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We previously developed single App knock-in mouse models of Alzheimer’s disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation ( App NL-G-F and App NL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations ( App G-F mice) and evaluated its characteristics. Amyloid β peptide (Aβ) pathology was exhibited by App G-F mice from 6 to 8 months of age and was accompanied by neuroinflammation. Aβ-secretase inhibitor, verubecestat, attenuated Aβ production in App G-F mice, but not in App NL-G-F mice, indicating that the App G-F mice are more suitable for preclinical studies of β-secretase inhibition given that most patients with AD do not carry the Swedish mutations. Comparison of isogenic App knock-in lines revealed that multiple factors, including elevated C-terminal fragment β (CTF-β) and humanization of Aβ might influence endosomal alterations in vivo. Thus, experimental comparisons between different isogenic App , knock-in mouse lines will provide previously unidentified insights into our understanding of the etiology of AD.

In an era when numerous disease-associated genes have been identified, determining the molecular mechanisms of complex diseases is still difficult. The CELF1 region was identified by genome-wide association studies as an Alzheimer’s disease (AD) risk locus. Using transcriptomics and cross-linking and immunoprecipitation sequencing (CLIP-seq), we found that CELF1, an RNA-binding protein, binds to KLC1 RNA and regulates its splicing. Analysis of two brain banks revealed that CELF1 expression is correlated with inclusion of KLC1 exons downstream of the CELF1-binding region identified by CLIP-seq. In AD, low CELF1 levels result in high levels of KLC1 splice variant E (KLC1_vE), an amyloid-β (Aβ) pathology-driving gene product. Cell culture experiments confirmed regulation of KLC1_vE by CELF1. Analysis of mouse strains with different propensities for Aβ accumulation confirmed that Klc1_vE drives Aβ pathology. Using omics methods, we revealedthe molecular pathway of a complex disease supported by human and mouse genetics.

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer’s disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed (1st generation models). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid β peptide (Aβ) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL–G–F and AppNL–F mice) were developed (2nd generation models). While these models are interesting given that they exhibit Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aβ metabolism and clearance because the Aβ in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aβ antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aβ pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aβ metabolism, and to identifying somatostatin receptor subtypes involved in Aβ degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the AppNL–F and Psen1P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the AppNL–G–F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aβ. Furthermore, a derivative AppG–F line devoid of Swedish mutations which can be utilized for preclinical studies of β-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aβ immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-KATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aβ deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

SUMMARYWe previously developed single App knock-in mouse models of Alzheimer’s disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL- G-F and AppNL-F mice). These models showed the development of amyloid β peptide (Aβ) pathology, neuroinflammation and cognitive impairment with aging. We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and some additional mutants to address the following two questions: [1] Do the Swedish mutations influence the mode of β-secretase inhibitor action in vivo? [2] Does the quantity of C-terminal fragment of amyloid precursor protein (APP) generated by β-secretase (CTF-β) affect endosomal properties as previously reported as well as other pathological events? Aβ pathology was exhibited by AppG-F mice from 6 to 8 months of age, and was accompanied by microglial and astrocyte activation. We found that a β-secretase inhibitor, verubecestat, inhibited Aβ production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of β-secretase inhibition given that most AD patients do not carry Swedish mutations. We also found that the quantity of CTF-β generated by various App knock-in mutants failed to correlate with endosomal alterations or enlargement, implying that CTF-β, endosomal abnormalities, or both are unlikely to play a major role in AD pathogenesis. This is the first AD mouse model ever described that recapitulates amyloid pathology in the brain without the presence of Swedish mutations and without relying on the overexpression paradigm. Thus, experimental comparisons between different App knock-in mouse lines will potentially provide new insights into our understanding of the etiology of AD.

We previously developed single App knock-in mouse models of Alzheimer’s disease (AD), harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). These models showed amyloid β peptide (Aβ) pathology, neuroinflammation and cognitive impairment in an age-dependent manner. The former line exhibits extensive pathology as early as 6 months but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The weakness of the latter model is that it may take as long as 18 months for the pathology to become prominent. We have thus generated a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

Our understanding of the physiological relevance of unique Damage-induced neuronal endopeptidase (DINE) [also termed Endothelin-converting enzyme-like 1 (ECEL1)] has recently expanded. DINE/ECEL1 is a type II membrane-bound metalloprotease, belonging to a family including the neprilysin (NEP) and endothelin-converting enzyme (ECE). The family members degrade and/or process peptides such as amyloid β and big-endothelins, which are closely associated with pathological conditions. Similar to NEP and ECE, DINE has been expected to play an important role in injured neurons as well as in developing neurons, because of its remarkable transcriptional response to neuronal insults and predominant neuronal expression from the embryonic stage. However, the physiological significance of DINE has long remained elusive. In the last decade, a series of genetically manipulated mice have driven research progress to elucidate the physiological aspects of DINE. The mice ablating Dine fail to arborize the embryonic motor axons in some subsets of muscles, including the respiratory muscles, and die immediately after birth. The abnormal phenotype of motor axons is also caused by one amino acid exchanges of DINE/ECEL1, which are responsible for distal arthrogryposis type 5 in a group of human congenital movement disorders. Furthermore, the mature Dine-deficient mice in which the lethality is rescued by genetic manipulation have shown the involvement of DINE in central nervous system regeneration. Here we describe recent research advances that DINE-mediated proteolytic processes are critical for nerve development, regeneration and pathogenesis, and discuss the future potential for DINE as a therapeutic target for axonal degeneration/disorder.

SummaryAlzheimer’s disease (AD) is a major cause of dementia, with the number of patients with this condition anticipated to exceed 50 million worldwide in the near future. Despite extensive research efforts, no effective measures are available to facilitate the prevention or treatment of AD, which is due in part to a lack of animal models able to closely replicate a human-like disease state. Here, we describe the generation of three mutant marmoset individuals in which exon 9 of PSEN1 gene product has been deleted (PSEN1-ΔE9). Such ΔE9 mutations have been reported to cause early on-set familial AD (references1–5). We used Transcription Activator-Like Effector Nuclease (TALEN) to destroy the 3’ splice site of exon 9 in the marmoset PSEN1 gene. To this end, TALEN exhibits high genome-editing efficacy, generates few off-target effects, and produces minimal mosaicism. Indeed, whole genome sequencing and other analyses illustrated an absence of off-target effects and an apparent absence of mosaicism. Fibroblasts obtained from newborn marmosets exhibited uncleaved full-length presenilin 1 protein (PS1) caused by the perturbation of PS1 endoproteolysis as well as an increased ratio of Aβ42/Aβ40 production, a signature of familial AD pathogenesis. To our knowledge, this is the first non-human primate model of familial AD. We intend to make our marmoset model available to the research community to facilitate the global fight against AD.

Alzheimer's disease (AD) is the most common neurodegenerative disease, leading to dementia in the aging human brain. Although a huge amount of research has been done to date, the pathogenesis of AD remains largely unknown. Given the dominance of mRNA splicing in the nervous system, it would be invaluable to precisely identify mis-splicing in the AD brain to understand more deeply how the gene network orchestrates the development of pathology in affected cells. In this brief manuscript, we first introduce the term mRNA splicing and discuss its abnormality in the AD brain. Then, we describe the technical challenges of the precise detection of mis-splicing, as well as their reasonable solutions. Finally, we discuss the future directions for splicing biology in AD.

Base Editor (BE) and Target-AID (activation-induced cytidine deaminase) are engineered genome-editing proteins composed of Cas9 and cytidine deaminases. These base-editing tools convert C:G base pairs to T:A at target sites. Here, we inject either BE or Target-AID mRNA together with identical single-guide RNAs (sgRNAs) into mouse zygotes, and compare the base-editing efficiencies of the two distinct tools in vivo. BE consistently show higher base-editing efficiency (10.0–62.8%) compared to that of Target-AID (3.4–29.8%). However, unexpected base substitutions and insertion/deletion formations are also more frequently observed in BE-injected mice or zygotes. We are able to generate multiple mouse lines harboring point mutations in the mouse presenilin 1 (Psen1) gene by injection of BE or Target-AID. These results demonstrate that BE and Target-AID are highly useful tools to generate mice harboring pathogenic point mutations and to analyze the functional consequences of the mutations in vivo., CRISPR-guided cytidine deaminases, including BE3 (Base Editor 3) and Target-AID (activation-induced cytidine deaminase), can covert C:G base pairs to T:A at target site. Here, the authors generate missense mutations of mouse Psen1 gene and find BE3 has higher editing efficiency than Target-AID.

Although, a number of pathogenic mutations have been found for Alzheimer’s disease (AD), only one protective mutation has been identified so far in humans. Here we identify possible protective deletion mutations in the 3′-UTR of the amyloid precursor protein (App) gene in mice. We use an App knock-in mouse model carrying a humanized Aβ sequence and three AD mutations in the endogenous App gene. Genome editing of the model zygotes using multiple combinations of CRISPR/Cas9 tools produces genetically mosaic animals with various App 3′-UTR deletions. Depending on the editing efficiency, the 3′-UTR disruption mitigates the Aβ pathology development through transcriptional and translational regulation of APP expression. Notably, an App knock-in mouse with a 34-bp deletion in a 52-bp regulatory element adjacent to the stop codon shows a substantial reduction in Aβ pathology. Further functional characterization of the identified element should provide deeper understanding of the pathogenic mechanisms of AD., To date, only one mutation in the gene for amyloid-beta precursor protein APP has been suggested to be protective against Alzheimer’s disease. Here, authors found using gene editing of a mutant App knock-in mouse line that deletion of the 3’UTR region is protective against amyloid-β accumulation in vivo, and subsequently identify a 52-bp element in the 3’UTR region that is responsible for this effect.

Neprilysin (NEP), a membrane-bound metalloprotease, has been shown to play an essential role in the clearance of amyloid beta (Aβ) peptides. Previous studies have reported that NEP expression is downregulated in the normal aging brain as well as in the Alzheimer's disease (AD) brain, providing evidence that the downregulation of NEP expression contributes to the age-dependent deposition of Aβ-containing plaques, a pathological hallmark of AD. However, the mechanisms underlying the downregulation remain unclear. In this study, we explored the relationship between DNA methylation status of CpG islands in the NEP promoter and its expression level in AD brains. We performed pyrosequencing analyses to detect the DNA methylation level in 31 postmortem AD brains and 40 normal control brains. All 30 CpG sites showed no clear difference in methylation level. To further focus on methylation changes specific to neuronal cells, we performed methylation array experiments using neuronal nuclei from postmortem brains and found no clear difference in the methylation level between AD and normal control samples. Our detailed analyses, with a substantial number of brain samples, provide the first convincing evidence that DNA methylation of the NEP promoter is not involved in AD development and progression.

Endothelin-converting enzyme-like 1 (ECEL1, also termed DINE in rodents), a membrane-bound metalloprotease, has been identified as a gene responsible for distal arthrogryposis (DA). ECEL1-mutated DA is generally characterized by ocular phenotypes in addition to the congenital limb contractures that are common to all DA subtypes. Until now, the consequences of the identified pathogenic mutations have remained incompletely understood because of a lack of detailed phenotypic analyses in relevant mouse models. In this study, we generated a new knock-in mouse strain that carries an ECEL1/DINE pathogenic G607S missense mutation, based on a previous study reporting atypical DA hindlimb phenotypes in two siblings with the mutation. We compared the morphological phenotypes of G607S knock-in mice with C760R knock-in mice that we previously established. Both C760R and G607S knock-in mouse embryos showed similar axonal arborization defects with normal trajectory patterns from the spinal cord to the target hindlimb muscles, as well as axon guidance defects of the abducens nerves. Intriguingly, distinct phenotypes in DINE protein localization and mRNA expression were identified in these knock-in mouse lines. For G607S, DINE mRNA and protein expression was decreased or almost absent in motor neurons. In the C760R mutant mice DINE was expressed and localized in the somata of motor neurons but not in axons. Our mutant mouse data suggest that ECEL1/DINE G607S and C760R mutations both lead to motor innervation defects as primary causes in ECEL1-mutated congenital contracture disorders. However, the functional consequences of the two mutations are distinct, with loss of axonal transport of ECEL1/DINE in C760R mutants and mRNA expression deficits in G607S mutants. Electronic supplementary material The online version of this article (10.1186/s40478-017-0486-9) contains supplementary material, which is available to authorized users.

We previously developed single App knock-in mouse models of Alzheimer's disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice, respectively). These models showed Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer's disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new third-generation mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

Alzheimer's disease (AD) is the most common neurodegenerative disease, leading to dementia in the aging human brain. Although a huge amount of research has been done to date, the pathogenesis of AD remains largely unknown. Given the dominance of mRNA splicing in the nervous system, it would be invaluable to precisely identify mis-splicing in the AD brain to understand more deeply how the gene network orchestrates the development of pathology in affected cells. In this brief manuscript, we first introduce the term mRNA splicing and discuss its abnormality in the AD brain. Then, we describe the technical challenges of the precise detection of mis-splicing, as well as their reasonable solutions. Finally, we discuss the future directions for splicing biology in AD.

The membrane-bound metalloprotease endothelin-converting enzyme-like 1 (ECEL1) has been newly identified as a causal gene of a specific type of distal arthrogryposis (DA). In contrast to most causal genes of DA, ECEL1 is predominantly expressed in neuronal cells, suggesting a unique neurogenic pathogenesis in a subset of DA patients with ECEL1 mutation. The present study analyzed developmental motor innervation and neuromuscular junction formation in limbs of the rodent homologue damage-induced neuronal endopeptidase (DINE)-deficient mouse. Whole-mount immunostaining was performed in DINE-deficient limbs expressing motoneuron-specific GFP to visualize motor innervation throughout the limb. Although DINE-deficient motor nerves displayed normal trajectory patterns from the spinal cord to skeletal muscles, they indicated impaired axonal arborization in skeletal muscles in the forelimbs and hindlimbs. Systematic examination of motor innervation in over 10 different hindlimb muscles provided evidence that DINE gene disruption leads to insufficient arborization of motor nerves after arriving at the skeletal muscle. Interestingly, the axonal arborization defect in foot muscles appeared more severe than in other hindlimb muscles, which was partially consistent with the proximal-distal phenotypic discordance observed in DA patients. Additionally, the number of innervated neuromuscular junction was significantly reduced in the severely affected DINE-deficient muscle. Furthermore, we generated a DINE knock-in (KI) mouse model with a pathogenic mutation, which was recently identified in DA patients. Axonal arborization defects were clearly detected in motor nerves of the DINE KI limb, which was identical to the DINE-deficient limb. Given that the encoded sequences, as well as ECEL1 and DINE expression profiles, are highly conserved between mouse and human, abnormal arborization of motor axons and subsequent failure of NMJ formation could be a primary cause of DA with ECEL1 mutation.

Lewy body disease (LBD) is characterized by accumulation of aggregated α-synuclein in the central nervous system as eosinophilic cytoplasmic inclusions called Lewy bodies. According to their distribution pattern, it is classified into brainstem LBD, limbic LBD and diffuse neocortical LBD. It has been reported that α-synuclein affects various points in the MAPK cascade but its relationship with FGF receptors, which are the most upstream of the pathway, has not been previously investigated. We discovered that among the four FGFRs, FGFR3 showed neuronal upregulation in LBD brains histopathologically. Further examination using neuron-specific methylome analysis revealed that the gene body of FGFR3 was hypermethylated in LBD, suggesting its increased transcription. Altered methylation was not observed in the non-neuronal genome. Altered methylation status was associated with the severity of α-synuclein pathology.

Significance To extract critical information from Alzheimer’s disease (AD) postmortem brains that may otherwise be lost, we chose to screen epigenetic signatures. Epigenome analysis is a robust methodology in terms of its cell type and gene specificity, suitability for high-throughput analysis, and resistance to postmortem degradation. Analysis of the neuron-specific methylome revealed a variety of differentially methylated genes, including BRCA1. We demonstrate the pathogenic relevance of compromised genomic integrity by analyzing the neuroprotective function of BRCA1 against amyloid β (Aβ)-induced DNA double-strand breaks. Furthermore, insolubility of BRCA1 under the presence of aggregated tau suggested the reason for its dysfunction despite enhanced expression. We provide insight into the pathomechanism of AD and demonstrate the potential of screening neuron-specific methylome to reveal new pathogenic contributors., Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by pathology of accumulated amyloid β (Aβ) and phosphorylated tau proteins in the brain. Postmortem degradation and cellular complexity within the brain have limited approaches to molecularly define the causal relationship between pathological features and neuronal dysfunction in AD. To overcome these limitations, we analyzed the neuron-specific DNA methylome of postmortem brain samples from AD patients, which allowed differentially hypomethylated region of the BRCA1 promoter to be identified. Expression of BRCA1 was significantly up-regulated in AD brains, consistent with its hypomethylation. BRCA1 protein levels were also elevated in response to DNA damage induced by Aβ. BRCA1 became mislocalized to the cytoplasm and highly insoluble in a tau-dependent manner, resulting in DNA fragmentation in both in vitro cellular and in vivo mouse models. BRCA1 dysfunction under Aβ burden is consistent with concomitant deterioration of genomic integrity and synaptic plasticity. The Brca1 promoter region of AD model mice brain was similarly hypomethylated, indicating an epigenetic mechanism underlying BRCA1 regulation in AD. Our results suggest deterioration of DNA integrity as a central contributing factor in AD pathogenesis. Moreover, these data demonstrate the technical feasibility of using neuron-specific DNA methylome analysis to facilitate discovery of etiological candidates in sporadic neurodegenerative diseases.

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first-generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD Second-generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD In this review, we evaluate different APP mouse models of AD, and review recent studies using the second-generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study. ispartof: EMBO Journal vol:36 issue:17 pages:2473-2487 ispartof: location:England status: published

Neuropeptides are crucial mediators in nervous and endocrine systems. Processing and degradation, the major regulatory mechanisms, of enzymes are essential for the control of these peptidergic intercellular signaling systems. Damage-induced neuronal endopeptidase (or endothelin converting enzyme-like1), a member of the neprilysin family, has recently been identified as an M13 zinc metalloprotease. Damage-induced neuronal endopeptidase mRNA expression is strikingly restricted to neurons, and is remarkably induced in response to various types of neuronal injuries, although its function and substrate remain unknown. To clarify the role of damage-induced neuronal endopeptidase, we examined the localization and ontogeny of damage-induced neuronal endopeptidase mRNA expression in the rat nervous system using in situ hybridization. Damage-induced neuronal endopeptidase mRNA was detected at embryonic day 12, and its expression restricted to the ventral region of the neural tube. Subsequently, expression was also apparent in primordia of the striatum, hypothalamus, and cranial motor nuclei during neural development. This specific distribution was relatively maintained in the adult brain, although expression levels became weaker. Expression of damage-induced neuronal endopeptidase was absent in the cerebral cortex, hippocampus, and cerebellum. In addition to prominent expression in CNS, intestinal and sensory ganglia and retina demonstrated transient intense damage-induced neuronal endopeptidase mRNA expression during the embryonic period that then declined, and disappeared after birth. The results indicated that damage-induced neuronal endopeptidase might play an important role in embryonic neural development, in particular in peripheral ganglia derived from the neural crest, and in some neurons originating from the basal plate such as the hypothalamus and cranial motor neurons.

The hallmark of Alzheimer's disease (AD) pathology is an accumulation of amyloid β (Aβ) and phosphorylated tau, which are encoded by the amyloid precursor protein (APP) and microtubule-associated protein tau (MAPT) genes, respectively. Less than 5% of all AD cases are familial in nature, i.e. caused by mutations in APP, PSEN1 or PSEN2. Almost all mutations found in them are related to an overproduction of Aβ1-42, which is prone to aggregation. While these genes are mutation free, their function, or those of related genes, could be compromised in sporadic AD as well. In this study, pyrosequencing analysis of post-mortem brains revealed aberrant CpG methylation in APP, MAPT and GSK3B genes of the AD brain. These changes were further evaluated by a newly developed in vitro-specific DNA methylation system, which in turn highlighted an enhanced expression of APP and MAPT. Cell nucleus sorting of post-mortem brains revealed that the methylation changes of APP and MAPT occurred in both neuronal and non-neuronal cells, whereas GSK3B was abnormally methylated in non-neuronal cells. Further analysis revealed an association between abnormal APP CpG methylation and apolipoprotein E e4 allele (APOE e4)-negative cases. The presence of a small number of highly methylated neurons among normal neurons contribute to the methylation difference in APP and MAPT CpGs, thus abnormally methylated cells could compromise the neural circuit and/or serve as 'seed cells' for abnormal protein propagation. Our results provide a link between familial AD genes and sporadic neuropathology, thus emphasizing an epigenetic pathomechanism for sporadic AD.

MicroRNA (miRNA) is a small non-coding RNA that regulates gene expression by degrading target mRNAs or inhibiting translation. Although many miRNAs play important roles in various conditions, it is unclear whether miRNAs are involved in motor nerve regeneration. In this study, we identified the possible implication of miR-124 in nerve regeneration using a mouse hypoglossal nerve injury model. The significant down-regulation of miR-124 was observed in injured hypoglossal motor neurons after nerve injury, and this transient down-regulation showed a clear inverse correlation with the up-regulation of KLF6 and STAT3, known as axon elongation factor and regeneration-associated molecules, respectively. Furthermore, the luciferase assay and in vitro gain of function methods supported that both genes could be potent targets of miR-124. These results suggest that injury-induced repression of miR-124 may be implicated in the regulation of expression of several injury-associated transcription factors, which are crucial for appropriate nerve regeneration.

Damage-induced neuronal endopeptidase (DINE) is a metalloprotease belonging to the neprilysin family. Expression of DINE mRNA is observed predominantly in subsets of neurons in the CNS and peripheral nervous system during embryonic development, as well as after axonal injury. However, the physiological function of DINE and its substrate remain unknown. We generated DINE-deficient mice to examine the physiological role of DINE. Shortly after birth, these mice died of respiratory failure resulting from a dysfunction of the diaphragm, which showed severe atrophy. As DINE was abundantly expressed in motor neurons and there was atrophy of the diaphragm, we analyzed the interaction between motor nerves and skeletal muscles in the DINE-deficient mice. Although there were no obvious deficiencies in numbers of motor neurons in the spinal cord or in the nerve trajectories from the spinal cord to the skeletal muscle in DINE-deficient mice, detailed histochemical analysis demonstrated a significant decrease of nerve terminal arborization in the diaphragm from embryonic day 12.5. In accordance with the decrease of final branching, the diaphragms from DINE-deficient mice exhibited only a few neuromuscular junctions. Similar changes in nerve terminal morphology were also apparent in other skeletal muscles, including the latissimus dorsi and the intercostal muscles. These data suggest that DINE is a crucial molecule in distal axonal arborization into muscle to establish neuromuscular junctions.

Using measurements of reflectance, transmittance, and the ellipsometric parameter D, we have determined the thickness, refractive index, and the absorption coefficient of various thin films and thin-film stacks. (D, the relative phase between the p- and s-polarized components, is measured for both reflected and transmitted light.) These optical measurements are performed with a specially designed system at the fixed wavelength of lambda = 633 nm over the 10 degrees -75 degrees range of angles of incidence. The examined samples, prepared by means of sputtering on fused-silica substrates, consist of monolayers and trilayers of various materials of differing thickness and optical constants. These samples, which are representative of the media of rewritable phase-change optical disks, include a dielectric mixture of ZnS and SiO(2), an amorphous film of the Ge(2)Sb(2.3)Te(5) alloy, and an aluminum chromium alloy film. To avoid complications arising from reflection and transmission losses at the air-substrate interface, the samples are immersed in an index-matching fluid that eliminates the contributions of the substrate to reflected and transmitted light. A computer program estimates the unknown parameters of the film(s) by matching the experimental data to theoretically calculated values. Although our system can be used for measurements over a broad range of wavelengths, we describe only the results obtained at lambda = 633 nm.

Nerve injury requires the expression of large ensembles of genes. The key molecular mechanism for this gene transcription regulation in injured neurons is poorly understood. Among many nerve injury-inducible genes, the gene encoding damage-induced neuronal endopeptidase (DINE) showed most marked expression response to various kinds of nerve injuries in central and peripheral nervous system neurons. This unique feature led us to examine the promoter region of the DINE gene and clarify both the injury-responsive element within the promoter and its related transcriptional machinery. This study showed that DINE promoter was activated by leukemia inhibitory factor and nerve growth factor withdrawal, which were pivotal for the up-regulation of DINE mRNA after nerve injury. The injury-inducible transcription factors such as activating transcription factor 3 (ATF3), c-Jun, and STAT3, which were located at the downstream of leukemia inhibitory factor and nerve growth factor withdrawal, seemed to be involved in the activation of the DINE promoter. Surprisingly, these transcription factors did not bind to the DINE promoter directly. Instead, the general transcription factor, Sp1, bound to a GC box within the promoter. ATF3, c-Jun, and STAT3 interacted with Sp1 and are associated with the GC box region of the DINE gene in injured neurons. These findings suggested that Sp1 recruit ATF3, c-Jun, and STAT3 to obtain the requisite synergistic effect. Of these transcription factors, ATF3 may be the most critical, because ATF3 is specifically expressed after nerve injury.

Readout signal, noise, and cross-track cross talk were investigated for edge detection in a phase-change optical data storage system. Both theoretical and experimental results indicate that edge detection has a performance superior to the conventional detection of reflectance variations, especially when the amorphous marks are shorter than the size of the focused spot. More than 50 dB of carrier to noise ratio for marks of 0.36 μm in length is obtained using light at a wavelength of 690 nm and an objective lens of 0.6 numerical aperture. Diffraction analysis on the cross talk has shown that, in the scheme of land-groove recording, there is no optimum groove depth which can cancel the cross talk from adjacent tracks.

Apolipoprotein E genotypes in 88 unrelated Japanese patients with NINCDS-ADRDA sporadic Alzheimer's disease (AD) were examined and compared with those of 93 healthy controls. Frequency of epsilon 4 allele was increased in patients with AD (31%) compared with controls (10%), as was reported previously. Individuals homozygous or heterozygous for the allele epsilon 4 had a 5.9-fold increased risk of AD. This tendency was more pronounced in early onset sporadic (= non-familial) type than late onset type. The relative risk was also greater for early onset type (RR = 11.7; 95% CI, 4.9-28.3) than late onset type (RR = 4.3; 95% CI, 2.1-8.8). Moreover, patients with homozygote for the allele epsilon 4 had a 14.7-fold increased risk of early onset sporadic AD (P0.005, chi 2 = 9.0, df = 1, 95% CI, 2.5-85.1). Our findings indicated that association of apolipoprotein epsilon 4 with sporadic Alzheimer's disease is more pronounced in early onset type than in late onset type.

We report results of measurements of the optical constants of the dielectric layer, reflecting layer, and phase-change layer used as the media of phase-change optical recording. The refractive index and the absorption coefficient k of these materials vary to some extent with the film thickness and with the film deposition environment. We report the observed variations of optical constants among samples of differing structure and among samples fabricated in different laboratories.

We demonstrated the possibility of high data rate recording on a DVD-RAM disk which utilizes Ge-Sb-Te phase-change materials. To ensure high transfer rate overwriting on the DVD, quadruple speed (44Mbps) recording at a linear velocity of 16.4 m/s was tested using a Sn-added Ge-Sn-Sb-Te material as the recording layer. Double speed (22Mbps) recording on the present 4.7GB DVD-RAM at a linear velocity of 8.2 m/s was also tested. A CNR of more than 53 dB and an erasability of more than 30 dB were obtained at each double, triple and quadruple speeds. In addition, by recording via 8-16 random modulation signals, a jitter of 9 percent or less and a direct overwrite performance of 100,000 cycles were confirmed.

Various technologies enabling to increase the storage capacity of rewritable phase-change optical disk have been proposed.

Two marked effects are obtained by forming a Ge-N interface layer on either side of Ge-Sb-Te recording layer. One effect is a suppression of atomic diffusion between Ge-Sb-Te layer and protective layers, ZnS-SiO2 representatively, which leads to a significant improvement in overwrite cyclability, and the other is the acceleration of crystallization process which leads to higher speed optical disks. A rapid-cooling type experimental disk with Ge-N layers on both sides of the Ge-Sb-Te recording layer proved to be capable of exceeding 105 cycle overwrites and a recording data rate 40 Mbps at linear velocity 12 m/s. The recording conditions: bit length 0.28 micrometer and track pitch 0.6 micrometer (L/G method) using laser source with a wavelength 658 nm and a numerical aperture 0.6 correspond to a capacity 4.7 GB/(phi) 120 mm.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Conventionally, readout signal is obtained by differential detection in magneto-optical storage or by direct integration of the reflected light in phase-change optical storage. Mark edges are usually determined by slicing the level detection signal at the standard level, suffering from intersymbol interference when reading densely recorded short marks. Edge detection is a direct optical detection for mark edges. The readout signal is the difference signal from a split detector. Theoretically, edge detection has advantages over conventional level detection, such as high contrast and ability to identify edges of densely spaced marks. These features need to be confirmed experimentally. In magneto-optical storage [1], edge-shift of short marks using edge detection was found to be lower than that using differential level detection [2]. But in other aspects, such as signal and noise levels, edge detection was inferior to differential level detection [2, 3]. In phase-change optical storage [4], theoretical analysis indicates that edge detection has a potential superiority over conventional detection (hereafter referred to as sum detection). Experimentally, edge detection noise level has been confirmed to be lower than sum detection, especially at low and high spatial frequencies. In this work we present results for edge detection readout signal, carrier-to-noise ratio (CNR), and cross-talk characteristics in the scheme of land-groove as well as comparison with sum detection for phase-change optical storage.