The Parkinson's Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway.

Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson's disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2.

The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.

Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-ß 42 (Aß42)/Aß40 and Aß43/Aß40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.

The first Japanese case of leukodystrophy with ovarian failure arising from novel compound heterozygous AARS2 mutations.

Even now, only a portion of leukodystrophy patients are correctly diagnosed, though various causative genes have been identified. In the present report, we describe a case of adult-onset leukodystrophy in a woman with ovarian failure. By whole-exome sequencing, a compound heterozygous mutation consisting of NM_020745.3 (AARS2_v001):c.1145C>A and NM_020745.3 (AARS2_v001):c.2255+1G>A was identified. Neither of the mutations has been previously reported, and this is the first report of alanyl-transfer RNA synthetase 2 mutation in Asia. We anticipate that further studies of the molecular basis of leukodystrophy will provide insight into its pathogenesis and hopefully lead to sophisticated diagnostic and treatment strategies.

PINK1 and Parkin complementarily protect dopaminergic neurons in vertebrates.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by selective dopaminergic cell loss in the substantia nigra, but its pathogenesis remains unclear. The recessively inherited familial PD genes PARK2 and PARK6 have been attributed to mutations in the Parkin and PTEN-induced kinase 1 (PINK1) genes, respectively. Recent reports suggest that PINK1 works upstream of Parkin in the same pathway to regulate mitochondrial dynamics and/or conduct autophagic clearance of damaged mitochondria. This phenomenon is preserved from Drosophila to human cell lines but has not been demonstrated in a vertebrate animal model in vivo. Here, we developed a medaka fish (Oryzias latipes) model that is deficient in Pink1 and Parkin. We found that despite the lack of a conspicuous phenotype in single mutants for Pink1 or Parkin, medaka that are deficient in both genes developed phenotypes similar to that of human PD: late-onset locomotor dysfunction, a decrease in dopamine levels and a selective degeneration of dopaminergic neurons. Further analysis also revealed defects in mitochondrial enzymatic activity as well as cell death. Consistently, PINK1 and Parkin double-deficient MEF showed a further decrease in mitochondrial membrane potential and mitochondrial complex I activity as well as apoptosis compared with single-deficient MEF. Interestingly, these mitochondrial abnormalities in Parkin-deficient MEF were compensated by exogenous PINK1, but not by disease-related mutants. These results suggest that PINK1 and Parkin work in a complementary way to protect dopaminergic neurons by maintaining mitochondrial function in vertebrates.

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1.

Amyloid-ß peptide ending at the 42nd residue (Aß42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aß42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aß42, possibly by enhancing its conversion to Aß38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.

[Supervised administration of Alzheimer's patients using information communication technology].

Drug adherence is central to the treatment of dementia, which might reduce compliance due to memory loss, particularly among home-based patients with dementia. In order to improve drug adherence, we suggest the efficient and effective supervised administration by use of information communication technology(ICT). ICT makes face-to-face real-time communication possible, and it also enables picture sharing. Therefore, it might be useful to apply ICT to controlling and supervising medication for patients with dementia to improve drug adherence. Accordingly, we enrolled patients who were supposed to take a newly prescribed anti-dementia patch containing the choline esterase inhibitor rivastigmine(Rivastach®)and investigated the effect of ICT-based intervention for drug adherence, emotional change, and cognitive change, utilizing Skype, a free communication software program. Scheduled Skype interventions increased drug adherence ratio, levels of subjective satisfaction, and instrumental activities of daily living(IADL). Furthermore, we can provide patients and their caregivers with a feeling of safety through regular bidirectional communication, as patients can easily consult medical staff regarding the adverse effects of newly prescribed drugs. Instead of frequent visits to their primary physicians, ICT-based communications can be used as a substitute for supervision of medication, given the availability of the telecommunication system. By directly connecting the medical institution to the home, we expect that this ICT-based system will expand into the geriatric care field, including the care of elderly individuals living alone.

Lipocalin-type prostaglandin D synthase: a glymphopathy marker in idiopathic hydrocephalus.

Idiopathic normal pressure hydrocephalus in elderly people is considered a form of glymphopathy caused by malfunction of the waste clearance pathway, called the glymphatic system. Tau is a representative waste material similar to amyloid-ß. During neurodegeneration, lipocalin-type prostaglandin D synthase (L-PGDS), a major cerebrospinal fluid (CSF) protein, is reported to act as a chaperone that prevents the neurotoxic aggregation of amyloid-ß. L-PGDS is also a CSF biomarker in idiopathic normal pressure hydrocephalus and significantly correlates with tau concentration, age, and age-related brain white matter changes detected by magnetic resonance imaging. To investigate this glymphopathy, we aimed to analyze white matter changes and contributing factors in vivo and their interactions ex vivo. Cerebrospinal tap tests were performed in 60 patients referred for symptomatic ventriculomegaly. Patients were evaluated using an idiopathic normal pressure hydrocephalus grading scale, mini-mental state examination, frontal assessment battery, and timed up-and-go test. The typical morphological features of high convexity tightness and ventriculomegaly were measured using the callosal angle and Evans index, and parenchymal white matter properties were evaluated with diffusion tensor imaging followed by tract-based spatial statistics. Levels of CSF biomarkers, including tau, amyloid-ß, and L-PGDS, were determined by ELISA, and their interaction, and localization were determined using immunoprecipitation and immunohistochemical analyses. Tract-based spatial statistics for fractional anisotropy revealed clusters that positively correlated with mini-mental state examination, frontal assessment battery, and callosal angle, and clusters that negatively correlated with age, disease duration, idiopathic normal pressure hydrocephalus grading scale, Evans index, and L-PGDS. Other parameters also indicated clusters that correlated with symptoms, microstructural white matter changes, and L-PGDS. Tau co-precipitated with L-PGDS, and colocalization was confirmed in postmortem specimens of neurodegenerative disease obtained from the human Brain Bank. Our study supports the diagnostic value of L-PGDS as a surrogate marker for white matter integrity in idiopathic normal pressure hydrocephalus. These results increase our understanding of the molecular players in the glymphatic system. Moreover, this study indicates the potential utility of enhancing endogenous protective factors to maintain brain homeostasis.

Distinct dendritic spine and nuclear phases of calcineurin activation after exposure to amyloid-ß revealed by a novel fluorescence resonance energy transfer assay.

Calcineurin (CaN) activation is critically involved in the regulation of spine morphology in response to oligomeric amyloid-ß (Aß) as well as in synaptic plasticity in normal memory, but no existing techniques can monitor the spatiotemporal pattern of CaN activity. Here, we use a spectral fluorescence resonance energy transfer approach to monitor CaN activation dynamics in real time with subcellular resolution. When oligomeric Aß derived from Tg2576 murine transgenic neurons or human AD brains were applied to wild-type murine primary cortical neurons, we observe a dynamic progression of CaN activation within minutes, first in dendritic spines, and then in the cytoplasm and, in hours, in the nucleus. CaN activation in spines leads to rapid but reversible morphological changes in spines and in postsynaptic proteins; longer exposure leads to NFAT (nuclear factor of activated T-cells) translocation to the nucleus and frank spine loss. These results provide a framework for understanding the role of calcineurin in synaptic alterations associated with AD pathogenesis.

Exercise is more effective than diet control in preventing high fat diet-induced ß-amyloid deposition and memory deficit in amyloid precursor protein transgenic mice.

Accumulating evidence suggests that some dietary patterns, specifically high fat diet (HFD), increase the risk of developing sporadic Alzheimer disease (AD). Thus, interventions targeting HFD-induced metabolic dysfunctions may be effective in preventing the development of AD. We previously demonstrated that amyloid precursor protein (APP)-overexpressing transgenic mice fed HFD showed worsening of cognitive function when compared with control APP mice on normal diet. Moreover, we reported that voluntary exercise ameliorates HFD-induced memory impairment and ß-amyloid (Aß) deposition. In the present study, we conducted diet control to ameliorate the metabolic abnormality caused by HFD on APP transgenic mice and compared the effect of diet control on cognitive function with that of voluntary exercise as well as that of combined (diet control plus exercise) treatment. Surprisingly, we found that exercise was more effective than diet control, although both exercise and diet control ameliorated HFD-induced memory deficit and Aß deposition. The production of Aß was not different between the exercise- and the diet control-treated mice. On the other hand, exercise specifically strengthened the activity of neprilysin, the Aß-degrading enzyme, the level of which was significantly correlated with that of deposited Aß in our mice. Notably, the effect of the combination treatment (exercise and diet control) on memory and amyloid pathology was not significantly different from that of exercise alone. These studies provide solid evidence that exercise is a useful intervention to rescue HFD-induced aggravation of cognitive decline in transgenic model mice of AD.

Optineurin suppression causes neuronal cell death via NF-κB pathway.

Mutations in more than 10 genes are reported to cause familial amyotrophic lateral sclerosis (ALS). Among these genes, optineurin (OPTN) is virtually the only gene that is considered to cause classical ALS by a loss-of-function mutation. Wild-type optineurin (OPTN(WT) ) suppresses nuclear factor-kappa B (NF-κB) activity, but the ALS-causing mutant OPTN is unable to suppress NF-κB activity. Therefore, we knocked down OPTN in neuronal cells and examined the resulting NF-κB activity and phenotype. First, we confirmed the loss of the endogenous OPTN expression after siRNA treatment and found that NF-κB activity was increased in OPTN-knockdown cells. Next, we found that OPTN knockdown caused neuronal cell death. Then, overexpression of OPTN(WT) or OPTN(E) (50K) with intact NF-κB-suppressive activity, but not overexpression of ALS-related OPTN mutants, suppressed the neuronal death induced by OPTN knockdown. This neuronal cell death was inhibited by withaferin A, which selectively inhibits NF-κB activation. Lastly, involvement of the mitochondrial proapoptotic pathway was suggested for neuronal death induced by OPTN knockdown. Taken together, these results indicate that inappropriate NF-κB activation is the pathogenic mechanism underlying OPTN mutation-related ALS. Among the genes for typical amyotrophic lateral sclerosis (ALS) phenotypes, optineurin (OPTN) is virtually the only gene in which a loss-of-function mutation is considered as the principal disease mechanism. We found that OPTN knockdown induced neuronal cell death via NF-κB activation. Furthermore, proapoptotic molecules such as p53 and Bax representing downstream targets of NF-κB are suggested to be involved in neuronal death.

Neuronal activity and secreted amyloid ß lead to altered amyloid ß precursor protein and presenilin 1 interactions.

Deposition of amyloid ß (Aß) containing plaques in the brain is one of the neuropathological hallmarks of Alzheimer's disease (AD). It has been suggested that modulation of neuronal activity may alter Aß production in the brain. We postulate that these changes in Aß production are due to changes in the rate-limiting step of Aß generation, APP cleavage by γ-secretase. By combining biochemical approaches with fluorescence lifetime imaging microscopy, we found that neuronal inhibition decreases endogenous APP and PS1 interactions, which correlates with reduced Aß production. By contrast, neuronal activation had a two-phase effect: it initially enhanced APP-PS1 interaction leading to increased Aß production, which followed by a decrease in the APP and PS1 proximity/interaction. Accordingly, treatment of neurons with naturally secreted Aß isolated from AD brain or with synthetic Aß resulted in reduced APP and PS1 proximity. Moreover, applying low concentration of Aß(42) to cultured neurons inhibited de novo Aß synthesis. These data provide evidence that neuronal activity regulates endogenous APP-PS1 interactions, and suggest a model of a product-enzyme negative feedback. Thus, under normal physiological conditions Aß may impact its own production by modifying γ-secretase cleavage of APP. Disruption of this negative modulation may cause Aß overproduction leading to neurotoxicity.

Presenilin-1 adopts pathogenic conformation in normal aging and in sporadic Alzheimer's disease.

Accumulation of amyloid-ß (Aß) and neurofibrillary tangles in the brain, inflammation and synaptic and neuronal loss are some of the major neuropathological hallmarks of Alzheimer's disease (AD). While genetic mutations in amyloid precursor protein and presenilin-1 and -2 (PS1 and PS2) genes cause early-onset familial AD, the etiology of sporadic AD is not fully understood. Our current study shows that changes in conformation of endogenous wild-type PS1, similar to those found with mutant PS1, occur in sporadic AD brain and during normal aging. Using a mouse model of Alzheimer's disease (Tg2576) that overexpresses the Swedish mutation of amyloid precursor protein but has normal levels of endogenous wild-type presenilin, we report that the percentage of PS1 in a pathogenic conformation increases with age. Importantly, we found that this PS1 conformational shift is associated with amyloid pathology and precedes amyloid-ß deposition in the brain. Furthermore, we found that oxidative stress, a common stress characteristic of aging and AD, causes pathogenic PS1 conformational change in neurons in vitro, which is accompanied by increased Aß42/40 ratio. The results of this study provide important information about the timeline of pathogenic changes in PS1 conformation during aging and suggest that structural changes in PS1/γ-secretase may represent a molecular mechanism by which oxidative stress triggers amyloid-ß accumulation in aging and in sporadic AD brain.

Presenilin regulates insulin signaling via a gamma-secretase-independent mechanism.

Presenilin (PS), a causative molecule of familial Alzheimer disease, acts as a crucial component of the γ-secretase complex, which is required to cleave type I transmembrane proteins such as amyloid precursor protein and Notch. However, it also functions through γ-secretase-independent pathways. Recent reports suggested that PS could regulate the expression level of cell surface receptors, including the PDGF and EGF receptors, followed by modulating their downstream pathways via γ-secretase-independent mechanisms. The main purpose of this study was to clarify the effect of PS on expression of the insulin receptor (IR) as well as on insulin signaling. Here, we demonstrate that PS inhibited IR transcription and reduced IR expression, and this was followed by down-regulation of insulin signaling. Moreover, we suggest that neither γ-secretase activity nor Wnt/ß-catenin signaling can reduce the expression of IR, but a PS-mediated increase in the intracellular Ca(2+) level can be associated with it. These results clearly indicate that PS can functionally regulate insulin signaling by controlling IR expression.

N-cadherin regulates p38 MAPK signaling via association with JNK-associated leucine zipper protein: implications for neurodegeneration in Alzheimer disease.

Synaptic loss, which strongly correlates with the decline of cognitive function, is one of the pathological hallmarks of Alzheimer disease. N-cadherin is a cell adhesion molecule essential for synaptic contact and is involved in the intracellular signaling pathway at the synapse. Here we report that the functional disruption of N-cadherin-mediated cell contact activated p38 MAPK in murine primary neurons, followed by neuronal death. We further observed that treatment with Aß(42) decreased cellular N-cadherin expression through NMDA receptors accompanied by increased phosphorylation of both p38 MAPK and Tau in murine primary neurons. Moreover, expression levels of phosphorylated p38 MAPK were negatively correlated with that of N-cadherin in human brains. Proteomic analysis of human brains identified a novel interaction between N-cadherin and JNK-associated leucine zipper protein (JLP), a scaffolding protein involved in the p38 MAPK signaling pathway. We demonstrated that N-cadherin expression had an inhibitory effect on JLP-mediated p38 MAPK signal activation by decreasing the interaction between JLP and p38 MAPK in COS7 cells. Also, this study demonstrated a novel physical and functional association between N-cadherin and p38 MAPK and suggested neuroprotective roles of cadherin-based synaptic contact. The dissociation of N-cadherin-mediated synaptic contact by Aß may underlie the pathological basis of neurodegeneration such as neuronal death, synaptic loss, and Tau phosphorylation in Alzheimer disease brain.

Gain of function by phosphorylation in Presenilin 1-mediated regulation of insulin signaling.

We have recently reported that Presenilin 1 (PS1), a causative gene of familial Alzheimer disease (AD), down-regulates the expression level of insulin receptor (IR) as well as its signaling through a γ-secretase-independent pathway. PS1 is phosphorylated by glycogen synthase kinase 3 ß at the serine 353 and 357 residues. The main purpose of the present study was to clarify the effect of PS1 phosphorylation on IR/insulin signaling. Here, we demonstrate that the pseudo-phosphorylation mutant of PS1 inhibited IR transcription and reduced IR expression compared with wild-type PS1. Importantly, there was a decrease in expression of IR in AD brains, and the phosphorylation ratio of PS1 was negatively correlated with IR level in human brain samples. In the data from mouse models of AD, IR reduction was not observed at the pre-Aß deposition stage but became apparent in that of post-Aß deposition. Together with our previous reports, these results suggest that phosphorylated PS1 can promote the down-regulation of insulin signaling, which may be a positive feed-forward mechanism inhibiting insulin signaling. As insulin resistance is reported to be a risk factor for sporadic AD, this PS1-mediated regulatory mechanism of brain insulin signaling may be causally associated with AD pathology.

Disseminated Nocardia farcinica infection in a patient with myasthenia gravis successfully treated by linezolid: a case report and literature review.

Nocardiosis is increasingly being diagnosed because of a growing population of immunocompromised hosts and improvements in the detection of Nocardia species in clinical laboratories. Historically, sulphonamides have been the first-line therapy for the treatment of nocardiosis, but sulphonamides tend to have a high rate of drug allergy in clinical settings. In this report, we described a disseminated Nocardia farcinica infection that occurred in a patient with myasthenia gravis who suffered from multiple drug allergies and was successfully treated using linezolid. We undertook a review of the literature of previously reported cases of nocardiosis treated with linezolid. To date, only 15 cases of nocardiosis treated with linezolid have been published. All cases exhibited long-term tolerance of linezolid, and 14 of 15 cases showed either an improvement in or complete clearance of the infection. According to the literature review, linezolid is an attractive alternative to trimethoprim-sulfamethoxazole for the treatment of disseminated nocardiosis, despite limited clinical evidence to support this claim.

[Support for the medication monitoring of patients with alzheimer's dementia by the interactive care system using IT].

In our laboratory, we draw up research aims to improve medication compliance in patients with dementia by video phone, and we have intervened in the cases of 3 patients to date. In this study, we focused on patients who are using a rivastigmine patch for Alzheimer' disease, which can be confirmed by video phone, to examine its efficacy. Specifically, by monitoring the effects of the treatment, skin side effects, of skin and usability for patients and caregivers, we monitor the dosing schedule to prevent interruption of self-medication, with the aim of improving compliance and treatment efficacy. We also consider methods of intervention for increasing the persistence rate of the rivastigmine patch and quality of life(QOL)by using the effectiveness of the video phone to focus on the symptoms of skin side effects. In addition, we examine the interventions that reduce the care burden and anxiety of caregivers by listening during the regular intervention.

Failure of DNA double-strand break repair by tau mediates Alzheimer's disease pathology in vitro.

DNA double-strand break (DSB) is the most severe form of DNA damage and accumulates with age, in which cytoskeletal proteins are polymerized to repair DSB in dividing cells. Since tau is a microtubule-associated protein, we investigate whether DSB is involved in tau pathologies in Alzheimer's disease (AD). First, immunohistochemistry reveals the frequent coexistence of DSB and phosphorylated tau in the cortex of AD patients. In vitro studies using primary mouse cortical neurons show that non-p-tau accumulates perinuclearly together with the tubulin after DSB induction with etoposide, followed by the accumulation of phosphorylated tau. Moreover, the knockdown of endogenous tau exacerbates DSB in neurons, suggesting the protective role of tau on DNA repair. Interestingly, synergistic exposure of neurons to microtubule disassembly and the DSB strikingly augments aberrant p-tau aggregation and apoptosis. These data suggest that DSB plays a pivotal role in AD-tau pathology and that the failure of DSB repair leads to tauopathy.

N-cadherin enhances APP dimerization at the extracellular domain and modulates Aß production.

Sequential processing of amyloid precursor protein (APP) by ß- and γ-secretase leads to the generation of amyloid-ß (Aß) peptides, which plays a central role in Alzheimer's disease pathogenesis. APP is capable of forming a homodimer through its extracellular domain as well as transmembrane GXXXG motifs. A number of reports have shown that dimerization of APP modulates Aß production. On the other hand, we have previously reported that N-cadherin-based synaptic contact is tightly linked to Aß production. In the present report, we investigated the effect of N-cadherin expression on APP dimerization and metabolism. Here, we demonstrate that N-cadherin expression facilitates cis-dimerization of APP. Moreover, N-cadherin expression led to increased production of Aß as well as soluble APPß, indicating that ß-secretase-mediated cleavage of APP is enhanced. Interestingly, N-cadherin expression affected neither dimerization of C99 nor Aß production from C99, suggesting that the effect of N-cadherin on APP metabolism is mediated through APP extracellular domain. We confirmed that N-cadherin enhances APP dimerization by a novel luciferase-complementation assay, which could be a platform for drug screening on a high-throughput basis. Taken together, our results suggest that modulation of APP dimerization state could be one of mechanisms, which links synaptic contact and Aß production.

The cleavage of N-cadherin is essential for chondrocyte differentiation.

The aggregation of chondroprogenitor mesenchymal cells into precartilage condensation represents one of the earliest events in chondrogenesis. N-cadherin is a key cell adhesion molecule implicated in chondrogenic differentiation. Recently, ADAM10-mediated cleavage of N-cadherin has been reported to play an important role in cell adhesion, migration, development and signaling. However, the significance of N-cadherin cleavage in chondrocyte differentiation has not been determined. In the present study, we found that the protein turnover of N-cadherin is accelerated during the early phase of chondrogenic differentiation in ATDC5 cells. Therefore, we generated the subclones of ATDC5 cells overexpressing wild-type N-cadherin, and two types of subclones overexpressing a cleavage-defective N-cadherin mutant, and examined the response of these cells to insulin stimulation. The ATDC5 cells overexpressing cleavage-defective mutants severely prevented the formation of cartilage aggregates, proteoglycan production and the induction of chondrocyte marker gene expression, such as type II collagen, aggrecan and type X collagen. These results suggested that the cleavage of N-cadherin is essential for chondrocyte differentiation.