Background
Neurocognitive impairment (NCI) is a prevalent and important co-morbidity in virologically suppressed people living with HIV (PWH), yet the underlying mechanisms remain elusive and effective treatments lacking. Given the limitations in models adequately replicating neuronal biology and injury in PWH, we explored the use of participant-derived directly induced neurons (iNs) for this purpose. iNs have been shown to retain age- and disease-related features of the donors, providing a unique opportunity to reveal novel aspects of neurodegenerative disorders. We hypothesized that the application of this system would allow us to determine potentially dysregulated genes in iNs derived from PWH compared to persons without HIV (PWOH).
Methods
Primary dermal fibroblasts were obtained from six PWH on suppressive cART (median age 53 years (range: 27 – 64); 83% Male; 50% White) and seven demographically matched PWOH (median age 55 years (range: 27 – 66); 71% Male; 57% White). Participant fibroblasts were transduced with two lentiviral vectors for the doxycycline-dependent expression of neuronal pioneer transcription factors NGN2, and ASCL1. Transdifferentiation into iNs was accomplished by treatment with doxycycline and a cocktail of small molecules. At day 21, live iNs were isolated by FACS sorting based on the neuronal marker PSA-NCAM. Neuronal identity was validated by immunocytochemistry and scRNA analysis. Bulk-RNAseq was performed to compare gene expression between iNs from PWH vs. PWOH.
Results
Transcriptomic analysis identified 29 (p-adj. < 0.05, log2fc ± 0.5) differentially expressed genes between iNs derived from PWH and PWOH. Of these, 16 genes were downregulated and 13 upregulated in PWH iNs (Fig 1). Protein-protein interaction network mapping suggests PWH iNs exhibit differences in extracellular matrix organization and synaptic transmission. Amongst our findings, IFI27 has previously been shown to be upregulated in the post-mortem brain samples of PWH compared to PWOH. In contrast is our novel finding that expression of the FOXL2NB-FOXL2-LINC01391 genome locus is reduced in PWH iNs and associated with the degree of NCI in the PWH cohort (p<0.05).
Conclusions
We have identified an iN gene signature of HIV through direct reprogramming of skin fibroblasts into neurons. Comprising 29 DEGs, this signature supports previous findings of HIV-1-related neuroinflammation while revealing additional, potentially novel mechanisms of NCI in PWH.