Kazuhiro Nakaso

PI3K is a key molecule in the Nrf2-mediated regulation of antioxidative proteins by hemin in human neuroblastoma cells

Oxidative stress and ferrous metabolism are important in the pathogenesis in Parkinsons disease. In dopaminergic neurons, several stress proteins are upregulated under oxidative stress. To clarify this mechanism, we investigated hemin‐related signal transduction and the induction of oxidative stress‐related proteins in SH‐SY5Y cells. We identified phosphatidylinositol 3‐kinase (PI3K) and Nrf2 as important molecules in the induction of heme oxygenase‐1, thioredoxin, and peroxiredoxin‐I. PI3K‐related signal controlled Nrf2 activation, and consequently, PI3K inhibitors blocked the nuclear translocation of Nrf2 and induction of stress proteins. These observations suggest that PI3K and Nrf2 are key molecules in maintaining suitable conditions under oxidative stress and ferrous metabolism.

Plasma homocysteine and MTHFR C677T genotype in levodopa-treated patients with PD

To the Editor: The paper by Yasui et al.1 showing elevated plasma homocysteine levels in levodopa-treated patients with PD is interesting and confirms other reports2 and our unpublished findings (table). It also shows that patients with the TT-genotype of the C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR) have especially high homocysteine levels. These findings have to be related to the metabolism of levodopa and methyl groups and may have importance in the future treatment of PD with levodopa. Homocysteine is the demethylated derivative of methionine and is only formed in numerous transmethylation reactions (e.g., catecholamine O-methyltransferase [COMT]) that use S-adenosylmethionine (SAM) as the methyl donor.3 Most administered levodopa is COMT-methylated to 3O-methyldopa and the methylation of 1000 mg of levodopa consumes 5 mMol of SAM methyls and produces 5 mMol of S-adenosylhomocysteine (SAH), which then is deadenosylated to homocysteine. In the study of Yasui et al., the average levodopa dose was 1500 mg. Therefore, it is most likely that levodopa treatment contributed to hyperhomocysteinemia in their patients with PD. In this context it is important to note that humans normally consume about 15 mMol/day of SAM methyls for methylation of endogenous compounds such as phospholipids, DNA, myeline, and proteins. About 10 mMol of these methyls are supplied from food, mainly as methionine and choline, and about 5 mMol are de novo synthesized in one-carbon (folate) metabolism and are transferred by a vitamin B12-dependent reaction to homocysteine, again forming methionine and SAM.4 As we can assume that levodopa-treated PD patients do not increase their intake of labile methyls, they must de novo synthesize all their extra methyls consumed for levodopa methylation. In the above 1000 mg example, the patient must increase his or her de novo synthesis of methyls by about 100%. If methyl neogenesis is hampered by poor folate intake, especially if the patient is a TT-homozygote for C677T/MTHFR polymorphism (about 12% of the Caucasian population), or has a low vitamin B12 level, production of SAM methyls may not be sufficient for both methylation of endogenous compounds and levodopa. This may lead to hypomethylation of essential neural components, and might explain the neuropsychiatric symptoms in folate and vitamin B12 deficiency.5 This mechanism may contribute to side effects of levodopa. If this is the case, early combination of levodopa with COMT inhibitors (tolcapone, entacapone) would prove to be beneficial. Moreover, studies on the relationship between complications to levodopa therapy in PD and homocysteine, folate, vitamin B12, and C677T/MTHFR genotypes are warranted.Plasma homocysteine and cysteine levels were measured in 90 patients with PD with the MTHFR C677T (T/T) genotype. The authors found that the levels of homocysteine-a possible risk factor for vascular disease-were elevated by 60% in levodopa-treated patients with PD, with the most marked elevation occurring in patients with the T/T genotype. Cysteine levels in subjects with PD did not differ from levels in control subjects. In the T/T genotype patients, homocysteine and folate levels were inversely correlated. Increased homocysteine might be related to levodopa, MTHFR genotype, and folate in PD.

Parkin Mediates Neuroprotection through Activation of IκB Kinase/Nuclear Factor-κB Signaling

Mutations in the parkin gene are a major cause of autosomal recessive Parkinsons disease. Here we show that the E3 ubiquitin ligase parkin activates signaling through the IκB kinase (IKK)/nuclear factor κB (NF-κB) pathway. Our analysis revealed that activation of this signaling cascade is causally linked to the neuroprotective potential of parkin. Inhibition of NF-κB activation by an IκB super-repressor or a kinase-inactive IKKβ interferes with the neuroprotective activity of parkin. Furthermore, pathogenic parkin mutants with an impaired neuroprotective capacity show a reduced ability to stimulate NF-κB-dependent transcription. Finally, we present evidence that parkin interacts with and promotes degradation-independent ubiquitylation of IKKγ/NEMO (NF-κB essential modifier) and TRAF2 [TNF (tumor necrosis factor) receptor-associated factor 2], two critical components of the NF-κB pathway. Thus, our results support a direct link between the neuroprotective activity of parkin and ubiquitin signaling in the IKK/NF-κB pathway.

Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells

Parkinsons disease (PD) is defined by the degeneration of nigral dopaminergic (DA) neurons and can be caused by monogenic mutations of genes such as parkin. The lack of phenotype in parkin knockout mice suggests that human nigral DA neurons have unique vulnerabilities. Here we generate induced pluripotent stem cells from normal subjects and PD patients with parkin mutations. We demonstrate that loss of parkin in human midbrain DA neurons greatly increases the transcription of monoamine oxidases and oxidative stress, significantly reduces DA uptake and increases spontaneous DA release. Lentiviral expression of parkin, but not its PD-linked mutant, rescues these phenotypes. The results suggest that parkin controls dopamine utilization in human midbrain DA neurons by enhancing the precision of DA neurotransmission and suppressing dopamine oxidation. Thus, the study provides novel targets and a physiologically relevant screening platform for disease-modifying therapies of PD.

Caffeine activates the PI3K/Akt pathway and prevents apoptotic cell death in a Parkinson's disease model of SH-SY5Y cells

Parkinsons disease (PD) is one of the most common neurodegenerative diseases. Recent epidemiological studies suggest that caffeine, one of the major components of coffee, has a protective effect against developing PD. However, the detailed mechanisms of how caffeine suppresses neuronal death have not been fully elucidated. We investigated the cytoprotective mechanisms of caffeine using human dopaminergic neuroblastoma SH-SY5Y cells as a PD model. Caffeine prevented the apoptotic cell death induced by serum/retinoic acid (RA) deprivation, MPP+, rotenone, and 6-OHDA in SH-SY5Y cells in a dose dependent manner. Caffeine lowered caspase-3 activity induced by serum/RA deprivation and 6-OHDA administration, and also decreased the number of apoptotic condensed and/or fragmented nuclei. Akt was phosphorylated 60 min after caffeine administration in a dose dependent manner; PI3K inhibitors, wortmannin and LY294002 canceled this cytoprotective effect of caffeine. On the other hand, MAPKs such as Erk1/2, p38, or JNK were not activated by caffeine. These results suggest that caffeine has a cytoprotective effect due to the activation of the PI3K/Akt pathways in SH-SY5Y cells.

Transcriptional activation of p62/A170/ZIP during the formation of the aggregates: possible mechanisms and the role in Lewy body formation in Parkinson's disease.

Formation of intracellular inclusion bodies due to defects in the protein degradation machinery is associated with the pathogenesis of neurodegenerative diseases. Sequestosomal protein p62/A170/ZIP, which is an oxidative stress-related protein and a ubiquitin-binding protein, is a component protein of Lewy bodies that are observed in patients with Parkinsons disease. The association of p62 with poly-ubiquitinated proteins may be an important step in the formation of intracellular protein aggregates like Lewy bodies. To study the role of p62 in the formation of protein aggregates in PC12 cells, we monitored the intracellular localizations of p62 and ubiquitinated proteins and the levels of both components during treatment with MG132, a proteasome inhibitor. In the early stage of aggregate formation, p62 did not always co-localize with ubiquitin. In contrast, these proteins were always co-localized in later stages. After the treatment of the cells with MG132, we found that the expression level of p62 increased due to the transcriptional activation of the gene and that higher molecular sizes of p62, corresponding to mono- and di-ubiquitinated formes, were also formed. Both the transcriptional inhibitor actinomycin D and an antisense oligonucleotide of p62 inhibited the MG132-mediated increase of p62, the sequestration of ubiquitinated proteins, and the enlargement of the aggregates. Furthermore, p62-positive aggregates were observed primarily in surviving cells. Together, these results suggest that p62 plays an important role in the protection of cells from the toxicity of misfolded proteins by enhancing aggregate formation especially in the later stages.

Association of the insertion/deletion polymorphism of the angiotensin I-converting enzyme gene in patients of migraine with aura

Recently, several angiotensin I-converting enzyme (ACE) inhibitors and an angiotensin II receptor blocker were demonstrated to have a clinically important prophylactic effect in migraine. ACE is one of the key enzymes in the rennin-angiotensin-aldosterone system, which modulates vascular tension and blood pressure. In humans, serum ACE levels are strongly genetically determined. Individuals who were homozygous for the deletion (D) allele showed increased ACE activity levels. To investigate the role of ACE polymorphism in headache, we analyzed the ACE insertion (I)/deletion (D) genotypes of 54 patients suffering from migraine with aura (MwA), 122 from migraine without aura, 78 from tension-type headache (TH), and 248 non-headache healthy controls. The ACE D allele were significantly more frequent in the MwA than controls (p<0.01). The incidence of the D/D genotype in MwA (25.9%) was significantly higher than that in controls (12.5%; p<0.01; odds ratio=5.26, 95% confidence interval: 1.69-16.34, adjusted for age and gender). No differences in the remaining groups were found. Our results support the conclusion that the D allele and the D/D genotype in the ACE gene is a genetic risk factor for Japanese MwA. There seems to be a possible relationship between ACE activity and the pathogenesis of migraine.

Increased plasma substance P and CGRP levels, and high ACE activity in migraineurs during headache-free periods

Abstract Substance P (SP), calcitonin gene‐related peptide (CGRP), and angiotensin converting enzyme (ACE) may have roles in trigeminovascular nociceptive mechanisms. We investigated interictal levels of SP, CGRP, ACE activity, and their correlation, in a sample of migraineurs. Forty‐one patients suffering from migraine with aura (MA), 54 without aura (MO), and 52 non‐headache subjects (controls) participated in this study. Blood samples were collected from cubital veins. Plasma levels of SP and CGRP were measured by enzyme immunoassay. Plasma ACE activities were measured spectrophotometrically. SP levels in MA (6.6 ± 3.7 pg/ml; mean ± SD) and MO (6.6 ± 3.2 pg/ml) were significantly higher than in controls (4.8 ± 2.4 pg/ml) (P < 0.01). CGRP levels in MA (18.8 ± 8.8 pg/ml) and MO (19.1 ± 9.4 pg/ml) were also significantly higher than in controls (13.4 ± 4.4 pg/ml) (P < 0.01). ACE activities in MA (34.6 ± 19.0 U/l) were significantly higher than in MO (25.3 ± 13.2 U/l) and controls (27.0 ± 20.4 U/l) (P < 0.05). There was a significant correlation between SP and CGRP levels (P < 0.05). In MA, SP and CGRP showed a tendency toward positive correlation, which was not significant. There was a weak, but significant positive correlation between SP levels and ACE activities (P < 0.01). However, a relationship between ACE activities and CGRP levels was not observed. The data suggest that SP, CGRP, and ACE are relevant to migraine pathophysiology, and that they may interact.

Huntington's disease–like 2 (HDL2) in North America and Japan

Huntingtons Disease–like 2 (HDL2) is a progressive, autosomal dominant, neurodegenerative disorder with marked clinical and pathological similarities to Huntingtons disease (HD). The causal mutation is a CTG/CAG expansion mutation on chromosome 16q24.3, in a variably spliced exon of junctophilin‐3. The frequency of HDL2 was determined in nine independent series of patients referred for HD testing or selected for the presence of an HD‐like phenotype in North America or Japan. The repeat length, ancestry, and age of onset of all North American HDL2 cases were determined. The results show that HDL2 is very rare, with a frequency of 0 to 15% among patients in the nine case series with an HD‐like presentation who do not have the HD mutation. HDL2 is predominantly, and perhaps exclusively, found in individuals of African ancestry. Repeat expansions ranged from 44 to 57 triplets, with length instability in maternal transmission detected in a repeat of 33 triplets. A younger age of onset is correlated with a longer repeat length (r2 = 0.29, p = 0.0098). The results further support the evidence that the repeat expansion at the chromosome 16q24.3 locus is the direct cause of HDL2 and provide preliminary guidelines for the genetic testing of patients with an HD‐like phenotype. Ann Neurol 2004

D‐β‐hydroxybutyrate protects dopaminergic SH‐SY5Y cells in a rotenone model of Parkinson's disease

It has been postulated that the pathogenesis of Parkinsons disease (PD) is associated with mitochondrial dysfunction. Rotenone, an inhibitor of mitochondrial complex I, provides models of PD both in vivo and in vitro. We investigated the neuroprotective effect of D‐β‐hydroxybutyrate (bHB), a ketone body, against rotenone toxicity by using SH‐SY5Y dopaminergic neuroblastoma cells. SH‐SY5Y cells, differentiated by all‐trans‐retinoic acid, were exposed to rotenone at concentrations ranging from 0 to 1,000 nM. We evaluated cellular oxidation reduction by the alamarBlue assay, viability by lactate dehydrogenase (LDH) assay, and survival/death ratio by live/dead assays. Exposure to rotenone for 48 hr oxidized cells and decreased their viability and survival rate in a concentration‐dependent manner. Pretreatment of cells with 8 mM bHB provided significant protection to SH‐SY5Y cells. Whereas rotenone caused the loss of mitochondrial membrane potential, released cytochrome c into the cytosol, and reduced cytochrome c content in mitochondria, addition of bHB blocked this toxic effect. bHB also attenuated the rotenone‐induced activation of caspase‐9 and caspase‐3. Administration of 0–10 mM 3‐nitropropionic acid, a complex II inhibitor, also decreased the reducing power of SH‐SY5Y cells measured by alamarBlue assay. Pretreatment with 8 mM bHB attenuated the decrease of alamarBlue fluorescence. These data demonstrated that bHB had a neuroprotective effect that supported the mitochondrial respiration system by reversing the inhibition of complex I or II. Ketone bodies, the alternative energy source in the mammalian brain, appear to have therapeutic potential in PD.