CLSPCOL rescues Alzheimer's disease mouse models.

Calmodulin-like skin protein (CLSP) inhibits Alzheimer's disease (AD)-related neurotoxicity. The activity of CLSP is reduced in AD. To restore the CLSP activity, we developed a hybrid peptide named CLSPCOL, consisting of CLSP(1-61) and the collagen-homologous region (COL) of adiponectin. It was previously shown that the CLSPCOL-mediated restoration of the reduced CLSP activity alleviated memory impairment and neuronal synaptic loss in APPswe/PS1dE9 double transgenic mice (APP/PS1 mice) at an advanced phase. Here, we examined whether CLSPCOL is effective against the memory impairment of the APP/PS1 mice at an early phase, and the memory impairment, caused by the temporal disturbance of the cholinergic neurotransmission, that mimics a part of AD-linked neuronal abnormality. The CLSPCOL-mediated restoration of the CLSP activity corrected the impairment in acquisition of fear-conditioned memory at an early-phase AD model. A single subcutaneous injection of CLSPCOL rescued the short-term working memory impairment, caused by subcutaneous injection of scopolamine. We have concluded that CLSPCOL is a promising disease-modifying therapeutic agent for not only the advanced phase but also the early-phase AD. It also serves as a symptomatic modifier of AD by potentiating the cholinergic neurotransmission.

Proline-arginine poly-dipeptide encoded by the C9orf72 repeat expansion inhibits adenosine deaminase acting on RNA.

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Unconventional translation of the hexanucleotide repeat expansion generates five dipeptide repeat proteins (DPRs). The molecular mechanism underlying the DPR-linked neurotoxicity is under investigation. In this study, using cell-based models, we show that poly-proline-arginine DPR (poly-PR), the most neurotoxic DPR in vitro, binds to adenosine deaminase acting on RNA (ADAR)1p110 and ADAR2 and inhibits their RNA editing activity. We further show that poly-PR impairs cellular stress response that is mediated by ADAR1p110. These results together suggest that the poly-PR-mediated inhibition of the ADAR activity contributes to C9-ALS/FTD-linked neurotoxicity.

Identification of highest neurotoxic amyloid-ß plaque type showing reduced contact with astrocytes.

Amyloid-ß (Aß) plaques are strongly associated with the development of Alzheimer's disease (AD). However, it remains unclear how morphological differences in Aß plaques determine the pathogenesis of Aß. Here, we categorized Aß plaques into four types based on the macroscopic features of the dense core, and found that the Aß-plaque subtype containing a larger dense core showed the strongest association with neuritic dystrophy. Astrocytes dominantly accumulated toward these expanded/dense-core-containing Aß plaques. Previously, we indicated that deletion of the mitochondrial ubiquitin ligase MITOL/MARCH5 triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. In this study, MITOL deficiency accelerated the formation of expanded/dense-core-containing Aß plaques, which showed reduced contacts with astrocytes, but not microglia. Our findings suggest that expanded/dense-core-containing Aß-plaque formation enhanced by the alteration of mitochondrial function robustly contributes to the exacerbation of Aß neuropathology, at least in part, through the reduced contacts between Aß plaques and astrocytes.

Mitochondrial ubiquitin ligase alleviates Alzheimer's disease pathology via blocking the toxic amyloid-ß oligomer generation.

Mitochondrial pathophysiology is implicated in the development of Alzheimer's disease (AD). An integrative database of gene dysregulation suggests that the mitochondrial ubiquitin ligase MITOL/MARCH5, a fine-tuner of mitochondrial dynamics and functions, is downregulated in patients with AD. Here, we report that the perturbation of mitochondrial dynamics by MITOL deletion triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. Notably, MITOL deletion in the brain enhanced the seeding effect of Aß fibrils, but not the spontaneous formation of Aß fibrils and plaques, leading to excessive secondary generation of toxic and dispersible Aß oligomers. Consistent with this, MITOL-deficient mice with Aß etiology exhibited worsening cognitive decline depending on Aß oligomers rather than Aß plaques themselves. Our findings suggest that alteration in mitochondrial morphology might be a key factor in AD due to directing the production of Aß form, oligomers or plaques, responsible for disease development.

Restoration of the reduced CLSP activity alleviates memory impairment in Alzheimer disease.

Calmodulin-like skin protein (CLSP), a secreted peptide, inhibits neuronal death in cell-based Alzheimer's disease (AD) models and transgenic overexpression of the CLSP gene suppresses synaptic loss and memory impairment in AD model mice, APPswe/PS1dE9 double transgenic mice (APP/PS1 mice). Despite the anticipated role of CLSP as an AD-suppressing factor, it remains unanswered whether the insufficiency of the CLSP activity is linked to the AD pathogenesis. In this study, we first show that adiponectin, a CLSP potentiator/protector, dominantly determines the CLSP activity in the central nervous system where there are sufficient concentrations of CLSP, higher concentrations of CLSP inhibitors such as apolipoprotein E, and smaller concentrations of adiponectin. We next show that both the levels of brain adiponectin and the intraneuronal levels of SH3BP5, an important effector of the CLSP signal, are reduced in both AD patients and APP/PS1 mice. Finally, the restoration of the CLSP activity by subcutaneous injection of a hybrid peptide named CLSPCOL consisting of CLSP(1-61) and the collagen-homologous region of adiponectin, which has more potent neuroprotective activity than CLSP, is insensitive to the suppression by the CLSP inhibitors, and is efficiently recruited into brains, alleviates dementia and synaptic loss in the aged APP/PS1 mice. Collectively, these results suggest that the reduction in the CLSP activity, likely caused by the reduction in the levels of adiponectin, leads to the insufficient protection of neurons from neurotoxicity in the AD brains and the restoration of the CLSP activity is a promising strategy for the treatment of AD.

[Practical training of pharmacokinetics with an web-based simulation application].

A computer simulation application on pharmacokinetics, which we developed using a software, named "Stella®", has been successfully used for the virtual training of pharmacokinetics at multiple medical schools. The training course using Stella® has encouraged the medical students to optimize drug administration for individual patients on the computers. Importantly, the virtual training is free of any concern on human and animal ethics. The simulation application has been freely provided for medical schools without any restrictions and charge. For many years, it has been under constant version-upgrade in response to updates of the operating systems (OS) of personal computers or the software. Very recently, major updates of the OS and the software, and the emergence of tablet- and smartphones-type computers have been prompting us to perform a major revision of the simulation application. Here, we introduce the new version of the "web-based" simulation application that is available through any device including personal computers, tablets, and smartphones irrespective of the OSs (Microsoft Windows and Macintosh, Android, and iOS), without any extra charge unless the modification is required. We believe that the new-version of web-based simulation application will be useful not only for medical, nursing and pharmacy students, but also for medical workers who need to simulate drug pharmacokinetics on the computers before they administer drugs to the patients.

C9-ALS/FTD-linked proline-arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation.

A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.

Calmodulin-like skin protein suppresses the increase in senescence-associated ß-galactosidase induced by hydrogen peroxide or ultraviolet irradiation in keratinocytes.

Calmodulin-like skin protein (CLSP) is a secreted peptide that is produced by skin keratinocytes and some related epithelial cells. It has previously been shown that CLSP is recruited via the bloodstream into the central nervous system where it likely exerts a neuroprotective effect against toxicity related to Alzheimer's disease (AD) by binding to the heterotrimeric humanin receptor and activating intracellular survival signaling. However, it remains to be elucidated whether secreted CLSP shows a protective effect in the skin tissues. In the current study, using primary keratinocytes treated with hydrogen peroxide (H2 O2 ) or exposed to ultraviolet (UV) irradiation as senescence models of keratinocytes, we addressed whether CLSP affects senescence in skin keratinocytes. We found that CLSP expression was upregulated by H2 O2 or UV in keratinocytes. Furthermore, co-incubation with recombinant CLSP reduced the increase in senescence-associated ß-galactosidase-positivity in keratinocytes that were induced by H2 O2 or UV. These results suggest that CLSP may function as a senescence-suppressing factor in keratinocytes.

The proline-arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat (DPR) proteins. Although it has been reported that DPR proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we have first confirmed that proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity and found that the poly-PR-induced neurotoxicity is repressed by the acceleration of ribosomal RNA synthesis. These results suggest that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. We have further identified DEAD-box RNA helicases as poly-PR-binding proteins, the functions of which are inhibited by poly-PR. The enforced reduction in the expression of DEAD-box RNA helicases causes impairment of ribosome biogenesis and neuronal cell death. These results together suggest that poly-PR causes neurotoxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

Calmodulin-like skin protein protects against spatial learning impairment in a mouse model of Alzheimer disease.

Humanin and calmodulin-like skin protein (CLSP) inhibits Alzheimer disease (AD)-related neuronal cell death via the heterotrimeric humanin receptor in vitro. It has been suggested that CLSP is a central agonist of the heterotrimeric humanin receptor in vivo. To investigate the role of CLSP in the AD pathogenesis in vivo, we generated mouse CLSP-1 transgenic mice, crossed them with the APPswe/PSEN1dE9 mice, a model mouse of AD, and examined the effect of CLSP over-expression on the pathological phenotype of the AD mouse model. We found that over-expression of the mouse CLSP-1 gene attenuated spatial learning impairment, the loss of a presynaptic marker synaptophysin, and the inactivation of STAT3 in the APPswe/PSEN1dE9 mice. On the other hand, CLSP over-expression did not affect levels of Aß, soluble Aß oligomers, or gliosis. These results suggest that the CLSP-mediated attenuation of memory impairment and synaptic loss occurs in an Aß-independent manner. The results of this study may serve as a hint to the better understanding of the AD pathogenesis and the development of AD therapy.

Calmodulin-like skin protein is downregulated in human cerebrospinal fluids of Alzheimer's disease patients with apolipoprotein E4; a pilot study using postmortem samples.

OBJECTIVE: Calmodulin-like skin protein (CLSP) is a secreted peptide that inhibits neuronal cell death, linked to Alzheimer's disease (AD), by binding to the heterotrimeric humanin receptor and activating an intracellular survival pathway. CLSP is only expressed in skin keratinocytes and related epithelial cells, circulates in the blood stream, and passes the blood-cerebrospinal fluid (CSF) barrier. In the current study, we addressed the issues as to whether CLSP functions in the central nervous system and whether the concentration of CLSP is reduced in the CSFs of AD patients. METHODS: Mice were intraperitoneally injected with 5 nmol of recombinant human CLSP. At 1h after the injection, the mice were sacrificed for the analysis of the existence of human CLSP in blood and interstitial fluid (ISF)-containing brain samples. Using postmortem CSF samples, we next determined the concentrations of CLSP in CSFs of human AD and control cases. RESULTS: Intraperitoneally administered recombinant human CLSP circulated in the blood stream and reached the brain interstitial fluid. The concentrations of CLSP in CSFs of human AD and control cases are sufficient to exhibit the CLSP activity. Although the concentrations of CLSP in CSFs were not significantly different between AD and control cases, the concentrations of CLSP are lower in the AD cases with the apolipoprotein E4 genotype than in the AD cases without the apolipoprotein E4 genotype. DISCUSSION: The first result indicates that CLSP enters the central nervous system through the blood-brain barrier. The second result suggests that CLSP functions in the human brains. The third result may exclude the possibility that the downregulation of the CLSP level is involved in the AD pathogenesis. The last result may contribute to the better understanding of the AD pathogenesis from the standpoint of the apolipoprotein E genotype.

hnRNPA1 autoregulates its own mRNA expression to remain non-cytotoxic.

Heterogeneous nuclear ribonucleoprotein (hnRNP)A1, a member of the hnRNP family, is involved in a variety of RNA metabolisms. The hnRNPA1 expression is altered in some human diseases and mutations of the hnRNPA1 gene cause amyotrophic lateral sclerosis and multisystem proteinopathy. It has been therefore assumed that the dysregulation of hnRNPA1 is linked to the pathogenesis of the diseases. However, the mechanism underlying the regulation of the hnRNPA1 expression remains unknown. In this study, using cell-based models, we have found that hnRNPA1 negatively regulates its own mRNA expression by inhibiting the intron10 splicing of hnRNPA1 pre-mRNA. This mechanism likely serves as an autoregulation of the hnRNPA1 expression. We have also found that a low-grade excess of hnRNPA1 expression causes cytotoxicity by activating the mitochondrial apoptosis pathway. Collectively, these data suggest that the level of hnRNPA1 is strictly controlled to be within a certain range by the mRNA autoregulation in the physiological condition so that the cytotoxicity-causative alteration of hnRNPA1 expression does not take place.

The Lysosomal Trafficking Transmembrane Protein 106B Is Linked to Cell Death.

A common genetic variation in the transmembrane protein 106B (TMEM106B) gene has been suggested to be a risk factor for frontotemporal lobar degeneration (FTLD) with inclusions of transactive response DNA-binding protein-43 (TDP-43) (FTLD-TDP), the most common pathological subtype in FTLD. Furthermore, previous studies have shown that TMEM106B levels are up-regulated in the brains of FTLD-TDP patients, although the significance of this finding remains unknown. In this study, we show that the overexpression of TMEM106B and its N-terminal fragments induces cell death, enhances oxidative stress-induced cytotoxicity, and causes the cleavage of TDP-43, which represents TDP-43 pathology, using cell-based models. TMEM106B-induced death is mediated by the caspase-dependent mitochondrial cell death pathways and possibly by the lysosomal cell death pathway. These findings suggest that the up-regulation of TMEM106B may increase the risk of FTLD by directly causing neurotoxicity and a pathological phenotype linked to FTLD-TDP.

An Alzheimer Disease-linked Rare Mutation Potentiates Netrin Receptor Uncoordinated-5C-induced Signaling That Merges with Amyloid ß Precursor Protein Signaling.

A missense mutation (T835M) in the uncoordinated-5C (UNC5C) netrin receptor gene increases the risk of late-onset Alzheimer disease (AD) and also the vulnerability of neurons harboring the mutation to various insults. The molecular mechanisms underlying T835M-UNC5C-induced death remain to be elucidated. In this study, we show that overexpression of wild-type UNC5C causes low-grade death, which is intensified by an AD-linked mutation T835M. An AD-linked survival factor, calmodulin-like skin protein (CLSP), and a natural ligand of UNC5C, netrin1, inhibit this death. T835M-UNC5C-induced neuronal cell death is mediated by an intracellular death-signaling cascade, consisting of death-associated protein kinase 1/protein kinase D/apoptosis signal-regulating kinase 1 (ASK1)/JNK/NADPH oxidase/caspases, which merges at ASK1 with a death-signaling cascade, mediated by amyloid ß precursor protein (APP). Notably, netrin1 also binds to APP and partially inhibits the death-signaling cascade, induced by APP. These results may provide new insight into the amyloid ß-independent pathomechanism of AD.

Glycolytic flux controls D-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes.

D-Serine is an essential coagonist with glutamate for stimulation of N-methyl-D-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control D-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the D-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with D-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of D-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls D-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus.

Nuclear TDP-43 causes neuronal toxicity by escaping from the inhibitory regulation by hnRNPs.

Dysregulation of transactive response DNA-binding protein-43 (TDP-43) is thought to be linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 normally localizes in the nucleus but its main localization shifts to the cytoplasm in most affected cells of ALS and FTLD patients. It is not yet known whether nuclear or cytoplasmic TDP-43 is responsible for TDP-43-induced neurotoxicity. In this study, we show that nuclear TDP-43 causes TDP-43 neurotoxicity. DNA/RNA-binding and dimerization of TDP-43 are both essential for TDP-43-induced cell death. Moreover, endogenous heterogeneous nuclear ribonucleoprotein-U (hnRNP-U) binds to TDP-43 and knocking-down of hnRNP-U induces neurotoxicity, whereas overexpression of hnRNP-U or hnRNP-A2 inhibits TDP-43-induced neurotoxicity. In addition, hnRNP-U inhibits TDP-43-mediated alterations in splicing of POLDIP3 mRNA. Altogether, these results suggest that nuclear TDP-43 becomes neurotoxic by escaping from the inhibitory regulation by hnRNPs.

Protective effects of Humanin and calmodulin-like skin protein in Alzheimer's disease and broad range of abnormalities.

Humanin is a 24-amino acid, secreted bioactive peptide that prevents various types of cell death and improves some types of cell dysfunction. Humanin inhibits neuronal cell death that is caused by a familial Alzheimer's disease (AD)-linked gene via binding to the heterotrimeric Humanin receptor (htHNR). This suggests that Humanin may play a protective role in AD-related pathogenesis. Calmodulin-like skin protein (CLSP) has recently been identified as a physiological agonist of htHNR with 10(5)-fold more potent anti-cell death activity than Humanin. Humanin has also shown to have protective effects against some metabolic disorders. In this review, the broad range of functions of Humanin and the functions of CLSP that have been characterized thus far are summarized.

Apollon/Bruce is upregulated by Humanin.

Humanin, a short bioactive peptide, inhibits a variety of cell deaths. Humanin-mediated inhibition of neuronal cell death, caused by an Alzheimer's disease (AD)-linked mutant gene occurs via binding of Humanin to its heterotrimeric Humanin receptor (htHNR), which results in the activation of the Janus-associated kinases (JAKs) and signal transducer and activator and transcription 3 (STAT3) signaling pathway. A previous study demonstrated that the Humanin-induced activation of the htHNR/JAK2/STAT3 signaling pathway leads to increased expression of SH3 domain-binding protein 5 (SH3BP5), which is an essential effector of Humanin's anti-cell death activity in some cultured neuronal cells. However, it remains unknown whether SH3BP5 is the sole effector of the Humanin signaling pathway via htHNR/JAKs/STAT3. Here we show that the Humanin signaling pathway via htHNR/JAKs/STAT3 increased the expression levels of mRNA and protein of Apollon/Bruce, an unusual member of the inhibitors of apoptosis proteins, and that overexpression of Apollon/Bruce inhibits neuronal death, caused by a London-type familial AD-linked mutant (V642I) of amyloid ß precursor protein. Overall, the results indicate that expression of Apollon/Bruce is upregulated by Humanin, and Apollon/Bruce could be an effector of Humanin in a context-dependent manner.