Makoto Urushitani

Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons

It has been shown that estrogen replacement in menopausal women is effective in slowing down the progression of cognitive impairment in Alzheimers disease. Although recent studies have demonstrated the neuroprotective effects of estrogen, the precise mechanism of neuroprotection has not been elucidated. In the present study, we show that the phosphatidylinositol 3‐kinase (PI3‐K) cascade is involved in the neuroprotective mechanism stimulated by estrogen. Exposure to glutamate reduced the viability of rat primary cortical neurons. Pretreatment with 10 nM 17β‐estradiol significantly attenuated the glutamate‐induced toxicity. This neuroprotective effect of 17β‐estradiol was blocked by co‐administration with LY294002, a selective PI3‐K inhibitor, but not by co‐administration with PD98059, a selective mitogen activated protein kinase kinase inhibitor. Pretreatment with ICI182780, a specific estrogen receptor antagonist, also blocked the neuroprotection. Immunoblotting assay revealed that treatment with 17β‐estradiol induced the phosphorylation of Akt/PKB, an effector immediately downstream of PI3‐K. These results suggest that PI3‐K mediates the neuroprotective effect of 17β‐estradiol against glutamate‐induced neurotoxicity. J. Neurosci. Res. 60:321–327, 2000

Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death.

Oxidative stress is important in the process of dopaminergic neuronal degeneration in Parkinsons disease. Recent studies suggest that estrogens have neuroprotective effects in neurodegenerative disorders, including Alzheimers disease. In the present study, we investigated neuroprotection against oxidative stress afforded by estradiol using primary neuronal culture of the rat ventral mesencephalon. Oxidative stress induced by glutamate, superoxide anions, and hydrogen peroxide caused significant neuronal death. Although simultaneous administration of 17β‐estradiol and glutamate did not show any significant effects, preincubation with 17β‐estradiol provided significant neuroprotection against glutamate‐induced neurotoxicity (ED50 was 50 μM for dopaminergic and 15 μM for nondopaminergic neurons). Neuroprotection occurred even after a brief preincubation with 17β‐estradiol and was not significantly blocked by either an estrogen receptor antagonist or a protein synthesis inhibitor. These findings indicate that the neuroprotection against glutamate neurotoxicity is mediated by neither estrogen receptors nor activation of genome transcription. Other steroids (corticosterone, testosterone, and cholesterol) did not provide significant neuroprotection against glutamate‐induced neurotoxicity. Furthermore, preincubation with 17β‐estradiol provided neuroprotection against neuronal death induced by both superoxide anions and hydrogen peroxide. Dichlorofluorescin diacetate, a marker of oxygen radicals, revealed that preincubation with 17β‐estradiol suppressed intracellular oxygen radicals induced by hydrogen peroxide. The biologically inactive stereoisomer of estradiol, 17α‐estradiol, provided neuroprotection against glutamate‐induced toxicity in dopaminergic neurons, as well as the17β isoform. 17α‐estradiol may be a potential therapeutic agent used to prevent dopaminergic neuronal death induced by oxidative stress in Parkinsons disease. J. Neurosci. Res. 54:707–719, 1998.

Stimulation of α4β2 nicotinic acetylcholine receptors inhibits β-amyloid toxicity

In a regenerative air preheater of the stationary cylindrical regenerator chamber type, a rotatable heat-exchange element carrier is provided at the cold end of the regenerator chamber. The chamber wall has an aperture opposite the carrier through which access can be had to the heat-exchange elements in the carrier for their radial installation or removal.

Neuroprotective mechanism of glial cell line-derived neurotrophic factor in mesencephalic neurons.

Glial cell line‐derived neurotrophic factor (GDNF) provides neuroprotection, but its neuroprotective mechanism has not been resolved. We investigated the neuroprotective mechanism of GDNF using primary culture of the rat mesencephalon. Bleomycin sulfate (BLM) and L‐buthionine‐[S,R]‐sulfoximine (BSO) caused apoptosis in both dopaminergic and nondopaminergic neurons, as revealed by the presence of chromatin condensation, and positive staining by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end‐labeling (TUNEL). GDNF preincubation blocked the neurotoxicity and reduced the number of the TUNEL‐positive cells caused by BLM and BSO exposure. In contrast, GDNF did not provide neuroprotection against glutamate toxicity, which was not accompanied by these apoptotic features. The neuroprotection was mediated by phosphatidylinositol 3‐kinase, an effector downstream from c‐Ret, because it was blocked by LY294002. GDNF pretreatment caused up‐regulation of Bcl‐2 and Bcl‐x. Furthermore, GDNF suppressed oxygen radical accumulation caused by BLM. Apoptosis induced by BLM and BSO was blocked by a caspase‐3 inhibitor. Caspase‐3 activity was elevated by BLM and suppressed by GDNF pretreatment. These findings indicate that GDNF has no effect on necrosis but exerts protection against apoptosis by activation of phosphatidylinositol 3‐kinase and the subsequent up‐regulation of Bcl‐2 and Bcl‐x, which suppresses accumulation of oxygen radicals followed by caspase‐3 activation.

Immunoreactivity of valosin-containing protein in sporadic amyotrophic lateral sclerosis and in a case of its novel mutant

BackgroundMutations in the valosin-containing protein (VCP) gene were first found to cause inclusion- body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD). Mutations in the VCP gene were later reported to occur in familial amyotrophic lateral sclerosis (ALS). But the role of VCP in the neurodegenerative processes that occur in ALS remains unknown. The purpose of the present study was to elucidate the role of VCP in the neurodegeneration seen in sporadic and VCP mutant ALS.ResultsImmunohistochemistry demonstrated that the frequency of distinct VCP-positive nuclei of spinal motor neurons of patients with sporadic ALS (SALS) and the ALS with VCP novel mutation (ALS-VCP, M158V) was increased, compared with that of the control cases. No VCP-positive inclusion bodies were observed in SALS patients, a ALS-VCP patient or in control subjects. Neuropathologic examination of the ALS-VCP case showed loss of motor neurons, the presence of Bunina bodies, and degeneration of the corticospinal tracts. Bunina bodies detected in this case were confirmed to show immunohistochemical and ultrastructural features similar to those previously described. Furthermore, neuronal intracytoplasmic inclusions immunopositive for TAR DNA-binding protein 43xa0kDa (TDP-43), phosphorylated TDP-43, ubiquitin (Ub), p62, and optineurin were identified in the spinal and medullary motoneurons, but not in the neocortex. Gene analysis of this ALS-VCP patient confirmed the de novo mutation of M158V, which was not found in control cases; and bioinformatics using several in silico analyses showed possible damage to the structure of VCP. Immunocytochemical study of cultured cells showed increased cytoplasmic translocation of TDP-43 in cells transfected with several mutant VCP including our patient’s compared with wild-type VCP.ConclusionThese findings support the idea that VCP is associated with the pathomechanism of SALS and familial ALS with a VCP mutation, presumably acting through a dominant-negative mechanism.

Neuroprotective effect of cyclic GMP against radical-induced toxicity in cultured spinal motor neurons.

We have previously reported that nitric oxide‐related cyclic guanosine‐3′,5′‐monophosphate (GMP) protected spinal nonmotor neurons, but not motor neurons against chronic glutamate‐induced toxicity, which is associated with selective motor neuronal death after glutamate stress. In this report, we investigated the effect of cyclic GMP against reactive oxygen species (ROS)‐induced toxicity in cultured neurons from embryonic rat spinal cords. Pretreatment with a cGMP analogue, 8‐bromoguanosine monophosphate (8br‐cGMP), for 12–24 hours protected both spinal motor neurons and nonmotor neurons against injury induced by either hydrogen peroxide (H2O2), or a glutathione depletor, L‐buthionine‐[S,R]‐sulfoximine (BSO). This protective effect was reversed by coadministration with the cGMP‐dependent protein kinase (PKG) inhibitor Arg‐Lys‐Arg‐Ala‐Arg‐Lys‐Glu. Interestingly, when cultures were exposed to BSO for 24 hours to allow irreversible inhibition of glutathione synthesis, 8br‐cGMP protected only nonmotor neurons. Our results indicate that cGMP attenuates oxidative injury to cultured spinal neurons, in a mechanism associated with glutathione synthesis. J. Neurosci. Res. 61:443–448, 2000.

Gradual cerebral hypoperfusion in spontaneously hypertensive rats induces slowly evolving white matter abnormalities and impairs working memory

Rats subjected to bilateral common carotid arteries (CCAs) occlusion or 2-vessel occlusion (2VO) have been used as animal models of subcortical ischemic vascular dementia (SIVD). However, these models possess an inherent limitation in that cerebral blood flow (CBF) drops sharply and substantially after ligation of CCAs without vascular risk factors and causative small vessel changes. We previously reported a novel rat model of 2-vessel gradual occlusion (2VGO) in which ameroid constrictors (ACs) were placed bilaterally in the CCAs of Wistar-Kyoto rats. To simulate SIVD pathology more closely, we applied ACs in spontaneously hypertensive rats (SHRs), which naturally develop small vessel pathology, and compared their phenotypes with SHR-2VO and sham-operated rats. The mortality rate of the SHR-2VGO was 0% while that of the SHR-2VO was 56.5%. The CBF of the SHR-2VO dropped to 50% of the baseline level at 3u2009h, whereas the SHR-2VGO showed a gradual CBF reduction reaching only 68% of the baseline level at seven days. The SHR-2VGO showed slowly evolving white matter abnormalities and subsequent spatial working memory impairments of a similar magnitude to the remaining SHR-2VO at 28 days. We suggest the SHR-2VGO robustly replicates selective aspects of the pathophysiology of SIVD with low mortality rate.

Proteasome dysfunction induces muscle growth defects and protein aggregation.

ABSTRACT The ubiquitin–proteasome and autophagy–lysosome pathways are the two major routes of protein and organelle clearance. The role of the proteasome pathway in mammalian muscle has not been examined in vivo. In this study, we report that the muscle-specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in profound muscle growth defects and a decrease in force production in mice. Specifically, developing muscles in conditional Rpt3-knockout animals showed dysregulated proteasomal activity. The autophagy pathway was upregulated, but the process of autophagosome formation was impaired. A microscopic analysis revealed the accumulation of basophilic inclusions and disorganization of the sarcomeres in young adult mice. Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways. The deletion of a component of the proteasome complex contributed to myofiber degeneration and weakness in muscle disorders that are characterized by the accumulation of abnormal inclusions.

A cysteine residue affects the conformational state and neuronal toxicity of mutant SOD1 in mice: relevance to the pathogenesis of ALS

We previously showed by in vitro experiments that the cysteine residue (Cys111) near the dimer interface is critical for monomerization and resultant aggregate formation of mutant Cu, Zn-superoxide dismutase (SOD1) protein, which is toxic to motor neurons in familial amyotrophic lateral sclerosis (ALS). To verify the importance of Cys111 in the mutant SOD1-associated ALS pathogenesis in vivo, we analyzed the disease phenotype of SOD1 transgenic mice harboring H46R mutation alone (H46R mice) or H46R/C111S double mutations (H46R/C111S mice). Behavioral, histological and biochemical analyses of the spinal cord showed that the onset and progression of the disease phenotype were delayed in H46R/C111S mice compared with H46R mice. We found that peroxidized Cys111 of H46R SOD1 plays a role in promoting formation of high molecular weight insoluble SOD1 species that is correlated with the progression of the motor neuron disease phenotype. These results support that Cys111 is a critical residue for the neuronal toxicity of mutant SOD1 in vivo, and the blockage of peroxidation of this residue in mutant SOD1 may constitute a future target for developing ALS treatment.

Activated caspase-9 immunoreactivity in glial and neuronal cytoplasmic inclusions in multiple system atrophy.

The mitochondria play an important role in apoptotic cell death, and the released cytochrome c from the mitochondria promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9, resulting in the activation of caspase-9 and the promotion of the apoptotic cascade. To investigate the role of mitochondria-dependent apoptotic cell death in patients with multiple system atrophy (MSA), we performed immunohistochemical studies on apoptosome-related proteins in formalin-fixed, paraffin-embedded sections from 8 normal subjects and 10 patients with MSA. We then performed double-labeling immunohistochemistry for activated caspase-9 and α-synuclein in some sections from 10 patients with MSA. In the brains with MSA, glial cytoplasmic inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs) were intensely immunoreactive for cytochrome c, Apaf-1 and caspase-9. Activated caspase-9 immunoreactivities were also confirmed to be densely localized to both GCIs and NCIs using two types of anti-cleaved caspase-9 antibodies. The semiquantitative analyses using the upper pontine sections double-immunostained with cleaved caspase-9 and α-synuclein demonstrated that approximately 80% of GCIs and NCIs were immunopositive for cleaved caspase-9. Our results suggest that the formation of the apoptosome accompanied by the activation of caspase-9 may occur in brains affected by MSA, and that a mitochondria-dependent apoptotic pathway may be partially associated with the pathogenesis of MSA.