A pilot investigation of the association between HIV-1 Vpr amino acid sequence diversity and the tryptophan-kynurenine pathway as a potential mechanism for neurocognitive impairment.

HIV infection compromises both the peripheral and central immune systems due to its pathogenic and neuropathogenic features. The mechanisms driving HIV-1 pathogenesis and neuropathogenesis involve a series of events, including metabolic dysregulation. Furthermore, HIV-subtype-specific variations, particularly alterations in the amino acid sequences of key viral proteins, are known to influence the severity of clinical outcomes in people living with HIV. However, the impact of amino acid sequence variations in specific viral proteins, such as Viral protein R (Vpr), on metabolites within the Tryptophan (Trp)-kynurenine (Kyn) pathway in people living with HIV remains unclear. Our research aimed to explore the relationship between variations in the Vpr amino acid sequence (specifically at positions 22, 41, 45, and 55, as these have been previously linked to neurocognitive function) and peripheral Trp-Kyn metabolites. Additionally, we sought to clarify the systems biology of Vpr sequence variation by examining the link between Trp-Kyn metabolism and peripheral inflammation, as a neuropathogenic mechanism. In this preliminary study, we analyzed a unique cohort of thirty-two (n = 32) South African cART naïve people living with HIV. We employed Sanger sequencing to ascertain blood-derived Vpr amino acid sequence variations and a targeted LC-MS/MS metabolomics platform to assess Trp-Kyn metabolites, such as Trp, Kyn, kynurenic acid (KA), and quinolinic acid (QUIN). Particle-enhanced turbidimetric assay and Enzyme-linked immunosorbent assays were used to measure immune markers, hsCRP, IL-6, suPAR, NGAL and sCD163. After applying Bonferroni corrections (p =.05/3) and adjusting for covariates (age and sex), only the Vpr G41 and A55 groups was nearing significance for higher levels of QUIN compared to the Vpr S41 and T55 groups, respectively (all p =.023). Multiple regression results revealed that Vpr amino acid variations at position 41 (adj R2 = 0.049, ß = 0.505; p =.023), and 55 (adj R2 = 0.126, ß = 0.444; p =.023) displayed significant associations with QUIN after adjusting for age and sex. Lastly, the higher QUIN levels observed in the Vpr G41 group were found to be correlated with suPAR (r =.588, p =.005). These results collectively underscore the importance of specific Vpr amino acid substitutions in influencing QUIN and inflammation (specifically suPAR levels), potentially contributing to our understanding of their roles in the pathogenesis and neuropathogenesis of HIV-1.

Ndufs4 KO mice: A model to study comorbid mood disorders associated with mitochondrial dysfunction.

The Ndufs4 knockout (KO) mouse is a validated and robust preclinical model of mitochondrial diseases (specifically Leigh syndrome), that displays a narrow window of relative phenotypical normality, despite its inherent mitochondrial complex I dysfunction and severe phenotype. Preclinical observations related to psychiatric comorbidities that arise in patients with mitochondrial diseases and indeed in Leigh syndrome are, however, yet to be investigated in this model. Strengthening this narrative is the fact that major depression and bipolar disorder are known to present with deficits in mitochondrial function. We therefore screened the behavioural profile of male and female Ndufs4 KO mice (relative to heterozygous; HET and wildtype; WT mice) between postnatal days 28 and 35 for locomotor, depressive- and anxiety-like alterations and linked it with selected brain biomarkers, viz. serotonin, kynurenine, and redox status in brain areas relevant to psychiatric pathologies (i.e., prefrontal cortex, hippocampus, and striatum). The Ndufs4 KO mice initially displayed depressive-like behaviour in the tail suspension test on PND31 but not on PND35 in the forced swim test. In the mirror box test, increased risk resilience was observed. Serotonin levels of KO mice, compared to HET controls, were increased on PND36, together with increased tryptophan to serotonin and kynurenine turnover. Kynurenine to kynurenic acid turnover was however decreased, while reduced versus oxidized glutathione ratio (GSH/GSSG) was increased. When considering the comorbid psychiatric traits of patients with mitochondrial disorders, this work elaborates on the neuropsychiatric profile of the Ndufs KO mouse. Secondly, despite locomotor differences, Ndufs4 KO mice present with a behavioural profile not unlike rodent models of bipolar disorder, namely variable mood states and risk-taking behaviour. The model may elucidate the bio-energetic mechanisms underlying mood disorders, especially in the presence of mitochondrial disease. Studies are however required to further validate the model's translational relevance.

Running from depression: the antidepressant-like potential of prenatal and pre-pubertal exercise in adolescent FSL rats exposed to an early-life stressor.

OBJECTIVE: We aimed to answer the questions of whether early-life (perinatal and/or juvenile) exercise can induce antidepressant-like effects in a validated rodent model of depression, and whether such early-life intervention could prevent or reverse the adverse effects of early-life stress in their offspring. METHODS: Male and female Flinders sensitive line rats born to a dam that exercised during gestation, or not, were either maternally separated between PND02 and 16 and weaned on PND17 or not. Half of these animals then underwent a fourteen-day low-intensity exercise regimen from PND22. Baseline depressive-like behaviour was assessed on PND21 and then reassessed on PND36, whereafter hippocampal monoamine levels, redox state markers and metabolic markers relevant to mitochondrial function were measured. RESULTS: Pre-pubertal exercise was identified as the largest contributing factor to the observed effects, where it decreased immobility time in the FST by 6%, increased time spent in the open arms of the EPM by 9%. Hippocampal serotonin and norepinephrine levels were also increased by 35% and 26%, respectively, whilst nicotinic acid was significantly decreased. CONCLUSION: These findings suggest that pre-pubertal low-intensity exercise induces beneficial biological alterations that could translate into antidepressant behaviour in genetically susceptible individuals.

Genetically predisposed and resilient animal models of depression reveal divergent responses to early-life adversity.

OBJECTIVE: Early-life adversity (ELA) is one of the strongest predictors of childhood depression that may be exacerbated by a genetic predisposition to develop depression. We therefore investigated the bio-behavioural effects of an early-life stressor in an accepted rodent model of depression. METHODS: The Flinders sensitive line (FSL) and resistant line (FRL) rats were subjected to an early-life stressor, whereafter their bio-behavioural response during pubertal onset was evaluated. Male and female pups were maternally separated for 3 h per day from postnatal day 02 (PND02) to 17, when they were also weaned. Control animals were left undisturbed, until weaning on PND21. Depressive-like behaviour was analysed on PND21 and reassessed on PND36. Hippocampal monoamine levels, markers of oxidative stress and metabolic markers implicating mitochondrial function were also measured. RESULTS: On PND21, the non-maternal separation and early weaning (non-MSEW) FSL rats spent 10% more time mobile than their FRL controls in the tail suspension test (TST) yet displayed increased depressive-like behaviour in the forced swim test (FST) on PND36. This depressive-like behaviour coincided with increased hippocampal norepinephrine levels, serotonin turnover and a dysfunctional redox state. Maternal separation and early weaning (MSEW) appeared to initially reduce early-life (PND21) depressive-like behaviour in the TST but then induced depressive-like behaviour on PND36 and increased norepinephrine levels more profoundly in the FRL rats. CONCLUSION: These findings highlight the need to further investigate the stress response pathway in these animals and that the absence or presence of genetic susceptibility may influence the presentation of ELA effects.

Phenylalanine metabolism and tetrahydrobiopterin bio-availability in COVID-19 and HIV.

Various metabolomics studies have reported increased phenylalanine serum concentrations in SARS-CoV-2 positive cases and have correlated increased phenylalanine with COVID-19 severity. In this study, we report similar results based upon metabolomics analysis of serum collected from a South African cohort of adults with confirmed COVID-19. The novelty of this study is the inclusion of HIV positive cases in the African context. We found that pre-existing HIV co-infection exacerbates the disruption of phenylalanine metabolism in COVID-19. What is lacking in literature is biological context and deeper understanding of perturbed phenylalanine metabolism in COVID-19. We delve deep into the metabolism of phenylalanine in COVID-19 and posit new insights for COVID-19 cases co-infected with HIV; namely, HIV-COVID-19 co-infected individuals do not have sufficient bioavailability of tetrahydrobiopterin (BH4). Hence, we identify BH4 as a potential supplement to alleviate/lessen COVID-19 symptoms.

Reviewing the mitochondrial dysfunction paradigm in rodent models as platforms for neuropsychiatric disease research.

Neuropsychiatric disorders have complex pathophysiological constructs, requiring translational models to improve our understanding thereof. Mitochondrial dysfunction, generally associated with neurodegenerative disorders, is gaining interest as a key factor in the etiology of psychiatric conditions because of the often comorbid psychiatric symptoms observed. Although translational models of psychiatric disorders, support mitochondrial involvement, these models do not have a dysfunctional bio-energetic system as primary construct. Here, we consider the construct, face, and predictive validity of mitochondrial models from a neuropsychiatric perspective, to identify novel animal models that can improve our understanding of the underlying bio-energetic mechanisms of these conditions and treatments.

Elucidating the Antimycobacterial Mechanism of Action of Decoquinate Derivative RMB041 Using Metabolomics.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), still remains one of the leading causes of death from a single infectious agent worldwide. The high prevalence of this disease is mostly ascribed to the rapid development of drug resistance to the current anti-TB drugs, exacerbated by lack of patient adherence due to drug toxicity. The aforementioned highlights the urgent need for new anti-TB compounds with different antimycobacterial mechanisms of action to those currently being used. An N-alkyl quinolone; decoquinate derivative RMB041, has recently shown promising antimicrobial activity against Mtb, while also exhibiting low cytotoxicity and excellent pharmacokinetic characteristics. Its exact mechanism of action, however, is still unknown. Considering this, we used GCxGC-TOFMS and well described metabolomic approaches to analyze and compare the metabolic alterations of Mtb treated with decoquinate derivative RMB041 by comparison to non-treated Mtb controls. The most significantly altered pathways in Mtb treated with this drug include fatty acid metabolism, amino acid metabolism, glycerol metabolism, and the urea cycle. These changes support previous findings suggesting this drug acts primarily on the cell wall and secondarily on the DNA metabolism of Mtb. Additionally, we identified metabolic changes suggesting inhibition of protein synthesis and a state of dormancy.

Elucidating the Antimycobacterial Mechanism of Action of Ciprofloxacin Using Metabolomics.

In the interest of developing more effective and safer anti-tuberculosis drugs, we used a GCxGC-TOF-MS metabolomics research approach to investigate and compare the metabolic profiles of Mtb in the presence and absence of ciprofloxacin. The metabolites that best describe the differences between the compared groups were identified as markers characterizing the changes induced by ciprofloxacin. Malic acid was ranked as the most significantly altered metabolite marker induced by ciprofloxacin, indicative of an inhibition of the tricarboxylic acid (TCA) and glyoxylate cycle of Mtb. The altered fatty acid, myo-inositol, and triacylglycerol metabolism seen in this group supports previous observations of ciprofloxacin action on the Mtb cell wall. Furthermore, the altered pentose phosphate intermediates, glycerol metabolism markers, glucose accumulation, as well as the reduction in the glucogenic amino acids specifically, indicate a flux toward DNA (as well as cell wall) repair, also supporting previous findings of DNA damage caused by ciprofloxacin. This study further provides insights useful for designing network whole-system strategies for the identification of possible modes of action of various drugs and possibly adaptations by Mtb resulting in resistance.

DNA Methylation Associated with Mitochondrial Dysfunction in a South African Autism Spectrum Disorder Cohort.

Autism spectrum disorder (ASD) is characterized by phenotypic heterogeneity and a complex genetic architecture which includes distinctive epigenetic patterns. We report differential DNA methylation patterns associated with ASD in South African children. An exploratory whole-epigenome methylation screen using the Illumina 450 K MethylationArray identified differentially methylated CpG sites between ASD and controls that mapped to 898 genes (P ≤ 0.05) which were enriched for nine canonical pathways converging on mitochondrial metabolism and protein ubiquitination. Targeted Next Generation Bisulfite Sequencing of 27 genes confirmed differential methylation between ASD and control in our cohort. DNA pyrosequencing of two of these genes, the mitochondrial enzyme Propionyl-CoA Carboxylase subunit Beta (PCCB) and Protocadherin Alpha 12 (PCDHA12), revealed a wide range of methylation levels (9-49% and 0-54%, respectively) in both ASD and controls. Three CpG loci were differentially methylated in PCCB (P ≤ 0.05), while PCDHA12, previously linked to ASD, had two significantly different CpG sites (P ≤ 0.001) between ASD and control. Differentially methylated CpGs were hypomethylated in ASD. Metabolomic analysis of urinary organic acids revealed that three metabolites, 3-hydroxy-3-methylglutaric acid (P = 0.008), 3-methyglutaconic acid (P = 0.018), and ethylmalonic acid (P = 0.043) were significantly elevated in individuals with ASD. These metabolites are directly linked to mitochondrial respiratory chain disorders, with a putative link to PCCB, consistent with impaired mitochondrial function. Our data support an association between DNA methylation and mitochondrial dysfunction in the etiology of ASD. Autism Res 2020, 13: 1079-1093. © 2020 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals, Inc. LAY SUMMARY: Epigenetic changes are chemical modifications of DNA which can change gene function. DNA methylation, a type of epigenetic modification, is linked to autism. We examined DNA methylation in South African children with autism and identified mitochondrial genes associated with autism. Mitochondria are power-suppliers in cells and mitochondrial genes are essential to metabolism and energy production, which are important for brain cells during development. Our findings suggest that some individuals with ASD also have mitochondrial dysfunction.

Metabolomics of Ndufs4-/- skeletal muscle: Adaptive mechanisms converge at the ubiquinone-cycle.

Leigh syndrome is one of the most common childhood-onset neurometabolic disorders resulting from a primary oxidative phosphorylation dysfunction and affecting mostly brain tissues. Ndufs4-/- mice have been widely used to study the neurological responses in this syndrome, however the reason why these animals do not display strong muscle involvement remains elusive. We combined biochemical strategies and multi-platform metabolomics to gain insight into the metabolism of both glycolytic (white quadriceps) and oxidative (soleus) skeletal muscles from Ndufs4-/- mice. Enzyme assays confirmed severely reduced (80%) CI activity in both Ndufs4-/- muscle types, compared to WTs. No significant alterations were evident in other respiratory chain enzyme activities; however, Ndufs4-/- solei displayed moderate decreases in citrate synthase (12%) and CIII (18%) activities. Through hypothesis-generating metabolic profiling, we provide the first evidence of adaptive responses to CI dysfunction involving non-classical pathways fueling the ubiquinone (Q) cycle. We report a respective 48 and 34 discriminatory metabolites between Ndufs4-/- and WT white quadriceps and soleus muscles, among which the most prominent alterations indicate the involvement of the glycerol-3-phosphate shuttle, electron transfer flavoprotein system, CII, and proline cycle in fueling the Q cycle. By restoring the electron flux to CIII via the Q cycle, these adaptive mechanisms could maintain adequate oxidative ATP production, despite CI deficiency. Taken together, our results shed light on the underlying pathogenic mechanisms of CI dysfunction in skeletal muscle. Upon further investigation, these pathways could provide novel targets for therapeutic intervention in CI deficiency and potentially lead to the development of new treatment strategies.