Y.K. Hayashi

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.

Primary collagen VI deficiency is the second most common congenital muscular dystrophy in Japan.

Objectives: To determine the frequency of primary collagen VI deficiency in congenital muscular dystrophy (CMD) in Japan and to establish the genotype-phenotype correlation. Methods: We performed immunohistochemistry for collagen VI in muscles from 362 Japanese patients with CMD, and directly sequenced the three collagen VI genes, COL6A1, COL6A2, and COL6A3, in patients found to have collagen VI deficiency. Results: In Japan, primary collagen VI deficiency accounts for 7.2% of congenital muscular deficiency. Among these patients, five had complete deficiency (CD) and 29 had sarcolemma-specific collagen VI deficiency (SSCD). We found two homozygous and three compound heterozygous mutations in COL6A2 and COL6A3 in all five patients with CD, and identified heterozygous missense mutations or in-frame small deletions in 21 patients with SSCD in the triple helical domain (THD) of COL6A1, COL6A2, and COL6A3. All mutations in SSCD were sporadic dominant. No genotype-phenotype correlation was seen. Conclusion: Primary collagen VI deficiency is the second most common CMD after Fukuyama type CMD in Japan. Dominant mutations located in the N-terminal side from the cysteine residue in the THD of COL6A1, COL6A2, and COL6A3 are closely associated with SSCD.

High frequency of calcification in basal ganglia on brain computed tomography images in Japanese older adults

To investigate the frequency of calcification in the basal ganglia and the dentate nuclei in the cerebellum, and compare the difference in age and area, we examined the brain computed tomography (CT) images of all patients in two representative university hospitals in Japan.

NOVEL FHL1 MUTATIONS IN FATAL AND BENIGN REDUCING BODY MYOPATHY

Reducing body myopathy (RBM) is a rare disorder characterized pathologically by the presence of intracytoplasmic inclusions strongly stained by menadione-NBT (nitroblue tetrazolium) staining in the absence of the substrate α-glycerophosphate. The causative gene for RBM was recently identified as FHL1 on chromosome Xq27 encoding four and a half LIM domains 1.1 FHL1 is a 32 kDa protein, composed of four LIM domains preceded by a single N-terminal zinc finger. FHL1 is highly expressed in skeletal muscle and heart. Here, we searched for FHL1 mutations in three sporadic cases2-4 and one familial case5 of RBM we previously reported. ### Methods. All clinical materials used in this study were obtained for diagnostic purpose with informed consent. Patient 1 and patient 2 have fatal infantile form,2,3 and patient 3 has adult-onset form.4 Patients 4 (son) and 5 (his mother) had familial cases.5 We directly sequenced all exons and their flanking intronic regions of FHL1 in the five RBM patients and 250 Japanese controls. Frozen muscle specimens were examined by immunohistochemistry and immunoblotting using standard technique. ### Results. We identified four novel mutations in FHL1 : a heterozygous missense mutation of c.449G>A (p.C150Y) in patient 1 and c.302G>T (p.C101F) in patient 2, an in-frame 9 bp deletion at c.304-312delAAGGGGTGC (p.102-104delKFC) in patient 3, and a hemizygous mutation c.310T>C (p.C104R) in patient 4. The mother (patient 5) had the same mutation in heterozygous mode. All mutations we identified are located in the second LIM domain of FHL1 (figure e-1 on the Neurology ® Web site at www.neurology.org). Immunohistochemical analysis of …

Nuclear changes in skeletal muscle extend to satellite cells in autosomal dominant Emery-Dreifuss muscular dystrophy/limb-girdle muscular dystrophy 1B

Autosomal forms of Emery-Dreifuss muscular dystrophy (AD-/AR-EDMD) and limb-girdle muscular dystrophy type 1B (LGMD1B) are caused by mutations in the gene encoding A-type lamins (LMNA). A-type lamins are major components of nuclear lamina and known to have important roles in maintaining nuclear integrity. LMNA mutations are also suggested to cause reduced myogenic differentiation potentials, implying that satellite cell nuclei in AD-EDMD/LGMD1B are likewise affected. We examined nuclear changes of skeletal muscles including satellite cells from four patients with AD-EDMD/LGMD1B by light and electron microscopy. We found that 92.5+/-5.0% of myonuclei had structural abnormalities, including shape irregularity and/or chromatin disorganization, and the presence of peri-/intranuclear vacuoles. Chromatin changes were also observed in 50% of the satellite cell nuclei. Increased number of Pax7-positive nuclei, but fewer number of MyoD-positive nuclei were seen on immunohistochemical analyses, suggesting functional alteration of satellite cells in addition to the nuclear morphological changes in AD-EDMD/LGMD1B.

Metallothionein-III prevents neuronal death and prolongs life span in amyotrophic lateral sclerosis model mice

Defect of metallothionein-III (MT-III) has been reported to be a contributor to the progression of amyotrophic lateral sclerosis (ALS). We explored the expression and effects of MT-III on the motor neurons of spinal cords of ALS model mice (G93A Cu/Zn superoxide dismutase (SOD-1) mutant-transgenic (Tg) mice) using a retrograde viral delivery system. Once-weekly injection of the adenovirus encoding LacZ or MT-III gene was started at the age of 20 weeks, which was the mean age of ALS onset. Gene expression was detected in the motor neurons of the lumbar spinal cord. At 160 days of age (14 days after injection), the mean numbers of Nissl-stained α neurons were 15.42±5.32, 16.50±1.35, and 24.75±4.01 in 5-μm sections of the lumbar hemispinal cord from the untreated group, LacZ group, and MT-III group, respectively. The mean durations of illness were 15.20±5.30 days, 10.33±4.27 days, and 25.71±7.67 days in the untreated group, LacZ group, and MT-III group, respectively. The mean life spans were 163.20±7.72 days, 159.50±3.27 days, and 178.14±12.97 days in the untreated group, LacZ group, and MT-III group, respectively. We demonstrated that MT-III prevents the loss of motor neurons of ALS model mice and prolongs the life span, even when the administration is started at the time of onset.

Reduced cell anchorage may cause sarcolemma-specific collagen VI deficiency in Ullrich disease.

Background: COL6 gene mutations are associated with Ullrich congenital muscular dystrophy (UCMD), which is clinically characterized by muscle weakness from early infancy, hyperlaxity of distal joints, and multiple proximal joint contractures. We previously reported that the majority of patients with UCMD have sarcolemma-specific collagen VI deficiency (SSCD). More recently, we found heterozygous COL6A1 glycine substitutions in patients with UCMD with SSCD. Objective: To elucidate how COL6A1 glycine mutation leads to SSCD. Methods: We evaluated the synthesis, formation, and binding of collagen VI to the extracellular matrix in fibroblasts with p.G284R mutation in COL6A1. Results: Collagen VI was normally secreted into the cultured medium in fibroblasts harboring p.G284R mutation. When the medium with normal collagen VI was added to collagen VI-deficient fibroblast culture, collagen VI bound surrounding the cells, while collagen VI with p.G284R mutation did not. Cell adhesion of fibroblasts with p.G284R mutation was markedly reduced similarly to that of collagen VI–deficient cells. Interestingly, this reduction in adhesion of the cells with p.G284R mutation was recovered by the addition of the medium with normal collagen VI, which would suggest a therapeutic strategy for a replacement therapy. Conclusion: Heterozygous glycine substitution in COL6A1 may cause decreased binding of collagen VI microfibrils to the extracellular matrix resulting in sarcolemma-specific collagen VI deficiency.

Association between metallothionein genes polymorphisms and sporadic amyotrophic lateral sclerosis in a Japanese population

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease that selectively affects motor neurons. Reactive oxygen species (ROS) are assumed to be involved in the pathogenesis of ALS. Metallothioneins (MTs) are self‐protective, multifunctional proteins that scavenge ROS. In particular, metallothionein‐III (MT‐III) has a strong scavenging effect on hydroxyl radicals. MTs have been suggested to have important roles in the pathophysiology of ALS. Therefore we investigated single nucleotide polymorphisms (SNPs) of the MT‐III and the metallothionein‐IIA (MT‐IIA) promoter region in 37 Japanese SALS cases and 206 sex‐matched healthy controls using polymerase chain reaction (PCR)‐direct sequencing or PCR‐temporal temperature gradient gel electrophoresis (TTGE). We detected no SNPs of the MT‐III gene in SALS cases and controls, and no detectable association between SALS phenotypes and a SNP of the MT‐IIA promoter region. We conclude that gene polymorphisms of MT‐IIA promoter region and MT‐III gene are not associated with SALS phenotypes in a Japanese population.

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.

High Levels of Copper, Zinc, Iron and Magnesium, but not Calcium, in the Cerebrospinal Fluid of Patients with Fahr's Disease.

Patients with marked calcification of the basal ganglia and cerebellum have traditionally been referred to as having Fahr’s disease, but the nomenclature has been criticized for including heterogeneous etiology. We describe 3 patients with idiopathic bilateral striatopallidodentate calcinosis (IBSPDC). The patients were a 24-year-old man with mental deterioration, a 57-year-old man with parkinsonism and dementia, and a 76-year-old woman with dementia and mild parkinsonism. The former 2 patients showed severe calcification of the basal ganglia and cerebellum, and the latter patient showed severe calcification of the cerebellum. We found significantly increased levels of copper (Cu), zinc (Zn), iron (Fe) and magnesium (Mg), using inductively coupled plasma mass spectrometry in the CSF of all these 3 patients. The increased levels of Cu, Zn, Fe and Mg reflect the involvement of metabolism of several metals and/or metal-binding proteins during the progression of IBSPDC. More numerous patients with IBSPDC should be examined in other races to clarify the common mechanism of the disease and to investigate the specific treatment.