Yasushi Takehisa

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.

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.

Mutation Analysis of the PINK1 Gene in 391 Patients With Parkinson Disease

OBJECTIVES To determine the frequency, distribution, and clinical features of Parkinson disease (PD) with PINK1 mutations. DESIGN Retrospective clinical and genetic review. SETTING University hospital. PATIENTS We performed extensive mutation analyses of PINK1 in 414 PD patients negative for parkin mutations (mean [SD] age at onset, 42.8 [14.3] years), including 391 unrelated patients (190 patients with sporadic PD and 201 probands of patients with familial PD) from 13 countries. RESULTS We found 10 patients with PD from 9 families with PINK1 mutations and identified 7 novel mutations (2 homozygous mutations [p.D297MfsX22 and p.W437R] and 5 single heterozygous mutations [p.A78V, p.P196QfsX25, p.M342V, p.W437R, and p.N542S]). No compound heterozygous mutations were found. The frequency of homozygous mutations was 4.26% (2 of 47) in families with autosomal recessive PD and 0.53% (1 of 190) in patients with sporadic PD. The frequency of heterozygous mutations was 1.89% (2 of 106) in families with potential autosomal dominant PD and 1.05% (2 of 190) in patients with sporadic PD. The mean (SD) age at onset in patients with single heterozygous mutations (53.6 [11.1] years; range, 39-69 years) was higher than that in patients with homozygous mutations (34.0 [20.3] years; range, 10-55 years). Myocardial iodine-123 metaiodobenzylguanidine uptake was low in patients with heterozygous mutations but not in those with homozygous mutations. CONCLUSIONS Our results suggest that homozygous PINK1 mutations tend to be diagnosed as the early-onset autosomal recessive form of PD. Single heterozygous mutations may contribute to the development of sporadic PD and also could be an additional genetic predisposition for developing familial PD. The reduced myocardial iodine-123 metaiodobenzylguanidine uptake observed in patients with single heterozygous PINK1 mutations is similar to that seen in patients with sporadic PD.

Two kinds of mitogen-activated protein kinase phosphatases, MKP-1 and MKP-3, are differentially activated by acute and chronic methamphetamine treatment in the rat brain.

Two functionally different MAP kinase phosphatases (MKPs) were investigated to clarify their roles in behavioral sensitization to methamphetamine (METH). MKP‐1 mRNA levels increased substantially by about 60–300% in a range of brain regions, including several cortices, the striatum and thalamus 0.5–1 h after acute METH administration. After chronic METH administration its increase was less pronounced, but a more than 50% increase was still seen in the frontal cortex. MKP‐1 protein levels also increased 3 h after acute or chronic METH administration. MKP‐3 mRNA levels increased by about 30–50% in several cortices, the striatum and hippocampus 1 h after acute METH administration, but only in the hippocampus CA1 after chronic METH administration. Pre‐treatment with the D1 dopamine receptor antagonist, SCH23390, attenuated the METH‐induced increase of MKP‐1 and MKP‐3 mRNA in every brain region, while pre‐treatment with the NMDA receptor antagonist, MK‐801, attenuated it in some regions. These findings suggest that in METH‐induced sensitization, MKP‐1 and MKP‐3 play important roles in the neural plastic modification in widespread brain regions in the earlier induction process, but in the later maintenance process, they do so only in restricted brain regions such as MKP‐1 in the frontal cortices and MKP‐3 in the hippocampus.

Association of the Neprilysin Gene with Susceptibility to Late-Onset Alzheimer’s Disease

Neprilysin (NEP), also known as neutral endopeptidase, enkephalinase, CD 10, and common acute lymphoblastic leukemia antigen, is a 97-kD protein. NEP can degrade amyloid β peptides, and its mRNA and protein levels are known to be reduced in the brains of patients with Alzheimer’s disease (AD), making the NEP gene a substantial candidate for an AD risk factor. We examined the genetic association of three NEP polymorphisms, a GT-repeat polymorphism and two single nucleotide polymorphisms (SNPs, –1075A>G and –1284G>C) in its promoter region, with AD in a Japanese case-control sample (240 patients and 163 controls). The GT-repeat polymorphism, but not the SNPs, was significantly associated with late-onset AD (p = 0.0007). Our findings suggest that the GT-repeat polymorphism in the promoter region of the NEP gene or some other unknown polymorphisms, which are in a linkage disequilibrium, confer a susceptibility to late-onset AD.

Lead content of brain tissue in diffuse neurofibrillary tangles with calcification (DNTC): the possibility of lead neurotoxicity.

Diffuse neurofibrillary tangles with calcification (DNTC) is a form of presenile dementia, characterized pathologically by fronto-temporal atrophy with neurofibrillary tangles (NFTs), neuropil threads and Fahr-type calcification, in which no senile plaques are observed. As already noted, chronic exposure to lead (Pb) might be one of the etiological factors of Fahr-type calcification. Until now, there have been no reports in which Pb concentration has been quantified in DNTC brains. We examined the concentration of Pb in fresh-frozen brain tissue and in 10% formalin-fixed brain tissue from six cases of DNTC, four cases of Alzheimers disease, and in nine non-demented elderly controls by flameless atomic absorption spectrometry, and demonstrated a high concentration of Pb in DNTC brains. Although it remains unclear how these findings are related to the formation of NFTs, they suggest that Pb neurotoxicity may be involved in the pathogenesis of DNTC.

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.

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.

PINK1, a gene product of PARK6, accumulates in α-synucleinopathy brains

α-Synucleinopathy is an entity of neurodegenerative diseases such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), that involves accumulation of α-synuclein in the brain. PINK1 (PTEN induced kinase 1) is a novel gene recently identified as causative in autosomal recessive early onset parkinsonism (PARK6). In the present study, we examined the localisation of PINK1 in the brains of patients with α-synucleinopathy and found PINK1 in glial cytoplasmic inclusions (GCIs) in MSA, as well as in Lewy bodies (LBs) in PD and DLB. These findings imply that PINK1 may be involved in the formation of LBs and GCIs, suggesting that PINK1 is one of the major pathological proteins in α-synucleinopathy. The cDNA of PINK1, corresponding to 112–520 amino acids of the protein, was subcloned in a vector pET30(a) with a His tag. Anti-PINK1 antibody was generated against recombinant His tagged PINK1 by immunising a rabbit. The obtained antibody was affinity purified. A postmortem brain sample from a normal patient was homogenised, subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a membrane. After blocking in Tris buffered saline with 5% dry milk, the membrane was incubated with anti-PINK1 antibody (1:1000). The membrane was then incubated with a secondary antibody (1:2500; Amersham, Buckinghamshire, UK), and visualised with an enhanced chemiluminescent substrate (Pierce, Rockford, Illinois, USA). Immunohistochemical analysis was carried out with paraffin embedded midbrain sections from patients with sporadic PD, …

No association between the insulin degrading enzyme gene and Alzheimer's disease in a Japanese population.

Susceptibility to Alzheimers disease (AD) is thought to be regulated by multiple genetic factors. Recently, three independent studies have reported that loci on chromosome 10q are linked with AD, and the insulin degrading enzyme (IDE; MIM 146680) gene located on chromosome 10q23‐q25; IDE is located close to the maker D10S583, which exhibits a maximum LOD score for late‐onset AD. We examined seven polymorphisms in the IDE gene, the marker D10S583 in the 5′ flanking region, and SNPs in introns 1, 3, 11, 20, 21, and 22 (rs#1999764, 1855915, 1970244, 538469, 551266, and 489517, respectively). Four SNPs in introns 3, 11, 20, and 22 did not exhibit any polymorphisms in the Japanese population that was studied. D10S583 and two SNPs in introns 1 and 21 did not exhibit a significant association with early‐ or late‐onset AD. In addition, no associations were observed for subgroups of AD grouped according to APOE status. The present study indicates that the IDE gene polymorphisms do not confer susceptibility to early‐ or late‐onset AD at least in a Japanese population.