (PGAM1), which is a glycolytic enzyme, in sera from multiple sclerosis (MS) patients by proteomics-based analysis. We further searched this autoantibody in sera from patients with other neurological diseases. The prevalence of the anti-PGAM1 antibody is much higher in patients with MS and neuromyelitis optica (NMO) than in those with other neurological diseases and in healthy controls. It was reported that the anti-PGAM1 antibody is frequently detected in patients with autoimmune hepatitis (AIH). Results of our study suggest that the anti-PGAM1 antibody is not only a marker of AIH but also a nonspecific marker of central nervous system autoimmune diseases.

Identification of antibodies as biological markers in serum from multiple sclerosis patients by immunoproteomic approach

We identified the antibody against mitochondrial heat shock protein 70 (mtHSP70) in serum from multiple sclerosis (MS) patients by proteomics-based analysis. The prevalence of the anti-mtHSP70 antibody is significantly higher in serum from MS patients than in serum from Parkinson disease patients, multiple cerebral infarction patients, infectious meningoencephalitis patients, and healthy controls (HCs) (68% sensitivity; 74% specificity). We studied the clinical features and magnetic resonance imaging findings of MS patients with the anti-mtHSP70 antibody. As a result, there were no significant differences between the anti-mtHSP70-antibody-positive and -negative MS patients. Additionally, in our comprehensive analysis of the prevalence of both the anti-mtHSP70 antibody and the anti-phosphoglycerate mutase 1 (PGAM1) antibody, which was previously reported by us to also show a higher prevalence in serum from MS patients, the positivity rates of both these antibodies were significantly higher in serum from MS patients than in serum from patients with other neurological diseases and from HCs; moreover, the specificity of this combination assay was higher than that of the assay of only one antibody (57% sensitivity; 93% specificity). Results of our study suggest that not only the anti-PGAM1 antibody but also the anti-mtHSP70 antibody is good diagnostic markers of MS and the combination of both these antibodies is useful for a more specific diagnosis of MS.