Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia.

Vascular endothelial growth factor (VEGF) is reported to play a neuroprotective role through a VEGF receptor, fetal liver kinase-1 (Flk-1) in vitro. We investigated whether reduction of Flk-1 could induce motor neuron loss in rat spinal cord by inhibiting the expression of Flk-1 in rat spinal cord using antisense oligodeoxynucleotides (ODNs) against the Flk-1 receptor. Rat spinal cord was repetitively exposed to 12% hypoxia, and the change of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway was examined. Intrathecal infusion of Flk-1 antisense ODNs for 7 days suppressed almost completely Flk-1 expression in the lumbar segment of the spinal cord and was followed by a hypoxic challenge with 12% oxygen for 1 h that was repeated for 7 more days. In the lumbar segment, we observed that reduced Flk-1 expression and hypoxic challenge for 7 days resulted in approximately 50% loss of motor neurons, in which the activation of Akt and ERK, that is, increased levels of phosphorylated-Akt and of phosphorylated-ERK by hypoxia, was markedly inhibited. In contrast, the reduction of Flk-1 expression alone did not induce motor neuron loss. These results suggest that VEGF exerts its protective effect on motor neurons against hypoxia-induced toxicity by the Flk-1 receptor through the PI3-K/Akt and the MEK/ERK signaling pathways.

Glutamate enhances caspase-3 immunoreactivity in cultured spinal cord neurons of newborn rats.

The role of glutamate in the mechanism of spinal neuron death is not fully understood. With addition of glutamate to primary culture of 11-day-old rat spinal cord, the number of caspase-3 positive small neurons of the dorsal horn greatly increased at 6-24 h in contrast to the case with vehicle. The addition of glutamate made caspase-3 immunoreactivity stronger in the cytoplasm of large motor neurons in the ventral horn. The present results show that excessive amount of glutamate enhances apoptotic pathway through caspase-3 in cultured spinal neurons of newborn rat.

Glial cell line-derived neurotrophic factor protein prevents motor neuron loss of transgenic model mice for amyotrophic lateral sclerosis.

Effects of glial cell line-derived neurotrophic factor (GDNF) were studied in transgenic (Tg) mice model for amyotrophic lateral sclerosis. GDNF protein or vehicle was injected three times a week from 35 weeks of age into the right gastrocnemius muscle of Tg mice carrying mutant human Cu/Zn superoxide dismutase gene, and histological analysis was performed at 46 weeks. Clinical data showed a tendency of improvement, but was not significantly different between the two animal groups. In contrast, total number of and phospho-Akt (p-Akt) positive large motor neurons in the treated side was significantly more preserved in GDNF-treated group than in vehicle group (p < 0.05). Immunoreactivity of phospho-ERK and active caspases-3 and -9 showed no difference. These results indicate that the intramuscular injection of GDNF protein prevented motor neuron loss while preserving survival p-Akt signal and without affecting caspase activations, suggesting a future possibility for the therapy of the disease.

Ventral root avulsion leads to downregulation of GluR2 subunit in spinal motoneurons in adult rats.

It has been observed that motor neuron death is induced in adult rats by ventral root avulsion which involves pulling out the spinal cord root. Since motor neurons are reported to be selectively vulnerable to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated injury in vitro, we investigated changes in the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-receptor subunits in rat spinal motor neurons after ventral root avulsion. The L4-L5 ventral roots of adult Sprague-Dawley rats were avulsed by an extravertebral extraction procedure. After an appropriate survival time, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-receptor subunits were detected immunohistochemically in the L4-L5 segments. Ventral root avulsion resulted in a 60% loss of motor neurons by 14 days after surgery. GluR2 labeling in motor neurons was markedly decreased after avulsion, but before the onset of motor neuron death, while the GluR1 and GluR4 labeling of motor neurons remained unchanged. Intrathecal administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-receptor antagonists rescued a significant number of injured motor neurons from cell death. In contrast, N-methyl-D-aspartate-receptor antagonists did not prevent motor neuron death. Since the presence of GluR2 subunit renders heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors Ca(2+)-impermeable, the downregulation of GluR2 may result in increased formation of GluR2-lacking, Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors in motor neurons and could contribute to motor neuron death after ventral root avulsion.

A case of paroxysmal kinesigenic dyskinesia with spastic paraparesis.

We report the first case of paroxysmal kinesigenic dyskinesia (PKD) with spastic paraparesis. A 17-year-old male began to show a dystonic posture in both his upper limbs when walking at age 12 years. Neurological examination revealed bilateral talipes cavus, spasticity in all extremities with general hyperreflexia and pathological reflexes. On starting to walk, he showed a dystonic posture in bilateral maniphalanx, wrists, elbows, and toes. Magnetic resonance imaging (MRI) revealed high T2-weighted signal intensity in bilateral pyramidal tract. Although the combination of pyramidal and the basal ganglia disorders is very rare, the present case suggests an inter-relation of the pyramidal and the basal ganglia systems.

Tardive decrease of astrocytic glutamate transporter protein in transgenic mice with ALS-linked mutant SOD1.

The expressions of glutamate transporter proteins were immunocytochemically examined in the spinal cord of transgenic mice harboring a Gly93 --> Ala (G93A) mutant human SOD1 gene. Astroglial EAAT2 protein level was preserved in the ventral horn even after the beginning of paralysis, and finally decreased at terminal stage of the disease (35 weeks of age), when neuronal EAAT3 protein level was also decreased. In contrast, glial fibrillary acidic protein (GFAP) immunoreactivity progressively increased from 25 weeks of age in the ventral horn. The present results show interesting dissociative expressions of astroglial proteins EAAT2 and GFAP in the same ventral horn, but suggest not an early and primary role of EAAT2 in the motoneuronal death of this model.

Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents motor neuron loss of transgenic model mice for amyotrophic lateral sclerosis.

Effects of adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) were studied in transgenic (Tg) mice model for amyotrophic lateral sclerosis (ALS). Adenoviral vector containing GDNF gene (Ad-GDNF), E. coli lacZ (Ad-LacZ), or vehicle was injected once a week from 35 weeks of age into the right gastrocnemius muscle of Tg mice carrying mutant human Cu/Zn superoxide dismutase (SOD1) gene, and histological analysis was performed at 46 W. Clinical data showed a tendency of improvement, but was not significantly different among the three animal groups. In contrast, total number of and phospho-Akt (p-Akt) positive large motor neurons in the treated side was significantly preserved in Ad-GDNF-treated group than in vehicle- and Ad-LacZ-treated groups (*p < 0.05). Immunoreactivity of phospho-ERK (p-ERK) and active caspases-3 and -9 showed no difference. These results indicate that the Ad-GDNF treatment prevented motor neuron loss with preserving survival p-Akt signal and without affecting caspase activations, suggesting a future possibility for the therapy of the disease.

Early decrease of the immunophilin FKBP 52 in the spinal cord of a transgenic model for amyotrophic lateral sclerosis.

Expressions of immunophilin FKBP-12 and FKBP-52 were examined in the spinal cord of transgenic mice with an ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) gene. The immunoreactivity of FKBP-12 was present predominantly in the cytoplasm, but did not show a difference between age-matched wild type and transgenic (Tg) mice at 25 and 35 weeks. In contrast, the immunoreactivity of FKBP-52 was predominantly present in the nucleus, which progressively declined only in the Tg mice as early as an early presymptomatic stage at 25 weeks of age in the anterior horn neurons. The present result suggests that the downregulation of FKBP-52 may be involved in the pathogenesis in the early stages of amyotrophic lateral sclerosis (ALS).

Early decrease of redox factor-1 in spinal motor neurons of presymptomatic transgenic mice with a mutant SOD1 gene.

Oxidative stress has been proposed to play a pivotal role in pathogenesis of both sporadic and familial amyotrophic lateral sclerosis (ALS). Expression of DNA repair enzyme redox factor-1 (Ref-1) protein was examined in the spinal cord of transgenic mice with an ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) gene. Immunoblotting and immunocytochemical analyses showed that the most spinal motor neurons lost the immunoreactivity for Ref-1 in the early presymptomatic stage that preceded significant loss of the neurons. The present result suggests that an early impairment of DNA repair in the spinal motor neurons may account for the mutant SOD1-mediated motor neuronal death in this model.