Elevation of MCP-1 and MCP-1/VEGF ratio in cerebrospinal fluid of amyotrophic lateral sclerosis patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with progressive cell death of upper and lower motor neurons. In this study, we measured monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) levels in cerebrospinal fluid (CSF) and serum by enzyme-linked immunosorbent assay (ELISA) in 42 ALS patients, and compared these levels with those of control subjects with other neurodegenerative disorders or with those of normal controls. MCP-1 levels in CSF were significantly higher in ALS patients than in the control group. VEGF levels in CSF tended to be lower in ALS patients than in the control group, but not significantly. A positive correlation was found between MCP-1 levels in CSF of ALS patients and the total Norris scale. The elevation of MCP-1/VEGF ratio in CSF was more specific to ALS patients compared to other neurological diseases such as Parkinson's disease (PD) and spinocerebellar ataxia (SCA) and to controls. Our data suggested that both MCP-1 levels and MCP-1/VEGF ratio in CSF may be useful markers for the clinical diagnosis of ALS.

Increased ER stress during motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis.

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. ER stress is also thought to be involved in the pathogenesis of neurodegenerative diseases. In this study, we investigated whether ER stress is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) using the anterior part of the lumbar spinal cord of transgenic mice carrying a mutation (G93A) in the superoxide dismutase 1 (SOD1) gene. Western blot and immunohistochemical analyses demonstrated that the expressions of p-PERK and p-eIF2alpha were increased in the microsome fraction (P3) of the lumbar spinal cord at the pre-symptomatic age of 12 weeks (12W), while the expression of activated caspase-12 was increased in the cytoplasmic fraction (S3) of the lumbar spinal cord at both the pre-symptomatic age of 12W and the late symptomatic age of 20W. In contrast, GRP78 did not show any increases in the microsome fraction (P3) of the lumbar spinal cord at either the pre-symptomatic or symptomatic ages. Thus, the present results strongly suggest that the balance between anti- and pro-apoptotic proteins related to ER stress is impaired from the pre-symptomatic stage in this ALS mouse model, and that this imbalance may be related to the pathogenesis of motor neuron degeneration in ALS.

Therapeutic benefit of intrathecal injection of insulin-like growth factor-1 in a mouse model of Amyotrophic Lateral Sclerosis.

Insulin-like growth factor (IGF)-1 has been shown to have a protective effect on motor neurons both in vitro and in vivo, but has limited efficacy in patients with amyotrophic lateral sclerosis (ALS) when given subcutaneously. To examine the possible effectiveness of IGF-1 in a mouse model of familial ALS, transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) with a G93A mutation were treated by continuous IGF-1 delivery into the intrathecal space of the lumbar spinal cord. We found that the intrathecal administration of IGF-1 improved motor performance, delayed the onset of clinical disease, and extended survival in the G93A transgenic mice. Furthermore, it increased the expression of phosphorylated Akt and ERK in spinal motor neurons, and partially prevented motor neuron loss in these mice. Taken together, the results suggest that direct administration of IGF-1 into the intrathecal space may have a therapeutic benefit for ALS.

Beneficial effects of intrathecal IGF-1 administration in patients with amyotrophic lateral sclerosis.

OBJECTIVES: There is currently no effective pharmacological treatment for amyotrophic lateral sclerosis (ALS). In a transgenic mouse model of ALS, intrathecal infusion of insulin-like growth factor (IGF)-1 showed a promising increase in survival. We performed a double-blind clinical trial to assess the effect of intrathecal administration of IGF-1 on disease progression in patients with ALS. METHODS: Nine patients with ALS were randomly assigned to receive either a high dose (3 microg/kg of body weight) or low dose (0.5 microg/kg of body weight) of IGF-1 every 2 weeks for 40 weeks. The outcome measurements were the rate of decline of bulbar and limb functions (Norris scales) and forced vital capacity. RESULTS: The high-dose treatment slowed a decline of motor functions of the ALS patients in total Norris and limb Norris scales, but not in bulbar Norris or vital capacity. The intrathecal administration of IGF-1 had a modest but significant beneficial effect in ALS patients without any serious adverse effects. DISCUSSION: Intrathecal IGF-1 treatment could provide an effective choice for ALS although further studies in more patients are needed to confirm its efficacy and optimize dosages of IGF-1.

Hypoxic induction of vascular endothelial growth factor is selectively impaired in mice carrying the mutant SOD1 gene.

Localization and hypoxic induction of vascular endothelial growth factor (VEGF) was examined in the spinal cord of transgenic mice carrying a mutation in the superoxide dismutase 1 gene. Immunohistochemical and immunofluorescent study demonstrated that VEGF is mainly expressed in motor neurons before and after hypoxia. Baseline expression of VEGF was higher in transgenic (Tg) mice than in wild-type (Wt) littermates. However, VEGF was hardly induced after hypoxia in Tg mice, whereas Wt mice showed an approximate nine-fold increase. Impaired VEGF induction was evident in Tg mice at 12 weeks of age, when they were still presymptomatic. In contrast, baseline and hypoxic expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor did not differ between Tg and Wt mice. Thus, the present study demonstrates that hypoxic induction of VEGF in Tg mice is selectively impaired from a very early stage, suggesting profound involvement in the pathogenesis of motor neuron degeneration in this animal model of amyotrophic lateral sclerosis.

Change in superoxide dismutase 1 protein localization towards mitochondria: an immunohistochemical study in transgenic G93A mice.

Localization of superoxide dismutase1 (SOD1) and mitochondrial glucose-regulated protein 75 (Grp75), were examined in the spinal cords of transgenic (Tg) mice expressing human mutant SOD1 protein (G93A) and wild-type (Wt) controls at 8, 20 and 32 weeks. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons. Colocalization of Grp75 and SOD1 signals was demonstrated in Wt and presymptomatic Tg animals, while it was lost in Tg mice at a symptomatic age. The present results suggest that loss of SOD1 protein from mitochondria could contribute to motor neuron damage.

Sustained induction of survival p-AKT and p-ERK signals after transient hypoxia in mice spinal cord with G93A mutant human SOD1 protein.

Expression of survival p-AKT and p-ERK signals was examined by immunohistochemistry and Western blotting in the lumbar spinal cord of 12-week-old presymptomatic mice with human mutant G93A SOD1 gene (transgenic, Tg) and their wild-type (Wt) littermates during normoxia, and 0 and 6 h after 2 h of 9% hypoxia. During normoxia, a stronger p-AKT signal was detected in the nucleus of the motor neurons of Tg animals. At 0 h of recovery from 2 h of hypoxia, both p-AKT and p-ERK signals were induced at a slightly lower level in Tg (1.1-1.2-fold) compared to those of Wt (1.2-1.5-fold) animals. At 6 h of recovery, both p-AKT and p-ERK signals were sustained in the lumbar spinal motor neurons of Tg animals, while those in Wt animals quickly returned to baseline level. As a control, at 6 h of recovery, the hippocampus of Tg animals showed significantly sustained p-AKT levels, but not p-ERK levels, compared to Wt. The current results suggest that the presence of mutant SOD1 alters survival p-AKT and p-ERK signals, possibly to compensate for the acquired gain-of-function of the mutant protein.

Age-related changes in peroxisomal membrane protein 70 and superoxide dismutase 1 in transgenic G93A mice.

Peroxisomal membrane protein 70 (PMP70) and Cu/Zn superoxide dismutase (SOD1) were examined in the spinal cords of transgenic (Tg) mice expressing a human mutant SOD1 protein (G93A) and their age-matched controls at 8, 20 and 32 weeks by immunohistochemistry. At pre-symptomatic 20 weeks and symptomatic 32 weeks, PMP70 was reduced in the cytoplasm of motor neurons in Tg animals and increased in glial cells in anterior horn at late age. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons at 32 weeks. It is conceivable that reduction of PMP70 might underlie decrease in peroxisomal functions and increase in oxidative stress that is well documented in this animal model.

Single-nucleotide polymorphisms in uncoding regions of ALS2 gene of Japanese patients with autosomal-recessive amyotrophic lateral sclerosis.

ALS2 is an autosomal recessive form of amyotrophic lateral sclerosis (AR-ALS) with juvenile onset, and has been mostly found in North African and Middle Eastern countries. Deletion mutations in the coding exons of a new gene ALS2, encoding a protein with guanine-nucleotide exchange factor (GEF) domains, have recently been identified in ALS2 patients. These mutations are predicted to cause a loss of protein function, indicating that ALS2 is the causative gene underlying ALS2. To examine whether ALS2 is mutated in Japanese ALS patients sharing some characteristics of ALS2, we analyzed ALS2 gene from three patients with AR-ALS. While no deletion mutation was detected in the coding regions of ALS2 gene, several single-nucleotide polymorphisms (SNPs) that have been found in healthy controls as well as in Tunisian ALS2 patients were found mostly in intronic regions of the gene. These results suggest that deletion mutations in ALS2 gene detected in ALS2 patients seem to be uncommon in Japanese AR-ALS, and that SNPs in uncoding regions might possibly be relevant to predisposition to ALS.

Cauda equina syndrome caused by idiopathic sacral epidural lipomatosis.

The patient, who was a non-obese woman with no predisposing conditions of lipomatosis, slowly developed cauda equina syndrome. Spinal magnetic resonance imaging (MRI) presented mass lesion of high intensity on T1-weighted image (WI) and an intermediate signal intensity in T2 WI in the epidural space of S1 to coccyges. It has been reported that most idiopathic epidural lipomatosis (IEDL) is observed in obese men, and all cases have involved the thoracic or lumbar region. This is the first report of a patient with cauda equina syndrome caused by idiopathic sacral epidural lipomatosis (EDL).

[A case of carbon monoxide intoxication presenting subacute dementia as the initial symptom].

We reported a 67-year-old woman who had developed abnormal behavior and dementia from January 21, 2001 and deteriorated to akinetic mutism on February 15. T2-weighted magnetic resonance imaging showed high intensity in bilateral globus pallidus and a rapid spreading of diffuse high intensity in bilateral deep white matter. Later on, we got an important information that she had used a little coal stove three times about three weeks before presenting the initial symptom. She was diagnosed as carbon monoxide intoxication and treated with hyperbaric oxygen from March 1. A remarkable improvement on dementia and motor disability was observed. We conclude that this case is the first reported example of a case of intermittent carbon monoxide intoxication presenting subacute dementia as an initial symptom.

[A case of neuro-Behçet's disease with repeating isolated thoracic spinal cord lesion].

We reported a 45-year-old man who had repeated isolated thoracic spinal cord lesion on MRI in the clinical course of seven years. He had transient bilateral plantar numbness and urinary retention on December, 1994. Then, spastic paraplegia, total anesthesia of feet, and severe sphincter disturbance struck him on May, 1995. He was diagnosed as incomplete Behçet's disease and neuro-Behçet's disease on June, 1995 because of recurrent oral aphta, genital ulceration, and foliculitis. T2-weighted magnetic resonance imaging showed high intensity enhanced with Gd-DTPA in thoracic spinal cord from Th 5 to 8 level. Any other abnormal lesion on brain or spinal MRI was not observed. He was treated with corticosteroids and recovered incompletely. Another two big attacks occurred to him. No new lesion but thoracic spinal cord lesion was observed. We conclude that this case is the first reported example of neuro-Behçet's disease with repeating isolated thoracic spinal cord lesion.

Intrathecal injection of epidermal growth factor and fibroblast growth factor 2 promotes proliferation of neural precursor cells in the spinal cords of mice with mutant human SOD1 gene.

We investigated three steps of neural precursor cell activation--proliferation, migration, and differentiation--in amyotrophic lateral sclerosis spinal cord treated with intrathecal infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) into the lumbar spinal cord region of normal and symptomatic transgenic (Tg) mice with a mutant human Cu/Zn superoxide dismutase (SOD1) gene. We observed that 5-bromodeoxyuridine (BrdU) + nestin double-labeled neural precursor cells increased in the spinal cords of Tg mice compared with non-Tg mice, with a much greater increase produced by EGF and FGF2 treatment. The number of BrdU + nestin double-labeled cells was larger than that of BrdU + ionized calcium-binding adapter molecule-1 (Iba1), BrdU + glial fibrillary acidic protein (GFAP), or BrdU + highly polysialylated neural cell adhesion molecule (PSA-NCAM) double-labeled cells, but none expressed neuronal nuclear antigen (NeuN). On further analysis of the gray matter of Tg mice, the number of BrdU + nestin and BrdU + PSA-NCAM double-labeled cells increased more in the ventral horns than the dorsal horns, which was again greatly enhanced by EGF and FGF2 treatment. Because neural precursor cells reside close to the ependyma of central canal, the present study suggests that proliferation and migration of neural precursor cells to the ventral horns is greatly activated in symptomatic Tg mice and is further enhanced by EGF and FGF2 treatment and, furthermore, that the neural precursor cells preferentially differentiate into neuronal precursor cells instead of astrocytes in Tg mice with EGF and FGF2 treatment.

Unique cerebellar-cerebral form of autosomal recessive ataxia.

We describe a unique condition affecting two siblings with a form of progressive spinocerebellar ataxia. After a period of very slowly progressive ataxia, the patients developed an extremely accelerated progression of the condition which consisted of cerebellar ataxia, seizure, progressive dementia and spastic tetraparesis. Age of onset was variable at 7 to 18 years. Brain magnetic resonance image (MRI) showed marked atrophy of the cerebellum and cerebrum with strikingly preserved brainstem dimensions. Biochemical or molecular genetic analysis was performed in an elder sister and her parents to exclude known forms of familial spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy (DRPLA), progressive myoclonic epilepsy, and some metabolic disorders which could have a similar phenotype. The mode of inheritance appears to be autosomal recessive. We think that the affected siblings may have a new type of autosomal recessive cerebellar ataxia.

Prevention of spinal motor neuron death by insulin-like growth factor-1 associating with the signal transduction systems in SODG93A transgenic mice.

The role of insulin-like growth factor-1 (IGF-1) in amyotrophic lateral sclerosis (ALS) and its mechanism of action are important from both pathogenic and therapeutic points of view. The present study investigated the changes of IGF-1Rbeta and the key intracellular downstream protein insulin receptor substrate-1 (IRS-1) by using SOD1(G93A) transgenic mice with continuous intrathecal IGF-1 treatment. The number of lumbar spinal motor neurons was preserved with IGF-1 treatment in a dose-dependent manner. The numbers of immunopositive motor neurons for IGF-1Rbeta and IRS-1 were not significantly different between wild-type and Tg mice with vehicle treatment, whereas treatment of Tg mice with IGF-1 decreased the numbers of immunopositive motor neurons in a dose-dependent manner. On the other hand, the ratio of immunopositive motor neurons per total living motor neurons in vehicle-treated mice was greatly increased in Tg mice with vehicle treatment compared with wild-type mice. With IGF-1 treatment, the ratio was dramatically decreased in a dose-dependent manner. These results suggest that IGF-1 treatment prevents motor neuron loss by affecting the signal transduction system through IGF-1R and the main downstream signal, IRS-1.