Efficacy of oligodendrocyte precursor cells as delivery vehicles for single-chain variable fragment to misfolded SOD1 in ALS rat model.

Superoxide dismutase1 (SOD 1) mutation is a leading cause of familial amyotrophic lateral sclerosis (ALS). Growing evidence suggests that antibody therapy against misfolded SOD1 protein can be therapeutic. However, the therapeutic effects are limited, partly because of the delivery system. Therefore, we investigated the efficacy of oligodendrocyte precursor cells (OPCs) as a drug delivery vehicle of single-chain variable fragments (scFv). Using a Borna disease virus vector that is pharmacologically removable and episomally replicable in the recipient cells, we successfully transformed wild-type OPCs to secrete scFv of a novel monoclonal antibody (D3-1), specific for misfolded SOD1. Single intrathecal injection of OPCs scFvD3-1, but not OPCs alone, significantly delayed disease onset and prolonged the lifespan of ALS rat models expressing SOD1 H46R . The effect of OPC scFvD3-1 surpassed that of a 1 month intrathecal infusion of full-length D3-1 antibody alone. scFv-secreting OPCs suppressed neuronal loss and gliosis, reduced levels of misfolded SOD1 in the spinal cord, and suppressed the transcription of inflammatory genes, including Olr1, an oxidized low-density lipoprotein receptor 1. The use of OPCs as a delivery vehicle for therapeutic antibodies is a new option for ALS in which misfolded protein and oligodendrocyte dysfunction are implicated in the pathogenesis.

In vivo analysis of aggregation propensity of low levels of mislocalized TDP-43 on cytopathological and behavioral phenotype of ALS/FTLD.

Mislocalization and aggregate formation of TAR DNA-biding protein of 43kD (TDP-43) in the cytoplasm are signatures of amyotrophic lateral sclerosis(ALS) and frontotemporal lobar degeneration (FTLD). However, the role of two cytopathologies in ALS/FTLD pathogenesis is unclear. This study aims to elucidate the difference in their causality of TDP-43 in ALS/FTLD in vivo, using transgenic mice expressing human TDP-43 with defective nuclear localizing signals in neurons (Cyto-TDP) and those with aggregation propensity (Cyto-aggTDP). The expression levels of both proteins are less than half of endogenous TDP-43. Despite the low amount of Cyto-aggTDP, the TDP-43 phosphorylation is more evident than Cyto-TDP. Histopathological study showed accelerated astrogliosis in the anterior cerebral cortex of both mice. Cyto-aggTDP mice demonstrated significant but faint loss of neurons in the perirhinal(PERI) and ectorhinal(ECT) areas and higher Iba1-staining in the spinal cord than aged control. Despite the lack of locomotor dysfunctions in both mice, the open-field test showed enhanced exploratory behavior, indicating that the perpetual mislocalization of TDP-43 may suffice to trigger FTLD behavior. Besides, the aggregation propensity of TDP-43 promotes phosphorylation, but its role in the clinicopathological phenotype may not be primary.

Conformational change of RNA-helicase DHX30 by ALS/FTD-linked FUS induces mitochondrial dysfunction and cytosolic aggregates.

Genetic mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). Although mitochondrial dysfunction and stress granule have been crucially implicated in FUS proteinopathy, the molecular basis remains unclear. Here, we show that DHX30, a component of mitochondrial RNA granules required for mitochondrial ribosome assembly, interacts with FUS, and plays a crucial role in ALS-FUS. WT FUS did not affect mitochondrial localization of DHX30, but the mutant FUS lowered the signal of mitochondrial DHX30 and promoted the colocalization of cytosolic FUS aggregates and stress granule markers. The immunohistochemistry of the spinal cord from an ALS-FUS patient also confirmed the colocalization, and the immunoelectron microscope demonstrated decreased mitochondrial DHX30 signal in the spinal motor neurons. Subcellular fractionation by the detergent-solubility and density-gradient ultracentrifugation revealed that mutant FUS also promoted cytosolic mislocalization of DHX30 and aggregate formation. Interestingly, the mutant FUS disrupted the DHX30 conformation with aberrant disulfide formation, leading to impaired mitochondrial translation. Moreover, blue-native gel electrophoresis revealed an OXPHOS assembly defect caused by the FUS mutant, which was similar to that caused by DHX30 knockdown. Collectively, our study proposes DHX30 as a pivotal molecule in which disulfide-mediated conformational change mediates mitochondrial dysfunction and cytosolic aggregate formation in ALS-FUS.

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.

Synaptic Vesicle Protein 2B Negatively Regulates the Amyloidogenic Processing of AßPP as a Novel Interaction Partner of BACE1.

BACKGROUND: Given that amyloid-ß (Aß) peptide is produced and released at synapses, synaptic Aß is one of the promising therapeutic targets to prevent synaptic dysfunction in Alzheimer's disease (AD). Although Aß production begins with the cleavage of the amyloid-ß protein precursor (AßPP) by ß-site AßPP cleaving enzyme 1 (BACE1), the mechanism on how BACE1 is involved in AßPP processing at synapses remains unclear. OBJECTIVE: This study aimed to identify novel BACE1 interacting proteins regulating Aß production at the synapse. METHODS: BACE1 interacting proteins were pulled down using a mass spectrometry-based proteomics of wild-type (WT) rat brain synaptoneurosome lysates utilizing anti-BACE1 antibody. Then, a novel BACE1 interactor was identified and characterized using experimental systems that utilized transfected cells and knockout (KO) mice. RESULTS: Synaptic vesicle protein 2B (SV2B) was identified as a novel presynaptic interaction partner of BACE1. In HEK293 cells, co-overexpression of SV2B with BACE1 significantly reduced the sAßPPß and Aß levels released in the media; thus, SV2B overexpression negatively affected the AßPP cleavage by BACE1. Compared with those of WT mice, the hippocampal lysates of SV2B knockout mice had significantly elevated Aß levels, whereas the ß-secretase activity and the AßPP and BACE1 protein levels remained unchanged. Finally, a fractionation assay revealed that BACE1 was mislocalized in SV2B KO mice; hence, SV2B may be involved in BACE1 trafficking downregulating the amyloidogenic pathway of AßPP. CONCLUSION: SV2B has a novel role of negatively regulating the amyloidogenic processing of AßPP at the presynapses.

Two-Point Dynamic Observation of Alzheimer's Disease Cerebrospinal Fluid Biomarkers in Idiopathic Normal Pressure Hydrocephalus.

BACKGROUND: Extensive research into cerebrospinal fluid (CSF) biomarkers was performed in patients with idiopathic normal pressure hydrocephalus (iNPH). Most prior research into CSF biomarkers has been one-point observation. OBJECTIVE: To investigate dynamic changes in CSF biomarkers during routine tap test in iNPH patients. METHODS: We analyzed CSF concentrations of tau, amyloid-ß (Aß) 42 and 40, and leucine rich α-2-glycoprotein (LRG) in 88 consecutive potential iNPH patients who received a tap test. We collected two-point lumbar CSF separately at the first 1 ml (First Drip (FD)) and at the last 1 ml (Last Drip (LD)) during the tap test and 9 patients who went on to receive ventriculo-peritoneal shunt surgery each provided 1 ml of ventricular CSF (VCSF). RESULTS: Tau concentrations were significantly elevated in LD and VCSF compared to FD (LD/FD = 1.22, p = 0.003, VCSF/FD = 2.76, p = 0.02). Conversely, Aß42 (LD/FD = 0.80, p < 0.001, VCSF/FD = 0.38, p = 0.03) and LRG (LD/FD = 0.74, p < 0.001, VCSF/FD = 0.09, p = 0.002) concentrations were significantly reduced in LD and VCSF compared to FD. Gait responses to the tap test and changes in cognitive function in response to shunt were closely associated with LD concentrations of tau (p = 0.02) and LRG (p = 0.04), respectively. CONCLUSIONS: Dynamic changes were different among the measured CSF biomarkers, suggesting that LD of CSF as sampled during the tap test reflects an aspect of VCSF contributing to the pathophysiology of iNPH and could be used to predict shunt effectiveness.

Degradation of amyloid ß peptide by neprilysin expressed from Borna disease virus vector.

Accumulation of amyloid ß (Aß40 and Aß42) in the brain is a characteristic of Alzheimer's disease (AD). Because neprilysin (NEP) is a major Aß-degrading enzyme, NEP delivery in the brain is a promising gene therapy for AD. Borna disease virus (BoDV) vector enables long-term transduction of foreign genes in the central nerve system. Here, we evaluated the proteolytic ability of NEP transduced by the BoDV vector and found that the amounts of Aß40 and Aß42 significantly decreased, which suggests that NEP expressed from the BoDV vector is functional to degrade Aß.

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.

High Fat Diet Enhances ß-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting ß-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation.

Obesity and type 2 diabetes are risk factors of Alzheimer's disease (AD). We reported that a high fat diet (HFD) promotes amyloid precursor protein (APP) cleavage by ß-site APP cleaving enzyme 1 (BACE1) without increasing BACE1 levels in APP transgenic mice. However, the detailed mechanism had remained unclear. Here we demonstrate that HFD promotes BACE1/Adaptor protein-2 (AP-2)/clathrin complex formation by increasing AP-2 levels in APP transgenic mice. In Swedish APP overexpressing Chinese hamster ovary (CHO) cells as well as in SH-SY5Y cells, overexpression of AP-2 promoted the formation of BACE1/AP-2/clathrin complex, increasing the level of the soluble form of APP ß (sAPPß). On the other hand, mutant D495R BACE1, which inhibits formation of this trimeric complex, was shown to decrease the level of sAPPß. Overexpression of AP-2 promoted the internalization of BACE1 from the cell surface, thus reducing the cell surface BACE1 level. As such, we concluded that HFD may induce the formation of the BACE1/AP-2/clathrin complex, which is followed by its transport of BACE1 from the cell surface to the intracellular compartments. These events might be associated with the enhancement of ß-site cleavage of APP in APP transgenic mice. Here we present evidence that HFD, by regulation of subcellular trafficking of BACE1, promotes APP cleavage.

Idiopathic normal pressure hydrocephalus has a different cerebrospinal fluid biomarker profile from Alzheimer's disease.

The diagnosis of idiopathic normal pressure hydrocephalus (iNPH) is sometimes complicated by concomitant Alzheimer's disease (AD) pathology. The purpose of the present study is to identify an iNPH-specific cerebrospinal fluid (CSF) biomarker dynamics and to assess its ability to differentiate iNPH from AD. Total tau (t-tau), tau phosphorylated at threonine 181 (p-tau), amyloid-ß (Aß) 42 and 40, and leucine-rich α-2-glycoprotein (LRG) were measured in 93 consecutive CSF samples consisting of 55 iNPH (46 tap test responders), 20 AD, 11 corticobasal syndrome, and 7 spinocerebeller disease. Levels of t-tau and p-tau were significantly decreased in iNPH patients especially in tap test responders compared to AD. Correlation was observed between Mini-Mental State Examination scores and Aß42 in AD (R = 0.44) and mildly in iNPH (R = 0.28). Although Aß42/40 ratio showed no significant difference between iNPH and AD (p = 0.08), the levels of Aß40 and Aß42 correlated positively with each other in iNPH (R = 0.73) but much less in AD (R = 0.26), suggesting that they have discrete amyloid clearance and pathology. LRG levels did not differ between the two. Thus, our study shows that although CSF biomarkers of iNPH patients can be affected by concomitant tau and/or amyloid pathology, CSF t-tau and p-tau are highly useful for differentiation of iNPH and AD.

Continuation of exercise is necessary to inhibit high fat diet-induced ß-amyloid deposition and memory deficit in amyloid precursor protein transgenic mice.

High fat diet (HFD) is prevalent in many modern societies and HFD-induced metabolic condition is a growing concern worldwide. It has been previously reported that HFD clearly worsens cognitive function in amyloid precursor protein (APP) transgenic mice. On the other hand, we have demonstrated that voluntary exercise in an enriched environment is an effective intervention to rescue HFD-induced ß-amyloid (Aß) deposition and memory deficit. However, it had been unclear whether consumption of HFD after exercising abolished the beneficial effect of exercise on the inhibition of Alzheimer's disease (AD) pathology. To examine this question, we exposed wild type (WT) and APP mice fed with HFD to exercise conditions at different time periods. In our previous experiment, we gave HFD to mice for 20 weeks and subjected them to exercise during weeks 10-20. In the present study, mice were subjected to exercise conditions during weeks 0-10 or weeks 5-15 while being on HFD. Interestingly, we found that the effect of exercise during weeks 0-10 or weeks 5-15 on memory function was not abolished in WT mice even if they kept having HFD after finishing exercise. However, in APP transgenic mice, HFD clearly disrupted the effect of exercise during weeks 0-10 or weeks 5-15 on memory function. Importantly, we observed that the level of Aß oligomer was significantly elevated in the APP mice that exercised during weeks 0-10: this might have been caused by the up-regulation of Aß production. These results provide solid evidence that continuation of exercise is necessary to rescue HFD-induced aggravation of cognitive decline in the pathological setting of AD.

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.

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.

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.

Chronic cerebral hypoperfusion accelerates amyloid beta deposition in APPSwInd transgenic mice.

Chronic cerebral ischemia may accelerate clinicopathological changes in Alzheimer's disease. We have examined whether chronic cerebral hypoperfusion accelerates amyloid beta deposition in amyloid protein precursor transgenic (APP-Tg) mouse. At 5, 8, and 11 months of age, C57Bl/6J male mice overexpressing a mutant form of the human APP bearing the both Swedish (K670N/M671L) and the Indiana (V717F) mutations (APPSwInd) and their litterrmates were subjected to either sham operation or bilateral carotid artery stenosis (BCAS) using microcoils with an internal diameter of 0.18 mm (short-period group). One month after the sham operation or BCAS, these animals were examined by immunohistochemistry for glial fibrillary acidic protein, amyloid beta(1-40) (Abeta(1-40)), amyloid beta(1-42) (Abeta(1-42)), as well as Western blotting and filter assay for Abeta. Another batch of the littermates of APPSwInd mice were subjected to either sham operation or BCAS at 3 months and were examined in the same manner after survival for 9 months (long-period group). In the BCAS-treated group, the white matter was rarefied and astroglia was proliferated. Amyloid beta(1-40) immunoreactivity was found in a few axons in the white matter after BCAS, whereas Abeta(1-42) was accumulated in the scattered cortical neurons and the axons at ages of 6 months and thereafter in the short- and long-period groups. In the neuropil, both Abeta(1-40) and Abeta(1-42) were deposited in the sham-operated and BCAS-treated mice at ages of 9 and 12 months. There were no differences between the short-period group at ages of 12 months and the long-period group. Filter assay showed an increase of Abeta fibrils in the extracellular enriched fraction. Taken together, chronic cerebral hypoperfusion increased Abeta fibrils and induced Abeta deposition in the intracellular compartment and, therefore, may accelerate the pathological changes of Alzheimer's disease.