Investigating Functional Convergence of ASD Risk Factor Genes Using a CRISPR-Based Approach.
Our research investigated the functional roles of high-confidence autism-associated genes using the Perturb-seq approach in human stem cell-derived neuronal tissues. Despite these genes' diverse functions, we uncovered how they converge on common neurodevelopmental pathways that contribute to ASD. We identified "functional convergence" across different neuronal subtypes and stages of differentiation. Additionally, we demonstrated that the convergence signal increases with maturation. Importantly, we highlighted metabolism and mitochondrial biology as one of the key convergence pathways across different developmental stages.
- Fernandez Garcia*, M., Retallick-Townsley*, K., Pruitt*, A., Davidson*, E. A., Balafkan*, N., Warrell, J., ... & Brennand, K. (2026). Transcriptomic and phenotypic convergence of neurodevelopmental disorder risk genes in vitro and in vivo. Nature Neuroscience, 1-16.
Characterization of Mitochondrial Biology During Early Development in human stem cell derived cardiomyocytes.
This study investigated the remodeling of mitochondria during the differentiation of hPSCs into mesodermal lineages, particularly cardiac progenitors and functional cardiomyocytes. It revealed that while mitochondrial mass decreases during differentiation, mitochondrial activity and efficiency—particularly ATP-linked respiration—increase. Notably, the study demonstrated that mitochondrial behavior is cell-lineage-specific, with distinct differences between neuronal and cardiac lineages. My contributions included conceptualizing the study, designing and conducting experiments, performing data analysis, and drafting the manuscript. This research is particularly relevant to the proposed project, as it underscores my expertise in mitochondrial biology and stem cell differentiation, directly supporting Aim 1 and 2.
- Balafkan N*†, Mostafavi S*, Pettersen IKN, Siller R, Sullivan G, Bindoff LA†.(2021) Distinct mitochondrial remodeling during early cardiomyocyte development in a human-based stem cell model. Frontiers in Cell and Developmental Biology, 9:744777.
Establishing A Microscale Cardiomyocyte Differentiation System to Study Mitochondrial Disorders Using a Human-Based Stem Cell Model.
I developed a scalable method to differentiate hPSCs into functional cardiomyocytes in microplates, significantly enhancing reproducibility and throughput for large-scale studies. In addition, we used induced pluripotent stem cell (iPSC)-derived neural stem cells from individuals with POLG mutations to model disease-specific mitochondrial dysfunction. This research identified mitochondrial DNA depletion, complex I deficiency, and increased reactive oxygen species production, effectively replicating the pathological mechanisms observed in postmortem brain tissues. My contributions included experimental design, execution, data analysis, and manuscript preparation. These studies highlight my expertise in generating functional cardiomyocytes and neurons and assessing mitochondrial function, skills that are directly applicable to the proposed project’s investigation of mitochondrial dysfunction in neurodevelopmental and cardiac lineages.
- Balafkan N, Mostafavi S, Schubert M, …, Sullivan G, & Bindoff LA. (2020). A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates. Scientific reports, 10.1:18498.
- Liang KX, Kristiansen CK, Mostafavi S, …, Balafkan N, Hong Y, Siller R, Sullivan G, & Bindoff L A (2020). Disease‐specific phenotypes in iPSC‐derived neural stem cells with POLG mutations. EMBO molecular medicine, 12.10.
Molecular Pathogenesis of Mitochondrial Dysfunction in Neurological Disorders.
In a series of studies, we investigated the molecular mechanisms underlying neurodegeneration in individuals with POLG mutations, a known cause of mitochondrial disease. Using postmortem brain tissues, we demonstrated that POLG mutations cause early mitochondrial DNA depletion and progressive somatic mutations, impairing neuronal respiration and leading to chronic neurodegeneration and acute focal neuronal loss, particularly in the cerebellar and dopaminergic systems. Remarkably, we observed severe nigrostriatal degeneration, with dopaminergic neuronal loss in the substantia nigra surpassing that seen in idiopathic Parkinson’s disease, yet without clinical signs of Parkinsonism. Additionally, we explored the relationship between CAG repeat variations in the POLG1 gene and increased Parkinson's disease risk, further underscoring mitochondrial dysfunction's role in neurodegeneration and relevance to precision medicine. My role included conducting molecular studies of microdissected neurons and analyzing mitochondrial DNA deletions and respiratory chain dysfunction.
- Balafkan N, Tzoulis C, Müller B, Haugarvoll K, Tysnes OB, Larsen, JP, & Bindoff LA. (2012). Number of CAG repeats in POLG1 and its association with Parkinson disease in the Norwegian population. Mitochondrion 12.6 (2012):640-643.
- Tzoulis C, Tran GT, Coxhead J, Bertelsen B, Lilleng PK, Balafkan N,... & Bindoff LA (2014). Molecular pathogenesis of polymerase gamma–related neurodegeneration. Annals of neurology, 76(1), 66–81.
- Tzoulis C, Tran GT, Schwarzlmüller T, Specht K, Haugarvoll K, Balafkan N,... & Bindoff LA. (2013). Severe nigrostriatal degeneration without clinical parkinsonism in patients with polymerase gamma mutations. Brain, 136(8), 2393–2404.