Tomoki Nakamizo

α7 Nicotinic Receptor Transduces Signals to Phosphatidylinositol 3-Kinase to Block A β-Amyloid-induced Neurotoxicity

Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathogenesis of Alzheimers disease (AD). Here we show the signal transduction mechanism involved in nicotinic receptor-mediated protection against β-amyloid-enhanced glutamate neurotoxicity. Nicotine-induced protection was suppressed by an α7 nicotinic receptor antagonist (α-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were increased by nicotine. The α7 nicotinic receptor was physically associated with the PI3K p85 subunit and Fyn. These findings indicate that the α7 nicotinic receptor transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect. This might form the basis of a new treatment for AD.

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

Nongenomic antiapoptotic signal transduction by estrogen in cultured cortical neurons

Estrogen replacement therapy in menopausal women has been suggested to be beneficial in preventing the progression of cognitive impairment in Alzheimer disease. We demonstrated previously that the phosphatidylinositol 3‐kinase (PI3‐K)/Akt signal transduction pathway plays a pivotal role on the neuroprotection provided by 17β‐estradiol against acute glutamate toxicity. In the present study, we investigated the mechanism of neuroprotection against apoptosis because acute glutamate toxicity predominantly induced necrosis. 17β‐estradiol provided neuroprotection against apoptosis induced by staurosporine. This neuroprotection was inhibited by pretreatment with a PI3‐K inhibitor, LY294002. An estrogen receptor specific antagonist, ICI182780, also suppressed the neuroprotection provided by 17β‐estradiol. Western blotting analysis demonstrated that treatment with 17β‐estradiol induced the phosphorylation of Akt within 5 min, which was suppressed by pretreatment with LY294002 and ICI182780. Furthermore, 17β‐estradiol induced phosphorylation of the cAMP response element binding protein (CREB) at Ser133 within 15 min and then upregulated Bcl‐2 in a PI3‐K/Akt‐dependent manner. Because CREB is known to be a transcription factor for Bcl‐2, these results suggest that 17β‐estradiol exerts its antiapoptotic effects by CREB phosphorylation and Bcl‐2 upregulation via nongenomic activation of the PI3‐K/Akt pathway in cultured cortical neurons. J. Neurosci. Res. 64:466–475, 2001.

Estradiol protects dopaminergic neurons in a MPP+Parkinson’s disease model

The prevalence of Parkinsons disease is higher in males than in females. Although the reason for this gender difference is not clear, the level of female steroid hormones or their receptors may be involved in the pathogenesis. The estrogen receptor subtype expressed in the midbrain is limited to the novel beta subtype, whose role in the central nervous system has not been resolved. We demonstrated that ligand-activated estrogen receptor beta suppressed dopaminergic neuronal death in an in vitro Parkinsons disease model which uses 1-methyl-4-phenylpyridinium ions (MPP(+)). MPP(+) treatment caused the upregulation of c-Jun amino-terminal kinase (JNK) and dopaminergic neuronal death, the latter being blocked by curcumin, an inhibitor of the c-Jun/AP-1 cascade. 17alpha- and 17beta-estradiol both protected dopaminergic neurons from MPP(+)-induced neuronal death and this was blocked by a pure antagonist of the estrogen receptor, ICI 182,780, but not by an inhibitor of estrogen receptor dimerization, YP537. These data indicated that the neuroprotection provided by 17alpha-estradiol was via inhibitory transcriptional regulation at the activator protein-1 (AP-1) site mediated by estrogen receptor beta. Thus, 17alpha-estradiol is a suitable candidate for neuroprotective therapy of Parkinsons disease because it is associated with few undesirable feminizing effects.

Protective effect of dopamine D2 agonists in cortical neurons via the phosphatidylinositol 3 kinase cascade

Glutamate, one of the excitatory neurotransmitters, contributes to the neuronal death associated with neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease, and with ischemia. In Alzheimers disease brains, there is a decreased number of dopamine D2 receptors, which might cause neuronal dysfunction or death. In the present study, bromocriptine exerted a protective effect against glutamate‐induced cytotoxicity in rat cortical neurons. This neuroprotective effect was mediated via D2 receptors, because it was attenuated by domperidone, a D2 dopaminergic receptor antagonist. Another dopamine D2 agonist, quinpirole, also protected cells against glutamate toxicity. D2 agonists protected cells from calcium influx, nitric oxide, and peroxynitrite toxicity, which are thought to be the mediators of glutamate toxicity. The phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) inhibited this neuroprotective effect of bromocriptine, in contrast to the mitogen‐activated protein kinase kinase (MAPKK) inhibitor (PD98059), which did not counter the protective effect. Furthermore, Akt protein kinase, which is an effector of PI3K, was activated by bromocriptine, and the antiapoptotic protein Bcl‐2 was up‐regulated by bromocriptine treatment. These results suggest that D2 dopaminergic receptor activation plays an important role in neuroprotection against glutamate cytotoxicity and that the up‐regulation of Bcl‐2 expression via the PI3K cascade is, at least partially, involved in this effect.

Phosphodiesterase inhibitors are neuroprotective to cultured spinal motor neurons.

We have previously reported that cyclic guanosine‐3′,5′‐monophosphate (cGMP) protects spinal motor neurons against acute reactive oxygen species (ROS)‐induced toxicity but not against chronic ROS‐induced or glutamate (Glu)‐induced toxicity. In this study, we investigated the effects of phosphodiesterase (PDE) inhibitors on the survival of cultured spinal motor neurons. Selective PDE5 inhibitors (dipyridamole, T‐1032, and zaprinast) as well as a nonselective PDE inhibitor (aminophylline) protected motor and nonmotor neurons against both acute ROS‐induced and chronic Glu‐induced neurotoxicity, whereas selective inhibitors of PDE1–4 offered no protection. 8‐Bromo‐cGMP (8br‐cGMP), a cGMP analogue, protected both motor and nonmotor neurons against acute ROS‐induced toxicity but protected only nonmotor neurons against chronic Glu‐induced toxicity. This neuroprotection was blocked by KT5823, a cGMP‐dependent protein kinase (PKG) inhibitor. Immunohistochemical staining confirmed that PDE5 and PKG are located in almost all rat lumbar spinal neurons. Furthermore, semiquantitative analysis of the immunostaining intensity revealed that PDE5 was more abundant in motor neurons than in nonmotor neurons. Our results suggest that this difference in the amount of PDE5 may be responsible for the vulnerability of motor neurons to chronic excitotoxicity. In addition, the results of this study raise the possibility that PDE5 inhibitors might be used as a treatment for amyotrophic lateral sclerosis.

Protection of cultured spinal motor neurons by estradiol

Estrogens have been reported to exert neuroprotection in the brain, but there have been no reports of such neuroprotection in spinal motor neurons, the neurons selectively involved in amyotrophic lateral sclerosis (ALS). In this study, we demonstrated that 17β-estradiol and its biologically inactive stereoisomer, 17α-estradiol, prevented glutamate- and nitric oxide (NO)-induced selective motor neuronal death observed in primary cultures of the rat spinal cord. The dose of estradiols required for motor neuron protection was greatly reduced by co-administration with glutathione. The results of this study shows that estradiol protects spinal motor neurons from excitotoxic insults in vitro, and may have application as a treatment for ALS.

Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS

A dominant mutation in the gene for copper‐zinc superoxide dismutase (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis. Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Exploiting both affinity purification and mass spectrometry, we identified a novel interaction between heat‐shock protein 105 (Hsp105) and mutant SOD1. We detected this interaction both in spinal cord extracts of mutant SOD1G93A transgenic mice and in cultured neuroblastoma cells. Expression of Hsp105, which is found in mouse motor neurons, was depressed in the spinal cords of SOD1G93A mice as disease progressed, while levels of expression of two other heat‐shock proteins, Hsp70 and Hsp27, were elevated. Moreover, Hsp105 suppressed the formation of mutant SOD1‐containing aggregates in cultured cells. These results suggest that techniques that raise levels of Hsp105 might be promising tools for alleviation of the mutant SOD1 toxicity.

Effects of mitochondrial dysfunction on glutamate receptor-mediated neurotoxicity in cultured rat spinal motor neurons

Glutamate-induced excitotoxicity is implicated as playing a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS), and mitochondrial dysfunction is also found in ALS patients. We investigated the relationship between glutamate excitotoxicity and mitochondrial dysfunction elicited by rotenone (a complex I inhibitor), malonate (a complex II inhibitor), or antimycin (a complex III inhibitor), in primary cultures of the embryonic rat spinal cord. Rotenone and malonate induced relatively selective toxicity against motor neurons as compared to non-motor neurons, whereas antimycin caused non-selective toxicity. The toxicity of rotenone was prevented by a non-N-methyl-D-aspartate (NMDA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by an NMDA receptor antagonist, 5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). The toxicity of malonate was blocked by both CNQX and MK-801. The toxicity of antimycin was affected by neither CNQX nor MK-801. When mitochondrial complex I was mildly inhibited by a sub-lethal concentration of rotenone, AMPA-induced motor neuron death was significantly exacerbated. A sub-lethal concentration of malonate exacerbated both NMDA- and AMPA-induced motor neuron death. These data suggest that mitochondrial dysfunction predisposes motor neurons to ionotropic glutamate receptor-mediated excitotoxicity.

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.