Biology subjects

Sahagun, A.

Publications and source records attributed to Sahagun, A..

8 recordsLinked to original sources

bioRxiv2026

An isogenic single-cell atlas of familial Parkinson's disease mutations reveals convergent changes in dopamine neurons

Summary/AbstractFamilial Parkinsons disease (PD) is caused by mutations in more than twenty genes that affect diverse cellular pathways, including mitochondrial quality control, lysosomal function, and vesicular trafficking. A central question is how mutations impacting these distinct pathways converge to cause the selective degeneration of dopamine neurons. Human pluripotent stem cell (hPSC)-based disease models provide a valuable system to study this; however, systematic comparison of the pathogenic effects of different mutations has been limited by genetic background variability. To address this, we generated an isogenic single-cell transcriptomic atlas of fourteen familial PD mutations comprising more than 200,000 hPSC-derived midbrain specified cells. Integrated analysis revealed mutation-specific transcriptional signatures alongside shared dysregulated genes and modules that converge on mitochondrial homeostasis, endolysosomal degradation, and iron/ferroptosis pathways. Differentially expressed genes were significantly enriched for PD GWAS-implicated genes in dopamine neurons, bridging monogenic and sporadic PD genetic risk and highlighting a shared downstream state across multiple mutations. Finally, cells with a DNAJC6 mutation, which is associated with juvenile-onset parkinsonism, exhibited alteration of neurodevelopmental and psychiatric disorder risk genes, providing a transcriptional correlate for neurodevelopmental features observed in early-onset PD. Together, this resource enables molecular stratification of familial PD mutations and provides a foundational benchmarking data set.

genomics↗

bioRxiv2024

Comparison of Alternative pre-mRNA Splicing and Gene Expression Patterns in Midbrain Lineage Cells Carrying Familial Parkinson's Disease Mutations

Parkinsons disease (PD) arises from genetic and environmental factors. Human genetics has identified mutations in [~]20 inherited familial genes linked to monogenic forms of PD. To investigate the effects of individual familial PD mutations, human pluripotent embryonic stem cells (hPSCs) carrying 12 distinct familial PD mutations were differentiated into midbrain lineage cells, including dopaminergic (mDA) neurons. Global gene expression and pre-mRNA splicing patterns were analyzed in midbrain cultures carrying pathogenic PD mutations in the PRKN, SNCA, LRRK2, PINK1, DNAJC6, FBXO7, SYNJ1, DJ1, VPS13C, ATP13A2 and GBA1 genes. We have grouped the analysis of these familial PD mutations to genes expressed in mDA neurons and whose pre-mRNA splicing changes are linked to known PD defects in transport, cytoskeleton, lysosomes and mitochondria. Importantly, we have also shown that subsets of these splicing changes overlap with changes found in PD patient postmortem brains. Mutation-specific pre-mRNA isoforms may function as both diagnostic biomarkers for familial PD-associated genotypes and promising therapeutic targets.

neuroscience↗

bioRxiv2024

iSCORE-PD: an isogenic stem cell collection to research Parkinson Disease

Parkinsons disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer a unique experimental platform to advance our understanding of PD etiology by enabling the generation of disease-relevant cell types carrying patient mutations along with isogenic control cells. To facilitate this approach, we generated a collection of 65 human stem cell lines genetically engineered to harbor high risk or causal variants in genes associated with PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G/FS, SYNJ1 R258Q/FS, VPS13C A444P/FS, VPS13C W395C/FS, GBA1 IVS2+1/FS). All mutations were introduced into a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using different genome editing techniques. To ensure the genetic integrity of these cell lines, we implemented rigorous quality controls, including whole-genome sequencing of each line. Our analysis of the genetic variation in this cell line collection revealed that while genome editing, particularly using CRISPR/Cas9, can introduce rare off-target mutations, the predominant source of genetic variants arises from routine cell culture and are fixed in cell lines during clonal isolation. The observed genetic variation was minimal compared to that typically found in patient-derived iPSC experiments and predominantly affected non-coding regions of the genome. Importantly, our analysis outlines strategies for effectively managing genetic variation through stringent quality control measures and careful experimental design. This systematic approach ensures the high quality of our stem cell collection, highlights advantages of prime editing over conventional CRISPR/Cas9 methods and provides a roadmap for the generation of gene-edited hPSC collections at scale in an academic setting. Our iSCORE-PD collection represents an easily accessible and valuable platform to study PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.

neuroscience↗

bioRxiv2022

Neuronal Activity Alters Neuron to OPC Synapses

The mechanisms that drive the timing and specificity of oligodendrocyte myelination during development, or remyelination after injury or immune attack are not well understood. Recent work has shown that oligodendrocyte progenitors receive synapses from neurons, providing a potential mechanism for neuronal-glial communication. We hypothesize that these connections are important both for correct myelination of neurons during development and for myelination during neuronal plasticity. We utilized chemogenetic tools and viral monosynaptic circuit tracing to analyze these neuroglial connections and to examine OPC proliferation, myelination, synapse formation, and neuronal-glial connectivity after increasing or decreasing neuronal activity in vivo. We found that increasing neuronal activity increased OPC activation, but not proliferation. We also found that altering neuronal activity altered neuronal-glial synaptic connections: while it did not impact the total number of neuronal inputs, or the number of inhibitory neuronal inputs, it did alter the number of excitatory neuron to OPC connections. We also found that increasing or decreasing neuronal activity impacted the ratio of excitatory and inhibitory synapses. Our data show that neuronal activity affects OPC activation, neuronal synapse formation onto OPCs, as well as the types of neuronal inputs to OPCs, indicating that neuronal activity is important for OPC circuit composition and function.

neuroscience↗

bioRxiv2022

Physical and functional convergence of the autism risk genes Scn2a and Ank2 in neocortical pyramidal cell dendrites

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding NaV1.2 to the dendritic membrane of mouse neocortical neurons, and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a+/- conditions. Thus, these results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can be etiological to neurodevelopmental disorder pathophysiology.

neuroscience↗

bioRxiv2022

CRISPR activation rescues abnormalities in SCN2A haploinsufficiency-associated autism spectrum disorder

The majority of autism spectrum disorder (ASD) risk genes are associated with ASD due to haploinsufficiency, where only one gene copy is functional. Here, using SCN2A haploinsufficiency, a major risk factor for ASD, we show that increasing the expression of the existing functional SCN2A allele with CRISPR activation (CRISPRa) can provide a viable therapeutic approach. We first demonstrate therapeutic potential by showing that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency. Next, using an rAAV-CRISPRa based treatment, we restore electrophysiological deficits in both Scn2a heterozygous mice and human stem-cell-derived neurons. Our results provide a novel therapeutic approach for numerous ASD-associated genes and demonstrate that rescue of Scn2a haploinsufficiency, even at adolescent stages, can ameliorate neurodevelopmental phenotypes.

neuroscience↗

bioRxiv2021

Paradoxical hyperexcitability from NaV1.2 sodium channel loss in neocortical pyramidal cells

Loss-of-function variants in the gene SCN2A, which encodes the sodium channel NaV1.2, are strongly associated with autism spectrum disorder and intellectual disability. An estimated 20-30% of children with these variants are co-morbid for epilepsy, with altered neuronal activity originating in neocortex, a region where NaV1.2 channels are expressed predominantly in excitatory pyramidal cells. This is paradoxical, as sodium channel loss in excitatory cells would be expected to dampen neocortical activity rather than promote seizure. Here, we examined pyramidal neurons lacking NaV1.2 channels and found that they were intrinsically hyperexcitable, firing high-frequency bursts of action potentials (APs) despite decrements in AP size and speed. Compartmental modeling and dynamic clamp recordings revealed that NaV1.2 loss prevented potassium channels from properly repolarizing neurons between APs, increasing overall excitability by allowing neurons to reach threshold for subsequent APs more rapidly. This cell-intrinsic mechanism may therefore account for why SCN2A loss-of-function can paradoxically promote seizure.

neuroscience↗

bioRxiv2020

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse neocortex

ObjectiveGenetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human neocortex. MethodsRNA-seq data from 176 human dorsolateral prefrontal cortex samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples. ResultsIn the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A a synchronized 5N/5A transition occurs between 24 post-conceptual weeks (2nd trimester) and six years of age. In mice, the equivalent 5N/5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories. SignificanceSplice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.

genetics↗