Extracellular Vesicle-Enriched miRNA-Biomarkers Show Improved Utility for Detecting Alzheimer's Disease Dementia and Medial Temporal Atrophy.

Background: Emerging diagnostic modalities suggest that miRNA profiles within extracellular vesicles (EVs) isolated from peripheral blood specimens may provide a non-invasive diagnostic alternative for dementia and neurodegenerative disorders. Given that EVs confer a protective environment against miRNA enzymatic degradation, the miRNAs enriched in the EV fraction of blood samples could serve as more stable and clinically relevant biomarkers compared to those obtained from serum. Objective: To compare miRNAs isolated from EVs versus serum in blood taken from Alzheimer's disease (AD) dementia patients and control cohorts. Methods: We compared 25 AD patients to 34 individuals who exhibited no cognitive impairments (NCI). Subjects were Singapore residents with Chinese heritage. miRNAs purified from serum versus blood-derived EVs were analyzed for associations with AD dementia and medial temporal atrophy detected by magnetic resonance imaging. Results: Compared to serum-miRNAs, we identified almost twice as many EV-miRNAs associated with AD dementia, and they also correlated more significantly with medial temporal atrophy, a neuroimaging marker of AD-brain pathology. We further developed combination panels of serum-miRNAs and EV-miRNAs with improved performance in identifying AD dementia. Dominant in both panels was miRNA-1290. Conclusions: This data indicates that miRNA profiling from EVs offers diagnostic superiority. This underscores the role of EVs as vectors harboring prognostic biomarkers for neurodegenerative disorders and suggests their potential in yielding novel biomarkers for AD diagnosis.

Localized APP expression results in progressive network dysfunction by disorganizing spike timing.

Progressive cognitive decline in Alzheimer's disease could either be caused by a spreading molecular pathology or by an initially focal pathology that causes aberrant neuronal activity in a larger network. To distinguish between these possibilities, we generated a mouse model with expression of mutant human amyloid precursor protein (APP) in only hippocampal CA3 cells. We found that performance in a hippocampus-dependent memory task was impaired in young adult and aged mutant mice. In both age groups, we then recorded from the CA1 region, which receives inputs from APP-expressing CA3 cells. We observed that theta oscillation frequency in CA1 was reduced along with disrupted relative timing of principal cells. Highly localized pathology limited to the presynaptic CA3 cells is thus sufficient to cause aberrant firing patterns in postsynaptic neuronal networks, which indicates that disease progression is not only from spreading pathology but also mediated by progressively advancing physiological dysfunction.

FUS-dependent microRNA deregulations identify TRIB2 as a druggable target for ALS motor neurons.

MicroRNAs (miRNAs) modulate mRNA expression, and their deregulation contributes to various diseases including amyotrophic lateral sclerosis (ALS). As fused in sarcoma (FUS) is a causal gene for ALS and regulates biogenesis of miRNAs, we systematically analyzed the miRNA repertoires in spinal cords and hippocampi from ALS-FUS mice to understand how FUS-dependent miRNA deregulation contributes to ALS. miRNA profiling identified differentially expressed miRNAs between different central nervous system (CNS) regions as well as disease states. Among the up-regulated miRNAs, miR-1197 targets the pro-survival pseudokinase Trib2. A reduced TRIB2 expression was observed in iPSC-derived motor neurons from ALS patients. Pharmacological stabilization of TRIB2 protein with a clinically approved cancer drug rescues the survival of iPSC-derived human motor neurons, including those from a sporadic ALS patient. Collectively, our data indicate that miRNA profiling can be used to probe the molecular mechanisms underlying selective vulnerability, and TRIB2 is a potential therapeutic target for ALS.

APP family member dimeric complexes are formed predominantly in synaptic compartments.

BACKGROUND: The amyloid precursor protein (APP), a key player in Alzheimer's disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. RESULTS: We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls. CONCLUSIONS: Together, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

Evidence for a clathrin-independent endocytic pathway for APP internalization in the neuronal somatodendritic compartment.

Amyloid precursor protein (APP) internalization via clathrin-/dynamin-mediated endocytosis (CME) mediated by its YENPTY motif into endosomes containing ß-secretase is proposed to be critical for amyloid-beta (Aß) production. Here, we show that somatodendritic APP internalization in primary rodent neurons is not blocked by inhibiting dynamin or mutating the YENPTY motif, in contrast to non-neuronal cell lines. These phenomena, confirmed in induced human neurons under dynamin inhibition, occur during basal conditions and chemical long-term-depression stimulus, pointing to a clathrin-independent internalization pathway for somatodendritic APP. Mutating the YENPTY motif does not alter APP recycling, degradation, or endolysosomal colocalization. However, both dynamin inhibition and the YENPTY mutant significantly decrease secreted Aß in neurons, suggesting that internalized somatodendritic APP may not constitute a major source of Aß. Interestingly, like APP, somatodendritic low-density lipoprotein receptor (LDLR) internalization does not require its CME motif. These results highlight intriguing differences in neuronal internalization pathways and refine our understanding of Aß production and secretion.

Differential effects of familial Alzheimer's disease-causing mutations on amyloid precursor protein (APP) trafficking, proteolytic conversion, and synaptogenic activity.

The amyloid precursor protein (APP) is a key player in Alzheimer`s disease (AD) and the precursor of the Aß peptide, which is generated by consecutive cleavages of ß- and γ-secretases. Familial Alzheimer's disease (FAD) describes a hereditary subgroup of AD that represents a low percentage of AD cases with an early onset of the disease. Different APP FAD mutations are thought to have qualitatively different effects on its proteolytic conversion. However, few studies have explored the pathogenic and putative physiological differences in more detail. Here, we compared different FAD mutations, located at the ß- (Swedish), α- (Flemish, Arctic, Iowa) or γ-secretase (Iberian) cleavage sites. We examined heterologous expression of APP WT and FAD mutants in non-neuronal cells and their impact on presynaptic differentiation in contacting axons of co-cultured neurons. To decipher the underlying molecular mechanism, we tested the subcellular localization, the endocytosis rate and the proteolytic processing in detail by immunoprecipitation-mass spectrometry. Interestingly, we found that only the Iberian mutation showed altered synaptogenic function. Furthermore, the APP Iowa mutant shows significantly decreased α-secretase processing which is in line with our results that APP carrying the Iowa mutation was significantly increased in early endosomes. However, most interestingly, immunoprecipitation-mass spectrometry analysis revealed that the amino acid substitutions of APP FAD mutants have a decisive impact on their processing reflected in altered Aß profiles. Importantly, N-terminally truncated Aß peptides starting at position 5 were detected preferentially for APP Flemish, Arctic, and Iowa mutants containing amino acid substitutions around the α-secretase cleavage site. The strongest change in the ratio of Aß40/Aß42 was observed for the Iberian mutation while APP Swedish showed a substantial increase in Aß1-17 peptides. Together, our data indicate that familial AD mutations located at the α-, ß-, and γ-secretase cleavage sites show considerable differences in the underlying pathogenic mechanisms.

Chronic cerebral hypoperfusion alters the CypA-EMMPRIN-gelatinase pathway: Implications for vascular dementia.

Chronic cerebral hypoperfusion (CCH) is postulated to underlie multiple pathophysiological processes in vascular dementia (VaD), including extracellular matrix dysfunction. While several extracellular matrix proteins, namely cyclophilin A (CypA), extracellular matrix metalloproteinase inducer (EMMPRIN) and gelatinases (matrix metalloproteinases, MMP-2 and -9) have been investigated in acute stroke, their involvement in CCH and VaD remains unclear. In this study, CypA-EMMPRIN-gelatinase proteins were analysed in a clinical cohort of 36 aged, cognitively unimpaired subjects and 48 VaD patients, as well as in a bilateral carotid artery stenosis mouse model of CCH. Lower CypA and higher EMMPRIN levels were found in both VaD serum and CCH mouse brain. Furthermore, gelatinases were differentially altered in CCH mice and VaD patients, with significant MMP-2 increase in CCH brain and serum, whilst serum MMP-9 was elevated in VaD but reduced in CCH, suggesting complex CypA-EMMPRIN-gelatinase regulatory mechanisms. Interestingly, subjects with cortical infarcts had higher serum MMP-2, while white matter hyperintensities, cortical infarcts and lacunes were associated with higher serum MMP-9. Taken together, our data indicate that perturbations of CypA-EMMPRIN signalling may be associated with gelatinase-mediated vascular sequelae, highlighting the potential utility of the CypA-EMMPRIN-gelatinase pathway as clinical biomarkers and therapeutic targets in VaD.

Enhanced cleavage of APP by co-expressed Bace1 alters the distribution of APP and its fragments in neuronal and non-neuronal cells.

BACKGROUND: Alzheimer's disease amyloid-beta peptides (Aß) are generated via sequential cleavage of the amyloid precursor protein (APP) by ß-secretase (Bace1) and γ-secretase. Though the precise subcellular location(s) of Bace1-mediated APP cleavage remains unresolved, current models suggest APP internalization into Bace1-containing endosomes is a critical step. However, direct evidence for this model is lacking, and previous reports that probed the APP/Bace1 interaction (using co-expressed APP and Bace1 differentially labeled with fluorescent protein tags) did not determine if APP fluorescence originated from full-length APP (fl-APP) molecules that had internalized from the cell surface pool. METHODS: We adapted the bungarotoxin-ligand (BTX) system to label surface APP and track internalized fluorescent APP/BTX puncta in rodent primary neurons co-expressing fluorescently-tagged Bace1. Subsequently, we employed imaging and biochemical-based approaches to measure N- and C-terminal APP epitope levels in primary neurons, N2a neuroblastoma, and HeLa cell lines. RESULTS: We hypothesized that surface-labeled APP/BTX puncta would, upon internalization, colocalize with fluorescently-tagged Bace1. Unexpectedly, we observed a dramatic loss of internalized APP in co-transfected neurons and ~ 80-90% loss of surface-resident fl-APP, which we also observed in HeLa and N2a cells. Loss of surface fl-APP could be reversed by a Bace1 inhibitor, suggesting that enhanced Bace1-mediated APP cleavage was responsible for the altered processing and mis-sorting. Importantly, in a C-terminally-tagged APP construct, the majority of C-terminal fluorescence was preserved in HeLa cells despite the loss of N-terminal APP signal. This phenomenon was not only recapitulated in cultured neurons, but also showed a progressive disappearance of the APP N-terminal tag, reflecting continual cleavage of fl-APP by Bace1 away from the cell body. CONCLUSIONS: Our results strongly suggested that in APP/Bace1 co-expression approaches, there was significant early and aberrant Bace1-mediated APP cleavage that perturbed fl-APP trafficking from the secretory pathway onwards, resulting in a substantial loss of surface fl-APP, which in turn led to a marked reduction in APP internalization. In C-terminally-tagged APP constructs, a large fraction of the APP fluorescence signal therefore likely arose from fluorescently-tagged ß-C-terminal-fragment (ß-CTF) or downstream proteolytic derivatives instead of fl-APP. Thus, care is needed in interpreting results where APP is detected only with a C-terminal tag in the presence of Bace1 co-expression, and previous findings may need to be reinterpreted if it is unclear whether fl-APP is present in normal physiological levels.

SINgapore GERiatric intervention study to reduce physical frailty and cognitive decline (SINGER)-pilot: A feasibility study.

INTRODUCTION: The SINGER pilot randomized controlled trial aims to examine the feasibility and acceptability of the Finnish Geriatric Intervention Study (FINGER) multi-domain lifestyle interventions compared to Singaporean adaptations. METHODS: Seventy elderly participants were recruited and randomized into FINGER (n = 36) or SINGER (n = 34) interventions; involving physical exercise, cognitive training, diet, and vascular risk factors management, for 6 months. RESULTS: Both intervention groups were equally feasible and acceptable with participants completing at least 80% of the interventions. Body strength improved in both groups (Pupper body = .04, P lower body = .06, P core = .05). More participants in the SINGER group attained good blood pressure control at month-6 compared to FINGER (41% vs 19%; P = .06). DISCUSSION: This study is the first to compare the feasibility of multi-domain interventions adapted to local culture with the FINGER interventions. The findings will be utilized for a larger study to provide evidence for the efficacy of multi-domain lifestyle interventions in preventing cognitive decline.

Machine learning classifies predictive kinematic features in a mouse model of neurodegeneration.

Motor deficits are observed in Alzheimer's disease (AD) prior to the appearance of cognitive symptoms. To investigate the role of amyloid proteins in gait disturbances, we characterized locomotion in APP-overexpressing transgenic J20 mice. We used three-dimensional motion capture to characterize quadrupedal locomotion on a treadmill in J20 and wild-type mice. Sixteen J20 mice and fifteen wild-type mice were studied at two ages (4- and 13-month). A random forest (RF) classification algorithm discriminated between the genotypes within each age group using a leave-one-out cross-validation. The balanced accuracy of the RF classification was 92.3 ± 5.2% and 93.3 ± 4.5% as well as False Negative Rate (FNR) of 0.0 ± 0.0% and 0.0 ± 0.0% for the 4-month and 13-month groups, respectively. Feature ranking algorithms identified kinematic features that when considered simultaneously, achieved high genotype classification accuracy. The identified features demonstrated an age-specific kinematic profile of the impact of APP-overexpression. Trunk tilt and unstable hip movement patterns were important in classifying the 4-month J20 mice, whereas patterns of shoulder and iliac crest movement were critical for classifying 13-month J20 mice. Examining multiple kinematic features of gait simultaneously could also be developed to classify motor disorders in humans.

Dysfunction in nonsense-mediated decay, protein homeostasis, mitochondrial function, and brain connectivity in ALS-FUS mice with cognitive deficits.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of the same disease spectrum of adult-onset neurodegenerative diseases that affect the motor and cognitive functions, respectively. Multiple common genetic loci such as fused in sarcoma (FUS) have been identified to play a role in ALS and FTD etiology. Current studies indicate that FUS mutations incur gain-of-toxic functions to drive ALS pathogenesis. However, how the disease-linked mutations of FUS affect cognition remains elusive. Using a mouse model expressing an ALS-linked human FUS mutation (R514G-FUS) that mimics endogenous expression patterns, we found that FUS proteins showed an age-dependent accumulation of FUS proteins despite the downregulation of mouse FUS mRNA by the R514G-FUS protein during aging. Furthermore, these mice developed cognitive deficits accompanied by a reduction in spine density and long-term potentiation (LTP) within the hippocampus. At the physiological expression level, mutant FUS is distributed in the nucleus and cytosol without apparent FUS aggregates or nuclear envelope defects. Unbiased transcriptomic analysis revealed a deregulation of genes that cluster in pathways involved in nonsense-mediated decay, protein homeostasis, and mitochondrial functions. Furthermore, the use of in vivo functional imaging demonstrated widespread reduction in cortical volumes but enhanced functional connectivity between hippocampus, basal ganglia and neocortex in R514G-FUS mice. Hence, our findings suggest that disease-linked mutation in FUS may lead to changes in proteostasis and mitochondrial dysfunction that in turn affect brain structure and connectivity resulting in cognitive deficits.

World-Wide FINGERS Network: A global approach to risk reduction and prevention of dementia.

Reducing the risk of dementia can halt the worldwide increase of affected people. The multifactorial and heterogeneous nature of late-onset dementia, including Alzheimer's disease (AD), indicates a potential impact of multidomain lifestyle interventions on risk reduction. The positive results of the landmark multidomain Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) support such an approach. The World-Wide FINGERS (WW-FINGERS), launched in 2017 and including over 25 countries, is the first global network of multidomain lifestyle intervention trials for dementia risk reduction and prevention. WW-FINGERS aims to adapt, test, and optimize the FINGER model to reduce risk across the spectrum of cognitive decline-from at-risk asymptomatic states to early symptomatic stages-in different geographical, cultural, and economic settings. WW-FINGERS aims to harmonize and adapt multidomain interventions across various countries and settings, to facilitate data sharing and analysis across studies, and to promote international joint initiatives to identify globally implementable and effective preventive strategies.

Caspase Activation and Caspase-Mediated Cleavage of APP Is Associated with Amyloid ß-Protein-Induced Synapse Loss in Alzheimer's Disease.

Amyloid ß-protein (Aß) toxicity is hypothesized to play a seminal role in Alzheimer's disease (AD) pathogenesis. However, it remains unclear how Aß causes synaptic dysfunction and synapse loss. We hypothesize that one mechanism of Aß-induced synaptic injury is related to the cleavage of amyloid ß precursor protein (APP) at position D664 by caspases that release the putatively cytotoxic C31 peptide. In organotypic slice cultures derived from mice with a knock-in mutation in the APP gene (APP D664A) to inhibit caspase cleavage, Aß-induced synaptic injury is markedly reduced in two models of Aß toxicity. Loss of dendritic spines is also attenuated in mice treated with caspase inhibitors. Importantly, the time-dependent dendritic spine loss is correlated with localized activation of caspase-3 but is absent in APP D664A cultures. We propose that the APP cytosolic domain plays an essential role in Aß-induced synaptic damage in the injury pathway mediated by localized caspase activation.

Presymptomatic Increase of an Extracellular RNA in Blood Plasma Associates with the Development of Alzheimer's Disease.

The extracellular RNAs (exRNAs) from human biofluid have recently been systematically characterized. However, the correlations of biofluid exRNA levels and human diseases remain largely untested. Here, considering the unmet need for presymptomatic biomarkers of sporadic Alzheimer's disease (AD), we leveraged the recently developed SILVER-seq (small-input liquid volume extracellular RNA sequencing) technology to generate exRNA profiles from a longitudinal collection of human plasma samples. These 164 plasma samples were collected from research subjects 70 years or older with up to 15 years of clinical follow-up prior to death and whose clinical diagnoses were confirmed by pathological analysis of their post mortem brains. The exRNAs of AD-activated genes and transposons in the brain exhibited a concordant trend of increase in AD plasma in comparison with age-matched control plasma. However, when we required statistical significance with multiple testing adjustments, phosphoglycerate dehydrogenase (PHGDH) was the only gene that exhibited consistent upregulation in AD brain transcriptomes from 3 independent cohorts and an increase in AD plasma as compared to controls. We validated PHGDH's serum exRNA and brain protein expression increases in AD by using 5 additional published cohorts. Finally, we compared the time-course exRNA trajectories between "converters" and controls. Plasma PHGDH exRNA exhibited presymptomatic increases in each of the 11 converters during their transitions from normal to cognitive impairment but remained stable over the entire follow-up period in 8 out of the 9 control elderly subjects. These data suggest the potential utilities of plasma exRNA levels for screening and longitudinal exRNA changes as a presymptomatic indication of sporadic AD.

PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

FUS-mediated dysregulation of Sema5a, an autism-related gene, in FUS mice with hippocampus-dependent cognitive deficits.

Pathological fused in sarcoma (FUS) inclusions are found in 10% of patients with frontotemporal dementia and those with amyotrophic lateral sclerosis (ALS) carrying FUS mutations. Current work indicates that FUS mutations may incur gain-of-toxic functions to drive ALS pathogenesis. However, how FUS dysfunction may affect cognition remains elusive. Using a mouse model expressing wild-type human FUS mimicking the endogenous expression pattern and level within the central nervous system, we found that they developed hippocampus-mediated cognitive deficits accompanied by an age-dependent reduction in spine density and long-term potentiation in their hippocampus. However, there were no apparent FUS aggregates, nuclear envelope defects and cytosolic FUS accumulation. These suggest that these proposed pathogenic mechanisms may not be the underlying causes for the observed cognitive deficits. Unbiased transcriptomic analysis identified expression changes in a small set of genes with preferential expression in the neurons and oligodendrocyte lineage cells. Of these, we focused on Sema5a, a gene involved in axon guidance, spine dynamics, Parkinson's disease and autism spectrum disorders. Critically, FUS binds directly to Sema5a mRNA and regulates Sema5a expression in a FUS-dose-dependent manner. Taken together, our data suggest that FUS-driven Sema5a deregulation may underlie the cognitive deficits in FUS transgenic mice.

Cerebral transcriptome analysis reveals age-dependent progression of neuroinflammation in P301S mutant tau transgenic male mice.

Aggregation of the microtubule-associated protein, tau, can lead to neurofibrillary tangle formation in neurons and glia which is the hallmark of tauopathy. The cellular damage induced by the formation of neurofibrillary tangles leads to neuroinflammation and consecutive neuronal death. However, detailed observation of transcriptomic changes under tauopathy together with the comparison of age-dependent progression of neuroinflammatory gene expressions mediated by tau overexpression is required. Employing RNA sequencing on PS19 transgenic mice that overexpress human mutant tau harboring the P301S mutation, we have examined the effects of age-dependent tau overexpression on transcriptomic changes of immune and inflammatory responses in the cerebral cortex. Compared to age-matched wild type control, P301S transgenic mice exhibit significant transcriptomic alterations. We have observed age-dependent neuroinflammatory gene expression changes in both wild type and P301S transgenic mice where tau overexpression further promoted the expression of neuroinflammatory genes in 10-month old P301S transgenic mice. Moreover, functional gene network analyses (gene ontology and pathway enrichment) and prospective target protein interactions predicted the potential involvement of multiple immune and inflammatory pathways that may contribute to tau-mediated neuronal pathology. Our current study on P301S transgenic mice model revealed for the first time, the differences of gene expression patterns in both early and late stage of tau pathology in cerebral cortex. Our analyses also revealed that tau overexpression alone induces multiple inflammatory and immune transcriptomic changes and may provide a roadmap to elucidate the targets of anti-inflammatory therapeutic strategy focused on tau pathology and related neurodegenerative diseases.

Systemic toxicity of di (2-ethylhexyl) adipate (DEHA) in rats following 28-day intravenous exposure.

Di (2-ethylhexyl) adipate (DEHA) is a potential plasticizer alternative for di-2-ethylhexyl phthalate (DEHP). Toxicity of DEHA has been studied mostly via oral exposure but not assessed after repeated intravenous exposure. The present study shows the toxicity effects after intravenous administration for 28 consecutive days and the reversibility of the effects following a 14-day recovery period. The study was conducted under GLP conditions. Four groups of rats (15/sex/group) each received either vehicle or DEHA in vehicle (100, 200, or 450 mg/kg/day). Criteria for evaluation included clinical observations, body weight, food consumption, clinical pathology (hematology, serum chemistry, coagulation, urinalyses), gross (necropsy) evaluation, organ weight and histopathological evaluation. There were no DEHA-related changes in all the endpoints evaluated at 100 or 200 mg/kg/day. There were no test article-related changes in clinical pathology or gross necropsy observation at 450 mg/kg/day. At the high-dose, DEHA-related findings included clinical observations, decreased body weight gain and food consumption, increased liver weight in females associated with minimal hepatocellular hypertrophy, and decreased thymus weight in males and females without histopathology findings. All these findings were completely reversible within a 14-day recovery period. Therefore, the 200 mg/kg/day dose is considered to be the No-Observed-Effect Level (NOEL).

The ALS-FTD-linked gene product, C9orf72, regulates neuronal morphogenesis via autophagy.

Mutations in C9orf72 leading to hexanucleotide expansions are the most common genetic causes for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A phenotype resembling ALS and FTD is seen in transgenic mice overexpressing the hexanucleotide expansions, but is absent in C9orf72-deficient mice. Thus, the exact function of C9orf72 in neurons and how loss of C9orf72 may contribute to neuronal dysfunction remains to be clearly defined. Here, we showed that primary hippocampal neurons cultured from c9orf72 knockout mice have reduced dendritic arborization and spine density. Quantitative proteomic analysis identified C9orf72 as a component of the macroautophagy/autophagy initiation complex composed of ULK1-RB1CC1-ATG13-ATG101. The association was mediated through the direct interaction with ATG13 via the isoform-specific carboxyl-terminal DENN and dDENN domain of C9orf72. Furthermore, c9orf72 knockout neurons showed reduced LC3-II puncta accompanied by reduced ULK1 levels, suggesting that loss of C9orf72 impairs basal autophagy. Conversely, wild-type neurons treated with a ULK1 kinase inhibitor showed a dose-dependent reduction of dendritic arborization and spine density. Furthermore, expression of the long isoform of human C9orf72 that interacts with the ULK1 complex, but not the short isoform, rescues autophagy and the dendritic arborization phenotypes of c9orf72 knockout neurons. Taken together, our data suggests that C9orf72 has a cell-autonomous role in neuronal and dendritic morphogenesis through promotion of ULK1-mediated autophagy.

Microinterventional endocapsular nucleus disassembly: novel technique and results of first-in-human randomised controlled study.

AIM: To assess the safety and efficacy of microinterventional endocapsular nuclear fragmentation in moderate to severe cataracts. METHODS: This was a prospective single-masked multisurgeon interventional randomised controlled trial (ClinicalTrials.gov NCT02843594) where 101 eyes of 101 subjects with grade 3‒4+ nuclear cataracts were randomised to torsional phacoemulsification alone (controls) or torsional phacoemulsification with adjunctive endocapsular nuclear fragmentation using a manual microinterventional nitinol filament loop device (miLOOP group). Outcome measures were phacoemulsification efficiency as measured by ultrasound energy (cumulative dispersed energy (CDE) units) and fluidics requirements (total irrigation fluid used) as well as incidence of intraoperative and postoperative complications. RESULTS: Only high-grade advanced cataracts were enrolled with more than 85% of eyes with baseline best corrected visual acuity (BCVA) of 20/200 or worse in either group. Mean CDE was 53% higher in controls (32.8±24.9 vs 21.4±13.1 with miLOOP assistance) (p=0.004). Endothelial cell loss after surgery was low and similar between groups (7‒8%, p=0.561) One-month BCVA averaged 20/27 Snellen in miLOOP eyes and 20/24 in controls. No direct complications were caused by the miLOOP. In two cases, capsular tears occurred during IOL implantation and in all remaining cases during phacoemulsification, with none occurring during the miLOOP nucleus disassembly part of the procedure. CONCLUSIONS: Microinterventional endocapsular fragmentation with the manual, disposable miLOOP device achieved consistent, ultrasound-free, full-thickness nucleus disassembly and significantly improved overall phaco efficiency in advanced cataracts. TRIAL REGISTRATION NUMBER: NCT02843594.