Neuronal Subtype-Specific Modeling of Alzheimer’s Disease by Direct Neuronal Reprogramming of Patient Fibroblasts

My laboratory studies genetic networks underlying neurogenesis and develops cell fate reprogramming approaches using microRNAs (miR-9/9* and miR-124) to generate human neurons by directly converting (reprogramming) adult fibroblasts. The primary purpose of developing the direct neuronal conversion approaches is to establish a patient neuron-based platform to model and study adult-onset disorders such as Alzheimer’s disease (AD) and Huntington’s disease. With support from Cure Alzheimer’s Fund (CAF), we have successfully addressed several vital considerations for modeling neurodegenerative disorders. One question relates to the age information stored in the original fibroblasts when cells transition to neurons via direct reprogramming. We previously carried out in-depth analyses of age-associated marks, including DNA-methylation-based epigenetic clock analysis, transcriptome, miRNA profiles and cellular properties (reactive oxygen species, DNA damage and telomere lengths) in human neurons converted from fibroblasts across the age spectrum. We learned that the age information stored in the starting fibroblasts propagated during neuronal conversion, resulting in neurons that reflect fibroblast donors’ age, a feature highly instrumental to modeling adult-onset neurodegenerative disorders. Extending these findings, our goals pertinent to the CAF grant have been to leverage direct neuronal reprogramming as a system to model AD.

Neurodegenerative disorders often affect different subtypes of neurons differentially. It is critical to have control for what types of neurons can be generated by direct neuronal reprogramming. Toward this goal, we have carried out studies to dissect mechanisms underlying how miR-9/9* and miR-124 (miR-9/9*-124) elicit reprogramming of human fibroblasts to neurons. Supported by CAF, we previously performed genomic analyses of changes in chromatin accessibility in response to miR-9/9*-124. We learned that miR-9/9*-124 led to an extensive reconfiguration of the chromatin state poised to receive inputs from subtype-defining transcription factors. In a recently published study (a new publication for the current round of progress report), we conducted a follow-up study in which we looked into cellular dynamics of reprogramming cells at a single-cell resolution. Our findings indicated that the miRNA-mediated neuronal conversion occurs through distinct steps where miR-9/9*-124 orchestrate the erasure of fibroblast identity first, followed by the adoption of the neuronal identity in sequence. During the later stage of miRNA-induced neuronal state, subtype-defining transcription factors promote the maturation of converting neurons into a specific neuronal subtype. This study provides us with fundamental insights into how neuron-specific degenerative phenotypes may arise during the neuronal conversion of patient fibroblasts.

Taking our experiences in modeling Huntington’s diseases with patient-derived striatal medium spiny neurons (MSNs) we are actively pursuing AD-modeling studies using fibroblast samples from familial and late-onset AD patients. We hope to submit three manuscripts for publication within this year based on the results we have so far. One study relates to our current AD modeling using miRNAs and NeuroD2 and MYT1L TFs, which produce glutamatergic cortical neurons; i) directly converting familial AD fibroblast samples generates neurons that display extracellular amyloid beta accumulation, ii) reprogrammed familial AD neurons show increased tau phosphorylation that appears to underly insoluble form of tau, and iii) most importantly, familial AD neurons display spontaneous neurodegeneration as cells acquire the neuronal identity.

We are currently extending these studies to fibroblast samples obtained from late-onset AD patients. Another study extends our expertise in subtype-specific neuronal reprogramming, in which we developed miRNA-based reprogramming methods specific for serotonergic neurons and upper-layer cortical neurons. Finally, we are in the process of preparing a manuscript that describes the tau isoform expression in human neurons reprogrammed from adult fibroblasts. Based on the results obtained, reprogrammed neurons from adult fibroblasts express tau 3R and 4R isoforms at a ratio similar to what is detected in the human adult brain, both transcriptionally and protein level-wise.