Mutations in the AAA+ ATPase p97 cause multisystem proteinopathy 1, which includes amyotrophic lateral sclerosis; however, the pathogenic mechanisms that contribute to motor neuron loss remain obscure. Here, we use two induced pluripotent stem cell models differentiated into spinal motor neurons to investigate how p97 mutations perturb the motor neuron proteome. Using quantitative proteomics, we find that motor neurons harboring the p97 R155H mutation have deficits in the selective autophagy of lysosomes (lysophagy). p97 R155H motor neurons are unable to clear damaged lysosomes and have reduced viability. Lysosomes in mutant motor neurons have increased pH compared with wild-type cells. The clearance of damaged lysosomes involves UBXD1-p97 interaction, which is disrupted in mutant motor neurons. Finally, inhibition of the ATPase activity of p97 using the inhibitor CB-5083 rescues lysophagy defects in mutant motor neurons. These results add to the evidence that endo-lysosomal dysfunction is a key aspect of disease pathogenesis in p97-related disorders.

Dopaminergic neurons of the substantia nigra exist in a persistent state of vulnerability resulting from high baseline oxidative stress, high-energy demand, and broad unmyelinated axonal arborisations. Impairments in the storage of dopamine compound this stress because of cytosolic reactions that transform the vital neurotransmitter into an endogenous neurotoxicant, and this toxicity is thought to contribute to the dopamine neuron degeneration that occurs Parkinson's disease. We have previously identified synaptic vesicle glycoprotein 2C (SV2C) as a modifier of vesicular dopamine function, demonstrating that genetic ablation of SV2C in mice results in decreased dopamine content and evoked dopamine release in the striatum. Here, we adapted a previously published in vitro assay utilising false fluorescent neurotransmitter 206 (FFN206) to visualise how SV2C regulates vesicular dopamine dynamics and determined that SV2C promotes the uptake and retention of FFN206 within vesicles. In addition, we present data indicating that SV2C enhances the retention of dopamine in the vesicular compartment with radiolabelled dopamine in vesicles isolated from immortalised cells and from mouse brain. Further, we demonstrate that SV2C enhances the ability of vesicles to store the neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) and that genetic ablation of SV2C results in enhanced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced vulnerability in mice. Together, these findings suggest that SV2C functions to enhance vesicular storage of dopamine and neurotoxicants and helps maintain the integrity of dopaminergic neurons.

Mitochondria morphology and function, and their quality control by mitophagy, are essential for heart function. We investigated whether these are influenced by time of the day (TOD), sex, and fed or fasting status, using transmission electron microscopy (EM), mitochondrial electron transport chain (ETC) activity, and mito-QC reporter mice. We observed peak mitochondrial number at ZT8 in the fed state, which was dependent on the intrinsic cardiac circadian clock, as hearts from cardiomyocyte-specific BMAL1 knockout (CBK) mice exhibit different TOD responses. In contrast to mitochondrial number, mitochondrial ETC activities do not fluctuate across TOD, but decrease immediately and significantly in response to fasting. Concurrent with the loss of ETC activities, ETC proteins were decreased with fasting, simultaneous with significant increases of mitophagy, mitochondrial antioxidant protein SOD2, and the fission protein DRP1. Fasting-induced mitophagy was lost in CBK mice, indicating a direct role of BMAL1 in regulating mitophagy. This is the first of its kind report to demonstrate the interactions between sex, fasting, and TOD on cardiac mitochondrial structure, function and mitophagy. These studies provide a foundation for future investigations of mitochondrial functional perturbation in aging and heart diseases.

The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of renal cell carcinoma-RCC (clear cell histology) and promotes tumor progression. L-2HG is structurally similar to α-ketoglutarate (α-KG). Therefore, L-2HG can competitively inhibit enzymes that utilize α-KG as a cofactor including α-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH), resulting in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line has revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. The findings were consistent in the patient samples where high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions than that of the low L-2HG renal tumors and the patient-matched normal kidneys. Consistently, 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2HGDH KO and wild-type (WT) mice revealed less serine content in the absence of L2HGDH, further confirming that high L-2HG suppresses serine biosynthesis in vivo. We found that L-2HG-mediated inhibition of the α-KG-dependent histone demethylase KDM4C silences ATF4 transcription. ATF4 is a master regulator of amino acid biosynthetic genes including PHGDH and PSAT1. Using ATF4 gain of function analysis, we confirmed that high L-2HG causes the suppression of PHGDH and PSAT1 in an ATF4-dependent manner. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N⁶-methyladenosine (m6A) via inhibiting α-KG-dependent RNA demethylases ALKBH5 and FTO. In the setting of high L-2HG, m6A is enriched in the 3’-UTR region of transcripts including PSAT1. Using mutational analysis, we demonstrate that L-2HG promotes m6A accumulation at a specific site within the 3’UTR of PSAT1 that silences its translation. In accord with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione in high L-2HG RCC which supports both proliferation and resistance to oxidative stress. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism through a bimodal mechanism via remodeling of both the epigenome and epitranscriptome. This results in a serine liability in the setting of raised L-2HG. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Citation Format: Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Hayley Widden, Michelle Johnson, Juan Liu, Yasaman Heidarian, Nader Mahmoudzadeh, Devin Absher, Han-Fei Ding, David Crosman, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmar, Jason Tennessen, Sunil Sudarshan. L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3705.

Table S1. List of all 94 Oncolead cell lines tested with corresponding IC50 values for NCI-737 and NCI-006.

Combined Supplementary Data file containing: Figure S1. Proliferation assays using NCI-737 and NCI-006 in an expanded panel of EWS cell lines. Figure S2. Effects of additional siRNA sequences against LDHA and LDHB in an expanded panel of EWS cell lines. Figure S3. Effects of additional siRNA sequences against EWS-FLI1 on LDHA and LDHB and additional ChIP-seq data. Figure S4. LDH expression, basal activity and in vitro early time course inhibition in EWS cell lines. Figure S5. Steady state levels of pyruvate and lactate and 13C-tracing experiments from 13C-U-glucose. Figure S6. Effects of NCI-737 and NCI-006 on ECAR measurements. Figure S7. Effects of NCI-737 on OCR measurements. Figure S8. Effects of NCI-737 on ECAR measurements of expanded panel of EWS cell lines. Figure S9. Results of oral dosing of LDH inhibitors in EWS xenograft models. Figure S10. Results of IV pilot studies of LDH inhibitors in EWS xenograft models. Figure S11. Additional growth rate and pharmacokinetic data for EWS xenograft models. Figure S12. Additional growth rate and necrosis data for EWS xenograft models. Figure S13. Depiction of of changes in lactate and pyruvate peaks and lactate/pyruvate ratio by hyperpolarized 13C-MR spectroscopy in EWS xenograft models. Figure S14. Measurements of toxicity in animals treated with NCI-737.

A description of additional supplemental methods

BackgroundEnfortumab vedotin (EV) is an antibody-drug conjugate approved for patients with treatment-refractory advanced urothelial carcinoma (aUC), however data on biomarkers of response is lacking.MethodsWe retrospectively identified all aUC patients at our institution who received EV monotherapy and had next-generation sequencing (NGS) data available. Patients were considered responders if they had a complete response or partial response on restaging scans during treatment. Observed response rate (ORR) was evaluated by local investigator and compared between responders and non-responders using Chi-squared test. A univariable analysis was conducted using the Cox proportional hazard test to assess for associations between baseline characteristics and most common somatic alterations (in ≥10% of patients) with patient survival outcomes [progression-free survival (PFS) and overall survival (OS)]. Somatic alterations were then individually evaluated in separate multivariate models while accounting for patient and clinical characteristics using Cox regression models.ResultsAmong 29 patients treated with EV monotherapy, 27 had available NGS data. Median age was 70, 24 (83%) were men, 19 (62%) were Caucasian, 15 (52%) had pure urothelial histology and 22 (76%) had primary tumor in the bladder. ORR was 41%, and PFS and OS for the overall cohort were 5.1 months and 10.2 months. Responders were enriched among patients with TP53, KDM6A and MDM2 alterations. Patients with these alterations, as well as those with composite TP53/MDM2 alterations (alterations in either TP53 or MDM2), also had increased ORR with EV treatment compared to patients without these alterations. In the univariable analysis, baseline albumin level ≥ 3.0g/dL and presence of composite TP53/MDM2 alterations were associated with a prolonged OS. Baseline ECOG 0/1, TP53 alterations and TP53/MDM2 alterations were associated with a prolonged PFS. In the multivariable analysis, TP53 and TP53/MDM2 alterations were genomic markers predictive of improved PFS after accounting for the relevant clinical characteristics.ConclusionIn this single-center retrospective analysis of aUC patients treated with EV, presence of TP53 or MDM2 somatic alterations, lower ECOG PS scores (ECOG 0 or 1) and higher albumin levels (≥3 g/dL) were associated with improved outcomes with EV treatment. Prospective and external validation of these findings in larger cohorts is warranted.

You have accessJournal of UrologyCME1 Apr 2023PD17-08 EPIGENETIC AND EPITRANSCRIPTOMIC REPROGRAMMING IN RENAL CANCER BY ONCOMETABOLITE L-2HG CREATES METABOLIC VULNERABILITY Garrett Brinkley, Anirban Kundu, Hyeyoung Nam, Suman Karki, Richard Kirkman, Hayley Widden, Michelle Johnson, Juan Liu, Yasaman Heidarian, Nader Mahmoudzadeh, Devin Absher, Han-Fei Ding, David Crosman, William Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmar, Jason Tennessen, and Sunil Sudarshan Garrett BrinkleyGarrett Brinkley More articles by this author , Anirban KunduAnirban Kundu More articles by this author , Hyeyoung NamHyeyoung Nam More articles by this author , Suman KarkiSuman Karki More articles by this author , Richard KirkmanRichard Kirkman More articles by this author , Hayley WiddenHayley Widden More articles by this author , Michelle JohnsonMichelle Johnson More articles by this author , Juan LiuJuan Liu More articles by this author , Yasaman HeidarianYasaman Heidarian More articles by this author , Nader MahmoudzadehNader Mahmoudzadeh More articles by this author , Devin AbsherDevin Absher More articles by this author , Han-Fei DingHan-Fei Ding More articles by this author , David CrosmanDavid Crosman More articles by this author , William PlaczekWilliam Placzek More articles by this author , Jason LocasaleJason Locasale More articles by this author , Dinesh RakhejaDinesh Rakheja More articles by this author , Victor Darley-UsmarVictor Darley-Usmar More articles by this author , Jason TennessenJason Tennessen More articles by this author , and Sunil SudarshanSunil Sudarshan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The oncometabolite L-2-hydroxyglutarate (L-2HG) is elevated in a large percentage of the most common form of RCC (clear cell histology) and promotes tumor progression. Structurally similar to α-ketoglutarate (α-KG), L-2HG can competitively inhibit enzymes that utilize α-KG as a cofactor including α-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. METHODS: Patient samples, cell lines, Crisper-Cas9 L2HGDH knock out whole body C57-B6 mice, and xenograft models of nude mice were used. Data were analyzed via real-time PCR, western blot, luciferase assays, untargeted metabolomics, and [U-13C] Glucose labeling. RESULTS: RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH) which results in their high L-2HG levels. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. Furthermore, patient samples with high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions (both mRNA and protein) than that of the low L-2HG renal tumors and the patient-matched normal kidneys. In vitro 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2HGDH KO and wild-type mice revealed less serine content in the absence of L2HGDH. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N⁶-methyladenosine (m6A) via inhibiting α-KG-dependent RNA demethylases ALKBH5 and FTO. In the setting of high L-2HG, m6A is enriched in the 3’-UTR region of transcripts including PSAT1. In accord with these data, high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione in high L-2HG RCC which supports both proliferation and resistance to oxidative stress. CONCLUSIONS: The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism through a bimodal mechanism via remodeling of both the epigenome and epitranscriptome. This results in a serine liability in the setting of raised L-2HG. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Source of Funding: The research reported in this article was supported by the National Institutes of Health (R01CA200653 and I01BX002930 to S.S.; F30CA232397 and T32GM008361 to G.B.) and in part by the University of Alabama at Birmingham O’Neal Comprehensive Cancer Center (P30CA013148). Tumor genotyping was supported in part by the University of Texas Health Science Center at San Antonio Mays Cancer Center Genomics Shared Resource Facility (P30CA054174). J.M.T. is supported by a Maximizing Investigators’ Research Award from the National Institute of General Medical Sciences (R35GM119557) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e498 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Garrett Brinkley More articles by this author Anirban Kundu More articles by this author Hyeyoung Nam More articles by this author Suman Karki More articles by this author Richard Kirkman More articles by this author Hayley Widden More articles by this author Michelle Johnson More articles by this author Juan Liu More articles by this author Yasaman Heidarian More articles by this author Nader Mahmoudzadeh More articles by this author Devin Absher More articles by this author Han-Fei Ding More articles by this author David Crosman More articles by this author William Placzek More articles by this author Jason Locasale More articles by this author Dinesh Rakheja More articles by this author Victor Darley-Usmar More articles by this author Jason Tennessen More articles by this author Sunil Sudarshan More articles by this author Expand All Advertisement PDF downloadLoading ...

The 2021 VCP Scientific Conference took place virtually from September 9–10, 2021. This conference, planned and organized by the nonprofit patient advocacy group Cure VCP Disease, Inc. (https://www.curevcp.org), was the first VCP focused meeting since the 215th ENMC International Workshop VCP-related multi-system proteinopathy in 2016 (Evangelista et al., 2016). Mutations in VCP cause a complex and heterogenous disease termed inclusion body myopathy (IBM) with Paget's disease of the bone (PDB) and frontotemporal dementia (FTD) (IBMPFD), or multisystem proteinopathy 1 (MSP-1) Kimonis (n.d.), Kovach et al. (2001), Kimonis et al. (2000). In addition, VCP mutations also cause other age-related neurodegenerative disorders including amyptrophic lateral sclerosis (ALS), Parkinsonism, Charcot-Marie type II-B, vacuolar tauopathy among others (Korb et al., 2022). The objectives of this conference were as follows: (1) to provide a forum that facilitates sharing of published and unpublished information on physiological roles of p97/VCP, and on how mutations of VCP lead to diseases; (2) to bolster understanding of mechanisms involved in p97/VCP-relevant diseases and to enable identification of therapeutics to treat these conditions; (3) to identify gaps and barriers of further discoveries and translational research in the p97/VCP field; (4) to set a concrete basic and translational research agenda for future studies including crucial discussions on biomarker discoveries and patient longitudinal studies to facilitate near-term clinical trials; (5) to accelerate cross-disciplinary research collaborations among p97/VCP researchers; (6) to enable attendees to learn about new tools and reagents with the potential to facilitate p97/VCP research; (7) to assist trainees in propelling their research and to foster mentorship from leaders in the field; and (8) to promote diversity and inclusion of under-represented minorities in p97/VCP research as diversity is critically important for strong scientific research. Given the range of topics, the VCP Scientific Conference brought together over one hundred and forty individuals representing a diverse group of research scientists, trainees, medical practitioners, industry representatives, and patient advocates. Twenty-five institutions with individuals from thirteen countries attended this virtual meeting. In this report, we summarize the major topics presented at this conference by a range of experts.

Accumulation of cytoplasmic inclusions containing fused in sarcoma (FUS), an RNA/DNA-binding protein, is a common hallmark of frontotemporal lobar degeneration and amyotrophic lateral sclerosis neuropathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 aired with mass spectrometry to investigate whether N-terminal phosphorylation alters the FUS protein-protein interaction network (interactome), and subsequently, FUS function. We report the first analysis comparing the interactomes of three FUS variants: homeostatic wildtype FUS (FUS WT), phosphomimetic FUS (FUS PM; a proxy for N-terminally phosphorylated FUS), and the toxic FUS proline 525 to leucine mutant (FUS P525L) that causes juvenile amyotrophic lateral sclerosis. We found that the phosphomimetic FUS interactome is uniquely enriched for a group of cytoplasmic proteins that mediate mRNA metabolism and translation, as well as nuclear proteins involved in the spliceosome and DNA repair functions. Furthermore, we identified and validated the RNA-induced silencing complex RNA helicase MOV10 as a novel interacting partner of FUS. Finally, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady-state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the interactome and function of FUS.

The effects of topically applied 12-O-tetradecanoylphorbol-13-acetate (TPA), acetone, and ethanol on systemic immune function were analyzed in SSIN mice. A total of one, four, or eight dorsal applications of solvent (50–300 μl/treatment, 2×/week) affected neither the overall cellularity of the spleen nor the relative proportions of splenic cells expressing CD4, CD8, or Ig surface markers. In contrast, overall cellularities of the spleen increased and relative T cell content of the spleen decreased in mice treated multiple times with TPA (2 μg/application in 0.2 ml acetone). The development of splenic B cells secreting IgM against sheep red blood cells (SRBC, a T-cell-dependent antigen) was retarded, and the overall duration of IgM synthesis was decreased, in mice immunized 1 hr after the last of four applications (>200 μl) of either solvent. Comparable retardations occurred in mice immunized as late as 7 days after termination of solvent treatment. However, solvent effects on the development of antibody-forming cells were not observed after eight topical applications or when TNP-LPS (a T-cell-independent antigen) was used as the immunogen. The effects of TPA on the development of IgM-secreting B cells were indistinguishable from those of the solvent used for its application. Although serum hemagglutination titers to SRBC correlated with the relative numbers of splenic B cells producing IgM,in vitroproliferative responses to B and T cell mitogens were not predictive of the effects of solvents or TPA on the development of antibody-secreting cells. Collectively, these studies demonstrate that topically applied acetone and ethanol can systemically modulate humoral immunity and emphasize the need for inclusion of nontreated controls when assessing the potential immunomodulatory activities of agents dissolved in acetone or ethanol.

The accumulation of neurotoxic proteins characteristic of age-related neurodegenerative pathologies such as Alzheimer’s and Parkinson’s diseases is associated with the perturbation of metabolism, bioenergetics, and mitochondrial quality control. One approach to exploit these interactions therapeutically is to target the pathways that regulate metabolism. In this respect, the nutrient-sensing hexosamine biosynthesis pathway is of particular interest since it introduces a protein post-translational modification known as O-GlcNAcylation, which modifies different proteins in control versus neurodegenerative disease postmortem brains. A potent inhibitor of the O-GlcNAcase enzyme that removes the modification from proteins, Thiamet G (TG), has been proposed to have potential benefits in Alzheimer’s disease. We tested whether key factors in the O-GlcNAcylation are correlated with mitochondrial electron transport and proteins related to the autophagy/lysosomal pathways in the cortex of male and female mice with and without exposure to TG (10 mg/kg i.p.). Mitochondrial complex activities were measured in the protein homogenates, and a panel of metabolic, autophagy/lysosomal proteins and O-GlcNAcylation enzymes were assessed by either enzyme activity assay or by western blot analysis. We found that the networks associated with O-GlcNAcylation enzymes and activities with mitochondrial parameters, autophagy-related proteins as well as neurodegenerative disease-related proteins exhibited sex and TG dependent differences. Taken together, these studies provide a framework of interconnectivity for multiple O-GlcNAc-dependent pathways in mouse brain of relevance to aging and sex/age-dependent neurodegenerative pathogenesis and response to potential therapies.

O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer’s disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson’s disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.

The proper storage and release of monoamines contributes to a wide range of neuronal activity. Here, we examine the effects of altered vesicular monoamine transport in the nematode Caenorhabditis elegans. The gene cat-1 is responsible for the encoding of the vesicular monoamine transporter (VMAT) in C. elegans and is analogous to the mammalian vesicular monoamine transporter 2 (VMAT2). Our laboratory has previously shown that reduced VMAT2 activity confers vulnerability on catecholamine neurons in mice. The purpose of this article was to determine whether this function is conserved and to determine the impact of reduced VMAT activity in C. elegans. Here we show that deletion of cat-1/VMAT increases sensitivity to the neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) as measured by enhanced degeneration of dopamine neurons. Reduced cat-1/VMAT also induces changes in dopamine-mediated behaviors. High-resolution mass spectrometry-based metabolomics in the whole organism reveals changes in amino acid metabolism, including tyrosine metabolism in the cat-1/VMAT mutants. Treatment with MPP+ disrupted tryptophan metabolism. Both conditions altered glycerophospholipid metabolism, suggesting a convergent pathway of neuronal dysfunction. Our results demonstrate the evolutionarily conserved nature of monoamine function in C. elegans and further suggest that high-resolution mass spectrometry-based metabolomics can be used in this model to study environmental and genetic contributors to complex human disease.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative disorders that are thought to exist on a clinical and pathological spectrum. FTD and ALS are linked by shared genetic causes (e.g. C9orf72 hexanucleotide repeat expansions) and neuropathology, such as inclusions of ubiquitinated, misfolded proteins (e.g. TAR DNA-binding protein 43; TDP-43) in the CNS. Furthermore, some genes that cause FTD or ALS when mutated encode proteins that localize to the lysosome or modulate endosome-lysosome function, including lysosomal fusion, cargo trafficking, lysosomal acidification, autophagy, or TFEB activity. In this review, we summarize evidence that lysosomal dysfunction, caused by genetic mutations (e.g. C9orf72, GRN, MAPT, TMEM106B) or toxic-gain of function (e.g. aggregation of TDP-43 or tau), is an important pathogenic disease mechanism in FTD and ALS. Further studies into the normal function of many of these proteins are required and will help uncover the mechanisms that cause lysosomal dysfunction in FTD and ALS. Mutations or polymorphisms in genes that encode proteins important for endosome-lysosome function also occur in other age-dependent neurodegenerative diseases, including Alzheimer's (e.g. APOE, PSEN1, APP) and Parkinson's (e.g. GBA, LRRK2, ATP13A2) disease. A more complete understanding of the common and unique features of lysosome dysfunction across the spectrum of neurodegeneration will help guide the development of therapies for these devastating diseases.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressively enlarging cysts. Here we elucidate the interplay between oxidative stress, mitochondrial dysfunction, and metabolic derangement using two mouse models of PKD1 mutation, PKD1RC/null and PKD1RC/RC. Mouse kidneys with PKD1 mutation have decreased mitochondrial complexes activity. Targeted proteomics analysis shows a significant decrease in proteins involved in the TCA cycle, fatty acid oxidation (FAO), respiratory complexes, and endogenous antioxidants. Overexpressing mitochondrial-targeted catalase (mCAT) using adeno-associated virus reduces mitochondrial ROS, oxidative damage, ameliorates the progression of PKD and partially restores expression of proteins involved in FAO and the TCA cycle. In human ADPKD cells, inducing mitochondrial ROS increased ERK1/2 phosphorylation and decreased AMPK phosphorylation, whereas the converse was observed with increased scavenging of ROS in the mitochondria. Treatment with the mitochondrial protective peptide, SS31, recapitulates the beneficial effects of mCAT, supporting its potential application as a novel therapeutic for ADPKD.

O-GlcNAcylation is a protein posttranslational modification that results in the addition of O-GlcNAc to Ser/Thr residues. Since its discovery in the 1980s, it has been shown to play an important role in a broad range of cellular functions by modifying nuclear, cytosolic, and mitochondrial proteins. The addition of O-GlcNAc is catalyzed by O-GlcNAc transferase (OGT), and its removal is catalyzed by O-GlcNAcase (OGA). Levels of protein O-GlcNAcylation change in response to nutrient availability and metabolic, oxidative, and proteotoxic stress. OGT and OGA levels, activity, and target engagement are also regulated. Together, this results in adaptive and, on occasions, detrimental responses that affect cellular function and survival, which impact a broad range of pathologies and aging. Over the past several decades, approaches and tools to aid the investigation of the regulation and consequences of protein O-GlcNAcylation have been developed and enhanced. This review is divided into two sections: 1) We will first focus on current standard and advanced technical approaches for assessing enzymatic activities of OGT and OGT, assessing the global and specific protein O-GlcNAcylation and 2) we will summarize in vivo findings of functional consequences of changing protein O-GlcNAcylation, using genetic and pharmacological approaches.

Calorie restriction (CR) promotes healthy ageing in diverse species. Recently, it has been shown that fasting for a portion of each day has metabolic benefits and promotes lifespan. These findings complicate the interpretation of rodent CR studies, in which animals typically eat only once per day and rapidly consume their food, which collaterally imposes fasting. Here we show that a prolonged fast is necessary for key metabolic, molecular and geroprotective effects of a CR diet. Using a series of feeding regimens, we dissect the effects of calories and fasting, and proceed to demonstrate that fasting alone recapitulates many of the physiological and molecular effects of CR. Our results shed new light on how both when and how much we eat regulate metabolic health and longevity, and demonstrate that daily prolonged fasting, and not solely reduced caloric intake, is likely responsible for the metabolic and geroprotective benefits of a CR diet.

The mortality rates among patients who initially survive sepsis are, in part, associated with a high risk of secondary lung infections and respiratory failure. Given that phagolysosomes are important for intracellular killing of pathogenic microbes, we investigated how severe lung infections associated with post-sepsis immunosuppression affect phagolysosome biogenesis. In mice with P. aeruginosa -induced pneumonia, we found a depletion of both phagosomes and lysosomes, as evidenced by decreased amounts of microtubule associated protein light chain 3-II (LC3-II) and lysosomal-associated membrane protein (LAMP1). We also found a loss of transcription factor E3 (TFE3) and transcription factor EB (TFEB), which are important activators for transcription of genes encoding autophagy and lysosomal proteins. These events were associated with increased expression of ZKSCAN3, a repressor for transcription of genes encoding autophagy and lysosomal proteins. Zkscan3 −/− mice had increased expression of genes involved in the autophagy-lysosomal pathway along with enhanced killing of P. aeruginosa in the lungs, as compared to wild-type mice. These findings highlight the involvement of ZKSCAN3 in response to severe lung infection, including susceptibility to secondary bacterial infections due to immunosuppression.