Glycyl-L-Histidyl-L-Lysine (GHK) Prevents Copper- and Zinc-Induced Protein Aggregation and CNS Cell Death in vitro.

Common features of neurodegenerative diseases are oxidative and inflammatory imbalances as well as the misfolding of proteins. An excess of free metal ions can be pathological and contribute to cell death, but only copper and zinc strongly promote protein aggregation. Herein we demonstrate that the endogenous copper binding tripeptide Glycyl-L-Histidyl-L-Lysine (GHK) has the ability to bind to and reduce copper redox activity, and to prevent copper and zinc induced cell death in vitro. In addition, GHK prevents copper- and zinc-induced BSA aggregation and reverses aggregation through resolubilizing the protein. We further demonstrate the enhanced toxicity of copper during inflammation and the ability of GHK to attenuate this toxicity. Finally, we investigated the effects of copper on enhancing paraquat toxicity and report a protective effect of GHK. We therefore conclude that GHK has potential as a cytoprotective compound with regards to copper and zinc toxicity, with positive effects on protein solubility and aggregation that warrants further investigation in the treatment of neurodegenerative diseases.

CD163+ macrophages monitor enhanced permeability at the blood-dorsal root ganglion barrier.

In dorsal root ganglia (DRG), macrophages reside close to sensory neurons and have largely been explored in the context of pain, nerve injury, and repair. However, we discovered that most DRG macrophages interact with and monitor the vasculature by sampling macromolecules from the blood. Characterization of the DRG vasculature revealed a specialized endothelial bed that transformed in molecular, structural, and permeability properties along the arteriovenous axis and was covered by macrophage-interacting pericytes and fibroblasts. Macrophage phagocytosis spatially aligned with peak endothelial permeability, a process regulated by enhanced caveolar transcytosis in endothelial cells. Profiling the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover, and function. CD163+ macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation, and were conserved in mouse and man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit.

Phenotyping of Macrophages After Radiolabeling and Safety of Intra-arterial Transplantation Assessed by SPECT/CT and MRI.

Cell therapy is an integral modality of regenerative medicine. Macrophages are known for their sensitivity to activation stimuli and capability to recruit other immune cells to the sites of injury and healing. In addition, the route of administration can impact engraftment and efficacy of cell therapy, and modern neuro-interventional techniques provide the possibility for selective intra-arterial (IA) delivery to the central nervous system (CNS) with very low risk. The effects of radiolabelling and catheter transport on differentially activated macrophages were evaluated. Furthermore, the safety of selective IA administration of these macrophages to the rabbit brain was assessed by single-photon emission computed tomography/computed tomography (SPECT/CT) and ultra-high-field (9.4 T) magnetic resonance imaging (MRI). Cells were successfully labeled with (111In)In-(oxinate)3 and passed through a microcatheter with preserved phenotype. No cells were retained in the healthy rabbit brain after IA administration, and no adverse events could be observed either 1 h (n = 6) or 24 h (n = 2) after cell administration. The procedure affected both lipopolysaccharide/gamma interferon (LPS/IFNγ) activated cells and interleukin 4 (IL4), interleukin 10 (IL10)/transforming growth factor beta 1 (TGFß1) activated cells to some degree. The LPS/IFNγ activated cells had a significant increase in their phagocytotic function. Overall, the major impact on the cell phenotypes was due to the radiolabeling and not passage through the catheter. Unstimulated cells were substantially affected by both radiolabeling and catheter administration and are hence not suited for this procedure, while both activated macrophages retained their initial phenotypes. In conclusion, activated macrophages are suitable candidates for targeted IA administration without adverse effects on normal, healthy brain parenchyma.

Antigen receptor stimulation induces purifying selection against pathogenic mitochondrial tRNA mutations.

Pathogenic mutations in mitochondrial (mt) tRNA genes that compromise oxidative phosphorylation (OXPHOS) exhibit heteroplasmy and cause a range of multisyndromic conditions. Although mitochondrial disease patients are known to suffer from abnormal immune responses, how heteroplasmic mtDNA mutations affect the immune system at the molecular level is largely unknown. Here, in mice carrying pathogenic C5024T in mt-tRNAAla and in patients with mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) syndrome carrying A3243G in mt-tRNALeu, we found memory T and B cells to have lower pathogenic mtDNA mutation burdens than their antigen-inexperienced naive counterparts, including after vaccination. Pathogenic burden reduction was less pronounced in myeloid compared with lymphoid lineages, despite C5024T compromising macrophage OXPHOS capacity. Rapid dilution of the C5024T mutation in T and B cell cultures could be induced by antigen receptor-triggered proliferation and was accelerated by metabolic stress conditions. Furthermore, we found C5024T to dysregulate CD8+ T cell metabolic remodeling and IFN-γ production after activation. Together, our data illustrate that the generation of memory lymphocytes shapes the mtDNA landscape, wherein pathogenic variants dysregulate the immune response.

Inhibition of pyruvate dehydrogenase kinase 4 ameliorates kidney ischemia-reperfusion injury by reducing succinate accumulation during ischemia and preserving mitochondrial function during reperfusion.

Ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury (AKI), is still without effective therapies. Succinate accumulation during ischemia followed by its oxidation during reperfusion leads to excessive reactive oxygen species (ROS) and severe kidney damage. Consequently, the targeting of succinate accumulation may represent a rational approach to the prevention of IR-induced kidney injury. Since ROS are generated primarily in mitochondria, which are abundant in the proximal tubule of the kidney, we explored the role of pyruvate dehydrogenase kinase 4 (PDK4), a mitochondrial enzyme, in IR-induced kidney injury using proximal tubule cell-specific Pdk4 knockout (Pdk4ptKO) mice. Knockout or pharmacological inhibition of PDK4 ameliorated IR-induced kidney damage. Succinate accumulation during ischemia, which is responsible for mitochondrial ROS production during reperfusion, was reduced by PDK4 inhibition. PDK4 deficiency established conditions prior to ischemia resulting in less succinate accumulation, possibly because of a reduction in electron flow reversal in complex II, which provides electrons for the reduction of fumarate to succinate by succinate dehydrogenase during ischemia. The administration of dimethyl succinate, a cell-permeable form of succinate, attenuated the beneficial effects of PDK4 deficiency, suggesting that the kidney-protective effect is succinate-dependent. Finally, genetic or pharmacological inhibition of PDK4 prevented IR-induced mitochondrial damage in mice and normalized mitochondrial function in an in vitro model of IR injury. Thus, inhibition of PDK4 represents a novel means of preventing IR-induced kidney injury, and involves the inhibition of ROS-induced kidney toxicity through reduction in succinate accumulation and mitochondrial dysfunction.

Enhancement of the SESN2-SHP cascade by melatonin ameliorates hepatic gluconeogenesis by inhibiting the CRBN-BTG2-CREBH signaling pathway.

Melatonin is involved in the regulation of various biological functions. Here, we explored a novel molecular mechanism by which the melatonin-induced sestrin2 (SESN2)-small heterodimer partner (SHP) signaling pathway protects against fasting- and diabetes-mediated hepatic glucose metabolism. Various key gene expression analyses were performed and multiple metabolic changes were assessed in liver specimens and primary hepatocytes of mice and human participants. The expression of the hepatic cereblon (CRBN) and b-cell translocation gene 2 (BTG2) genes was significantly increased in fasting mice, diabetic mice, and patients with diabetes. Overexpression of Crbn and Btg2 increased hepatic gluconeogenesis by enhancing cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CREBH), whereas this phenomenon was prominently ablated in Crbn null mice and Btg2-silenced mice. Interestingly, melatonin-induced SESN2 and SHP markedly reduced hepatic glucose metabolism in diabetic mice and primary hepatocytes, and this protective effect of melatonin was strikingly reversed by silencing Sesn2 and Shp. Finally, the melatonin-induced SESN2-SHP signaling pathway inhibited CRBN- and BTG2-mediated hepatic gluconeogenic gene transcription via the competition of BTG2 and the interaction of CREBH. Mitigation of the CRBN-BTG2-CREBH axis by the melatonin-SESN2-SHP signaling network may provide a novel therapeutic strategy to treat metabolic dysfunction due to diabetes.

Enhanced Ca2+-channeling complex formation at the ER-mitochondria interface underlies the pathogenesis of alcohol-associated liver disease.

Ca2+ overload-induced mitochondrial dysfunction is considered as a major contributing factor in the pathogenesis of alcohol-associated liver disease (ALD). However, the initiating factors that drive mitochondrial Ca2+ accumulation in ALD remain elusive. Here, we demonstrate that an aberrant increase in hepatic GRP75-mediated mitochondria-associated ER membrane (MAM) Ca2+-channeling (MCC) complex formation promotes mitochondrial dysfunction in vitro and in male mouse model of ALD. Unbiased transcriptomic analysis reveals PDK4 as a prominently inducible MAM kinase in ALD. Analysis of human ALD cohorts further corroborate these findings. Additional mass spectrometry analysis unveils GRP75 as a downstream phosphorylation target of PDK4. Conversely, non-phosphorylatable GRP75 mutation or genetic ablation of PDK4 prevents alcohol-induced MCC complex formation and subsequent mitochondrial Ca2+ accumulation and dysfunction. Finally, ectopic induction of MAM formation reverses the protective effect of PDK4 deficiency in alcohol-induced liver injury. Together, our study defines a mediatory role of PDK4 in promoting mitochondrial dysfunction in ALD.

Pyruvate Dehydrogenase Kinase 4 Deficiency Increases Tumorigenesis in a Murine Model of Bladder Cancer.

Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial isozyme in the PDK family (PDK1-4) partially responsible for phosphorylation of pyruvate dehydrogenase (PDH). Phosphorylation of PDH is thought to result in a pro-proliferative shift in metabolism that sustains growth of cancer cells. Previous data from our lab indicate the pan-PDK inhibitor dichloroacetate (DCA) or acute genetic knockdown of PDK4 blocks proliferation of bladder cancer (BCa) cells. The goal of this study was to determine the role of PDK4 in an in vivo BCa model, with the hypothesis that genetic depletion of PDK4 would impair formation of BCa. PDK4-/- or WT animals were exposed to N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 16 weeks, and tumors were allowed to develop for up to 7 additional weeks. PDK4-/- mice had significantly larger tumors at later time points. When animals were treated with cisplatin, PDK4-/- animals still had larger tumors than WT mice. PDK4 expression was assessed in human tissue and in mice. WT mice lost expression of PDK4 as tumors became muscle-invasive. Similar results were observed in human samples, wherein tumors had less expression of PDK4 than benign tissue. In summary, PDK4 has a complex, multifunctional role in BCa and may represent an underrecognized tumor suppressor.

The Role of Pyruvate Metabolism in Mitochondrial Quality Control and Inflammation.

Pyruvate metabolism, a key pathway in glycolysis and oxidative phosphorylation, is crucial for energy homeostasis and mitochondrial quality control (MQC), including fusion/fission dynamics and mitophagy. Alterations in pyruvate flux and MQC are associated with reactive oxygen species accumulation and Ca2+ flux into the mitochondria, which can induce mitochondrial ultrastructural changes, mitochondrial dysfunction and metabolic dysregulation. Perturbations in MQC are emerging as a central mechanism for the pathogenesis of various metabolic diseases, such as neurodegenerative diseases, diabetes and insulin resistance-related diseases. Mitochondrial Ca2+ regulates the pyruvate dehydrogenase complex (PDC), which is central to pyruvate metabolism, by promoting its dephosphorylation. Increase of pyruvate dehydrogenase kinase (PDK) is associated with perturbation of mitochondria-associated membranes (MAMs) function and Ca2+ flux. Pyruvate metabolism also plays an important role in immune cell activation and function, dysregulation of which also leads to insulin resistance and inflammatory disease. Pyruvate metabolism affects macrophage polarization, mitochondrial dynamics and MAM formation, which are critical in determining macrophage function and immune response. MAMs and MQCs have also been intensively studied in macrophage and T cell immunity. Metabolic reprogramming connected with pyruvate metabolism, mitochondrial dynamics and MAM formation are important to macrophages polarization (M1/M2) and function. T cell differentiation is also directly linked to pyruvate metabolism, with inhibition of pyruvate oxidation by PDKs promoting proinflammatory T cell polarization. This article provides a brief review on the emerging role of pyruvate metabolism in MQC and MAM function, and how dysfunction in these processes leads to metabolic and inflammatory diseases.

Activation of TRPV1 receptor facilitates myelin repair following demyelination via the regulation of microglial function.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel that is activated by capsaicin (CAP), the main component of chili pepper. Despite studies in several neurological diseases, the role of TRPV1 in demyelinating diseases remains unknown. Herein, we reported that TRPV1 expression was increased within the corpus callosum during demyelination in a cuprizone (CPZ)-induced demyelination mouse model. TRPV1 deficiency exacerbated motor coordinative dysfunction and demyelination in CPZ-treated mice, whereas the TRPV1 agonist CAP improved the behavioral performance and facilitated remyelination. TRPV1 was predominantly expressed in Iba1+ microglia/macrophages in human brain sections of multiple sclerosis patients and mouse corpus callosum under demyelinating conditions. TRPV1 deficiency decreased microglial recruitment to the corpus callosum, with an associated increase in the accumulation of myelin debris. Conversely, the activation of TRPV1 by CAP enhanced the recruitment of microglia to the corpus callosum and potentiated myelin debris clearance. Using real-time live imaging we confirmed an increased phagocytic function of microglia following CAP treatment. In addition, the expression of the scavenger receptor CD36 was increased, and that of the glycolysis regulators Hif1a and Hk2 was decreased. We conclude that TRPV1 is an important regulator of microglial function in the context of demyelination and may serve as a promising therapeutic target for demyelinating diseases such as multiple sclerosis.

Inhibition of Pyruvate Dehydrogenase Kinase 4 in CD4+ T Cells Ameliorates Intestinal Inflammation.

BACKGROUND & AIMS: Despite recent evidence supporting the metabolic plasticity of CD4+ T cells, it is uncertain whether the metabolic checkpoint pyruvate dehydrogenase kinase (PDK) in T cells plays a role in the pathogenesis of colitis. METHODS: To investigate the role of PDK4 in colitis, we used dextran sulfate sodium (DSS)-induced colitis and T-cell transfer colitis models based on mice with constitutive knockout (KO) or CD4+ T-cell-specific KO of PDK4 (Pdk4fl/flCD4Cre). The effect of PDK4 deletion on T-cell activation was also studied in vitro. Furthermore, we examined the effects of a pharmacologic inhibitor of PDK4 on colitis. RESULTS: Expression of PDK4 increased during colitis development in a DSS-induced colitis model. Phosphorylated PDHE1α, a substrate of PDK4, accumulated in CD4+ T cells in the lamina propria of patients with inflammatory bowel disease. Both constitutive KO and CD4+ T-cell-specific deletion of PDK4 delayed DSS-induced colitis. Adoptive transfer of PDK4-deficient CD4+ T cells attenuated murine colitis, and PDK4 deficiency resulted in decreased activation of CD4+ T cells and attenuated aerobic glycolysis. Mechanistically, there were fewer endoplasmic reticulum-mitochondria contact sites, which are responsible for interorganelle calcium transfer, in PDK4-deficient CD4+ T cells. Consistent with this, GM-10395, a novel inhibitor of PDK4, suppressed T-cell activation by reducing endoplasmic reticulum-mitochondria calcium transfer, thereby ameliorating murine colitis. CONCLUSIONS: PDK4 deletion from CD4+ T cells mitigates colitis by metabolic and calcium signaling modulation, suggesting PDK4 as a potential therapeutic target for IBD.

Noncanonical PDK4 action alters mitochondrial dynamics to affect the cellular respiratory status.

Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to electron transport chain (ETC) toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here, we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with ETC toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Importantly, we observed that the PDK4-mediated regulation of mitochondrial fission was independent of its canonical function, i.e., inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Phosphoproteomic screen for PDK4 substrates, followed by nonphosphorylatable and phosphomimetic mutations of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. Conversely, inhibition of the PDK4-SEPT2 axis could restore the balance in mitochondrial dynamics and reinvigorates cellular respiration in mitochondrial fusion factor, mitofusin 2-deficient cells. Furthermore, PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.

Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation.

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA-approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS-challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using ß-glucan-coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid-specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS-like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid-specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.

Inhibition of colony stimulating factor-1 receptor (CSF-1R) as a potential therapeutic strategy for neurodegenerative diseases: opportunities and challenges.

Microglia are specialized dynamic immune cells in the central nervous system (CNS) that plays a crucial role in brain homeostasis and in disease states. Persistent neuroinflammation is considered a hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and primary progressive multiple sclerosis (MS). Colony stimulating factor 1-receptor (CSF-1R) is predominantly expressed on microglia and its expression is significantly increased in neurodegenerative diseases. Cumulative findings have indicated that CSF-1R inhibitors can have beneficial effects in preclinical neurodegenerative disease models. Research using CSF-1R inhibitors has now been extended into non-human primates and humans. This review article summarizes the most recent advances using CSF-1R inhibitors in different neurodegenerative conditions including AD, PD, HD, ALS and MS. Potential challenges for translating these findings into clinical practice are presented.

An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion.

BACKGROUND: Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. METHODS: Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies. RESULTS: We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro. CONCLUSIONS: Our results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

Interpenetrating gallol functionalized tissue adhesive hyaluronic acid hydrogel polarizes macrophages to an immunosuppressive phenotype.

Innovative scaffold designs that modulate the local inflammatory microenvironment through favorable macrophage polarization and suppressing oxidative stress are needed for successful clinical translation of regenerative cell therapies and graft integration. We herein report derivation of a hydrazone-crosslinked gallol functionalized hyaluronic acid (HA-GA)-based hydrogel that displayed outstanding viscoelastic properties and immunomodulatory characteristics. Grafting of 6% gallol (GA) to a HA-backbone formed an interpenetrative network by promoting an additional crosslink between the gallol groups in addition to hydrazone crosslinking. This significantly enhanced the mechanical stability and displayed shear-thinning/self-healing characteristics, facilitated tissue adhesive properties to porcine tissue and also displayed radical scavenging properties, protecting encapsulated fibroblasts from peroxide challenge. The THP-1 human macrophage cell line or primary bone-marrow-derived murine macrophages cultured within HA-GA gels displayed selective polarization to a predominantly anti-inflammatory phenotype by upregulating IL4ra, IL-10, TGF-ß, and TGF-ßR1 expression when compared with HA-HA gels. Conversely, culturing of pro-inflammatory activated primary murine macrophages in HA-GA gels resulted in a significant reduction of pro-inflammatory TNF-α, IL-1ß, SOCS3 and IL-6 marker expression, and upregulated expression of anti-inflammatory cytokines including TGF-ß. Finally, when the gels were implanted subcutaneously into healthy mice, we observed infiltration of pro-inflammatory myeloid cells in HA-HA gels, while immunosuppressive phenotypes were observed within the HA-GA gels. Taken together these data suggest that HA-GA gels are an ideal injectable scaffold for viable immunotherapeutic interventions. STATEMENT OF SIGNIFICANCE: Host immune response against the implanted scaffolds that are designed to deliver stem cells or therapeutic proteins in vivo significantly limits the functional outcome. For this reason, we have designed immunomodulatory injectable scaffolds that can favorably polarize the recruited macrophages and impart antioxidant properties to suppress oxidative stress. Specifically, we have tailored a hyaluronic acid-based extracellular matrix mimetic injectable scaffold that is grafted with immunomodulatory gallol moiety. Gallol functionalization of hydrogel not only enhanced the mechanical properties of the scaffold by forming an interpenetrating network but also induced antioxidant properties, tissue adhesive properties, and polarized primary murine macrophages to immunosuppressive phenotype. We believe such immunoresponsive implants will pave the way for developing the next-generation of biomaterials for regenerative medicine applications.

MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells.

T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not up-regulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1low phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1high and MTH1low activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.

A Two-to-Five Year Follow-Up of a Pediatric Acute-Onset Neuropsychiatric Syndrome Cohort.

Little is known about the long-term prognosis of children with pediatric acute-onset neuropsychiatric syndrome (PANS). Out of the 46 eligible patients from the Karolinska PANS cohort, 34 consented to participate in a follow-up (median 3.3 years). Participants underwent a thorough clinical evaluation and were classified according to their clinical course. Resulting groups were compared on clinical characteristics and laboratory test results. We observed significant reductions in clinician-rated PANS symptom severity and improved general function. Two patients were classified as remitted, 20 as relapsing-remitting, and 12 as having a chronic-static/progressive course. The latter group had an earlier onset, greater impairment and received more pharmacological and psychological treatments. Although remission was rare, the majority of children with PANS were significantly improved over the follow-up period but a non-negligible minority of patients displayed a chronic-static/progressive course and required additional treatments. The proposed definitions of flare and clinical course may be useful in future clinical trials.

Pyruvate dehydrogenase kinase 1 and 2 deficiency reduces high-fat diet-induced hypertrophic obesity and inhibits the differentiation of preadipocytes into mature adipocytes.

Obesity is now recognized as a disease. This study revealed a novel role for pyruvate dehydrogenase kinase (PDK) in diet-induced hypertrophic obesity. Mice with global or adipose tissue-specific PDK2 deficiency were protected against diet-induced obesity. The weight of adipose tissues and the size of adipocytes were reduced. Adipocyte-specific PDK2 deficiency slightly increased insulin sensitivity in HFD-fed mice. In studies with 3T3-L1 preadipocytes, PDK2 and PDK1 expression was strongly increased during adipogenesis. Evidence was found for epigenetic induction of both PDK1 and PDK2. Gain- and loss-of-function studies with 3T3-L1 cells revealed a critical role for PDK1/2 in adipocyte differentiation and lipid accumulation. PDK1/2 induction during differentiation was also accompanied by increased expression of hypoxia-inducible factor-1α (HIF1α) and enhanced lactate production, both of which were absent in the context of PDK1/2 deficiency. Exogenous lactate supplementation increased the stability of HIF1α and promoted adipogenesis. PDK1/2 overexpression-mediated adipogenesis was abolished by HIF1α inhibition, suggesting a role for the PDK-lactate-HIF1α axis during adipogenesis. In human adipose tissue, the expression of PDK1/2 was positively correlated with that of the adipogenic marker PPARγ and inversely correlated with obesity. Similarly, PDK1/2 expression in mouse adipose tissue was decreased by chronic high-fat diet feeding. We conclude that PDK1 and 2 are novel regulators of adipogenesis that play critical roles in obesity.