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[Preprint]. 2024 Oct 22:2024.10.22.619617.
doi: 10.1101/2024.10.22.619617.

A Transcriptional Signature of Induced Neurons Differentiates Virologically Suppressed People Living With HIV from People Without HIV

Philipp N Ostermann  1 , Youjun Wu  2 , Scott A Bowler  1 , Mohammad Adnan Siddiqui  3 , Alberto Herrera  1 , Mega Sidharta  2 , Kiran Ramnarine  2 , Samuel Martínez-Meza  1 , Leslie Ann St Bernard  1 , Douglas F Nixon  1 , R Brad Jones  1 , Masahiro Yamashita  3 , Lishomwa C Ndhlovu  1 , Ting Zhou  2 , Teresa H Evering  1
Affiliations

A Transcriptional Signature of Induced Neurons Differentiates Virologically Suppressed People Living With HIV from People Without HIV

Philipp N Ostermann et al. bioRxiv. .

Abstract

Neurocognitive impairment is a prevalent and important co-morbidity in virologically suppressed people living with HIV (PLWH), yet the underlying mechanisms remain elusive and treatments lacking. Here, we explored for the first time, use of participant-derived directly induced neurons (iNs) to model neuronal biology and injury in PLWH. iNs retain age- and disease-related features of the donors, providing unique opportunities to reveal novel aspects of neurological disorders. We obtained primary dermal fibroblasts from six virologically suppressed PLWH (range: 27 - 64 years, median: 53); 83% Male; 50% White) and seven matched people without HIV (PWOH) (range: 27 - 66, median: 55); 71% Male; 57% White). iNs were generated using transcription factors NGN2 and ASCL1, and validated by immunocytochemistry and single-cell-RNAseq. Transcriptomic analysis using bulk-RNAseq identified 29 significantly differentially expressed genes between iNs from PLWH and PWOH. Of these, 16 genes were downregulated and 13 upregulated in PLWH iNs. Protein-protein interaction network mapping indicates that iNs from PLWH exhibit differences in extracellular matrix organization and synaptic transmission. IFI27 was upregulated in iNs from PLWH, which complements independent post-mortem studies demonstrating elevated IFI27 expression in PLWH-derived brain tissue, indicating that iN generation reconstitutes this pathway. Finally, we observed that expression of the FOXL2NB-FOXL2-LINC01391 genome locus is reduced in iNs from PLWH and negatively correlates with neurocognitive impairment. Thus, we have identified an iN gene signature of HIV through direct reprogramming of skin fibroblasts into neurons revealing novel mechanisms of neurocognitive impairment in PLWH.

Keywords: HAND; HIV; HIV-related neurocognitive impairment; IFI27; aging; directly converted neurons; iNs; induced neurons; neurodegenerative disease; transdifferentiation.

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Figures

Fig. 1
Fig. 1. Transdifferentiation of skin fibroblasts derived from people living with HIV and controls generates induced neurons.
(A) Scheme illustrating study outline. (B) Important participant information divided into people living with HIV-1 (PLWH) and without HIV-1 (PWOH). (C) Age distribution of the two groups of the study cohort. Statistical significance tested with unpaired, two-tailed t-test. Data presented as individual data points with mean ± SD and p-value. (D) UMAP plot showing the sample origin of each data point during scRNA analysis. (E) Gene expression patterns of fibroblast marker gene COL1A1 (a) and neuronal marker gene TUBB3 (b). (F) Percentage of cells from scRNA analysis depicted in (D-E) that express the annotated neuronal and fibroblast marker genes. Data presented as individual data points with mean ± SD. (G) Microscopic image of dermal fibroblasts before transdifferentiation. (H) Microscopic image after immunocytochemistry of induced neurons (iNs) 3-days post-FACS and stained for TUBB3 (TUJ1), MAP2 and nuclei (DAPI). Single channel images are provided in Fig. S1F (G-H) Scale bars are 20 μm. (I) MA plot based on bulk-RNA sequencing showing differential gene expression of all iNs samples compared to UNA fibroblasts. (J) PCA plot showing clustering of iNs- and UNA fibroblast-derived RNA samples after bulk-analysis. (K) Top 5 ranked gene ontology (GO) terms of Biological Processes (a-b) and Cellular Component (c-d) associated with the significantly up- (a and c) and downregulated genes (b and d) in iNs compared to UNA fibroblasts.
Fig. 2
Fig. 2. PLWH-derived iNs exhibit statistically significant differentially expressed genes compared to iNs from PWOH
(A) Volcano plot showing the 29 statistically significant (p-adj. < 0.05, log2fc > +/− 0.5) differentially expressed genes (DEGs) in PLWH-derived iNs compared to PWOH-derived iNs following bulk-RNA analysis iNs. Y-axis plots the p-adj. values and the dotted line indicates the selected cut-off of p-adj. < 0.05. (B) Heatmap showing the clustering of PLWH- vs. PWOH-derived iNs RNA samples based on expression levels of the 29 DEGs while displaying the log2 fold change and mean expression. (C) Venn diagram showing the numbers of DEGs between the PLWH- and PWOH-derived samples for the UNA fibroblasts and iNs. The gene symbols of the six genes that are differentially expressed in both cell types are displayed in the grey box. (D-F) Gene expression levels in PLWH- vs. PWOH-derived iNs (a) and STRING protein association networks (b) of DOC2B (D), RPH3AL (E), and SORCS1 (F)(33, 34). (G-I) Gene expression levels in PLWH- vs. PWOH-derived iNs of DPP6 (G), COL23A1 (H), and FSTL5 (I). (J) Gene expression levels in PLWH- vs. PWOH-derived iNs and UNA fibroblasts of FOXL2NB. (D-J). Data presented as individual data points with mean ± SD and p-adj-value derived from the conducted Wald test corrected for false discovery rates (FDR) using the Benjamini-Hochberg method (see methods section). (K) Correlation of gene expression levels of FOXL2, FOXL2NB, and LINC01391 with neurocognitive impairment in the PLWH study group (n =6). Data points are individual values and lines depict linear regression functions.
Fig. 3
Fig. 3. IFI27 expression levels are increased in PLWH-derived iNs and post-mortem brain tissue samples compared to PWOH-derived samples
(A) IFI27 gene expression levels in PLWH- vs. PWOH-derived iNs (a) and post-mortem brain tissue samples (b-d). (B) STRING protein association network of IFI27(33, 34). (C) Scheme illustrating the concept of type I and II neurocognitive impairment in PLWH according to Gelman et al. together with the associated IFI27 expression (11) (D) IFI27 gene expression levels in PLWH IFI27high vs. IFI27low iNs (a) and type I vs. type II PLWH-derived post-mortem brain tissue samples (b-d). (E) Volcano plot showing the statistically significant (p-adj. < 0.05, log2fc > +/− 0.5) differentially expressed genes (DEGs) in PLWH IFI27high vs. IFI27low iNs based on our bulk-RNA analysis. (F) Top 2 ranked gene ontology (GO) terms of Biological Processes associated with the significantly up- (a) and downregulated genes (b) in PLWH IFI27high vs. IFI27low iNs. (G) CD4+ T cell counts in PLWH divided into IFI27high vs. IFI27low participants. Statistical significance tested with unpaired, two-tailed t-test (A b-d, D b-d, G) or derived from the conducted Wald test corrected for false discovery rates (FDR) using the Benjamini-Hochberg method on whole-transcriptome data (A a, D a). Data presented as individual data points with mean ± SD. IFI27 expression values in post-mortem brain tissue samples derived from Gelman et al.(11).
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