Biology subjects

Cammack, A. J.

Publications and source records attributed to Cammack, A. J..

10 recordsLinked to original sources

bioRxiv2025

U7 small nuclear RNA splice-switching therapeutics for STMN2 and UNC13A in Amyotrophic Lateral Sclerosis

TDP-43 nuclear depletion in amyotrophic lateral sclerosis (ALS) causes de-repression of cryptic exons (CEs) in multiple transcripts, including UNC13A and STMN2, disrupting synaptic transmission and neurite outgrowth. We developed a therapeutic U7 snRNA (tU7) approach that suppresses TDP-43-dependent mis-splicing, restores target gene expression, rescues neuronal functions in human iPSC-derived neurons, and shows target engagement in vivo, positioning tU7-mediated splicing correction as a promising therapeutic strategy for ALS.

neuroscience↗

bioRxiv2025

CRISPR/Cas9-mediated generation of a de novo C9ORF72 knock-in isogenic iPSC cell bank to model ALS and FTD

BackgroundThe pathogenic G4C2 repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies focused on delineating the underlying perturbed mechanisms resulting from this genetic mutation are often confounded by the heterogeneity present in current disease models, such as patient-derived iPSC lines, with estimations of up to 50% of the variation in iPSC cell phenotypes resulting from inter-individual differences. Isogenic models, in which the pathogenic mutation is introduced into a defined genetic background, offer a powerful approach to isolating mutation-specific effects and enable high-resolution comparison across distinct ALS/FTD-associated mutations. Such models are essential for uncovering convergent disease mechanisms and improving reproducibility in ALS/FTD research. MethodsA two-step scarless CRISPR/Cas9 genome editing strategy was used to generate isogenic human iPSC lines carrying a de novo knock-in of a disease-length G4C2 repeat expansion in the C9ORF72 locus. The resulting lines underwent thorough quality control and were differentiated into lower motor neurons and assessed for the presence of key ALS/FTD pathologies, including changes to C9ORF72 mRNA and protein expression, RNA foci and dipeptide repeat proteins. ResultsTwo C9ORF72 knock-in iPSC lines were generated with 631 and 600 G4C2 repeats, alongside an isogenic genome editing control line. The C9ORF72 G4C2 repeat expansion knock-in iPSC lines exhibit both loss-of-function and gain-of-function pathological features characteristic of ALS/FTD. Compared to the parental wild-type KOLF2.1J line and isogenic (wild-type) CRISPR control line, these exhibit a significant reduction in C9ORF72 mRNA and protein levels, the presence of RNA foci accumulation, and a marked increase in poly(GA) and poly(GP) dipeptide repeat protein levels in iPSCs and motor neurons. ConclusionsThis is one of the first reports of a successful knock-in of the pathogenic C9ORF72 G4C2 repeat expansion into a human iPSC line, establishing a genetically defined and physiologically relevant model of ALS/FTD. These isogenic lines recapitulate both key loss- and gain-of-function disease pathologies, providing a crucial complement to existing patient-derived iPSC banks. By eliminating confounding genetic background variability, these cell lines will enable more precise interrogation of C9ORF72-linked pathomechanisms and offer a robust platform for comparative studies across the ALS and FTD spectrum, mechanistic investigations, and future therapeutic targeting with enhanced translational relevance.

neuroscience↗

bioRxiv2025

Prior epigenetic status predicts future susceptibility to seizures in mice

Wide variation of responses to identical stimuli presented to genetically inbred mice suggests the hypothesis that stochastic epigenetic variation during neurodevelopment can mediate such phenotypic differences. However , this hypothesis is largely untested since capturing pre-existing molecular states requires non-destructive, longitudinal recording. Therefore, we tested the potential of Calling Cards (CC) to record transient neuronal enhancer activity during postnatal development, and thereby associate epigenetic variation with a subsequent phenotypic presentation - degree of seizure response to the pro-convulsant pentylenetetrazol. We show that recorded differences in epigenetics at 243 loci predict a severe vs. mild response, and that these are enriched near genes associated with human epilepsy. We also validated pharmacologically a seizure -modifying role for two novel genes, Htr1f and Let7c. This proof-of-principle supports using CC broadly to discover predisposition loci for other neuropsychiatric traits and behaviors. Finally, as, human disease is also influenced by non-inherited factors, similar epigenetic predispositions are possible in humans.

genomics↗

bioRxiv2025

Generation of C9orf72 repeat knock-in iPSC lines for modelling ALS and FTD

Induced pluripotent stem cell (iPSC) models are powerful tools for neurodegenerative disease modelling, as they allow mechanistic studies in a human genetic environment and they can be differentiated into a range of neuronal and non-neuronal cells. However, these models come with inherent challenges due to line-to-line and clonal variability. To combat this issue, the iPSC Neurodegenerative Disease Initiative (iNDI) has generated an iPSC repository using a single clonal reference line, KOLF2.1J, into which disease-causing mutations and revertants are introduced via gene editing. Here we describe the generation and validation of lines carrying the most common causative mutation for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a repeat expansion in the C9orf72 gene, for the iNDI collection of neurodegenerative iPSC models. We demonstrate that these C9orf72 knock-in lines differentiate efficiently into neurons and display characteristic C9orf72-associated pathologies, including reduced C9orf72 levels and the presence of dipeptide repeat proteins (DPRs) and RNA foci, which increase in abundance over time in culture. These pathologies are not present in revertant cells lacking the repeat expansion. These repeat expansion and revertant cell lines are now available to academic and for-profit institutions through the JAX iPS cell repository and will help to facilitate and standardise iPSC-based ALS/FTD research.

neuroscience↗

bioRxiv2024

The Neurolipid Atlas: a lipidomics resource for neurodegenerative diseases uncovers cholesterol as a regulator of astrocyte reactivity impaired by ApoE4

Lipid changes in the brain have been implicated in many neurodegenerative diseases including Alzheimers Disease (AD), Parkinsons disease and Amyotrophic Lateral Sclerosis. To facilitate comparative lipidomic research across brain-diseases we established a data commons named the Neurolipid Atlas, that we have pre-populated with novel human, mouse and isogenic induced pluripotent stem cell (iPSC)-derived lipidomics data for different brain diseases. We show that iPSC-derived neurons, microglia and astrocytes display distinct lipid profiles that recapitulate in vivo lipotypes. Leveraging multiple datasets, we show that the AD risk gene ApoE4 drives cholesterol ester (CE) accumulation in human astrocytes recapitulating CE accumulation measured in the human AD brain. Multi-omic interrogation of iPSC-derived astrocytes revealed that cholesterol plays a major role in astrocyte interferon-dependent pathways such as the immunoproteasome and major histocompatibility complex (MHC) class I antigen presentation. We show that through enhanced cholesterol esterification ApoE4 suppresses immune activation of astrocytes. Our novel data commons, available at neurolipidatlas.com, provides a user-friendly tool and knowledge base for a better understanding of lipid dyshomeostasis in neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

The most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion in intron 1 of the C9orf72 gene. This repeat expansion undergoes bidirectional transcription to produce sense and antisense repeat RNA species. Both sense and antisense-derived repeat RNAs undergo repeat-associated non-AUG translation in all reading frames to generate five distinct dipeptide repeat proteins (DPRs). Importantly, toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs. This suggests targeting both sense and antisense repeat RNA may provide the most effective therapeutic strategy. The RNA-targeting CRISPR-Cas13 systems offer a promising avenue for simultaneous targeting of multiple RNA transcripts, as they mature their own guide arrays, thus allowing targeting of more than one RNA species from a single construct. We show that CRISPR-Cas13d originating from Ruminococcus flavefaciens (CasRx) can successfully reduce C9orf72 sense and antisense repeat transcripts and DPRs to background levels in HEK cells overexpressing C9orf72 repeats. CRISPR-CasRx also markedly reduced the endogenous sense and antisense repeat RNAs and DPRs in three independent C9orf72 patient-derived iPSC-neuron lines, without detectable off-target effects. To determine whether CRISPR-CasRx is effective in vivo, we treated two distinct C9orf72 repeat mouse models using AAV delivery and observed a significant reduction in both sense and antisense repeat-containing transcripts. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

neuroscience↗

bioRxiv2024

Neuronal polyunsaturated fatty acids are protective in FTD/ALS

We report a conserved transcriptomic signature of reduced fatty acid and lipid metabolism gene expression in human post-mortem ALS spinal cord and a Drosophila model of the most common genetic cause of FTD/ALS, a repeat expansion in C9orf72. To investigate lipid alterations, we performed lipidomics on C9FTD/ALS iPSC-neurons and post-mortem FTLD brain tissue. This revealed a common and specific reduction in phospholipid species containing polyunsaturated fatty acids (PUFAs). To determine whether this PUFA deficit contributes to neurodegeneration, we fed C9FTD/ALS flies PUFAs, which yielded a modest increase in survival. However, increasing PUFA levels specifically in neurons of the C9orf72 flies, by overexpressing fatty acid desaturase enzymes, led to a substantial extension of lifespan. Neuronal overexpression of fatty acid desaturases also suppressed stressor induced neuronal death in C9FTD/ALS patient iPSC-neurons. These data implicate neuronal fatty acid saturation in the pathogenesis of FTD/ALS and suggest that interventions to increase PUFA levels specifically within neurons will be beneficial.

neuroscience↗

bioRxiv2023

Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. Basic Protocol 1: Preparation and delivery of Calling Cards reagents Basic Protocol 2: Sample preparation Basic Protocol 3: Sequencing library preparation Basic Protocol 4: Library pooling and sequencing Basic Protocol 5: Data analysis

genomics↗

bioRxiv2019

Transposon-mediated, cell type-specific transcription factor recording in the mouse brain

Transcription factors (TFs) play a central role in the regulation of gene expression, controlling everything from cell fate decisions to activity dependent gene expression. However, widely-used methods for TF profiling in vivo (e.g. ChIP-seq) yield only an aggregated picture of TF binding across all cell types present within the harvested tissue; thus, it is challenging or impossible to determine how the same TF might bind different portions of the genome in different cell types, or even to identify its binding events at all in rare cell types in a complex tissue such as the brain. Here we present a versatile methodology, FLEX Calling Cards, for the mapping of TF occupancy in specific cell types from heterogenous tissues. In this method, the TF of interest is fused to a hyperactive piggyBac transposase (hypPB), and this bipartite gene is delivered, along with donor transposons, to mouse tissue via a Cre-dependent adeno-associated virus (AAV). The fusion protein is expressed in Cre-expressing cells where it inserts transposon "Calling Cards" near to TF binding sites. These transposons permanently mark TF binding events and can be mapped using high-throughput sequencing. Alternatively, unfused hypPB interacts with and records the binding of the super enhancer (SE)-associated bromodomain protein, Brd4. To demonstrate the FLEX Calling Card method, we first show that donor transposon and transposase constructs can be efficiently delivered to the postnatal day 1 (P1) mouse brain with AAV and that insertion profiles report TF occupancy. Then, using a Cre-dependent hypPB virus, we show utility of this tool in defining cell type-specific TF profiles in multiple cell types of the brain. This approach will enable important cell type-specific studies of TF-mediated gene regulation in the brain and will provide valuable insights into brain development, homeostasis, and disease.

neuroscience↗

bioRxiv2019

Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

In situ measurements of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

genomics↗