Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

Are there roles for brain cell senescence in aging and neurodegenerative disorders?

The term cellular senescence was introduced more than five decades ago to describe the state of growth arrest observed in aging cells. Since this initial discovery, the phenotypes associated with cellular senescence have expanded beyond growth arrest to include alterations in cellular metabolism, secreted cytokines, epigenetic regulation and protein expression. Recently, senescence has been shown to play an important role in vivo not only in relation to aging, but also during embryonic development. Thus, cellular senescence serves different purposes and comprises a wide range of distinct phenotypes across multiple cell types. Whether all cell types, including post-mitotic neurons, are capable of entering into a senescent state remains unclear. In this review we examine recent data that suggest that cellular senescence plays a role in brain aging and, notably, may not be limited to glia but also neurons. We suggest that there is a high level of similarity between some of the pathological changes that occur in the brain in Alzheimer's and Parkinson's diseases and those phenotypes observed in cellular senescence, leading us to propose that neurons and glia can exhibit hallmarks of senescence previously documented in peripheral tissues.

Evidence for miR-181 involvement in neuroinflammatory responses of astrocytes.

Inflammation is a common component of acute injuries of the central nervous system (CNS) such as ischemia, and degenerative disorders such as Alzheimer's disease. Glial cells play important roles in local CNS inflammation, and an understanding of the roles for microRNAs in glial reactivity in injury and disease settings may therefore lead to the development of novel therapeutic interventions. Here, we show that the miR-181 family is developmentally regulated and present in high amounts in astrocytes compared to neurons. Overexpression of miR-181c in cultured astrocytes results in increased cell death when exposed to lipopolysaccharide (LPS). We show that miR-181 expression is altered by exposure to LPS, a model of inflammation, in both wild-type and transgenic mice lacking both receptors for the inflammatory cytokine TNF-α. Knockdown of miR-181 enhanced LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8) and HMGB1, while overexpression of miR-181 resulted in a significant increase in the expression of the anti-inflammatory cytokine IL-10. To assess the effects of miR-181 on the astrocyte transcriptome, we performed gene array and pathway analysis on astrocytes with reduced levels of miR-181b/c. To examine the pool of potential miR-181 targets, we employed a biotin pull-down of miR-181c and gene array analysis. We validated the mRNAs encoding MeCP2 and X-linked inhibitor of apoptosis as targets of miR-181. These findings suggest that miR-181 plays important roles in the molecular responses of astrocytes in inflammatory settings. Further understanding of the role of miR-181 in inflammatory events and CNS injury could lead to novel approaches for the treatment of CNS disorders with an inflammatory component.

Common methodological pitfalls in ICI pneumonitis risk prediction studies.

Background: Pneumonitis is one of the most common adverse events induced by the use of immune checkpoint inhibitors (ICI), accounting for a 20% of all ICI-associated deaths. Despite numerous efforts to identify risk factors and develop predictive models, there is no clinically deployed risk prediction model for patient risk stratification or for guiding subsequent monitoring. We believe this is due to systemic suboptimal approaches in study designs and methodologies in the literature. The nature and prevalence of different methodological approaches has not been thoroughly examined in prior systematic reviews. Methods: The PubMed, medRxiv and bioRxiv databases were used to identify studies that aimed at risk factor discovery and/or risk prediction model development for ICI-induced pneumonitis (ICI pneumonitis). Studies were then analysed to identify common methodological pitfalls and their contribution to the risk of bias, assessed using the QUIPS and PROBAST tools. Results: There were 51 manuscripts eligible for the review, with Japan-based studies over-represented, being nearly half (24/51) of all papers considered. Only 2/51 studies had a low risk of bias overall. Common bias-inducing practices included unclear diagnostic method or potential misdiagnosis, lack of multiple testing correction, the use of univariate analysis for selecting features for multivariable analysis, discretization of continuous variables, and inappropriate handling of missing values. Results from the risk model development studies were also likely to have been overoptimistic due to lack of holdout sets. Conclusions: Studies with low risk of bias in their methodology are lacking in the existing literature. High-quality risk factor identification and risk model development studies are urgently required by the community to give the best chance of them progressing into a clinically deployable risk prediction model. Recommendations and alternative approaches for reducing the risk of bias were also discussed to guide future studies.

Interrogation of brain miRNA and mRNA expression profiles reveals a molecular regulatory network that is perturbed by mutant huntingtin.

Emerging evidence indicates that microRNAs (miRNAs) may play an important role in the pathogenesis of Huntington's disease (HD). To identify the individual miRNAs that are altered in HD and may therefore regulate a gene network underlying mutant huntingtin-induced neuronal dysfunction in HD, we performed miRNA array analysis combined with mRNA profiling in the cerebral cortex from N171-82Q HD mice. Expression profiles of miRNAs as well as mRNAs in HD mouse cerebral cortex were analyzed and confirmed at different stages of disease progression; the most significant changes of miRNAs in the cerebral cortex were also detected in the striatum of HD mice. Our results revealed a significant alteration of miR-200 family members, miR-200a, and miR-200c in the cerebral cortex and the striatum, at the early stage of disease progression in N171-82Q HD mice. We used a coordinated approach to integrate miRNA and mRNA profiling, and applied bioinformatics to predict a target gene network potentially regulated by these significantly altered miRNAs that might be involved in HD disease progression. Interestingly, miR-200a and miR-200c are predicted to target genes regulating synaptic function, neurodevelopment, and neuronal survival. Our results suggest that altered expression of miR-200a and miR-200c may interrupt the production of proteins involved in neuronal plasticity and survival, and further investigation of the involvement of perturbed miRNA expression in HD pathogenesis is warranted, and may lead to reveal novel approaches for HD therapy.

Correlation Between Early Endpoints and Overall Survival in Non-Small-Cell Lung Cancer: A Trial-Level Meta-Analysis.

Early endpoints, such as progression-free survival (PFS), are increasingly used as surrogates for overall survival (OS) to accelerate approval of novel oncology agents. Compiling trial-level data from randomized controlled trials (RCTs) could help to develop a predictive framework to ascertain correlation trends between treatment effects for early and late endpoints. Through trial-level correlation and random-effects meta-regression analysis, we assessed the relationship between hazard ratio (HR) OS and (1) HR PFS and (2) odds ratio (OR) PFS at 4 and 6 months, stratified according to the mechanism of action of the investigational product. Using multiple source databases, we compiled a data set including 81 phase II-IV RCTs (35 drugs and 156 observations) of patients with non-small-cell lung cancer. Low-to-moderate correlations were generally observed between treatment effects for early endpoints (based on PFS) and HR OS across trials of agents with different mechanisms of action. Moderate correlations were seen between treatment effects for HR PFS and HR OS across all trials, and in the programmed cell death-1/programmed cell death ligand-1 and epidermal growth factor receptor trial subsets. Although these results constitute an important step, caution is advised, as there are some limitations to our evaluation, and an additional patient-level analysis would be needed to establish true surrogacy.

The role of machine learning in clinical research: transforming the future of evidence generation.

BACKGROUND: Interest in the application of machine learning (ML) to the design, conduct, and analysis of clinical trials has grown, but the evidence base for such applications has not been surveyed. This manuscript reviews the proceedings of a multi-stakeholder conference to discuss the current and future state of ML for clinical research. Key areas of clinical trial methodology in which ML holds particular promise and priority areas for further investigation are presented alongside a narrative review of evidence supporting the use of ML across the clinical trial spectrum. RESULTS: Conference attendees included stakeholders, such as biomedical and ML researchers, representatives from the US Food and Drug Administration (FDA), artificial intelligence technology and data analytics companies, non-profit organizations, patient advocacy groups, and pharmaceutical companies. ML contributions to clinical research were highlighted in the pre-trial phase, cohort selection and participant management, and data collection and analysis. A particular focus was paid to the operational and philosophical barriers to ML in clinical research. Peer-reviewed evidence was noted to be lacking in several areas. CONCLUSIONS: ML holds great promise for improving the efficiency and quality of clinical research, but substantial barriers remain, the surmounting of which will require addressing significant gaps in evidence.

TLR2 activation inhibits embryonic neural progenitor cell proliferation.

Toll-like receptors (TLRs) play essential roles in innate immunity, and increasing evidence indicates that these receptors are expressed in neurons, astrocytes, and microglia in the brain, where they mediate responses to infection, stress, and injury. To address the possibility that TLR2 heterodimer activation could affect progenitor cells in the developing brain, we analyzed the expression of TLR2 throughout mouse cortical development, and assessed the role of TLR2 heterodimer activation in neuronal progenitor cell (NPC) proliferation. TLR2 mRNA and protein was expressed in the cortex in embryonic and early postnatal stages of development, and in cultured cortical NPC. While NPC from TLR2-deficient and wild type embryos had the same proliferative capacity, TLR2 activation by the synthetic bacterial lipopeptides Pam(3)CSK(4) and FSL1, or low molecular weight hyaluronan, an endogenous ligand for TLR2, inhibited neurosphere formation in vitro. Intracerebral in utero administration of TLR2 ligands resulted in ventricular dysgenesis characterized by increased ventricle size, reduced proliferative area around the ventricles, increased cell density, an increase in phospho-histone 3 cells, and a decrease in BrdU(+) cells in the sub-ventricular zone. Our findings indicate that loss of TLR2 does not result in defects in cerebral development. However, TLR2 is expressed and functional in the developing telencephalon from early embryonic stages and infectious agent-related activation of TLR2 inhibits NPC proliferation. TLR2-mediated inhibition of NPC proliferation may therefore be a mechanism by which infection, ischemia, and inflammation adversely affect brain development.

Functional MRI and response inhibition in children exposed to cocaine in utero. Preliminary findings.

This study investigated the potential long-term effects of cocaine exposure on brain functioning using fMRI in school-aged children. The sample included 12 children with prenatal cocaine exposure and 12 non-exposed children (8-9 years old). Groups did not differ on IQ, socioeconomic status, or perinatal risk factors. A response inhibition task was administered during an fMRI scan using a 1.5-T MRI system. Task performance did not differentiate groups, but groups were differentiated by patterns of task-related brain activity. Cocaine-exposed children showed greater activation in the right inferior frontal cortex and caudate during response inhibition, whereas non-exposed children showed greater activations in temporal and occipital regions. These preliminary findings suggest that prenatal cocaine may affect the development of brain systems involved in the regulation of attention and response inhibition.

Recruitment of anterior and posterior structures in lexical-semantic processing: an fMRI study comparing implicit and explicit tasks.

Previous studies examining explicit semantic processing have consistently shown activation of the left inferior frontal gyrus (IFG). In contrast, implicit semantic processing tasks have shown activation in posterior areas including the superior temporal gyrus (STG) and the middle temporal gyrus (MTG) with less consistent activation in the IFG. These results raise the question whether the functional role of the IFG is related to those processes needed to make a semantic decision or to processes involved in the extraction and analysis of meaning. This study examined neural activation patterns during a semantic judgment task requiring overt semantic analysis, and then compared these activation patterns to previously obtained results using the same semantically related and unrelated word pairs in a lexical decision task which required only implicit semantic processing (Rissman, J., Eliassen, J. C., & Blumstein, S. E. (2003). An event-related fMRI investigation of implicit semantic priming. Journal of Cognitive Neuroscience, 15, 1160-1175). The behavioral results demonstrated that the tasks were equivalent in difficulty. fMRI results indicated that the IFG and STG bilaterally showed greater activation for semantically unrelated than related word pairs across the two tasks. Comparison of the two task types across conditions revealed greater activation for the semantic judgment task only in the STG bilaterally and not in the IFG. These results suggest that the pre-frontal cortex is recruited similarly in the service of both the lexical decision and semantic judgment tasks. The increased activation in the STG in the semantic judgment task reflects the greater depth of semantic processing required in this task and indicates that the STG is not simply a passive store of lexical-semantic information but is involved in the active retrieval of this information.

An event-related fMRI investigation of phonological-lexical competition.

This study explored the neural correlates of phonological-lexical competition and frequency on word recognition. An event-related fMRI experiment was conducted using an auditory lexical decision task in which word and nonword stimuli varied in terms of neighborhood density (high and low). Word stimuli also varied in terms of frequency (high and low). Behavioral results were similar to those of Luce and Pisoni [Luce, P. A., & Pisoni, D. B. (1998). Recognizing spoken words: The neighborhood activation model. Ear and Hearing, 19, 1-36], with the reaction time data showing a main effect of word frequency and density as well as a significant interaction effect between these two factors. fMRI results revealed an overall greater neural response for high-density compared to low-density words in the left supramarginal gyrus, consistent with the view that there are greater demands on phonological processing under conditions of increased phonological-lexical competition. The comparison between high and low frequency words revealed greater activation for high frequency words in both anterior and posterior left middle temporal gyrus. A significant interaction between density and frequency was found in lateral and medial frontal structures. This frontal activation may reflect the greater computational resources required in integrating frequency and density information in order to access a word. Overall, these findings demonstrate the sensitivity of neural structures to different properties of the lexicon.

Extracellular Vesicle-Associated Aß Mediates Trans-Neuronal Bioenergetic and Ca2+-Handling Deficits in Alzheimer's Disease Models.

Alzheimer's Disease (AD) is an age-related neurodegenerative disorder in which aggregation-prone neurotoxic amyloid ß-peptide (Aß) accumulates in the brain. Extracellular vesicles (EVs) are small 50-150 nanometer membrane vesicles that have recently been implicated in the prion-like spread of self-aggregating proteins. Here we report that EVs isolated from AD patient CSF and plasma, from the plasma of two AD mouse models, and from the medium of neural cells expressing familial AD presenilin 1 mutations, destabilize neuronal Ca2+ homeostasis, impair mitochondrial function, and sensitize neurons to excitotoxicity. EVs contain a relatively low amount of Aß but have an increased Aß42/ Aß40 ratio; the majority of Aß is located on the surface of the EVs. Impairment of lysosome function results in increased generation EVs with elevated Aß42 levels. EVs may mediate transcellular spread of pathogenic Aß species and that impair neuronal Ca2+ handling and mitochondrial function, and may thereby render neurons vulnerable to excitotoxicity.

Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2.

During neuronal differentiation, use of an alternative splice site on the rat telomere repeat-binding factor 2 (TRF2) mRNA generates a short TRF2 protein isoform (TRF2-S) capable of derepressing neuronal genes. However, the RNA-binding proteins (RBPs) controlling this splicing event are unknown. Here, using affinity pull-down analysis, we identified heterogeneous nuclear ribonucleoproteins H1 and H2(HNRNPH) as RBPs specifically capable of interacting with the spliced RNA segment (exon 7) of Trf2 pre-mRNA. HNRNPH proteins prevent the production of the short isoform of Trf2 mRNA, as HNRNPH silencing selectively elevates TRF2-S levels. Accordingly, HNRNPH levels decline while TRF2-S levels increase during neuronal differentiation. In addition, CRISPR/Cas9-mediated deletion of hnRNPH2 selectively accelerates the NGF-triggered differentiation of rat pheochromocytoma cells into neurons. In sum, HNRNPH is a splicing regulator of Trf2 pre-mRNA that prevents the expression of TRF2-S, a factor implicated in neuronal differentiation.

Combination therapy with lenalidomide and nanoceria ameliorates CNS autoimmunity.

OBJECTIVE: Multiple sclerosis (MS) is a debilitating neurological disorder involving an autoimmune reaction to oligodendrocytes and degeneration of the axons they ensheath in the CNS. Because the damage to oligodendrocytes and axons involves local inflammation and associated oxidative stress, we tested the therapeutic efficacy of combined treatment with a potent anti-inflammatory thalidomide analog (lenalidomide) and novel synthetic anti-oxidant cerium oxide nanoparticles (nanoceria) in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. METHODS: C57BL/6 mice were randomly assigned to a control (no EAE) group, or one of the four myelin oligodendrocyte glycoprotein-induced EAE groups: vehicle, lenalidomide, nanoceria, or lenalidomide plus nanoceria. During a 23 day period, clinical EAE symptoms were evaluated daily, and MRI brain scans were performed at 11-13 days and 20-22 days. Histological and biochemical analyses of brain tissue samples were performed to quantify myelin loss and local inflammation. RESULTS: Lenalidomide treatment alone delayed symptom onset, while nanoceria treatment had no effect on symptom onset or severity, but did promote recovery; lenalidomide and nanoceria each significantly attenuated white matter pathology and associated inflammation. Combined treatment with lenalidomide and nanoceria resulted in a near elimination of EAE symptoms, and reduced white matter pathology and inflammatory cell responses to a much greater extent than either treatment alone. INTERPRETATION: By suppressing inflammation and oxidative stress, combined treatment with lenalidomide and nanoceria can reduce demyelination and associated neurological symptoms in EAE mice. Our preclinical data suggest a potential application of this combination therapy in MS.

Telomere shortening in neurological disorders: an abundance of unanswered questions.

Telomeres, ribonucleoprotein complexes that cap eukaryotic chromosomes, typically shorten in leukocytes with aging. Aging is a primary risk factor for neurodegenerative disease (ND), and a common assumption has arisen that leukocyte telomere length (LTL) can serve as a predictor of neurological disease. However, the evidence for shorter LTL in Alzheimer's and Parkinson's patients is inconsistent. The diverse causes of telomere shortening may explain variability in LTL between studies and individuals. Additional research is needed to determine whether neuronal and glial telomeres shorten during aging and in neurodegenerative disorders, if and how LTL is related to brain cell telomere shortening, and whether telomere shortening plays a causal role in or exacerbates neurological disorders.

HuD regulates coding and noncoding RNA to induce APP→Aß processing.

The primarily neuronal RNA-binding protein HuD is implicated in learning and memory. Here, we report the identification of several HuD target transcripts linked to Alzheimer's disease (AD) pathogenesis. HuD interacted with the 3' UTRs of APP mRNA (encoding amyloid precursor protein) and BACE1 mRNA (encoding ß-site APP-cleaving enzyme 1) and increased the half-lives of these mRNAs. HuD also associated with and stabilized the long noncoding (lnc)RNA BACE1AS, which partly complements BACE1 mRNA and enhances BACE1 expression. Consistent with HuD promoting production of APP and APP-cleaving enzyme, the levels of APP, BACE1, BACE1AS, and Aß were higher in the brain of HuD-overexpressing mice. Importantly, cortex (superior temporal gyrus) from patients with AD displayed significantly higher levels of HuD and, accordingly, elevated APP, BACE1, BACE1AS, and Aß than did cortical tissue from healthy age-matched individuals. We propose that HuD jointly promotes the production of APP and the cleavage of its amyloidogenic fragment, Aß.

Molecular control of the amount, subcellular location, and activity state of translation elongation factor 2 in neurons experiencing stress.

Eukaryotic elongation factor 2 (eEF-2) is an important regulator of the protein translation machinery whereby it controls the movement of the ribosome along the mRNA. The activity of eEF-2 is regulated by changes in cellular energy status and nutrient availability and by posttranslational modifications such as phosphorylation and mono-ADP-ribosylation. However, the mechanisms regulating protein translation under conditions of cellular stress in neurons are unknown. Here we show that when rat hippocampal neurons experience oxidative stress (lipid peroxidation induced by exposure to cumene hydroperoxide; CH), eEF-2 is hyperphosphorylated and ribosylated, resulting in reduced translational activity. The degradation of eEF-2 requires calpain proteolytic activity and is accompanied by accumulation of eEF-2 in the nuclear compartment. The subcellular localization of both native and phosphorylated forms of eEF-2 is influenced by CRM1 and 14.3.3, respectively. In hippocampal neurons p53 interacts with nonphosphorylated (active) eEF-2, but not with its phosphorylated form. The p53-eEF-2 complexes are present in cytoplasm and nucleus, and their abundance increases when neurons experience oxidative stress. The nuclear localization of active eEF-2 depends upon its interaction with p53, as cells lacking p53 contain less active eEF-2 in the nuclear compartment. Overexpression of eEF-2 in hippocampal neurons results in increased nuclear levels of eEF-2 and decreased cell death after exposure to CH. Our results reveal novel molecular mechanisms controlling the differential subcellular localization and activity state of eEF-2 that may influence the survival status of neurons during periods of elevated oxidative stress.

Dietary energy intake modifies brainstem autonomic dysfunction caused by mutant α-synuclein.

Parkinson's disease (PD) patients often exhibit impaired regulation of heart rate by the autonomic nervous system (ANS) that may precede motor symptoms in many cases. Results of autopsy studies suggest that brainstem pathology, including the accumulation of α-synuclein, precedes damage to dopaminergic neurons in the substantia nigra in PD. However, the molecular and cellular mechanisms responsible for the early dysfunction of brainstem autonomic neurons are unknown. Here we report that mice expressing a mutant form of α-synuclein that causes familial PD exhibit aberrant autonomic control of the heart characterized by elevated resting heart rate and an impaired cardiovascular stress response, associated with reduced parasympathetic activity and accumulation of α-synuclein in the brainstem. These ANS abnormalities occur early in the disease process. Adverse effects of α-synuclein on the control of heart rate are exacerbated by a high energy diet and ameliorated by intermittent energy restriction. Our findings establish a mouse model of early dysregulation of brainstem control of the cardiovascular system in PD, and further suggest the potential for energy restriction to attenuate ANS dysfunction, particularly in overweight individuals.