Jackson Laboratory

Endometriosis is a chronic gynecological disorder marked by the growth of endometrial-like tissue outside the uterus, often leading to pelvic pain, inflammation, and infertility. Despite its global prevalence, diagnosis remains delayed, and effective non-surgical treatments are lacking. While recent transcriptomic studies have identified mRNA transcript changes in ectopic lesions, the contribution of alternative pre-mRNA splicing, a key posttranscriptional regulatory layer, remains largely unknown. In this study, we performed a comprehensive analysis of alternative splicing (AS) events in endometriotic lesions using transcriptomic data. We uncovered distinct alterations in alternative splicing events are associated with endometriosis, highlighting a previously underappreciated layer of gene regulation. Specifically, we discovered that AS events, including exon skipping (SE) and intron retention (IR), were more prevalent than other events, and altered AS events correlated with transcriptomic variation in lesions. We identified two genes, GALNT7 and ZNF28, with significantly reduced exon inclusion in epithelial cells of lesions, potentially resulting in decreased levels of mature transcripts. Functional assays showed that knockdown of GALNT7 and ZNF28, or of critical exons within these genes, increased cellular proliferation, supporting their potential roles as growth‑suppressive genes in endometriotic cells. Together, this study broadens our understanding of transcriptomic dysregulation and highlights misregulated alternative splicing as a potential contributor to endometriotic cell growth and disease progression.

While induced pluripotent stem cells (iPSCs) have gained popularity in studying neurodegenerative diseases, the heterogeneity of stem cells used across studies impacts cross-study comparison. The iPSC Neurodegenerative Disease Initiative (iNDI) selected the KOLF2.1J cell line and prioritized its use as a reference standard for studying the effects of pathogenic variants on cell biology due to its stability and neutral neurodegenerative disease genetic risk. This cell line, and its derivatives expressing over 100 variants related to Alzheimer’s disease, Parkinson’s disease, and other neurological diseases, are available for academic and industry access. Current genomic data analyses are limited by the use of a human reference genome that does not capture the complete genetic background of a given iPSC line. While in the future this issue may be partially mitigated by the creation of a comprehensive human pangenome, previous work has shown that generating custom genomes is of value both to characterize the variation present and to serve as a more appropriate genomic reference. Here, we generated and characterized a custom complete genome assembly from KOLF2.1J. Mapping of sequencing reads to a personalized diploid assembly results in more comprehensive mapping compared to traditional linear references (i.e GRCh38). In addition, we provide a comprehensive custom gene annotation along with isoform expression and differential methylation analyses across multiple cell types. The assembly and all additional data is browsable and publicly available. This resource will enable more accurate investigation of the KOLF2.1J cell line and any genomics data generated compared to using traditional generalized references, while also serving as a foundational approach for establishing custom reference assemblies for other high-value iPSC lines.

Detecting antigen-specific lymphocytes is crucial for immune monitoring in vaccination, infection, cancer and autoimmunity. However, their low frequency and dispersed distribution make reliable detection challenging. We developed a strategy exploiting the functions of tissue-resident memory T cells (TRMs) to locally concentrate target circulating immune cells in the skin, enabling their non-invasive sampling using a microneedle (MN) skin patch. TRMs were first induced by antigen sensitization and subsequently restimulated by intradermal inoculation of the same antigen to trigger the 'alarm' and immune recruitment functions of these cells, leading to accumulation of antigen-specific T cells from the circulation over several days. In mouse vaccination models, MN patches with optimized hydrogel coatings effectively isolated thousands of live antigen-specific lymphocytes and innate immune cells. In a human participant with allergic contact dermatitis, allergen-induced TRM stimulation followed by MN application recovered diverse lymphocyte populations absent from untreated skin sites. These results establish TRM restimulation coupled with MN sampling as a non-invasive platform to monitor both local and systemic antigen-specific immune responses across disease or vaccination settings.

Although prime editing (PE) can effect virtually any specified local change to genomic DNA in living systems, its efficient application currently requires extensive optimization of prime editing guide RNA (pegRNA) sequences. We present OptiPrime, a machine learning model of PE efficiency based on our current understanding of the mechanism of prime editing. OptiPrime achieves state-of-the-art accuracy on PE efficiency prediction and also enables prediction of nicking guide RNA (PE3) and dual pegRNA (twinPE) outcomes. We validated that OptiPrime has learned the determinants of mammalian mismatch repair (MMR), and is therefore well suited for nominating MMR-evasive silent edits that improve PE efficiency. We demonstrate the utility of OptiPrime in a variety of prospective therapeutic contexts, including in primary human and mouse cells. Finally, we show how OptiPrime can be used to achieve highly streamlined and efficient in vivo correction of a pathogenic mutation in the brain of a mouse model of KIF1A-associated neurological disorder.

Age-related skeletal muscle decline (sarcopenia) is a major contributor to frailty and mortality during aging, yet the extent to which sex shapes muscle aging and its response to dietary interventions remains poorly understood. Here, we use the short-lived vertebrate Nothobranchius furzeri (African turquoise killifish; ATK) to investigate how sex and intermittent fasting (IF) interact to regulate lifespan and skeletal-muscle aging. We establish and optimize an IF regimen that significantly extends lifespan in both male and female killifish, albeit with classical trade-offs including reduced growth and reproductive output. Despite these costs, IF markedly improves swimming performance in aged animals of both sexes. Structural analyses of killifish on a normal diet reveal pronounced sexual dimorphism in muscle aging. Males exhibit age-associated myofiber hyperplasia, whereas females maintain fiber number but undergo hypertrophic remodeling. IF partially reverses both phenotypes, restoring a more youthful fiber size distribution in both males and females. Single-nucleus RNA sequencing uncovers sex-specific remodeling of muscle-fiber composition in killifish on a normal diet, with females displaying an age-associated shift toward oxidative slow-twitch fibers that is reversed by IF, while males show relatively stable fiber-type proportions under normal and IF feeding regimens. Cell-cell communication analyses further reveal a global decline in intercellular signaling with age, alongside sex-specific restoration of distinct pathways under IF, including axon guidance and IGF signaling in females and metabolic ANGPTL signaling in males. Finally, bulk transcriptomic profiling demonstrates that aging follows largely sexually dimorphic molecular trajectories, whereas IF induces both sex-specific and shared responses. Notably, under IF, both sexes exhibit upregulation of ribosome biogenesis and genes supporting myofibrillar organization and contraction, likely underlying preserved muscle function. Together, these findings demonstrate that IF promotes longevity and muscle health through conserved anabolic mechanisms alongside sex-specific cellular and molecular rejuvenation strategies. Our work highlights the importance of incorporating sex as a biological variable when designing dietary interventions to promote healthy aging.

Neurodegenerative diseases affect 1 in 12 people globally and remain incurable. Central to their pathogenesis is a loss of neuronal protein maintenance and the accumulation of protein aggregates with ageing1,2. Here we engineered bioorthogonal tools3 that enabled us to tag the nascent neuronal proteome and study its turnover with ageing, its propensity to aggregate and its interaction with microglia. We show that neuronal protein half-life approximately doubles on average between 4-month-old and 24-month-old mice, with the stability of individual proteins differing among brain regions. Furthermore, we describe the aged neuronal 'aggregome', which encompasses 1,726 proteins, nearly half of which show reduced degradation with age. The aggregome includes well-known proteins linked to diseases and numerous proteins previously not associated with neurodegeneration. Notably, we demonstrate that neuronal proteins accumulate in aged microglia, with 54% also displaying reduced degradation and/or aggregation with age. Among these proteins, synaptic proteins are highly enriched, which suggests that there is a cascade of events that emerge from impaired synaptic protein turnover and aggregation to the disposal of these proteins, possibly through microglial engulfment of synapses. These findings reveal the substantial loss of neuronal proteome maintenance with ageing, which could be causal for age-related synapse loss and cognitive decline.

There are ~ 60 clustered protocadherin (cPcdh) isoforms expressed from three gene clusters (Pcdha, Pcdhb, Pcdhg) arrayed in tandem across nearly 1 Mb in mammals. cPcdhs are homophilic cell adhesion molecules (CAMs) critical for a host of neural developmental functions consistent with a role in cell-cell recognition. Indeed, isoforms make recognition modules in combination to generate recognition diversity far exceeding the ~ 60 individual CAMs. However, there is also growing evidence for specialized functions for specific isoforms, particularly the C-type isoforms found at the 3' ends of the Pcdha cluster (αC1 and αC2) and at the 3' end of the Pcdhg cluster (γC3, γC4, and γC5). We have previously described unique roles for γC3 in dendrite arborization in the cerebral cortex and neural circuit formation in the spinal cord, as well as for γC4 in neuronal survival. Here we report a new mouse mutant specifically targeting the Pcdhgc5 exon encoding γC5. Unlike the rest of the Pcdhg cluster, expression of this isoform does not begin until postnatal stages of mouse development, increasing in the second week of life, suggesting specialized roles. We found significant expression changes in gene pathways involved in synaptic activity, learning and memory, and cognition. Despite this, we saw no major disruption in the cerebral cortex in neuronal organization, survival, dendritic arborization, or synaptic protein expression in these mutants. This new model will be an important tool for future studies delineating specific functions for γC5.