Brain transcriptomic, metabolic and mitohormesis properties associated with N-propargylglycine treatment: A prevention strategy against neurodegeneration.

INTRODUCTION: There is an urgent need for new or repurposed therapeutics that protect against or significantly delay the clinical progression of neurodegenerative diseases, such as Huntington's disease (HD), Parkinson's disease and Alzheimer's disease. In particular, preclinical studies are needed for well tolerated and brain-penetrating small molecules capable of mitigating the proteotoxic mitochondrial processes that are hallmarks of these diseases. We identified a unique suicide inhibitor of mitochondrial proline dehydrogenase (Prodh), N-propargylglycine (N-PPG), which has anticancer and brain-enhancing mitohormesis properties, and we hypothesize that induction of mitohormesis by N-PPG protects against neurodegenerative diseases. We carried out a series of mouse studies designed to: i) compare brain and metabolic responses while on oral N-PPG treatment (50 mg/kg, 9-14 days) of B6CBA wildtype (WT) and short-lived transgenic R6/2 (HD) mice; and ii) evaluate potential brain and systemwide stress rebound responses in WT mice 2 months after cessation of extended mitohormesis induction by well-tolerated higher doses of N-PPG (100-200 mg/kg x 60 days). WT and HD mice showed comparable global evidence of N-PPG induced brain mitohormesis characterized by Prodh protein decay and increased mitochondrial expression of chaperone and Yme1l1 protease proteins. Interestingly, transcriptional analysis (RNAseq) showed partial normalization of HD whole brain transcriptomes toward those of WT mice. Comprehensive metabolomic profiles performed on control and N-PPG treated blood, brain, and kidney samples revealed expected N-PPG-induced tissue increases in proline levels in both WT and HD mice, accompanied by surprising parallel increases in hydroxyproline and sarcosine. Two months after cessation of the higher dose N-PPG stress treatments, WT mouse brains showed robust rebound increases in Prodh protein levels and mitochondrial transcriptome responses, as well as altered profiles of blood amino acid-related metabolites. Our HD and WT mouse preclinical findings point to the brain penetrating and mitohormesis-inducing potential of the drug candidate, N-PPG, and provide new rationale and application insights supporting its further preclinical testing in various models of neurodegenerative diseases characterized by loss of mitochondrial proteostasis.

Proteomic Analysis of Huntington's Disease Medium Spiny Neurons Identifies Alterations in Lipid Droplets.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin (HTT) gene. The resulting polyglutamine (polyQ) tract alters the function of the HTT protein. Although HTT is expressed in different tissues, the medium-spiny projection neurons (MSNs) in the striatum are particularly vulnerable in HD. Thus, we sought to define the proteome of human HD patient-derived MSNs. We differentiated HD72-induced pluripotent stem cells and isogenic controls into MSNs and carried out quantitative proteomic analysis. Using data-dependent acquisitions with FAIMS for label-free quantification on the Orbitrap Lumos mass spectrometer, we identified 6323 proteins with at least two unique peptides. Of these, 901 proteins were altered significantly more in the HD72-MSNs than in isogenic controls. Functional enrichment analysis of upregulated proteins demonstrated extracellular matrix and DNA signaling (DNA replication pathway, double-strand break repair, G1/S transition) with the highest significance. Conversely, processes associated with the downregulated proteins included neurogenesis-axogenesis, the brain-derived neurotrophic factor-signaling pathway, Ephrin-A:EphA pathway, regulation of synaptic plasticity, triglyceride homeostasis cholesterol, plasmid lipoprotein particle immune response, interferon-γ signaling, immune system major histocompatibility complex, lipid metabolism, and cellular response to stimulus. Moreover, proteins involved in the formation and maintenance of axons, dendrites, and synapses (e.g., septin protein members) were dysregulated in HD72-MSNs. Importantly, lipid metabolism pathways were altered, and using quantitative image analysis, we found that lipid droplets accumulated in the HD72-MSN, suggesting a deficit in the turnover of lipids possibly through lipophagy. Our proteomics analysis of HD72-MSNs identified relevant pathways that are altered in MSNs and confirm current and new therapeutic targets for HD.

N-Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties.

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood-brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p < 0.05). Given this in vivo evidence of brain bioavailability and neural mitohormesis induction, N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

Modulating FKBP5/FKBP51 and autophagy lowers HTT (huntingtin) levels.

Current disease-modifying therapies for Huntington disease (HD) focus on lowering mutant HTT (huntingtin; mHTT) levels, and the immunosuppressant drug rapamycin is an intriguing therapeutic for aging and neurological disorders. Rapamycin interacts with FKBP1A/FKBP12 and FKBP5/FKBP51, inhibiting the MTORC1 complex and increasing cellular clearance mechanisms. Whether the levels of FKBP (FK506 binding protein) family members are altered in HD models and if these proteins are potential therapeutic targets for HD have not been investigated. Here, we found levels of FKBP5 are significantly reduced in HD R6/2 and zQ175 mouse models and human HD isogenic neural stem cells and medium spiny neurons derived from induced pluripotent stem cells. Moreover, FKBP5 interacts and colocalizes with HTT in the striatum and cortex of zQ175 mice and controls. Importantly, when we decreased FKBP5 levels or activity by genetic or pharmacological approaches, we observed reduced levels of mHTT in our isogenic human HD stem cell model. Decreasing FKBP5 levels by siRNA or pharmacological inhibition increased LC3-II levels and macroautophagic/autophagic flux, suggesting autophagic cellular clearance mechanisms are responsible for mHTT lowering. Unlike rapamycin, the effect of pharmacological inhibition with SAFit2, an inhibitor of FKBP5, is MTOR independent. Further, in vivo treatment for 2 weeks with SAFit2, results in reduced HTT levels in both HD R6/2 and zQ175 mouse models. Our studies establish FKBP5 as a protein involved in the pathogenesis of HD and identify FKBP5 as a potential therapeutic target for HD.Abbreviations : ACTB/ß-actin: actin beta; AD: Alzheimer disease; BafA1: bafilomycin A1; BCA: bicinchoninic acid; BBB: blood brain barrier; BSA: bovine serum albumin; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FKBPs: FK506 binding proteins; HD: Huntington disease; HTT: huntingtin; iPSC: induced pluripotent stem cells; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MES: 2-ethanesulfonic acid; MOPS: 3-(N-morphorlino)propanesulfonic acid); MSN: medium spiny neurons; mHTT: mutant huntingtin; MTOR: mechanistic target of rapamycin kinase; NSC: neural stem cells; ON: overnight; PD: Parkinson disease; PPIase: peptidyl-prolyl cis/trans-isomerases; polyQ: polyglutamine; PPP1R1B/DARPP-32: protein phosphatase 1 regulatory inhibitor subunit 1B; PTSD: post-traumatic stress disorder; RT: room temperature; SQSTM1/p62: sequestosome 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST:Tris-buffered saline, 0.1% Tween 20; TUBA: tubulin; ULK1: unc-51 like autophagy activating kinase 1; VCL: vinculin; WT: littermate controls.

FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16INK4a increase.

Neurodegenerative diseases (ND) have been linked to the critical process in aging-cellular senescence. However, the temporal dynamics of cellular senescence in ND conditions is unresolved. Here, we show senescence features develop in human Huntington's disease (HD) neural stem cells (NSCs) and medium spiny neurons (MSNs), including the increase of p16INK4a , a key inducer of cellular senescence. We found that HD NSCs reprogram the transcriptional targets of FOXO3, a major cell survival factor able to repress cell senescence, antagonizing p16INK4a expression via the FOXO3 repression of the transcriptional modulator ETS2. Additionally, p16INK4a promotes cellular senescence features in human HD NSCs and MSNs. These findings suggest that cellular senescence may develop during neuronal differentiation in HD and that the FOXO3-ETS2-p16INK4a axis may be part of molecular responses aimed at mitigating this phenomenon. Our studies identify neuronal differentiation with accelerated aging of neural progenitors and neurons as an alteration that could be linked to NDs.