Kenzo Terashita

Amyloid-beta causes memory impairment by disturbing the JAK2/STAT3 axis in hippocampal neurons.

Elevation of intracranial soluble amyloid-β (Aβ) levels has been implicated in the pathogenesis of Alzheimers disease (AD). Intracellular events in neurons, which lead to memory loss in AD, however, remain elusive. Humanin (HN) is a short neuroprotective peptide abolishing Aβ neurotoxicity. Recently, we found that HN derivatives activate the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling axis. We here report that an HN derivative named colivelin completely restored cognitive function in an AD model (Tg2576) by activating the JAK2/STAT3 axis. In accordance, immunofluorescence staining using a specific antibody against phospho- (p-) STAT3 revealed that p-STAT3 levels in hippocampal neurons age-dependently decreased in both AD model mice and AD patients. Intracerebroventricular administration of Aβ1–42 downregulated p-STAT3 whereas passive immunization with anti-Aβ antibody conversely restored hippocampal p-STAT3 levels in Tg2576 mice, paralleling the decrease in the brain Aβ burden. Aβ1–42 consistently modulated p-STAT3 levels in primary neurons. Pharmacological inhibition of the JAK2/STAT3 axis not only induced significant loss of spatial working memory by downregulating an acetylcholine-producing enzyme choline acetyltransferase but also desensitized the M1-type muscarinic acetylcholine receptor. Thus, we propose a novel theory accounting for memory impairment related to AD: Aβ-dependent inactivation of the JAK2/STAT3 axis causes memory loss through cholinergic dysfunction. Our findings provide not only a novel pathological hallmark in AD but also a novel target in AD therapy.

Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-related insults

An unbiased functional screening with brain cDNA library from an Alzheimers disease (AD) brain identified a novel 24-residue peptide Humanin (HN), which suppresses AD-related neurotoxicity. As the 1567-base cDNA containing the open reading frame (ORF) of HN is 99% identical to mitochondrial 16S ribosomal RNA as well as registered human mRNA, it was elusive whether HN is produced in vivo. Here, we raised anti-HN antibody and found that long cDNAs containing the ORF of HN (HN-ORF) produced the HN peptide in mammalian cells, dependent on the presence of full-length HN-ORF. Immunoblot analysis detected a 3-kDa protein with HN immunoreactivity in the testis and the colon in 3-week-old mice and in the testis in 12-week-old mice. HN immunoreactivity was also detected in an AD brain, but little in normal brains. This study suggests that HN peptide could be produced in vivo, and would provide a novel insight into the pathophysiology of AD.

Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death

Amyloid precursor protein (APP), the precursor of Aβ, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti‐APP antibody. The c‐Jun N‐terminal kinase (JNK) can mediate various neurotoxic signals, including Aβ neurotoxicity. However, the relationship of APP‐mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Aβ independent. Here we examined whether JNK is involved in APP‐mediated neuronal cell death and found that: (i) neuronal cell death by antibody‐bound APP was inhibited by dominant‐negative JNK, JIP‐1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK‐mediated cell death closely coincided with that of APP‐mediated cell death. Pertussis toxin (PTX) suppressed APP‐mediated cell death but not caJNK‐induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP‐mediated cytotoxicity. In the presence of PTX, the PTX‐resistant mutant of Gαo, but not that of Gαi, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP‐mediated neuronal cell death as a downstream signal transducer of Go.

A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease‐relevant insults

Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimers disease (AD)‐related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two‐hybrid screening, we identified TRIM11 as a novel HN‐interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B‐box domain, a coiled‐coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled‐coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S‐transferase‐fused or EGFP‐fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin‐mediated protein degradation pathways.

Development of a femtomolar-acting humanin derivative named colivelin by attaching activity-dependent neurotrophic factor to its N terminus: characterization of colivelin-mediated neuroprotection against Alzheimer's disease-relevant insults in vitro and in vivo.

Alzheimers disease (AD) is the most common cause of dementia. Humanin (HN) is a short bioactive peptide abolishing neuronal cell death induced by various familial AD (FAD)-causative genes and amyloid-β (Aβ) in vitro. It has been shown that HN suppresses memory impairment of mice induced by intracerebroventricular administration of Aβ. To potentiate the neuroprotective effect of HN, we synthesized a hybrid peptide named Colivelin composed of activity-dependent neurotrophic factor (ADNF) C-terminally fused to AGA-(C8R)HNG17, a potent HN derivative. Colivelin completely suppresses death induced by overexpressed FAD-causative genes and Aβ1-43 at a concentration of 100 fm, whereas AGA-(C8R)HNG17 does so at a concentration of 10 pm. Colivelin-induced neuroprotection has been confirmed to occur via two neuroprotective pathways: one mediated by Ca2+/calmodulin-dependent protein kinase IV, triggered by ADNF, and one mediated by signal transducer and activator of transcription 3, triggered by HN. In vivo animal studies have further indicated that intracerebroventricular administration of Colivelin not only completely suppresses impairment in spatial working memory induced by repetitive intracerebroventricular injection of Aβ25-35 or Aβ1-42, but also it antagonizes neuronal loss in the CA1 region of hippocampus induced by hippocampal injection of Aβ1-42. In addition, intraperitoneally administered Colivelin suppresses memory impairment caused by a muscarinic acetylcholine receptor antagonist, 3-quinuclidinyl benzilate, indicating that a substantial portion of intraperitoneally administered Colivelin passes through the blood-brain barrier and suppresses functional memory deficit. Thus, Colivelin might serve as a novel drug candidate for treatment of AD.

Two serine residues distinctly regulate the rescue function of Humanin, an inhibiting factor of Alzheimer's disease-related neurotoxicity: functional potentiation by isomerization and dimerization

The 24‐residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimers disease (AD) genes and Aβ. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post‐translational modification of Ser14 might play a role, we found that HN with d‐Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas d‐Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self‐dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by d‐isomerization of Ser14 and Ser7‐relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pm against 25 µm Aβ1–43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics.

Implanted cannula-mediated repetitive administration of Aβ25–35 into the mouse cerebral ventricle effectively impairs spatial working memory

Amyloid beta (Abeta) is closely related to the onset of Alzheimers disease (AD). To construct AD animal models, a bolus administration of a large dose of toxic Abeta into the cerebral ventricles of rodents has been performed in earlier studies. In parallel, a continuous infusion system via an osmotic pump into the cerebral ventricle has been developed to make a rat AD model. In this study, we developed a mouse AD model by repetitive administration of Abeta25-35 via a cannula implanted into the cerebral ventricle. Using this administration system, we reproducibly constructed a mouse with impaired spatial working memory. In accordance with the occurrence of the abnormal mouse behavior, we found that the number of choline acetyltransferase (ChAT)-positive neurons was reduced in paraventricular regions of brains of Abeta25-35-administered mice in a dose-dependent manner. Considering that the repetitive administration of a small dose of toxic Abeta via an implanted cannula leads to a brain status more resembling that of the AD patients than a bolus injection of a large dose of Abeta, and therapeutic as well as toxic agents are able to be repeatedly and reliably administered via an implanted cannula, we concluded that the implanted cannula-bearing AD mouse model is useful for development of new AD therapy.

Nasal Colivelin Treatment Ameliorates Memory Impairment Related to Alzheimer's Disease

Humanin (HN) and its derivatives, such as Colivelin (CLN), suppress neuronal death induced by insults related to Alzheimers disease (AD) by activating STAT3 in vitro. They also ameliorate functional memory impairment of mice induced by anticholinergic drugs or soluble toxic amyloid-β (Aβ) in vivo when either is directly administered into the cerebral ventricle or intraperitoneally injected. However, the mechanism underlying the in vivo effect remains uncharacterized. In addition, from the standpoint of clinical application, drug delivery methods that are less invasive and specific to the central nervous system (CNS) should be developed. In this study, we show that intranasally (i.n.) administered CLN can be successfully transferred to CNS via the olfactory bulb. Using several behavioral tests, we have demonstrated that i.n. administered CLN ameliorates memory impairment of AD models in a dose-responsive manner. Attenuation of AD-related memory impairment by HN derivatives such as CLN appears to be correlated with an increase in STAT3 phosphorylation levels in the septohippocampal region, suggesting that anti-AD activities of HN derivatives may be mediated by activation of STAT3 in vivo as they are in vitro. We further demonstrate that CLN treatment inhibits an Aβ induced decrease in the number of choline acetyltransferase (ChAT)-positive neurons in the medial septum. Combined with the finding that HN derivatives upregulate mRNA expression of neuronal ChAT and vesicular acetylcholine transporter (VAChT) in vitro, it is assumed that CLN may ameliorate memory impairment of AD models by supporting cholinergic neurotransmission, which is at least partly mediated by STAT3-mediated transcriptional upregulation of ChAT and VAChT.

Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways

Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.

Neuroprotective effect of activity-dependent neurotrophic factor against toxicity from familial amyotrophic lateral sclerosis-linked mutant SOD1 in vitro and in vivo

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease, affecting mostly middle‐aged people. There are no curative therapies for ALS. Several lines of evidence have supported the notion that the proapoptotic property of familial ALS (FALS)‐linked mutant Cu/Zn‐superoxide dismutase‐1 (SOD1) genes may play an important role in the pathogenesis of some FALS cases. Here we found that activity‐dependent neurotrophic factor (ADNF), a neurotrophic factor originally identified to have the anti‐Alzheimers disease (AD) activity, protected against neuronal cell death caused by FALS‐linked A4T‐, G85R‐ and G93R‐SOD1 in a dose‐responsive fashion. Notably, ADNF‐mediated complete suppression of SOD1 mutant‐induced neuronal cell death occurs at concentrations as low as 100 fM. ADNF maintains the neuroprotective activity even at concentrations of more than 1 nM. This is in clear contrast to the previous finding that ADNF loses its protective activity against neurotoxicity induced by AD‐relevant insults, including some familial AD genes and amyloid β peptide at concentrations of more than 1 nM. Characterization of the neuroprotective activity of ADNF against cell death caused by SOD1 mutants revealed that CaMKIV and certain tyrosine kinases are involved in ADNF‐mediated neuroprotection. Moreover, in vivo studies showed that intracerebroventricularly administered ADNF significantly improved motor performance of G93A‐SOD1 transgenic mice, a widely used model of FALS, although survival was extended only marginally. Thus, the neuroprotective activity of ADNF provides a novel insight into the development of curative drugs for ALS.