We hypothesized that competition between NRTI-triphosphate and endogenous deoxyribonucleoside triphosphate (dNTP) may lead to depletion of dNTP pools and mitochondrial dysfunction independent of Pol-γ inhibition. We collected peripheral blood mononuclear cells from 75 adults (25 cases: HIV-infected with mitochondrial toxicity, 25 HIV-infected positive controls, and 25 HIV-negative controls). We observed statistically significant individual and group differences in ribonucleotide (RN) and deoxyribonucleotide (dRN) pools. The median RN pool was 10062 (IQR, 7090 – 12590), 4360 (IQR, 3058 –6838), and 2968 (IQR, 2538 – 4436) pmol/106 cells for negative controls, positive controls, and cases, respectively. Cases had significantly higher absolute mtDNA copy number compared to negative controls (p<0.05). Cases had significantly higher expression of Pol-γ, nucleoside transporters, cellular kinases, and ABC compared to controls. Antiretroviral therapy perturbs ribonucleotide and deoxyribonucleotide pools. Depletion of RN and dRN pools may be associated with ART-induced mitochondrial toxicity independent of Pol-γ inhibition.

SUMMARY Adult mammalian central nervous system (CNS) trauma interrupts neural networks and, because axonal regeneration is minimal, neurological deficits persist. Repair via axonal growth is limited by extracellular inhibitors and cell-autonomous factors. Based on results from a screen in vitro , we evaluate nearly 400 genes through a large-scale in vivo regeneration screen. Suppression of 40 genes using viral-driven short hairpin RNAs (shRNAs) promotes retinal ganglion cell (RGC) axon regeneration after optic nerve crush (ONC), and most are validated by separate CRISPR-Cas9 editing experiments. Expression of these axon-regeneration-suppressing genes is not significantly altered by axotomy. Among regeneration-limiting genes, loss of the interleukin 22 (IL-22) cytokine allows an early, yet transient, inflammatory response in the retina after injury. Reduced IL-22 drives concurrent activation of signal transducer and activator of transcription 3 (Stat3) and dual leucine zipper kinase (DLK) pathways and upregulation of multiple neuron-intrinsic regeneration-associated genes (RAGs). Including IL-22, our screen identifies dozens of genes that limit CNS regeneration. Suppression of these genes in the context of axonal damage could support improved neural repair.

ObjectivesWe recently observed a decrease in deoxyribonucleotide (dNTP) pools in HIV‐infected individuals on antiretroviral therapy (ART). Alterations in dNTPs result in mutations in mitochondrial DNA (mtDNA) in cell culture and animal models. Therefore, we investigated whether ART is associated with mitochondrial genome sequence variation in peripheral blood mononuclear cells (PBMCs) of HIV‐infected treatment‐experienced individuals.MethodsIn this substudy of a case−control study, 71 participants were included: 22 ‘cases’, who were HIV‐infected treatment‐experienced patients with mitochondrial toxicity, 25 HIV‐infected treatment‐experienced patients without mitochondrial toxicity, and 24 HIV‐uninfected controls. Total DNA was extracted from PBMCs and purified polymerase chain reaction (PCR) products were subjected to third‐generation sequencing using the PacBio Single Molecule Real‐Time (SMRT) sequencing technology. The sequences were aligned against the revised Cambridge reference sequence for human mitochondrial DNA (NC_012920.1) for detection of variants.ResultsWe identified a total of 123 novel variants, 39 of them in the coding region. HIV‐infected treatment‐experienced patients with and without toxicity had significantly higher average numbers of mitochondrial variants per participant than HIV‐uninfected controls. We observed a higher burden of mtDNA large‐scale deletions in HIV‐infected treatment‐experienced patients with toxicity compared with HIV‐uninfected controls (P = 0.02). The frequency of mtDNA molecules containing a common deletion (mt.δ4977) was higher in HIV‐infected treatment‐experienced patients with toxicity compared with HIV‐uninfected controls (P = 0.06). There was no statistically significant difference in mtDNA variants between HIV‐infected treatment‐experienced patients with and without toxicity.ConclusionsThe frequency of mtDNA variants (mutations and large‐scale deletions) was higher in HIV‐infected treatment‐experienced patients with or without ART‐induced toxicity than in uninfected controls.

dWe found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-␥) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-␥ mutant binding affinity for dCTP is 8-fold less than that of the wild type. The R953C mutant shows a 4-fold decrease in discrimination of analog nucleotides relative to the wild type. R953 is located on the "O-helix" that forms the substrate deoxynucleoside triphosphate (dNTP) binding site; the interactions of R953 with E1056 and Y986 may stabilize the O-helix and affect polymerase activity.A ntiretroviral therapy (ART)-related toxicities predominantly manifest in mitochondrial dysfunction. A critical backbone of ART is nucleoside reverse transcriptase inhibitors (NRTIs). With widespread use of NRTIs, clinical manifestations such as lactic acidosis, lipodystrophy, peripheral neuropathies, cardiomyopathies, and pancytopenia were observed (1-3). These adverse effects of NRTIs were attributed to inhibition of the polymerase gamma enzyme (Pol-␥), responsible for mitochondrial DNA (mtDNA) replication (4). The role of mutant Pol-␥ variants in ART-related toxicity has not been systematically investigated. Only two Pol-␥ mutations (R964C and E1143G) have been associated with ART-induced mitochondrial toxicity (5, 6).We hypothesized that Pol-␥ mutations might predispose patients toward developing mitochondrial toxicity. We performed a retrospective analysis of data and specimens collected during a prospective, case-control study of ART-induced mitochondrial toxicity (i) to investigate whether Pol-␥ mutations are associated with ART-induced mitochondrial toxicity and (ii) to characterize the biochemical effect of these mutations, if any, on Pol-␥ activity. The details of the study design have been previously published (7,8). In brief, the cases comprised HIV-infected individuals identified by their HIV care providers as having symptoms consistent with ART-induced mitochondrial toxicity (2, 9). The study protocol was approved by the Institutional Review Board of the Yale School of Medicine. All participants gave their written informed consent before participation in the study.The study included 45 African Americans (15 HIV-infected individuals with mitochondrial toxicity [9], cases; 15 HIV-infected individuals without toxicity, positive controls; and 15 HIVuninfected individuals, negative controls). The demographic and clinical characteristics of participants are illustrated in Table S1 in the supplemental material. We amplified and sequenced the entire polymerase gamma (POLG) genome, comprising 22 exons, of the 45 study participants using 16 pairs of overlapping primers (see Table S2) and a previously described PCR protocol (10). We observed a heterozygous C2857T mutation in exon 18 (see Fig. S1 in the supplemental material) of the POLG catalytic active site, corresponding to a substitution of R953 in the wild type (WT) to cysteine, yielding mutant R953C (Fig. 1A), in one HIV-infected patient with mitochondrial toxicity and observed no mutations in the two control groups. The...

A case-control study of the effect of antiretroviral therapy (ART) on apoptosis pathway genes comprising 16 cases (HIV infected with mitochondrial toxicity) and 16 controls (HIV uninfected) was conducted. A total of 26 of 84 genes of the apoptosis pathway were differentially expressed. Two of the upregulated genes, DFFA and TNFRSF1A, classified 75% of study participants correctly as either a case or control. Thus, apoptosis may be in the causal pathway of ART-associated mitochondrial toxicity. These two genes could be markers for detecting and monitoring ARTinduced mitochondrial toxicity.