Biology subjects

Talbot, K.

Publications and source records attributed to Talbot, K..

5 recordsLinked to original sources

bioRxiv2025

Targeted BDNF upregulation via upstream open reading frame disruption

To understand the relative contributions of 5' UTR elements to translation, we performed a comprehensive analysis of upstream open reading frames (uORFs) across a representative 5' UTR. We selected the neurotrophin BDNF (Brain derived neurotrophic factor) as an exemplar as upregulation of this protein is a potential therapeutic approach for a plethora of neurodevelopmental, neurodegenerative, and neuropsychiatric disorder indications. Predicted uORFs were identified in 14 out of 17 BDNF RefSeq transcript isoforms, and experimentally confirmed to be exerting translation repression effects for five of these transcripts. These findings suggest that uORF elements play an important role in shaping the protein output from this locus. We explored several approaches to disrupt BDNF uORF function. Deletion of a 5' UTR exon in BDNF v11 (containing eight predicted uORFs), in order to simulate an exon skipping outcome, resulted in pronounced upregulation in a reporter construct system. This effect was found to be partially uORF-dependent, but was also dependent on the disruption of an RNA secondary structure element. However, this transcript variant was found to not be expressed in human brain. Conversely, direct disruption of a single uORF start codon in the widely expressed BDNF v4 transcript variant using an adenine base editing approach resulted in a [~]1.8-fold upregulation of endogenous BDNF protein expression in cell culture. This study describes novel BDNF regulatory mechanisms, and potential uORF-targeted modalities for therapeutic gene activation.

molecular biology↗

bioRxiv2023

The ALS-associated TDP-43M337V mutation dysregulates microglia-derived extracellular microRNAs in a sex-specific manner

Evidence suggests the presence of microglial activation and microRNA (miRNA) dysregulation in amyotrophic lateral sclerosis (ALS), the most common form of adult motor neuron disease. However, few studies have investigated whether the miRNA dysregulation may originate from microglia. Furthermore, TDP-43, involved in miRNA biogenesis, aggregates in tissues of [~]98% of ALS cases. Thus, this study aimed to determine whether expression of the ALS-linked TDP-43M337V mutation in a transgenic mouse model dysregulates microglia-derived miRNAs. RNA sequencing identified several dysregulated miRNAs released by transgenic microglia, and a differential miRNA release by lipopolysaccharide-stimulated microglia, which was more pronounced in cells from female mice. We validated the downregulation of two candidate miRNAs, miR-16-5p and miR-99a-5p by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and identified their predicted targets, which include primarily genes involved in neuronal development and function. These results suggest that altered TDP-43 function leads to changes in the miRNA population released by microglia in a sex dependent manner, which may in turn influence disease progression in ALS. This has important implications for the role of neuroinflammation in ALS pathology and could provide potential therapeutic targets.

neuroscience↗

bioRxiv2021

Modelling seeding and neuroanatomic spread of pathology in amyotrophic lateral sclerosis

The neurodegenerative disorder amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of upper and lower motor neurons, with pathological involvement of cerebral motor and, additionally, extra-motor areas, in a clinicopathological spectrum with frontotemporal dementia (FTD). A key unresolved question is whether the distribution of pathology in ALS is driven by molecular factors such as regional gene expression, by differential network vulnerability, or is a combination of both. A system of histopathological staging of ALS based on the regional burden of TDP-43 pathology observed in post mortem brains has been supported to some extent by analysis of distribution of in vivo structural MRI changes. In this paper, computational modelling using a Network Diffusion Model (NDM) was used to investigate whether a process of focal pathological seeding followed by structural network-based spread recapitulated post mortem histopathological staging and, secondly, whether this had any relationship to the pattern of expression of a panel of genes implicated in ALS across the healthy brain. Regionally parcellated T1-weighted MRI data from ALS patients (baseline n=79) was studied in relation to a healthy control structural connectome and a database of associated regional cerebral gene expression. The NDM provided strong support for a structural network-based basis for regional pathological spread in ALS, but no simple relationship to the spatial distribution of ALS-related genes in the healthy brain. Intriguingly, the critical seed regions for spread within the model were not within the primary motor cortex but basal ganglia, thalamus and insula, where NDM recapitulated aspects of the post mortem histopathological staging system. Within the ALS-FTD clinicopathological spectrum, non-primary motor structures may be among the earliest sites of cerebral pathology.

neuroscience↗

bioRxiv2019

Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair

The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 and non-homologous end-joining (NHEJ) by deleting the repeat region with the risk of creating indels and genomic instability. In this study we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/FTD patient using CRISPR/Cas9 genome editing and homology directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines, and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared to its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the KEGG ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9 mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

neuroscience↗

bioRxiv2018

ALS mice carrying pathological mutant TDP-43, but not mutant FUS, display axonal transport defects in vivo

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in the basic neuronal process of axonal transport have been identified in several ALS models. However, in vivo evidence of early/pre-symptomatic deficiencies in neuronal cargo trafficking remains limited, thus the pathogenic importance of axonal transport to the ALS disease spectrum remains to be fully resolved. We therefore analysed the in vivo dynamics of retrogradely transported, neurotrophin-containing signalling endosomes in motor neuron axons of two new mouse models of ALS that have mutations in different RNA processing genes (Tardbp and Fus). TDP-43M337V mice, which show neuromuscular pathology but no overt motor neuron loss, displayed in vivo perturbations in axonal transport that manifested between 1.5 and 3 months and preceded motor symptom onset. In contrast, signalling endosome transport remained largely unaffected in mutant Fus{Delta}14/+ mice, despite 20% motor neuron loss. These findings indicate that deficiencies in retrograde neurotrophin signalling and axonal transport are not common to all ALS-linked genes, and that there are inherent and mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.

neuroscience↗