Shingo Kariya

Humanin inhibits cell death of serum-deprived PC12h cells

Humanin (HN) and S14G HN (HNG) are recently discovered polypeptides that rescue cells from death induced by multiple different types of familial Alzheimers disease genes and by amyloid-&bgr;. However, the cytoprotective activity of these peptides against other cell death-inducing stimuli remains unclear. In this study, we demonstrated, using three different methods (MTS assay, caspase-3 assay, and detection of DNA fragmentation), that both HN and HNG protect PC12 cells from death elicited by serum deprivation. This implies the potential of the peptides to rescue cells from a broad spectrum, if not all, of cell death-inducing factors. Further investigations on HN may lead the possible application of this peptide as therapeutic agent for the treatment of other neurodegenerative diseases.

DNA single-strand break repair is impaired in aprataxin-related ataxia

Early‐onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive form of cerebellar ataxia. The causative protein for EAOH/AOA1, aprataxin (APTX), interacts with X‐ray repair cross‐complementing 1 (XRCC1), a scaffold DNA repair protein for single‐strand breaks (SSBs). The goal of this study was to prove the functional involvement of APTX in SSB repair (SSBR).

Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes

Humanin (HN) has been reported to be an endogenous peptide that exerts highly selective neuroprotection against cell death induced by various types of Alzheimers disease-related insults. We previously proposed the much broader cytoprotective potential of HN from the result that HN suppressed serum-deprivation-induced death of rat pheochromocytoma cells. In this study, we showed that HN also suppressed death of human lymphocytes cultured under serum-deprived condition. Further, we revealed, by assaying metabolic activity and survival rate, that HN was a potent factor capable of increasing the metabolic activity of individual serum-deprived lymphocytes. To our knowledge, there is no report described about a rescue factor that increases the metabolic activity of individual serum-deprived cells and prolongs their survival. This novel feature of HN may enable us to apply this peptide for the management of diseases involving poor metabolic activity, such as mitochondria-related disorders and brain ischemia.

Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent

Humanin (HN) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer’s disease-related insults. We have previously indicated that HN increases cellular ATP levels and speculated that this peptide may rescue energy-deficient cells in mitochondrial disorders. Here, we report, for the first time, increased HN expression in skeletal muscles from patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). HN was strongly positive in all ragged-red fibers (RRFs) and some non-RRFs, and most of them were type 1 fibers generally requiring higher energy than type 2 fibers. HN in these fibers was localized in mitochondria. HN expression was also increased in small arteries that strongly reacted for succinate dehydrogenase. Our experiments on muscular TE671 cells indicated the possibility that synthesized HN increases cellular ATP levels by directly acting on mitochondria. From these in vivo and in vitro findings, we propose that HN expression might be induced in response to the energy crisis within affected fibers and vessels in MELAS muscles and further be a possible therapeutic candidate for MELAS.

Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA

Humanin (HN) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis by mutant Alzheimers disease genes. This 24-residue peptide has been recently shown to suppress apoptosis by interfering with activation of Bcl-2-associated X protein (Bax) in cytosol. In the present study, we showed that HN increases ATP levels in human lymphocytes, muscular TE671 cells, and neural SKN-MC cells, and protects these cells from serum deprivation-induced apoptosis. The suppressed apoptotic death of serum-deprived cells would be explained by the anti-Bax effect of HN; however, HN also increased ATP levels of serum-supplemented cells (non-apoptotic cells), in which Bax is likely to be inactive. This result suggests the presence of a certain mechanism independent of Bax inactivation to increase ATP levels of cells under non-apoptotic condition. By treatment with HN, the ATP levels of lymphocytes from patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) associated with A3243G mutant mtDNA were increased as well, suggesting that HN is able to prevent cells in MELAS from falling into ATP deficiency. Our quantitative PCR findings indicated that the HN-induced increase in ATP may not be a consequence of mitochondrial proliferation, because HN rather suppressed mtDNA replication. This suppression may be important in the treatment of affected cells in MELAS, since the mutant mtDNAs that increase during compensatory mtDNA replication for ATP deficiency cause excessive formation of reactive oxygen species, leading to further energy crisis. We thus propose that HN, which increases cellular ATP levels without inducing mtDNA replication, may be suited for the treatment of MELAS.

Humanin attenuates apoptosis induced by DRPLA proteins with expanded polyglutamine stretches.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal-dominant neurodegenerative disorder caused by expansion of CAG repeats in the DRPLA gene, which codes for a polyglutamine (polyQ) stretch. The expanded polyQs are known to form intracellular aggregates and to confer neurotoxic activity. Recent studies have indicated that activation of apoptosis signal-regulating kinase 1 (ASK1) is involved in polyQ-induced apoptosis. Humanin (HN) is an endogenous peptide that inhibits neuronal cell death caused by mutant Alzheimer’s disease genes, and this neuroprotective factor has recently been reported to suppress apoptosis by inhibiting activation of ASK1. To test the anti-ASK1 effect of HN on polyQ neurotoxicity, we constructed neuronal PC12 cells expressing expanded polyQs under the control of the Tet-Off™ system. Using this cell line, we showed that HN suppresses apoptotic cell death induced by expanded polyQs. However, the suppression was incomplete, suggesting that polyQs also stimulate other pathogenic cascades unrelated to ASK1. We further showed that HN suppresses polyQ aggregate formation. This result implied the possibility that aggregation is also related to the polyQ-mediated cascade involving ASK1 activation. Although the details remain uncertain, our results suggest that ASK1 is potentially involved in pathogenesis of DRPLA and that HN might contribute partially to the suppression of neurodegeneration in polyQ diseases.

Increased vulnerability to L-DOPA toxicity in dopaminergic neurons From VMAT2 heterozygote knockout mice.

Parkinson’s disease (PD) is characterized by a preferential loss of dopaminergic neurons in the substantia nigra pars compacta. The etiology of PD remains unclear; however, generation of reactive oxygen species during oxidation of free dopamine (DA) in the cytoplasm might be one of the causes of selective dopaminergic neuron loss in PD. Vesicular monoamine transporter type 2 (VMAT2) proteins in nerve terminals take up and partition DA from neuronal cytoplasm into synaptic vesicles. Alterations of VMAT2 function might therefore cause cytoplasmic accumulation of free DA, which is toxic for dopaminergic neurons. We showed that dopaminergic neurons from VMAT2 heterozygous knockout mice were more vulnerable to the toxic effect of l-3,4-dihydroxyphenylalanine (l-DOPA, a DA precursor) than those from wild-type mice. Our results suggest that reduction of VMAT2 activity might attenuate the efficacy of l-DOPA therapy for patients with PD.

Cytoprotective effect of novel histone deacetylase inhibitors against polyglutamine toxicity

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurological disorder caused by a CAG repeat expansion in the DRPLA gene encoding polyglutamine (polyQ). Although previous experimental studies have demonstrated that histone deacetylase (HDAC) inhibitors are therapeutically active, known HDAC inhibitors have considerable adverse effects clinically. To identify new HDAC inhibitors for the treatment of DRPLA, we evaluated a new series of HDAC inhibitors, N-hydroxycarboxamides, with our drug screening system, which uses neuronal PC12 cells stably transfected with a part of the DRPLA gene. We found that two of four N-hydroxycarboxamides significantly reduced polyQ-induced cell death. The essential structure of these compounds is a hydroxamic acid residue, which is shared with trichostatin A, a known HDAC inhibitor. Although our study showed mild neuroprotective effects, further structural modification of compounds that retain this residue may decrease cytotoxicity and increase protective activity against polyQ toxicity.

Two novel spliced presenilin 2 transcripts in human lymphocyte with oxidant stress and brain.

We identified two novel spliced human presenilin 2 (PS2) transcripts. The first, PS2ΔEx3–7, lacked part ofexon 3, all of exons 4, 5, and 6, and part of exon 7, resulting in an in-frame shift and inclusion of the natural start codon and proteolytic region. This transcript was detected in cerebral cortex and peripheral lymphocytes. The second transcript, PS2ΔEx4, lacked exon 4, resulting in a frame shift and inclusion of the natural start codon, and was transcribed in peripheral lymphocytes and heart but not in brain. Quantitative RT-PCR analysis revealed that the PS2ΔEx3–7 significantly increased in lymphocytes treated with H2O2, suggesting that this transcript is a novel genetic marker that can be used to study the pathogenesis of Alzheimers disease.

Novel splice variants increase molecular diversity of aprataxin, the gene responsible for early-onset ataxia with ocular motor apraxia and hypoalbuminemia.

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is one of the most common forms of autosomal recessive cerebellar ataxia. We identified six new alternative transcripts produced by the aprataxin gene responsible for EAOH. Total eight transcripts encoded truncated proteins that were located within the nucleus or cytoplasm and showed different binding abilities to wild-type (WT) aprataxin. Thus, the alternative splicing increases the molecular diversity of aprataxin and the expression profiles of these transcripts in various tissues may be related to the tissue-specific phenotypes.