Silverman, G. A. | Search bioRxiv

bioRxiv2025

Sequelae and reversal of age-dependent alterations in mitochondrial dynamics via autophagy enhancement in reprogrammed human neurons

How aging of human neurons affects dynamics of essential organelle such as mitochondria and autophagosomes remains largely unknown. MicroRNA-induced directly reprogrammed neurons (miNs) derived from adult fibroblasts retain age-associated signatures of the donor, enabling the study of age-dependent features in human neurons, including longitudinal isogenic samples. Transcriptomic analysis revealed that neurons derived from elderly individuals are characterized by gene expression changes associated with the regulation of autophagosomes, lysosomes, and mitochondria, compared to young counterparts. To clarify these changes at the cellular level, we performed live-cell imaging of cellular organelles in miNs from donors of different ages. Older donor miNs exhibit decreased mitochondrial membrane potential, which surprisingly co-occurs with a significant increase in mitochondrial fission and fusion events. We posit that the increased fission and fusion of mitochondria may reflect age-dependent compensation for impaired mitochondrial turnover, perhaps due to changes in autophagy. We subsequently identified a significant decrease in autophagosome acidification in neurons derived from individuals >65 years compared to younger donors, and a corresponding age-dependent reduction in neuritic lysosomes resulting in fewer lysosomes available to acidify autophagosomes. This age-dependent deficit in autolysosome flux was rescued by promoting autophagosome generation through TFEB, which also reversed the age-dependent increase in mitochondrial fission and fusion and improved mitochondrial health. Partial organelle recovery occurred after inducing mitophagy or inhibiting mitochondrial fission. Together, this work reveals a mechanism by which aging reduces autophagic flux secondary to a loss of neuritic lysosomes, resulting in in mitochondria-intrinsic mechanisms to avoid loss of energy production.

neuroscience↗

bioRxiv2022

Functional analysis of a novel de novo variant in PPP5C associated with microcephaly, seizures, and developmental delay

We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C. This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI=1, o/e=0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the probands phenotypes.

genetics↗