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Higher serum urate concentration is associated with decreased risk of Parkinson’s disease (PD) as well as slower disease progression, but its relationship with severity of PD remains unclear. This study investigated whether changes in serum urate concentration over 5 years were associated with disease progression assessed by MDS-UPDRS Part III score, Hoehn and Yahr stage, or DaTscan imaging. Average serum urate concentration was stable over time and change in serum urate concentration did not correlate with worsening of measures of PD progression. These results suggest that serum urate concentration is not a monitoring biomarker of PD progression in early stages.

ObjectiveTo identify markers of resistance to developing Parkinson disease (PD) among LRRK2 mutation carriers (LRRK2+), we carried out metabolomic profiling in individuals with PD and unaffected controls (UC), with and without the LRRK2 mutation.MethodsPlasma from 368 patients with PD and UC in the LRRK2 Cohort Consortium (LCC), comprising 118 LRRK2+/PD+, 115 LRRK2+/UC, 70 LRRK2−/PD+, and 65 LRRK2−/UC, and CSF available from 68 of them, were analyzed by liquid chromatography with mass spectrometry. For 282 analytes quantified in plasma and CSF, we assessed differences among the 4 groups and interactions between LRRK2 and PD status, using analysis of covariance models adjusted by age, study site cohort, and sex, with p value corrections for multiple comparisons.ResultsPlasma caffeine concentration was lower in patients with PD vs UC (p < 0.001), more so among LRRK2+ carriers (by 76%) than among LRRK2− participants (by 31%), with significant interaction between LRRK2 and PD status (p = 0.005). Similar results were found for caffeine metabolites (paraxanthine, theophylline, 1-methylxanthine) and a nonxanthine marker of coffee consumption (trigonelline) in plasma, and in the subset of corresponding CSF samples. Dietary caffeine was also lower in LRRK2+/PD+ compared to LRRK2+/UC with significant interaction effect with the LRRK2+ mutation (p < 0.001).ConclusionsMetabolomic analyses of the LCC samples identified caffeine, its demethylation metabolites, and trigonelline as prominent markers of resistance to PD linked to pathogenic LRRK2 mutations, more so than to idiopathic PD. Because these analytes are known both as correlates of coffee consumption and as neuroprotectants in animal PD models, the findings may reflect their avoidance by those predisposed to develop PD or their protective effects among LRRK2 mutation carriers.

Two purines, caffeine and urate, have been associated with a reduced risk of idiopathic Parkinson's disease (PD) in multiple cohorts and populations. The Harvard Biomarkers Study (HBS) is a longitudinal study designed to accelerate the discovery and validation of molecular diagnostic and progression markers of early-stage PD. To investigate whether these 'reduced risk' factors are associated with PD within this cohort, we conducted a cross-sectional, case-control study in 566 subjects consisting of idiopathic PD patients and healthy controls. Caffeine intake as assessed by a validated questionnaire was significantly lower in idiopathic PD patients compared to healthy controls in males (mean difference -125 mg/day, p < 0.001) but not in females (mean difference -30 mg/day, p = 0.29). A strong inverse association was also observed with plasma urate levels both in males (mean difference -0.46 mg/dL, p = 0.017) and females (mean difference -0.45 mg/dL, p = 0.001). Both analyses stratified for sex and adjusted for age, body mass index, and either urate level or caffeine consumption, respectively. These results highlight the robustness of caffeine intake and urate as factors inversely associated with idiopathic PD.

ObjectiveTo investigate whether women and men with Parkinson disease (PD) differ in their biochemical and clinical responses to long-term treatment with inosine.MethodsThe Safety of Urate Elevation in Parkinson’s Disease (SURE-PD) trial enrolled 75 people with early PD and baseline serum urate below 6 mg/dL and randomized them to 3 double-blinded treatment arms: oral placebo or inosine titrated to produce mild (6.1–7.0 mg/dL) or moderate (7.1–8.0 mg/dL) serum urate elevation for up to 2 years. Parkinsonism, serum urate, and plasma antioxidant capacity were measured at baseline and repeatedly on treatment; CSF urate was assessed once, at 3 months. Here in secondary analyses results are stratified by sex.ResultsInosine produced an absolute increase in average serum urate from baseline that was 50% greater in women (3.0 mg/dL) than in men (2.0 mg/dL), consistent with expected lower baseline levels in women. Similarly, only among women was CSF urate significantly greater on mild or moderate inosine (+87% [p < 0.001] and +98% [p < 0.001], respectively) than on placebo (in contrast to men: +10% [p = 0.6] and +14% [p = 0.4], respectively). Women in the higher inosine dosing group showed a 7.0 Unified Parkinson’s Disease Rating Scale (UPDRS) points/year lower rate of decline vs placebo (p = 0.01). In women, slower rates of UPDRS change were associated with greater increases in serum urate (r = −0.52; p = 0.001), and with greater increases in plasma antioxidant capacity (r = −0.44; p = 0.006). No significant associations were observed in men.ConclusionsInosine produced greater increases in serum and CSF urate in women compared to men in the SURE-PD trial, consistent with the study's design and with preliminary evidence for slower clinical decline in early PD among women treated with urate-elevating doses of inosine.Clinicaltrials.gov identifierNCT00833690.Classification of evidenceThis study provides Class II evidence that inosine produced greater urate elevation in women than men and may slow PD progression in women.

Objective To identify markers of resistance to developing Parkinson disease (PD) among LRRK2 mutation carriers (LRRK2+), we carried out metabolomic profiling in individuals with PD and unaffected controls (UC), with and without the LRRK2 mutation. Methods Plasma from 368 patients with PD and UC in the LRRK2 Cohort Consortium (LCC), comprising 118 LRRK2+/PD+, 115 LRRK2+/UC, 70 LRRK2−/PD+, and 65 LRRK2−/UC, and CSF available from 68 of them, were analyzed by liquid chromatography with mass spectrometry. For 282 analytes quantified in plasma and CSF, we assessed differences among the 4 groups and interactions between LRRK2 and PD status, using analysis of covariance models adjusted by age, study site cohort, and sex, with p value corrections for multiple comparisons. Results Plasma caffeine concentration was lower in patients with PD vs UC (p < 0.001), more so among LRRK2+ carriers (by 76%) than among LRRK2− participants (by 31%), with significant interaction between LRRK2 and PD status (p = 0.005). Similar results were found for caffeine metabolites (paraxanthine, theophylline, 1-methylxanthine) and a nonxanthine marker of coffee consumption (trigonelline) in plasma, and in the subset of corresponding CSF samples. Dietary caffeine was also lower in LRRK2+/PD+ compared to LRRK2+/UC with significant interaction effect with the LRRK2+ mutation (p < 0.001). Conclusions Metabolomic analyses of the LCC samples identified caffeine, its demethylation metabolites, and trigonelline as prominent markers of resistance to PD linked to pathogenic LRRK2 mutations, more so than to idiopathic PD. Because these analytes are known both as correlates of coffee consumption and as neuroprotectants in animal PD models, the findings may reflect their avoidance by those predisposed to develop PD or their protective effects among LRRK2 mutation carriers.

Objective To test the safety, tolerability, and urate‐elevating capability of the urate precursor inosine taken orally or by feeding tube in people with amyotrophic lateral sclerosis (ALS). Methods This was a pilot, open‐label trial in 25 participants with ALS. Treatment duration was 12 weeks. The dose of inosine was titrated at pre‐specified time points to elevate serum urate levels to 7–8 mg/dL. Primary outcomes were safety (as assessed by the occurrence of adverse events [AEs]) and tolerability (defined as the ability to complete the 12‐week study on study drug). Secondary outcomes included biomarkers of oxidative stress and damage. As an exploratory analysis, observed outcomes were compared with a virtual control arm built using prediction algorithms to estimate ALSFRS‐R scores. Results Twenty‐four out of 25 participants (96%) completed 12 weeks of study drug treatment. One participant was unable to comply with study visits and was lost to follow‐up. Serum urate rose to target levels in 6 weeks. No serious AEs attributed to study drug and no AEs of special concern, such as urolithiasis and gout, occurred. Selected biomarkers of oxidative stress and damage had significant changes during the study period. Observed changes in ALSFRS‐R did not differ from baseline predictions. Interpretation Inosine appeared safe, well tolerated, and effective in raising serum urate levels in people with ALS. These findings, together with epidemiological observations and preclinical data supporting a neuroprotective role of urate in ALS models, provide the rationale for larger clinical trials testing inosine as a potential disease‐modifying therapy for ALS.

The therapeutic value of inhibiting translation of the amyloid precursor protein (APP) offers the possibility to reduce neurotoxic amyloid formation, particularly in cases of familial Alzheimer’s disease (AD) caused by APP gene duplications (Dup⁻APP) and in aging Down syndrome individuals. APP mRNA translation inhibitors such as the anticholinesterase phenserine, and high throughput screened molecules, selectively inhibited the uniquely folded iron-response element (IRE) sequences in the 5’untranslated region (5’UTR) of APP mRNA and this class of drug continues to be tested in a clinical trial as an anti-amyloid treatment for AD. By contrast, in younger age groups, APP expression is not associated with amyloidosis, instead it acts solely as a neuroprotectant while facilitating cellular ferroportin-dependent iron efflux. We have reported that the environmental metallotoxins Lead (Pb) and manganese (Mn) cause neuronal death by interfering with IRE dependent translation of APP and ferritin. The loss of these iron homeostatic neuroprotectants thereby caused an embargo of iron (Fe) export from neurons as associated with excess unstored intracellular iron and the formation of toxic reactive oxidative species (ROS). We propose that APP 5’UTR directed translation activators can be employed therapeutically to protect neurons exposed to high acute Pb and/or Mn exposure. Certainly, high potency APP translation activators, exemplified by the Food and Drug Administration (FDA) pre-approved M1 muscarinic agonist AF102B and high throughput-screened APP 5’UTR translation activators, are available for drug development to treat acute toxicity caused by Pb/Mn exposure to neurons. We conclude that APP translation activators can be predicted to prevent acute metal toxicity to neurons by a mechanism related to the 5’UTR specific yohimbine which binds and targets the canonical IRE RNA stem loop as an H-ferritin translation activator.

Peroxisome proliferator-activated receptor γcoactivator 1α(PGC-1α) is a central regulator of cellular and mitochondrial metabolism. Cellular bioenergetics are critically important in “energy-guzzling” neurons, but the components and wiring of the transcriptional circuit through whichPGC-1αregulates the neuronal electron transport chain have not been established. This information may be vital for restoring neuronal bioenergetics gene expression that is compromised during incipient Parkinson’s neuropathology and in aging-dependent brain diseases. Here we delineate a neuronal transcriptional circuit controlled by endogenousPGC-1α. We show that a feed-forward circuit of endogenous neuronalPGC-1αand the orphan nuclear estrogen-related receptorα(ERRα) activates the nuclear-encoded mitochondrial electron transport chain.PGC-1αnot onlytrans-activated expression ofERRα, but also coactivatedERRαtarget genes in complexes I, II, IV, and V of the neuronal electron transport chain via association with evolutionary conservedERRαpromoter binding motifs. Chemical activation of this transcriptional program induced transcription of the neuronal electron transport chain. These data highlight a neuronal transcriptional circuit regulated byPGC-1αthat can be therapeutically targeted for Parkinson’s and other neurodegenerative diseases.

Urate has emerged as a promising target for neuroprotection based on epidemiological observations, preclinical models, and early clinical trial results in multiple neurologic diseases, including Parkinson's disease (PD). This study investigates the astrocytic mechanism of urate's neuroprotective effect. Targeted biochemical screens of conditioned medium from urate- versus vehicle-treated astrocytes identified markedly elevated glutathione (GSH) concentrations as a candidate mediator of urate's astrocyte-dependent neuroprotective effects. Urate treatment also induced the nuclear translocation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and transcriptional activation of its key target genes in primary astrocytic cultures. Urate's neuroprotective effect was attenuated when GSH was depleted in the conditioned media either by targeting its synthesis or release by astrocytes. Overall, these results implicate GSH as the extracellular astrocytic factor mediating the protective effect of urate in a cellular model of PD. These results also show that urate can employ a novel indirect neuroprotective mechanism via induction of the Nrf2 signaling pathway, a master regulator of the response to oxidative stress, in astrocytes.

Inosine (hypoxanthine 9-beta-D-ribofuranoside), a purine nucleoside with multiple intracellular roles, also serves as an extracellular modulatory signal. On neurons, it can produce anti-inflammatory and trophic effects that confer protection against toxic influences in vivo and in vitro. The protective effects of inosine treatment might also be mediated by its metabolite urate. Urate in fact possesses potent antioxidant properties and has been reported to be protective in preclinical Parkinson's disease (PD) studies and to be an inverse risk factor for both the development and progression of PD. In this study we assessed whether inosine might protect rodent MES 23.5 dopaminergic cell line from oxidative stress in a cellular model of PD, and whether its effects could be attributed to urate. MES 23.5 cells cultured alone or in presence of enriched murine astroglial cultures MES 23.5-astrocytes co-cultures were pretreated with inosine (0.1-100 μM) for 24 h before addition of the oxidative stress inducer H₂O₂ (200 μM). Twenty-four hours later, cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or immunocytochemistry in pure and MES 23.5-astrocytes co-cultures, respectively. H₂O₂-toxic effect on dopaminergic cells was reduced when they were cultured with astrocytes, but not when they were cultured alone. Moreover, in MES 23.5-astrocytes co-cultures, indicators of free radical generation and oxidative damage, evaluated by nitrite (NO₂(-)) release and protein carbonyl content, respectively, were attenuated. Conditioned medium experiments indicated that the protective effect of inosine relies on the release of a protective factor from inosine-stimulated astrocytes. Purine levels were measured in the cellular extract and conditioned medium using high-performance liquid chromatography (HPLC) method. Urate concentration was not significantly increased by inosine treatment however there was a significant increase in levels of other purine metabolites, such as adenosine, hypoxanthine and xanthine. In particular, in MES 23.5-astrocytes co-cultures, inosine medium content was reduced by 99% and hypoxanthine increased by 127-fold. Taken together these data raise the possibility that inosine might have a protective effect in PD that is independent of any effects mediated through its metabolite urate.

The acute myeloid leukemia 1 (AML1, RUNX1) transcription factor is a key regulator of hematopoietic differentiation that forms multi-protein complexes with co-regulatory proteins. These complexes are assembled at target gene promoters in nuclear microenvironments to mediate phenotypic gene expression and chromatin related epigenetic modifications. Here, immunofluorescence microscopy and biochemical assays are used to show that RUNX1 associates with the human ATP-dependent SWI/SNF chromatin remodeling complex. The SWI/SNF subunits BRG1 and INI1 bind in vivo to RUNX1 target gene promoters (e.g., GMCSF, IL3, MCSF-R, MIP and p21). These interactions correlate with histone modifications characteristic of active chromatin, including acetylated H4 and dimethylated H3 Lysine 4. Down-regulation of RUNX1 by RNA interference diminishes the binding of BRG1 and INI1 at selected target genes. Taken together, our findings indicate that RUNX1 interacts with the human SWI/SNF complex to control hematopoietic-specific gene expression.

Nuclear microenvironments are architecturally organized subnuclear sites where the regulatory machinery for gene expression, replication, and repair resides. This compartmentalization is necessary to attain required stoichiometry for organization and assembly of regulatory complexes for combinatorial control. Combined and methodical application of molecular, cellular, biochemical, and in vivo genetic approaches is required to fully understand complexities of biological control. Here we provide methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.

RUNX1/AML1 is required for definitive hematopoiesis and is frequently targeted by chromosomal translocations in acute myeloid leukemia (AML). The t(8;21)-related AML1-ETO fusion protein blocks differentiation of myeloid progenitors. Here, we show by immunofluorescence microscopy that during interphase, endogenous AML1-ETO localizes to nuclear microenvironments distinct from those containing native RUNX1/AML1 protein. At mitosis, we clearly detect binding of AML1-ETO to nucleolar-organizing regions in AML-derived Kasumi-1 cells and binding of RUNX1/AML1 to the same regions in Jurkat cells. Both RUNX1/AML1 and AML1-ETO occupy ribosomal DNA repeats during interphase, as well as interact with the endogenous RNA Pol I transcription factor UBF1. Promoter cytosine methylation analysis indicates that RUNX1/AML1 binds to rDNA repeats that are more highly CpG methylated than those bound by AML1-ETO. Downregulation by RNA interference reveals that RUNX1/AML1 negatively regulates rDNA transcription, whereas AML1-ETO is a positive regulator in Kasumi-1 cells. Taken together, our findings identify a novel role for the leukemia-related AML1-ETO protein in epigenetic control of cell growth through upregulation of ribosomal gene transcription mediated by RNA Pol I, consistent with the hyper-proliferative phenotype of myeloid cells in AML patients.