Human iPSCs offer a controllable, human-specific system to study early mechanisms predisposing to Alzheimer’s disease (AD). We will dissect how the human-specific gene CHRFAM7A — an endogenous modulator of the alpha7 nicotinic acetylcholine receptor — shapes neurogenesis and interneuron (IN) lineage specification, maturation and vulnerability to amyloid-beta (Abeta). We will combine genomically defined iPSC panels (0/1/2 CHRFAM7A copies) with isogenic CHRFAM7A loss-of-function and add-back, harmonized 2D differentiations, molecular and imaging phenotyping, and standardized A? challenges to map causal relationships between CHRFAM7A dosage, alpha7 signaling and AD-relevant phenotypes. In a second phase, optimized protocols will be translated to 3D neuro-organoids and ventral–dorsal assembloids to model neuron migration and integration in a tissue-like context. The outcome will be a rigorous, human-relevant framework linking CHRFAM7A/alpha7 signaling to early AD trajectories.

Background and central hypothesis.
Developmental programs in human cortex — NPC fate, interneuron (IN) subtype specification, maturation and integration — are increasingly implicated in the network imbalance that precedes clinical Alzheimer’s disease (AD). CHRFAM7A, a human-specific partial duplication of CHRNA7, modulates alpha7-nAChR signaling and Ca²+ handling, but its causal role during human neurodevelopment and Abeta vulnerability is unresolved. We hypothesize that CHRFAM7A dosage calibrates alpha7 signaling to tune NPC proliferation/differentiation/migration and MGE/CGE IN trajectories, thereby shaping early Abeta responses.
Aim 1 — Genomic definition of CHRFAM7A in human iPSCs.
We will assemble a high-quality iPSC panel spanning 0/1/2 CHRFAM7A copies (copy number will be quantified by ddPCR/qPCR). In parallel, we will generate isogenic CHRFAM7A knockouts from a 2-copy line using CRISPR/Cas9 genome editing that preserves the intact CHRNA7 allele. Edited cells will undergo single-cell cloning, followed by PCR and amplicon sequencing to confirm on-target disruption, and allele-aware RT-qPCR to verify loss of CHRFAM7A transcripts with retention of CHRNA7 expression. For genetic rescue, we will create a KO+add-back line by inserting dupalpha7 cDNA under a controllable promoter at a safe-harbor locus. All lines (0/1/2-copy, KO, KO+add-back) will pass unified quality controls (pluripotency markers, normal karyotype, mycoplasma-free), be banked, and then compared side-by-side under identical NPC/MGE/CGE differentiation protocols.
Aim 2 — 2D differentiations of NPCs and INs.
Using standard operating procedure suitable for comparative biology, we will derive anterior NPCs by dual-SMAD inhibition, rosette selection and expansion in defined growth factors; and INs via dual-SMAD induction to anterior neuroectoderm, followed by SHH-pathway activation with WNT modulation and rostro-caudal cues to bias MGE (FGF8) versus CGE (retinoic acid ± Activin A/FGF19). IN progenitors will be banked at day ~21–28 and matured with neurotrophic support and enforced post-mitotic exit. Identity, purity and maturation will be verified with compact immunofluorescence/RT-qPCR panels and image-based morphology (neurite length; Sholl-derived dendritic complexity). Synaptic development will be tracked with a minimal set of presynaptic/postsynaptic (Synaptophysin, PSD95) markers to provide objective integration indices. Correct synaptic development and in vitro assessment of spontaneous functional neuronal activity will be validated following microelectrode array (MEA) analysis.
Aim 3 — ?7/CHRFAM7A axis and Abeta vulnerability in vitro.
We will quantify CHRNA7/CHRFAM7A expression, alpha-bungarotoxin binding, and simple alpha7 pharmacology (agonists/PAMs ± MLA) across the genotype series (0/1/2 copy, KO, add-back) processed under identical methods. Developmental readouts will emphasize NPC neurogenesis and proliferation (EdU/Ki67), early neuronal differentiation and morphological maturation (maturation markers analysis). A standardized, quality-controlled Abeta oligomer challenge will then be used to measure neuronal survival and neurite/synapse integrity. Rescue experiments with ?7 agonists/PAMs and dupalpha7 add-back will test causality. The deliverable is a coherent, human-specific map of how CHRFAM7A/alpha7 signaling affects lineage specification, maturation and Abeta sensitivity.
Aim 4 (Perspective) — Step-up to 3D neuro-organoids and assembloids.
Optimized 2D protocols will be translated to 3D systems aligned with the laboratory’s models: dorsal cortical organoids (NPC excitatory scaffolds) and ventral MGE/CGE organoids (IN progenitors). Each differentiation will be carried out independently using its specific protocol, followed by fusion of the resulting tissues. The assembly of ventral and dorsal brain organoids into ventral?dorsal assembloids will enable modeling of human interneuron migration and spatial positioning within a physiologically relevant, tissue-like environment. We will analyze readouts: cell identity and morphology, migration trajectories, and synaptic markers, with optional transcriptomics at a decisive timepoint to capture alpha7-dependent state shifts. To further validate physiological relevance, these assembloids will be integrated in vivo through transplantation into neonatal mouse cortex. This will enable assessment of interneuron/neuron survival, maturation, circuit integration, and long-range connectivity under a vascularized and dynamically active environment. It will also extend observation windows for long-term development and disease-relevant dynamics.
Methods integration and analytics : All experiments will be run in biological replicates across independent differentiations and, where relevant, multiple clones per genotype. We will also investigate genotype/sex interactions and sex-stratified secondary analyses (female/male iPSCs lines). Mixed-effects models will account for batch/clone effects; false discovery rates will be controlled for multi-marker panels. Standard procedures and analysis of notebooks will be versioned, with blinded image quantification and predefined acceptance criteria for differentiation batches and Abeta preparations.
Expected outcomes and impacts.
The project will produce: (i) a resolved genomic/expressional atlas of CHRFAM7A in the specific iPSCs cell line; (ii) causal evidence that CHRFAM7A dosage/perturbation impacting NPC development faith and MGE/CGE IN trajectories via alpha7; and (iii) a human-relevant link between CHRFAM7A/alpha7 signaling and early Abeta vulnerability. By anchoring organoid/assembloid readouts to rigorously benchmarked 2D phenotypes, the work will deliver transferable endpoints for future target validation in early AD.

Amyotrophic lateral sclerosis (ALS) affects 97% of patients through TDP-43 mislocalization and aggregation. Accumulating evidence demonstrates that TDP-43 localizes to mitochondria and binds key mRNAs, impairing respiratory complex function. However, the complete mitochondrial transcriptomic landscape disrupted by disease-associated TARDBP mutations is undefined. This project will identify a convergent “MitoTAR signature”, a core set of mitochondrial RNAs disrupted across different ALS mutations. Long-read RNA sequencing of mitochondria from patient iPSC-derived motor neurons via Oxford Nanopore will allow mapping of the full mitochondrial transcriptome. Functional validation via RNA knockdown and rescue will establish causality between specific RNA targets and metabolic dysfunction. In vivo validation in TDP-43 zebrafish models will test therapeutic targeting of this signature using mitochondria-directed small molecules. This work will define a novel therapeutic node for ALS treatment

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that affects both upper and lower motor neurons, causing disruptions in signal transmission from the brain to the muscles. These symptoms are fatal, leading to a median survival time following diagnosis of three to five years1. A pathological hallmark of ALS is the mislocalization and aggregation of TDP-43, which occurs in approximately 97% of all ALS patients2. TDP-43 is an RNA-binding protein that regulates splicing of critical genes like STMN2 and UNC13A, whose dysregulation causes downstream synaptic function deficits3–6.
Mitochondrial perturbation is a recognized feature of ALS pathogenesis. Recent evidence suggests that TDP-43 localizes to mitochondria where it binds mRNAs encoding respiratory Complex I subunits (ND3, ND6), causing complex disassembly and bioenergetic failure7. While progress of understanding TDP-43 mitochondrial mislocalization continues, several gaps remain. First, the functional consequences of TDP-43 mitochondrial localization remain narrowly defined8. Second, while mutation-specific differences in mitochondrial transport and nuclear transcriptomes have been observed9,10, a systematic link between individual mutations, their specific mitochondrial RNA targets, and their corresponding metabolic phenotypes is missing. Lastly, the causality gap remains unexplored. While blocking TDP-43 mitochondrial import can rescue toxicity, it is unclear if this localization is a primary driver of disease or a secondary consequence of cellular stress2,11,12.
Preliminary data from the Kabashi lab provides evidence supporting mutation-specific heterogeneity in patient iPSC-derived motor neurons. While one patient line recapitulates a previously reported glycolytic shift, two other patient lines display paradoxical increases in mitochondrial membrane potential (??m). Importantly, metabolic phenotype severity correlates with the quantitative burden of TDP-43 localized to the mitochondria, suggesting convergent RNA-level disruptions may underlie this heterogeneity.
Therefore, we hypothesize that the mislocalization of TDP-43 to mitochondria is a critical event that disrupts the mitochondrial transcriptome. We predict this disruption converges on a core set of mitochondrial RNAs, which drives metabolic dysfunction in motor neurons. Defining this convergent “MitoTAR signature” will establish the mitochondrial transcriptome as a novel and therapeutically actionable node in ALS.
Aim 1: Defining the Core Mitochondrial Transcriptomic Signature
Aim 1 serves as the foundation of this PhD project by utilizing in silico sequencing approaches to define the core mitochondrial signature of TDP-43 proteinopathy. We will isolate mitochondria from iPSC-derived motor neurons representing isogenic controls, TARDBP mutation carriers, and C9orf72 hexanucleotide repeat expansion carriers. Both mutations converge on TDP-43 dysfunction, as C9orf72 patients show impaired mitochondrial respiration and TDP-43 pathology. To enhance neuronal-specific signatures, we will include cortical organoid-derived neurons to provide an autonomous neuronal model complementing the motor neuron focus13. Mitochondrial RNA will undergo long-read sequencing via Oxford Nanopore PromethION, enabling full-length, single-molecule reads that capture complete transcript isoforms, a critical aspect given the role of TDP-43 in splicing regulation. Bioinformatic analysis will employ Seurat for clustering and differential expression alongside hdWGCNA to identify co-expression modules. We will analyze RNA processing, transcript abundance, and RNA editing patterns. Isoform-switching analysis will identify mutation-specific versus convergent alterations. Functional enrichment via Enrichr will map affected metabolic pathways. Top candidates will be validated using RT-qPCR and customized Nanostring nCounter codesets to generate a high-confidence “MitoTAR signature” atlas.
Aim 2: Establishing a Causal Role in Metabolic Dysfunction
This aim will determine the functional necessity of the top candidate RNAs identified in Aim 1. We will select 5-6 high-priority RNA targets based on differential expression strength and metabolic pathway enrichment. We will employ complementary loss-of-function (LOF) and gain-of-function (GOF) approaches in our iPSC-derived motor neurons. In our LOF approach, control motor neurons will be transfected with antisense oligonucleotides targeting candidate mitochondrial RNAs. We predict this will recapitulate patient metabolic phenotypes. In our GOF approach, patient motor neurons will receive expression constructs or ASOs to restore RNA function. We predict this will rescue metabolic dysfunction.
Functional outcomes will be assessed using established phenotyping platforms. Seahorse analysis will be utilized to measure mitochondrial respiration and glycolytic function, and live-cell imaging with TMRM will be utilized via IncuCyte to quantify changes in mitochondrial membrane potential. To confirm that the ASO-mediated rescue occurs without altering the percentage of mitochondria containing TDP-43, the Opera Phenix high-content imaging pipeline will be employed. This experimental design will definitely establish that observed metabolic dysfunction is a direct consequence of the specific RNA interaction instead of a secondary effect from reduced TDP-43 localization in mitochondria.
Aim 3: In vivo Validation and Therapeutic Testing
This aim will validate the pathophysiological relevance of the MitoTAR signature in vivo and test a direct therapeutic strategy. We will utilize a TDP-43 transgenic zebrafish model crossed with an Hb9:GFP line to fluorescently label motor neurons14–16. FACS-sorted spinal motor neurons will undergo RT-qPCR/ Nanostring to confirm MitoTAR signature preservation. We will then test therapeutic approaches utilizing mitochondria-targeted small molecules (e.g., SBT-272) or other compounds that modulate mitochondrial dynamics and reduce oxidative stress. We will utilize quantitative video tracking to measure the rescue of motor functions, such as swimming velocity17. The outcome of this aim will be in vivo validation of the MitoTAR signature and pre-clinical evidence for the efficacy of mitochondrial-targeted therapies in TDP-43-mediated ALS.
This investigation is paramount for defining a core pathogenic mechanism in ALS. By identifying the convergent “MitoTAR signature”, this work will move beyond a narrow focus on rare mutations to define a core pathogenic mechanism for TDP-43-driven metabolic failure relevant to the vast majority of ALS patients. Beyond fundamental mechanistic understanding, this research has significant translational potential. The MitoTAR signature itself would represent a novel set of therapeutic targets. By validating that direct mitochondrial support is an effective therapeutic strategy, this work will provide a robust rationale for new treatment opportunities for ALS and offer a promising alternative to targeting elusive upstream regulators.
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Our lab recently demonstrated that damaging mutations in the SMARCC2 gene were associated with bipolar disorders. Interestingly, numerous de-novo mutations in SMARCC2 have already been reported in individuals with other neurodevelopmental disorders, but no study has shown the pathophysiological impact of such mutations. Our project aims to understand the pathophysiological mechanisms resulting from mutations in the SMARCC2 gene by studying their impact on the early steps of brain development, structure, and function. In this project, the candidate will generate and characterize induced pluripotent stem cell-derived neurons, brain organoids and assembloids carrying or not mutations identified in patients. In addition to the phenotypic characterization, the candidate will conduct ATAC-Seq and single-cell RNA-seq experiments to identify the genes targeted by the SWI/SNF complex and those deregulated when SMARCC2 is mutated in affected individuals.

Bipolar disorder (BD) is a major public health concern for which a high heritability has been widely demonstrated. Recent genome-wide association studies (GWAS) on tens of thousands of subjects have reported several loci associated with the disease with an additive effect of many genetic vulnerability variants, each having a low effect size (1). However, common polymorphisms explain only 7% of the genetic variance in BD suggesting the major heritability could reside in highly penetrant rare damaging variants. Using multiplex families with early onset forms of BD, we have recently identified several genes with rare damaging variants segregating with the disease (2,3). An enrichment pathway analysis highlighted that these genes encoded proteins involved either in neural gene expression regulation or in neural development and morphogenesis or both, suggesting for the first time that familial BD with an early age of onset could be a neurodevelopmental disorder (3). Among the genes identified in these multiplex families, we observed that SMARCC2, encoding the SWI/SNF related, Matrix-Associated, actin dependent Regulator of Chromatin subfamily C member 2, showed a higher frequency of damaging variants in individuals with BD than in the general population (3). Interestingly, damaging variants in this gene had also been reported in individuals with a rare disease named Coffin-Siris syndrome and autism spectrum disorders, but no functional studies have been performed yet to assess the impact of these variants and explain the variability in the phenotypic outcome.
The chromatin remodeling complex SWI (switch mutants)/SNF (sucrose non-fermenting) plays an essential role in the regulation of gene expression. This ATP-dependent multiprotein complex, also known as BRG1-associated factor (BAF), is composed of 15 proteins that interact to modulate the nucleosome and change chromatin conformation and accessibility. Among these proteins, BAF170, a common core protein of the BAF complex, which directly interacts with BRG1, is encoded by SMARCC2. SMARCC2 plays a crucial role in embryogenesis and corticogenesis (4), suggesting that amino acid changes in its protein could result in brain abnormalities and contribute to the BD vulnerability as well as to other neurodevelopmental disorders.
Modeling bipolar disorders in animals is a real challenge to understand the pathophysiology of the disease, particularly for studying early stages of brain development. The aim of our project is thus to use neuron culture and brain organoids and assembloids to study early brain development and assess the impact of disease associated SMARCC2 mutations identified in individuals with BD. First, the candidate will study two BD-associated mutations to compare the phenotypic consistency of SMARCC2 mutations identified in patients. These mutations will be introduced in two commercial lines of human induced pluripotent stem cells (hIPSCs) that are already available in the lab, using CRISPR/Cas9 approach to have isogenic lines varying only for the mutations of interest. These mutated lines will be compared to the wild-type lines. Two hIPSC lines will be studied to confirm that the mutation effect is independent of the genetic background. Then, mutated and non-mutated hIPSCs will be differentiated in 2D-cell culture into human neurons and into 3D-cortical organoids to assess the impact of mutations on neuron and dendritic growth as well as on more complex structure. The candidate will compare the gene expression and protein level of SMARCC2 and its SWI/SNF complex partners between wild-type and mutants cells/organoids. The neurite length of differentiating neurons will be measured with live cell imaging using an Incucyte® Live Cell analysis instrument. In addition, immunocytochemistry will be performed on neurons after several days in culture using neuronal markers (e.g. TUJ1, DCX, MAP2) to monitor the neuronal differentiation. In parallel, a higher degree of organization and cortical differentiation as well as synapse characterization and quantification will be analyzed by immunohistochemistry on 3D-brain organoids. Organoids will be fixed at multiple steps of differentiation and incubate with undifferentiation markers (e.g., PAX6 and SOX2), neuron specific markers (e.g. MAP2, TUJ1) and synaptic specific markers (e.g. SV1, VLGUT1, VGLUT2, VGAT, GPHN).
Phenotypic rescue of mutated neurons and organoids will be tested using common mood regulators, e.g. lithium and valproic acid, and other psychotropic drugs that will be added to the cell culture media.
In parallel, the candidate will dedifferentiate hair follicle cells collected from families in which SMARCC2 mutations have been identified and will differentiate them in neurons and brain organoids in order to assess the impact of the patients’ genetic background on the phenotypic expression of the disease-associated mutation. We have already obtained the ethical and legal authorization to collect hair follicles in these families. These mutations will then be corrected in vitro in patient’s cells using CRISPR-Cas9 approach to demonstrate that the observed phenotype results from the mutation.

As a second step, as the SWI/SNF complex regulates the expression of many genes by modulating the chromatin architecture, the candidate will perform single nuclei Assay for Transposase Accessible Chromatin combined with sequencing (ATAC-Seq) as well as single cell RNA sequencing of IPSCs-derived brain organoids to identify the molecular pathways affected in mutated organoids. This will result in the identification of chromosomal loci for which the chromatin accessibility changes with the mutations in SMARCC2, as well as the genes and molecular pathways for which the expression is affected by these changes. The candidate will thus compare in a cell-type specific manner, the loci with open chromatin regions (OCRs) in the genome between organoids with and without SMARCC2 mutations.

Finally, as bipolar disorder is known to show impairment in cortico-striatal connectivity, the candidate will also differentiate hIPSC into region-specific brain organoids that resemble the developing human striatum and cortex. He or she will then assemble these organoids in three-dimensional cultures to form cortico-striatal assembloids and characterize axon projections and interneuron migration using viral tracing and functional assays (5). Then, the candidate will assess whether the SMARCC2 mutations affect the connectivity between the two brain areas, as observed in subjects with BD.

This project is already founded by the FondaMental Foundation, which have identified a sponsor for the running costs of the project. Altogether, these results should allow the identification of molecular pathways that are affected in individuals with mutations in SMARCC2 and should shed new light on the pathophysiological mechanisms underlying BD.

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