Natsue Shimizu

Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant, neurodegenerative disorder that affects the cerebellum and other areas of the central nervous system. We have devised a novel strategy, the direct identification of repeat expansion and cloning technique (DIRECT), which allows selective detection of expanded GAG repeats and cloning of the genes involved. By applying DIRECT, we identified an expanded CAG repeat of the gene for SCA2. CAG repeats of normal alleles range in size from 15 to 24 repeat units, while those of SCA2 chromosomes are expanded to 35 to 59 repeat units. The SCA2 cDNA is predicted to code for 1,313 amino acids — with the CAG repeats coding for a polyglutamine tract. DIRECT is a robust strategy for identification of pathologically expanded trinucleotide repeats and will dramatically accelerate the search for causative genes of neuropsychiatric diseases caused by trinucleotide repeat expansions.

Atrophy of the cerebellum and brainstem in dentatorubral pallidoluysian atrophy. Influence of CAG repeat size on MRI findings.

To elucidate how the size of the expanded CAG repeat of the gene for dentatorubral pallidoluysian atrophy (DRPLA) and other factors affect the atrophy of the brainstem and cerebellum, and the appearance of high-intensity signals on T2-weighted MRI of the cerebral white matter of patients with DRPLA, we quantitatively analyzed the MRI findings of 26 patients with DRPLA, the diagnosis of which was confirmed by molecular analysis of the DRPLA gene. When we classified the patients into two groups based on the size of the expanded CAG repeat of the DRPLA gene (group 1, number of CAG repeat units≥66; group 2, number of CAG repeat units ≤65), we found strong inverse correlations between the age at MRI and the areas of midsagittal structures of the cerebellum and brainstem in group 1 but not in group 2. Multiple regression analysis, however, revealed that both the patients age at MRI and the size of the expanded CAG repeat correlated with the areas of midsagittal structures. Involvement of the cerebral white matter as detected on T2-weighted images was observed more frequently in patients belonging to group 2 than in group 1 patients. Furthermore it was demonstrated that high-intensity signals can be detected on T2-weighted images of the cerebral white matter of patients with a largely expanded CAG repeat (group 1) in their thirties. These results suggest that patient age as well as the size of the expanded CAG repeat are related to the degree of atrophy of the brainstem and cerebellum, and the white matter changes in patients with DRPLA.

Increased interleukin-6 of skin and serum in amyotrophic lateral sclerosis

Studies of skin in amyotrophic lateral sclerosis (ALS) have demonstrated morphological and biochemical alterations. Interleukin-6 (IL-6) has been suggested to have a trophic effect in nerve cells and to have a direct pathogenic role in neurodegenerative central nervous system (CNS) disorders. However, little is known concerning IL-6 in ALS patients. We examined IL-6 immunoreactivity of biopsy specimens of skin and measured serum IL-6 levels from 11 ALS patients and 11 diseased control subjects. IL-6 immunoreactivity was markedly positive in the epidermis and dermal blood vessels and glands and was moderately positive in the reticular dermis in all ALS patients. These optical densities for IL-6 immunoreactivity in ALS patients were significantly higher than in control subjects, and were significantly increased with duration of illness. Serum IL-6 levels were detected in 8 (73%) of 11 ALS patients compared with only 1 (9%) of 11 diseased control subjects. Serum IL-6 levels were significantly correlated with duration of illness in ALS patients and immunoreactivity of IL-6 of the epidermis. These data suggest that the increased levels of serum IL-6 may reflect an increased IL-6 immunoreactivity of skin in ALS patients.

A clinical and genetic study in a large cohort of patients with spinocerebellar ataxia type 6

AbstractIn 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.

Loss of catecholaminergic neurons in the medullary reticular formation in myotonic dystrophy

Objective: To clarify the possible relation between the extent of involvement of catecholaminergic neurons and the presence of alveolar hypoventilation in patients with myotonic dystrophy (MyD). Background: Respiratory insufficiency has been reported frequently in MyD patients. Recent data support the hypothesis that this respiratory failure results from a primary dysfunction of the CNS. Methods: The authors performed a quantitative immunoreactive study of tyrosine hydroxylase immunoreactive (TH+) neurons linked to hypoventilation in the dorsal central medullary nucleus (DCMN), the ventral central medullary nucleus (VCMN), and the subtrigeminal medullary nucleus (SMN)-where the automatic respiratory center is thought to be located-in eight MyD patients and in 10 age-matched control subjects. Alveolar hypoventilation of the central type was present in three of the MyD patients but not in the remaining MyD patients or the control subjects. Results: The densities of TH+ neurons of the DCMN, the VCMN, and the SMN in MyD patients with hypoventilation were significantly lower than in those without hypoventilation (p < 0.02, p < 0.01, and p < 0.01, respectively) and control subjects (p < 0.01, p < 0.01, and p < 0.01, respectively). Conclusions: These data suggest that the loss of TH+ neurons of the DCMN, the VCMN, and the SMN is associated with the presence of hypoventilation in MyD and may be an important feature of MyD.

Decreased type IV collagen of skin and serum in patients with amyotrophic lateral sclerosis

Objective: To study type IV collagen of skin and serum in patients with ALS. Background: Collagen abnormalities of skin have been reported in ALS patients. However, little is known concerning type IV collagen in ALS. Methods: We studied type IV collagen immunoreactivity of skin and measured serum levels of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in patients with ALS and control subjects. Results: The basement membrane as well as blood vessels of skin in ALS patients was weakly positive for type IV collagen as compared with those of diseased control subjects. This weak immunostaining became more pronounced as ALS progressed. The optical density for type IV collagen immunoreactivity in ALS patients was significantly lower (p < 0.001) than in diseased control subjects and was significantly decreased with duration of illness(r = -0.85, p < 0.01). Serum 7S collagen levels in patients with ALS were significantly decreased (p < 0.01) as compared with those in diseased and healthy control subjects and were negatively and significantly associated with duration of illness (r= -0.81, p < 0.001). There was an appreciable positive correlation between concentrations of serum 7S collagen and the density for type IV collagen immunoreactivity in ALS patients (r = 0.81, p < 0.02). Conclusions: These data suggest that a metabolic alteration of type IV collagen may take place in the skin of ALS patients and that the decreased levels of serum 7S collagen may reflect a decreased type IV collagen immunoreactivity of skin in patients with ALS.

Collagen abnormalities in the spinal cord from patients with amyotrophic lateral sclerosis

During the last 10 years, we have demonstrated morphological and biochemical abnormalities of skin extracellular matrices in amyotrophic lateral sclerosis (ALS). However, currently little is known concerning collagen of the spinal cord in ALS. We measured the amount of collagen and characterized collagen at light and electron microscopic levels in posterior funiculus, posterior half of lateral funiculus and anterior horn of cervical enlargement of the spinal cord obtained from ten patients with ALS, 11 patients with other neurologic diseases (control group A), and ten patients without neurologic ones (control group B). In posterior half of lateral funiculus and anterior horn, (1) by light microscopy, there was no significant difference in vessel wall area between ALS patients and control groups A and B; (2) ultrastructurally, collagen bundles were more fragmented and widely separated, and the fibrils were randomly oriented in the perivascular space of capillaries in ALS patients, which were not observed in any areas of control groups or in posterior funiculus of ALS patients; and (3) the collagen contents in ALS were significantly lower (P<0.001 and P<0.001, respectively) than those in control groups A and B. Fragmented and widely separated collagen bundles in the interstitial tissue surrounding capillaries and markedly decreased amount of collagen in posterior half of lateral funiculus and in anterior horn of ALS could be related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS, that is, selective neuronal vulnerability in ALS.

Alterations of skin glycosaminoglycans in patients with ALS

Background and Objective: Collagen abnormalities of skin have been reported among patients with ALS. However, little is known concerning glycosaminoglycans of the skin in ALS. Our objective was to clarify morphologic and biochemical findings of skin glycosaminoglycans among patients with ALS. Methods: We performed morphologic studies and biochemical analysis of glycosaminoglycans of skin from 8 patients with ALS, 6 patients with other neurologic or muscular diseases (control group A), and 7 patients without neurologic disorders (control group B). Results: The wide spaces that separate collagen bundles reacted strongly with Alcian blue stain in skin from patients with ALS and stained more markedly as ALS progressed. Staining with Alcian blue was virtually eliminated by Streptomyces hyaluronidase. The content of hyaluronic acid was significantly higher (p < 0.001) among patients with ALS than in control groups A and B. There was a significant positive correlation between content of hyaluronic acid and duration of illness among patients with ALS (r = 0.88, p < 0.01). However, there was no significant difference in content of dermatan sulfate, chondroitin sulfate-4S, or chondroitin sulfate-6S between patients with ALS and control groups A and B. There was also an appreciable positive correlation between optical density of Alcian blue and content of hyaluronic acid among patients with ALS (r = 0.92, p < 0.01). Conclusions: The data suggest that a metabolic alteration of glycosaminoglycans related to the increased amount of hyaluronic acid may take place in the skin of patients with ALS.

Eye-head co-ordination in patients with Parkinsonism and cerebellar ataxia.

Eye-head co-ordination of patients with Parkinsonism and cerebellar ataxia was investigated and compared with that of normal subjects. In Parkinsonian patients eye-head co-ordination was of the same pattern as normals, with an accurate and stable gaze. Reaction times for both eyes and head, however, were prolonged. It was also noted that the contribution of head movements to gaze shift was abnormally large and that the gaze accuracy was decreased when the head was immobilised. In patients with cerebellar ataxia, gaze was dysmetric, often hypermetric, and was unstable during head movements. The contribution of head movements to gaze was also large. It is concluded that Parkinsonism and cerebellar diseases influence eye-head co-ordination differently.

Increased expression of insulin-like growth factor I in skin in amyotrophic lateral sclerosis

OBJECTIVES Insulin-like growth factor I (IGF-I) has potent effects on motor neuron survival and is being studied as a possible therapeutic agent for ALS. However, little is known concerning IGF-I in the skin of patients with amyotrophic lateral sclerosis (ALS). The aim was to evaluate IGF-I immunoreactivity of skin in patients with ALS. METHODS IGF-I immunoreactivity of skin from 18 patients with ALS and 16 controls was examined. RESULTS IGF-I immunoreactivity was markedly positive in the epidermis and dermal blood vessels and glands and was moderately positive in the reticular dermis in all patients with ALS. On the other hand, the epidermis and dermal blood vessels and glands and the reticular dermis showed a weak IGF-I immunoreactivity in controls. The optical density for IGF-I immunoreactivity of the epidermis and dermal blood vessels and glands, and the reticular dermis in patients with ALS was significantly higher than in diseased controls, and was significantly increased with duration of illness. CONCLUSIONS These data suggest that a metabolic alteration of IGF-I may take place in the skin of patients with ALS.