Transcriptome analysis indicates dominant effects on ribosome and mitochondrial function of a premature termination codon mutation in the zebrafish gene psen2

Published: July 13, 2020PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.Haowei Jiang, Stephen Martin Pederson, Morgan Newman, Yang Dong, Karissa Barthelson, Michael Lardell

Semiconductive and Photoconductive Properties of the Single Molecule Magnets Mn12_{12}-Acetate and Fe8_8Br8_8

Resistivity measurements are reported for single crystals of Mn$_{12}$-Acetate and Fe$_8$Br$_8$. Both materials exhibit a semiconductor-like, thermally activated behavior over the 200-300 K range. The activation energy, $E_a$, obtained for Mn$_{12}$-Acetate was 0.37 $\pm$ 0.05 eV, which is to be contrasted with the value of 0.55 eV deduced from the earlier reported absorption edge measurements and the range of 0.3-1 eV from intramolecular density of states calculations, assuming $2E_a$= $E_g$, the optical band gap. For Fe$_8$Br$_8$, $E_a$ was measured as 0.73 $\pm$ 0.1 eV, and is discussed in light of the available approximate band structure calculations. Some plausible pathways are indicated based on the crystal structures of both lattices. For Mn$_{12}$-Acetate, we also measured photoconductivity in the visible range; the conductivity increased by a factor of about eight on increasing the photon energy from 632.8 nm (red) to 488 nm (blue). X-ray irradiation increased the resistivity, but $E_a$ was insensitive to exposure.Comment: 7 pages, 8 figure

Effects of aging on the motor, cognitive and affective behaviors, neuroimmune responses and hippocampal gene expression

The known effects of aging on the brain and behavior include impaired cognition, increases in anxiety and depressive-like behaviors, and reduced locomotor activity. Environmental exposures and interventions also influence brain functions during aging. We investigated the effects of normal aging under controlled environmental conditions and in the absence of external interventions on locomotor activity, cognition, anxiety and depressive-like behaviors, immune function and hippocampal gene expression in C57BL/6 mice. Healthy mice at 4, 9, and 14 months of age underwent behavioral testing using an established behavioral battery, followed by cellular and molecular analysis using flow cytometry, immunohistochemistry, and quantitative PCR. We found that 14-month-old mice showed significantly reduced baseline locomotion, increased anxiety, and impaired spatial memory compared to younger counterparts. However, no significant differences were observed for depressive-like behavior in the forced-swim test. Microglia numbers in the dentate gyrus, as well as CD8+ memory T cells increased towards late middle age. Aging processes exerted a significant effect on the expression of 43 genes of interest in the hippocampus. We conclude that aging is associated with specific changes in locomotor activity, cognition, anxiety-like behaviors, neuroimmune responses and hippocampal gene expression.Gaurav Singhal, Julie Morgan, Magdalene C.Jawahar, Frances Corrigan, Emily J.Jaehn

Collaborative International Research in Clinical and Longitudinal Experience Study in NMOSD

OBJECTIVE: To develop a resource of systematically collected, longitudinal clinical data and biospecimens for assisting in the investigation into neuromyelitis optica spectrum disorder (NMOSD) epidemiology, pathogenesis, and treatment. METHODS: To illustrate its research-enabling purpose, epidemiologic patterns and disease phenotypes were assessed among enrolled subjects, including age at disease onset, annualized relapse rate (ARR), and time between the first and second attacks. RESULTS: As of December 2017, the Collaborative International Research in Clinical and Longitudinal Experience Study (CIRCLES) had enrolled more than 1,000 participants, of whom 77.5% of the NMOSD cases and 71.7% of the controls continue in active follow-up. Consanguineous relatives of patients with NMOSD represented 43.6% of the control cohort. Of the 599 active cases with complete data, 84% were female, and 76% were anti-AQP4 seropositive. The majority were white/Caucasian (52.6%), whereas blacks/African Americans accounted for 23.5%, Hispanics/Latinos 12.4%, and Asians accounted for 9.0%. The median age at disease onset was 38.4 years, with a median ARR of 0.5. Seropositive cases were older at disease onset, more likely to be black/African American or Hispanic/Latino, and more likely to be female. CONCLUSION: Collectively, the CIRCLES experience to date demonstrates this study to be a useful and readily accessible resource to facilitate accelerating solutions for patients with NMOSD

Meta-analysis of exome array data identifies six novel genetic loci for lung function

Background: Over 90 regions of the genome have been associated with lung function to date, many of which have also been implicated in chronic obstructive pulmonary disease. Methods: We carried out meta-analyses of exome array data and three lung function measures: forced expiratory volume in one second (FEV1), forced vital capacity (FVC) and the ratio of FEV1 to FVC (FEV1/FVC). These analyses by the SpiroMeta and CHARGE consortia included 60,749 individuals of European ancestry from 23 studies, and 7,721 individuals of African Ancestry from 5 studies in the discovery stage, with follow-up in up to 111,556 independent individuals. Results: We identified significant (P<2·8x10-7) associations with six SNPs: a nonsynonymous variant in RPAP1, which is predicted to be damaging, three intronic SNPs (SEC24C, CASC17 and UQCC1) and two intergenic SNPs near to LY86 and FGF10. Expression quantitative trait loci analyses found evidence for regulation of gene expression at three signals and implicated several genes, including TYRO3 and PLAU. Conclusions: Further interrogation of these loci could provide greater understanding of the determinants of lung function and pulmonary disease

Brain transcriptome analysis of a protein-truncating mutation in sortilin-related receptor 1 associated with early-onset familial alzheimer's disease indicates early effects on mitochondrial and ribosome function

BACKGROUND: The early cellular stresses leading to Alzheimer's disease (AD) remain poorly understood because we cannot access living, asymptomatic human AD brains for detailed molecular analyses. Sortilin-related receptor 1 (SORL1) encodes a multi-domain receptor protein genetically associated with both rare, early-onset familial AD (EOfAD) and common, sporadic, late-onset AD (LOAD). SORL1 protein has been shown to act in the trafficking of the amyloid β A4 precursor protein (AβPP) that is proteolysed to form one of the pathological hallmarks of AD, amyloid-β (Aβ) peptide. However, other functions of SORL1 in AD are less well understood. OBJECTIVE: To investigate the effects of heterozygosity for an EOfAD-like mutation in SORL1 on the brain transcriptome of young-adult mutation carriers using zebrafish as a model organism. METHODS: We performed targeted mutagenesis to generate an EOfAD-like mutation in the zebrafish orthologue of SORL1 and performed RNA-sequencing on mRNA isolated from the young adult brains of siblings in a family of fish either wild type (non-mutant) or heterozygous for the EOfAD-like mutation. RESULTS: We identified subtle differences in gene expression indicating changes in mitochondrial and ribosomal function in the mutant fish. These changes appear to be independent of changes in mitochondrial content or the expression of AβPP-related proteins in zebrafish. CONCLUSION: These findings provided evidence supporting that EOfAD mutations in SORL1 affect mitochondrial and ribosomal function and provide the basis for future investigation elucidating the nature of these effects.Karissa Barthelson, Stephen Martin Pederson, Morgana Newman, Michael Lardell

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer’s disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis

Background: Early-onset familial Alzheimer’s disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer’s disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1(Q96_K97del), in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. Results: We generated clutches of wild type and heterozygous psen1(Q96_K97del) 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. Conclusions: Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1(Q96_K97del) brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3′ untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1(Q96_K97del) EOfAD-like mutant brains.Yang Dong, Morgan Newman, Stephen M. Pederson, Karissa Barthelson, Nhi Hin and Michael Lardell

Genome-wide CRISPR screen identifies RACK1 as a critical host factor for flavivirus replication

ABSTRACT Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1’s crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.Byron Shue, Abhilash I. Chiramel, Berati Cerikan, Thu-Hien To, Sonja Frölich, Stephen M. Pederson, Emily N. Kirby, Nicholas S. Eyre, Ralf F. W. Bartenschlager, Sonja M. Best, and Michael R. Bear

3-dimensional analysis of vascular structure, function & receptor distribution using confocal laser scanning microscopy

No abstract available

Short-term environmental enrichment, and not physical exercise, alleviate cognitive decline and anxiety from middle age onwards without affecting hippocampal gene expression

Physical exercise (PE) and environmental enrichment (EE) have consistently been shown to modulate behavior and neurobiological mechanisms. The current literature lacks evidence to confirm the relationship between PE and EE, if any, and whether short-term treatment with PE, EE, or PE+EE could be considered to correct age-related behavioral deficits. Three-, 8-, and 13-month-old C57BL/6 mice were assigned to either PE, EE, or PE+EE treatment groups (n = 12-16/group) for 4 weeks before behavioral testing and were compared to controls. Differential effects of the treatments on various behaviors and hippocampal gene expression were measured using an established behavioral battery and high-throughput qPCR respectively. Short-term EE enhanced locomotor activity at 9 and 14 months of age, whereas the combination of PE and EE reduced locomotor activity in the home cage at 14 months. Short-term EE also was found to reverse the age-related increase in anxiety at 9 months and spatial memory deficits at 14 months of age. Conversely, short-term PE induced spatial learning impairment and depressive-like behavior at four months but showed no effects in 9- and 14-month-old mice. PE and PE+EE, but not EE, modified the expression of several hippocampal genes at 9 months of age compared with control mice. In conclusion, short-term EE may help to alleviate age-related cognitive decline and increase in anxiety, without altering hippocampal gene expression. On the contrary, PE is detrimental at a young age for both affective-like behaviors and spatial learning and memory but showed no effects at middle and late middle age despite hippocampal gene expression alterations.Gaurav Singhal, Julie Morgan, Magdalene C. Jawahar, Frances Corrigan, Emily J. Jaehne, Catherine Toben, James Breen, Stephen M. Pederson, Anthony J. Hannan, Bernhard T. Baun