Biology subjects

Bloom, G. S.

Publications and source records attributed to Bloom, G. S..

9 recordsLinked to original sources

bioRxiv2025

The use of Benzonase to produce ribosome footprints simplifies translational levels quantification by Ribo-seq

Gene expression quantification through genomics methods is crucial for understanding diverse biological contexts. Among these methods, ribosome profiling (Ribo-seq) stands out as a valuable tool for uncovering post-transcriptional gene expression regulation by providing a comprehensive view of the translatome. While current protocols are time-intensive with limited variations, we introduced the use of the Benzonase enzyme to generate ribosome footprints from a polysome-enriched fraction that exhibit expected characteristics in size, transcriptome mapping, and periodicity. Comparing translatome from Benzonase- and RNAse I-derived footprints reveals minimal differences underscoring Benzonases potential to streamline the protocol, reducing time, cost and bias. We further demonstrate Ribo-seq application in primary neuronal cultures, using Benzonase to digest the post-mitochondrial supernatant, thereby bypassing the labor-intensive ribosome/polysome purification step. The introduction of such protocol variations for Ribo-seq, especially for challenging low-input samples, offers a significant advancement of this resource for the community. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/642103v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@b5e325org.highwire.dtl.DTLVardef@153ccf9org.highwire.dtl.DTLVardef@b5f123org.highwire.dtl.DTLVardef@ea6bab_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Mitochondrial NADK2-dependent NADPH controls Tau oligomer uptake in human neurons

Alterations in NADH and NADPH metabolism are associated with aging, cancer, and Alzheimers Disease. Using 2P-FLIM imaging of the mitochondrial NAD(P)H in live human neurons and PS19 mouse brains, we show that tau oligomers (TauO) upregulate the mitochondrial de novo NADPH synthesis through NADK2. This process controls LRP1-mediated internalization of TauO, setting a vicious cycle for further TauO internalization. Thus, mitochondrial NADK2-dependent NADPH controls a key step in TauO toxicity.

neuroscience↗

bioRxiv2024

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APPSAA knock-in mouse model of Alzheimer disease

IntroductionReduced brain energy metabolism, mTOR dysregulation, and extracellular amyloid-{beta} oligomer (xcA{beta}O) buildup characterize AD; how they collectively promote neurodegeneration is poorly understood. We previously reported that xcA{beta}Os inhibit Nutrient-induced Mitochondrial Activity (NiMA) in cultured neurons. We now report NiMA disruption in vivo. MethodsBrain energy metabolism and oxygen consumption were recorded in APPSAA/+ mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. ResultsNiMA is inhibited in APPSAA/+ mice before other defects are detected in these amyloid-{beta}-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. GSK3{beta} signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3{beta} with lithium or TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APPSAA mice. ConclusionNiMA disruption in vivo occurs before histopathological changes and cognitive decline in APPSAA mice, and may represent an early stage in human AD.

neuroscience↗

bioRxiv2023

Structural and functional damage to neuronal nuclei caused by extracellular tau oligomers

INTRODUCTIONNeuronal nuclei are normally smoothly surfaced. In Alzheimers disease (AD) and other tauopathies, though, they often develop invaginations. We investigated mechanisms and functional consequences of neuronal nuclear invagination in tauopathies. METHODSNuclear invagination was assayed by immunofluorescence in brain, and in cultured neurons before and after extracellular tau oligomers (xcTauO) exposure. Nucleocytoplasmic transport was assayed in cultured neurons. Gene expression was investigated using nanoString nCounter technology and qRT-PCR. RESULTSInvaginated nuclei were twice as abundant in human AD as in cognitively normal adults, and were increased in mouse neurodegeneration models. In cultured neurons, nuclear invagination was induced by xcTauOs by an intracellular tau-dependent mechanism. xcTauOs impaired nucleocytoplasmic transport, increased histone H3 trimethylation at lysine 9 and altered gene expression, especially by increasing tau mRNA. DISCUSSIONxcTauOs may be a primary cause of nuclear invagination in vivo, and by extension, impair nucleocytoplasmic transport and induce pathogenic gene expression changes.

neuroscience↗

bioRxiv2022

Localization, induction, and cellular effects of tau-phospho-threonine 217

IntroductionTau phosphorylation at T217 is a promising AD biomarker, but its functional consequences were unknown. MethodsHuman brain and cultured mouse neurons were analyzed by immunoblotting and immunofluorescence for total tau, taupT217, taupT181, taupT231 and taupS396/pS404. dSTORM super resolution microscopy was used to localize taupT217 in cultured neurons. EGFP-tau was expressed in fibroblasts as wild type and T217E pseudo-phosphorylated tau, and fluorescence recovery after photobleaching (FRAP) reported tau turnover rates on microtubules. ResultsIn brain, taupT217 appears in neurons at Braak stages I-II, becomes more prevalent later and co-localizes partially with other phospho-tau epitopes. In cultured neurons taupT217, is increased by extracellular tau oligomers (xcTauOs), and is associated with developing post-synaptic sites. FRAP recovery was fastest for EGFP-tauT217E. ConclusionTaupT217 increases in brain as AD progresses and is induced by xcTauOs. Post-synaptic taupT217 suggests a role for T217 phosphorylation in synapse impairment. T217 phosphorylation reduces taus affinity for microtubules.

neuroscience↗

bioRxiv2021

Transcriptional and translational regulation of pathogenesis in Alzheimer's disease model mice

BackgroundDefining the cellular mechanisms that drive Alzheimers disease (AD) pathogenesis and progression will be aided by studies defining how gene expression patterns change during pre-symptomatic AD and the ensuing periods of steadily declining cognition. Previous studies have emphasized changes in transcriptional regulation, but not translational regulation, leaving the ultimate results of gene expression alterations relatively unexplored in the context of AD. ObjectiveTo identify genes whose expression might be regulated at the transcriptional, and especially at the translational levels in AD, we analyzed gene expression in cerebral cortex of two AD model mouse strains, CVN (APPSwDI;NOS2-/-) and Tg2576 (APPSw), and their companion wild type (WT) strains at 6 months of age by tandem RNA-Seq and Ribo-Seq (ribosome profiling). MethodsIdentical starting pools of bulk RNA were used for RNA-Seq and Ribo-Seq. Differential gene expression analysis was performed at the transcriptional and translational levels separately, and also at the translational efficiency level. Regulated genes were functionally evaluated by gene ontology tools. ResultsCompared to WT mice, AD model mice had similar levels of transcriptional regulation, but displayed differences in translational regulation. A specific microglial signature associated with early stages of A{beta} accumulation was up-regulated at both transcriptome and translatome levels in CVN mice. Although the two mice strains did not share many regulated genes, they showed common regulated pathways related to APP metabolism associated with neurotoxicity and neuroprotection. ConclusionThis work represents the first genome-wide study of brain translational regulation in animal models of AD, and provides evidence of a tight and early translational regulation of gene expression controlling the balance between neuroprotective and neurodegenerative processes in brain.

neuroscience↗

bioRxiv2021

Extracellular cues accelerate neurogenesis of induced pluripotent stem cell derived neurons

Neurogenesis is a complex process encompassing neuronal progenitor cell expansion/proliferation and differentiation, followed by neuron maturation. In vivo models are most commonly used to study neurogenesis; however, human induced pluripotent stem cell-derived (iPSC) neurons are increasingly used to establish cellular models of human neurological processes. Unfortunately, the differentiation and maturation of iPSC-derived neurons varies in methodology, is asynchronous, and has restricted experimental utility because of extended differentiation/maturation times. To accelerate and standardize iPS neuronal maturation, we differentiated and matured feeder layer-free iPSC-derived neuronal cultures under physiological oxygen levels (5%), and modified the underlying extracellular matrix and medium composition. Our results demonstrate that calretinin gene expression occurred earlier under our optimized iPS conditions and the corresponding "neurogenesis burst" associated with proliferative expansion occurred more synchronously, reliably emerging two and three weeks after differentiation. As expected, the expression of mature neuronal markers (i.e., NeuN+/Calbindin+) started at 4-weeks post-differentiation. qPCR microarray, western blot and single cell analyses using high content imaging indicated that 4-week iPS neuronal cultures were non-cycling with decreased expression of cyclin D1 and Ki67. Our data demonstrate that extracellular cues influence the kinetics of neurogenesis models and that feeder layer-free iPSC-derived neurogenesis can be reproducibly miniaturized.

neuroscience↗

bioRxiv2021

SOD1 Mediates Lysosome-to-Mitochondria Communication and its Dysregulation by Amyloid-β Oligomers

Altered mitochondrial DNA (mtDNA) occurs in neurodegenerative disorders like Alzheimers disease (AD); how mtDNA synthesis is linked to neurodegeneration is poorly understood. We discovered Nutrient-induced Mitochondrial Activity (NiMA), an inter-organelle signaling pathway where nutrient-stimulated lysosomal mTORC1 activity regulates mtDNA replication in neurons by a mechanism sensitive to amyloid-{beta} oligomers (A{beta}Os), a primary factor in AD pathogenesis. Using 5-ethynyl-2-deoxyuridine (EdU) incorporation into mtDNA of cultured neurons, along with photoacoustic and mitochondrial metabolic imaging of cultured neurons and mouse brains, we show these effects being mediated by mTORC1-catalyzed T40 phosphorylation of superoxide dismutase 1 (SOD1). Mechanistically, tau, another key factor in AD pathogenesis and other tauopathies, reduced the lysosomal content of the tuberous sclerosis complex (TSC), thereby increasing NiMA and suppressing SOD1 activity and mtDNA synthesis. A{beta}Os inhibited these actions. Dysregulation of mtDNA synthesis was observed in fibroblasts derived from TS patients, who lack functional TSC and elevated SOD1 activity was also observed in human AD brain. Together, these findings imply that tau and SOD1 couple nutrient availability to mtDNA replication, linking mitochondrial dysfunction to AD.

neuroscience↗

bioRxiv2020

Human Tau Isoform Aggregation and Selective Detection of Misfolded Tau from Post-Mortem Alzheimer's Disease Brains

Tau aggregates are present in a large number of neurodegenerative diseases known as "tauopathies", including Alzheimers disease (AD). As there are six human tau isoforms in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions, we tested whether tau isoforms behave differently in aggregation. We discovered that all six tau isoforms are capable of forming PHF-tau like filaments and the 3R tau isoforms aggregate significantly faster than their 4R counterparts. We further mapped key segments of tau isoforms that contribute to their aggregation kinetics, where it was determined that microtubule binding domains R2 and R3 were the major contributors to tau aggregation. To evaluate the feasibility of using the six recombinant tau isoforms as substrates to amplify misfolded tau, we demonstrated that full-length human tau isoforms can seed and detect misfolded tau from the post-mortem AD brain tissues with high specificity by an ultrasensitive technology termed real-time quaking-induced conversion (RT-QuIC). Mass spectrometric analysis of PHF-tau samples extracted from AD brains identified peptides corresponding to all major forms of human brain tau isoforms along with a consensus hyperphosphorylated peptide near the C-terminus. Together, our findings not only reveal new aggregation kinetic properties of human tau isoforms, support the development of methods to quantitatively measure misfolded human tau isoforms in AD brains, but also uncover the capability of full-length human tau isoforms as substrates for "prion-like" tau seeding by RT-QuIC assays that may be used for new biomarker development for AD and other tauopathy diagnosis.

pathology↗