Kazunori Miyazaki

Increased autophagy in transgenic mice with a G93A mutant SOD1 gene.

Autophagy, like the ubiquitin-proteasome system, is considered to play an important role in preventing the accumulation of abnormal proteins. Rat microtubule-associated protein 1 light chain 3 (LC3) is important for autophagy, and the conversion from LC3-I into LC3-II is accepted as a simple method for monitoring autophagy. We examined a SOD1G93A transgenic mouse model for amyotrophic lateral sclerosis (ALS) to consider a possible relationship between autophagy and ALS. In our study we analyzed LC3 and mammalian target of rapamycin (mTOR), a suppressor of autophagy, by immunoassays. The level of LC3-II, which is known to be correlated with the extent of autophagosome formation, was increased in SOD1G93A transgenic mice at symptomatic stage compared with non-transgenic or human wild-type SOD1 transgenic animals. Moreover, the ratio of phosphorylated mTOR/Ser2448 immunopositive motor neurons to total motor neurons was decreased in SOD1G93A-Tg mice. The present data show the possibility of increased autophagy in an animal model for ALS. And autophagy may be partially regulated by an mTOR signaling pathway in these animals.

Dissociation and protection of the neurovascular unit after thrombolysis and reperfusion in ischemic rat brain

In the ischemic brain, reperfusion with tissue plasminogen activator (tPA) sometimes causes catastrophic hemorrhagic transformation (HT); however, the mechanism remains elusive. Here, we show that the basement membrane, and not the endothelial cells, is vulnerable to ischemic/reperfusion injury with tPA treatment. We treated a spontaneously hypertensive rat model of middle cerebral artery occlusion (MCAO) with vehicle alone, tPA alone, or a free radical scavenger, edaravone, plus tPA. Light and electron microscopic analyses of each microvascular component revealed that the basement membrane disintegrated and became detached from the astrocyte endfeet in tPA-treated animals that showed HT. On the other hand, edaravone prevented the dissociation of the neurovascular unit, dramatically decreased the HT, and improved the neurologic score and survival rate of the tPA-treated rats. These results suggest that the basement membrane that underlies the endothelial cells is a key structure for maintaining the integrity of the neurovascular unit, and a free-radical scavenger can be a viable agent for inhibiting tPA-induced HT.

Disruption of neurovascular unit prior to motor neuron degeneration in amyotrophic lateral sclerosis

Recent reports suggest that functional or structural defect of vascular components are implicated in amyotrophic lateral sclerosis (ALS) pathology. In the present study, we examined a possible change of the neurovascular unit consisting of endothelium (PCAM‐1), tight junction (occludin), and basement membrane (collagen IV) in relation to a possible activation of MMP‐9 in ALS patients and ALS model mice. We found that the damage in the neurovascular unit was more prominent in the outer side and preferentially in the anterior horn of ALS model mice. This damage occurred prior to motor neuron degeneration and was accompanied by MMP‐9 up‐regulation. We also found the dissociation between the PCAM‐1‐positive endothelium and GFAP‐positive astrocyte foot processes in both humans and the animal model of ALS. The present results indicate that perivascular damage precedes the sequential changes of the disease, which are held in common between humans and the animal model of ALS, suggesting that the neurovascular unit is a potential target for therapeutic intervention in ALS.

Atorvastatin and pitavastatin improve cognitive function and reduce senile plaque and phosphorylated tau in aged APP mice.

In addition to simply reducing the serum level of cholesterol, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have various pleiotrophic effects such as reducing oxidative stress, neuroinflammation, and neurotoxicity. However, such a pleiotrophic effect has not been fully studied in a new statin (pitavastatin). We examined and compared the effects of two strong statins (atorvastatin, 30 mg/kg/day, p.o.; pitavastatin, 3mg/kg/day, p.o.) on the serum level of lipids, cognitive dysfunction, senile plaque (SP) and phosphorylated tau-positive dystrophic neuritis (pτDN) in amyloid precursor protein (APP) transgenic (Tg) mice from 5 months (M) of age to 20 M. These two statins improved behavioral memory and reduced the numbers of SP and pτDN at 15 and 20 M without affecting serum lipid levels, but preserved mice brain weight in pitavastatin group at 20 M. These protective effects of statins took 10 M from the beginning of treatment to show an improvement in the present model mice, and sensitivity to the statin treatment was linked to behavioral memory, SP and pτDN in this order. These findings suggest that early treatment with both atorvastatin and pitavastatin prevented subsequent worsening of cognitive function and the amyloidogenic process, probably due to pleiotrophic effects, suggesting a therapeutic potential for Alzheimers disease (AD).

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T3-S1 level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L4-5 level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.

Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosis

When fused with the protein transduction domain (PTD) derived from the human immunodeficiency virus TAT protein, proteins can cross the blood–brain barrier and cell membrane and transfer into several tissues, including the brain, making protein therapy feasible for various neurological disorders. We have constructed a powerful antiapoptotic modified Bcl‐XL protein (originally constructed from Bcl‐XL) fused with PTD derived from TAT (TAT‐modified Bcl‐XL), and, to examine its clinical effectiveness in a mouse model of familial amyotrophic lateral sclerosis (ALS), transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation were treated by intrathecal infusion of TAT‐modified Bcl‐XL. We demonstrate that intrathecally infused TAT‐fused protein was effectively transferred into spinal cord neurons, including motor neurons, and that intrathecal infusion of TAT‐modified Bcl‐XL delayed disease onset, prolonged survival, and improved motor performance. Histological studies show an attenuation of motor neuron loss and a decrease in the number of cleaved caspase 9‐, cleaved caspase 3‐, and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL)‐positive cells in the lumbar cords of TAT‐modified Bcl‐XL‐treated G93A mice. Our results indicate that intrathecal protein therapy using a TAT‐fused protein is an effective clinical tool for the treatment of ALS.

Atorvastatin and pitavastatin reduce senile plaques and inflammatory responses in a mouse model of Alzheimer's disease.

Abstract Objectives: To examine and compare the pleiotropic anti-inflammatory effects and the long-term effects of atorvastatin and pitavasatin in mouse model of Alzheimer’s disease (AD). Methods: We examined the effects of two strong statins on senile plaque (SP) size and inflammatory responses in the brain of an amyloid precursor protein (APP) transgenic (Tg) mouse. We gave the Tg mice either atorvastatin or pitavastatin from 5–20 months of age, and performed immunohistological analysis [SP area, monocyte chemotactic protein 1 (MCP-1)-positive neurons, ionized calcium-binding adaptor molecule 1 (Iba-1)-1-positive microglia, and tumor necrosis factor &alpha ( (TNF-α)-positive neurons] every 5 months. Results: In the APP-Tg mice treated with both statins, the number of MCP-1-positive neurons was reduced at 10 months, that of Iba-1-positive microglia was reduced at 15 months, and that of TNF-α-positive neurons and the mean total SP area decreased at 15–20 months, compared with APP-Tg mice with vehicle treatment. Discussion: The protective effect of these statins took 5 months to reach significance in these mice, and the order of sensitivity to statin treatment was MCP-1>Iba-1>TNF-α>SPs. Proinflammatory responses including MCP-1, Iba-1, and TNF-α preceded and possibly contributed to SP formation. Pitavastatin has the same significant pleiotrophic effect to prevent and ameliorate inflammation and also has a long-term effect compared with atorvastatin, and both of them have high potential for a preventative approach in patients at risk of AD.

Early decrease of mitochondrial DNA repair enzymes in spinal motor neurons of presymptomatic transgenic mice carrying a mutant SOD1 gene

Growing evidence has recently shown that mutant SOD1 accumulate in the mitochondria and cause vacuolation in transgenic mice carrying mutant SOD1, an animal model of amyotrophic lateral sclerosis (ALS). In this study, the expressions of DNA repair enzymes, oxoguanine glycosylase 1 (ogg1), DNA polymerase beta (polbeta), and DNA polymerase gamma (polgamma) were examined in transgenic mice with an ALS-linked mutant SOD1 gene, a valuable model for human ALS. In presymptomatic Tg mice, the nuclear form of ogg1 was upregulated, whereas mitochondrial ogg1 remained at the same level. DNA polymerase was selectively downregulated in the mitochondria. This study suggests an impaired protective mechanism against oxidative stress in mitochondria. The expressions of these enzymes are predominant in spinal motor neurons, suggesting a mechanism of selective motor neuron death in this animal model of ALS.

Early and progressive impairment of spinal blood flow–glucose metabolism coupling in motor neuron degeneration of ALS model mice

The exact mechanism of selective motor neuron death in amyotrophic lateral sclerosis (ALS) remains still unclear. In the present study, we performed in vivo capillary imaging, directly measured spinal blood flow (SBF) and glucose metabolism, and analyzed whether if a possible flow—metabolism coupling is disturbed in motor neuron degeneration of ALS model mice. In vivo capillary imaging showed progressive decrease of capillary diameter, capillary density, and red blood cell speed during the disease course. Spinal blood flow was progressively decreased in the anterior gray matter (GM) from presymptomatic stage to 0.80-fold of wild-type (WT) mice, 0.61 at early-symptomatic, and 0.49 at end stage of the disease. Local spinal glucose utilization (LSGU) was transiently increased to 1.19-fold in anterior GM at presymptomatic stage, which in turn progressively decreased to 0.84 and 0.60 at early-symptomatic and end stage of the disease. The LSGU/SBF ratio representing flow—metabolism uncoupling (FMU) preceded the sequential pathological changes in the spinal cord of ALS mice and was preferentially found in the affected region of ALS. The present study suggests that this early and progressive FMU could profoundly involve in the whole disease process as a vascular factor of ALS pathology, and could also be a potential target for therapeutic intervention of ALS.

Impaired antioxydative Keap1/Nrf2 system and the downstream stress protein responses in the motor neuron of ALS model mice

The Kelch-like ECH-associated protein 1 (Keap1)/Nuclear erythroid 2-related factor 2 (Nrf2) system is the major cellular defense mechanism under oxidative stress, but the role in motor neuron degeneration under amyotrophic lateral sclerosis (ALS) pathology has not yet been fully elucidated. Here we examined temporal and spatial changes of Keap1, Nrf2, and their downstream stress response proteins heme oxgenase-1 (HO-1), glutathione, thioredoxin (TRX), and heat shock protein 70 (HSP70) throughout the course of motor neuron (MN) degeneration in the spinal cord of ALS model mice. Keap1 protein levels progressively decreased in the MN and anterior lumbar cord of ALS mice to 63% at early symptomatic 14 weeks and 58% at end symptomatic 18 weeks, while Nrf2 dramatically increased in the anterior lumbar cord with accumulation in the MN nucleus to 229% at 14 weeks and 471% at 18 weeks when glial like cells became also positive. In contrast, downstream stress response proteins such as HO-1, glutathione, TRX, and HSP70 showed only a small increase in MN with a significant increase to 149% to 280% in the number of glial-like cells after symptomatic 14 weeks. Our present observation suggests that MN selectively lost inductions of these important downstream protective proteins without regard to the Keap1/Nrf2 system activation, which could be a pivotal mechanism of neurodegenerative processes of ALS.