Lactate receptor HCAR1 regulates neurogenesis and microglia activation after neonatal hypoxia-ischemia.

Neonatal cerebral hypoxia-ischemia (HI) is the leading cause of death and disability in newborns with the only current treatment being hypothermia. An increased understanding of the pathways that facilitate tissue repair after HI may aid the development of better treatments. Here, we study the role of lactate receptor HCAR1 in tissue repair after neonatal HI in mice. We show that HCAR1 knockout mice have reduced tissue regeneration compared with wildtype mice. Furthermore, proliferation of neural progenitor cells and glial cells, as well as microglial activation was impaired. Transcriptome analysis showed a strong transcriptional response to HI in the subventricular zone of wildtype mice involving about 7300 genes. In contrast, the HCAR1 knockout mice showed a modest response, involving about 750 genes. Notably, fundamental processes in tissue repair such as cell cycle and innate immunity were dysregulated in HCAR1 knockout. Our data suggest that HCAR1 is a key transcriptional regulator of pathways that promote tissue regeneration after HI.

Hypoxic-ischaemic brain injury is the most common cause of disability in newborn babies. This happens when the blood supply to the brain is temporarily blocked during birth and cells do not receive the oxygen and nutrients they need to survive. Cooling the babies down after the hypoxic-ischemic attack (via a technique called hypothermic treatment) can to some extent reduce the damage caused by the injury. However, doctors still need new drugs that can protect the brain and improve its recovery after the injury has occurred. Research in mice suggests that a chemical called lactate might help the brain to recover. Lactate is produced by muscles during hard exercise to provide energy to cells when oxygen levels are low. Recent studies have shown that it can also act as a signalling molecule that binds to a receptor called HCAR1 (short for hydroxycarboxylic acid receptor) on the surface of cells. However, it is poorly understood what role HCAR1 plays in the brain and whether it helps the brain recover from a hypoxic-ischaemic injury. To investigate, Kennedy et al. compared newborn mice with and without the gene that codes for HCAR1 that had undergone a hypoxic-ischaemic brain injury. While HCAR1 did not protect the mice from the disease, it did help their brains to heal. Mice with the gene for HCAR1 partly recovered some of their damaged brain tissue six weeks after the injury. Their cells switched on thousands of genes involved in the immune system and cell cycle, resulting in new brain cells being formed that could repopulate the injured areas. In contrast, the brain tissue of mice lacking HCAR1 barely produced any new cells. These findings suggest that HCAR1 may help with brain recovery after hypoxia-ischemia in newborn mice. This could lead to the development of drugs that might reduce or repair brain damage in newborn babies. However, further studies are needed to investigate whether HCAR1 has the same effect in humans.

Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.

Poly(ADP-ribose) polymerase (PARP) enzymes initiate (mt)DNA repair mechanisms and use nicotinamide adenine dinucleotide (NAD+) as energy source. Prolonged PARP activity can drain cellular NAD+ reserves, leading to de-regulation of important molecular processes. Here, we provide evidence of a pathophysiological mechanism that connects mtDNA damage to cardiac dysfunction via reduced NAD+ levels and loss of mitochondrial function and communication. Using a transgenic model, we demonstrate that high levels of mice cardiomyocyte mtDNA damage cause a reduction in NAD+ levels due to extreme DNA repair activity, causing impaired activation of NAD+-dependent SIRT3. In addition, we show that myocardial mtDNA damage in combination with high dosages of nicotinamideriboside (NR) causes an inhibition of sirtuin activity due to accumulation of nicotinamide (NAM), in addition to irregular cardiac mitochondrial morphology. Consequently, high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and instability of mtDNA.

The Lactate Receptor HCA1 Is Present in the Choroid Plexus, the Tela Choroidea, and the Neuroepithelial Lining of the Dorsal Part of the Third Ventricle.

The volume, composition, and movement of the cerebrospinal fluid (CSF) are important for brain physiology, pathology, and diagnostics. Nevertheless, few studies have focused on the main structure that produces CSF, the choroid plexus (CP). Due to the presence of monocarboxylate transporters (MCTs) in the CP, changes in blood and brain lactate levels are reflected in the CSF. A lactate receptor, the hydroxycarboxylic acid receptor 1 (HCA1), is present in the brain, but whether it is located in the CP or in other periventricular structures has not been studied. Here, we investigated the distribution of HCA1 in the cerebral ventricular system using monomeric red fluorescent protein (mRFP)-HCA1 reporter mice. The reporter signal was only detected in the dorsal part of the third ventricle, where strong mRFP-HCA1 labeling was present in cells of the CP, the tela choroidea, and the neuroepithelial ventricular lining. Co-labeling experiments identified these cells as fibroblasts (in the CP, the tela choroidea, and the ventricle lining) and ependymal cells (in the tela choroidea and the ventricle lining). Our data suggest that the HCA1-containing fibroblasts and ependymal cells have the ability to respond to alterations in CSF lactate in body-brain signaling, but also as a sign of neuropathology (e.g., stroke and Alzheimer's disease biomarker).

Brain energy rescue: an emerging therapeutic concept for neurodegenerative disorders of ageing.

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner - a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes.

Blood lactate dynamics in awake and anaesthetized mice after intraperitoneal and subcutaneous injections of lactate-sex matters.

Lactate treatment has shown a therapeutic potential for several neurological diseases, including Alzheimer's disease. In order to optimize the administration of lactate for studies in mouse models, we compared blood lactate dynamics after intraperitoneal (IP) and subcutaneous (SC) injections. We used the 5xFAD mouse model for familial Alzheimer's disease and performed the experiments in both awake and anaesthetized mice. Blood glucose was used as an indication of the hepatic conversion of lactate. In awake mice, both injection routes resulted in high blood lactate levels, mimicking levels reached during high-intensity training. In anaesthetized mice, SC injections resulted in significantly lower lactate levels compared to IP injections. Interestingly, we observed that awake males had significantly higher lactate levels than awake females, while the opposite sex difference was observed during anaesthesia. We did not find any significant difference between transgenic and wild-type mice and therefore believe that our results can be generalized to other mouse models. These results should be considered when planning experiments using lactate treatment in mice.

Targeting NAD+ in translational research to relieve diseases and conditions of metabolic stress and ageing.

Nicotinamide adenine dinucleotide (NAD+) plays a fundamental role in life and health through the regulation of energy biogenesis, redox homeostasis, cell metabolism, and the arbitration of cell survival via linkages to apoptosis and autophagic pathways. The importance of NAD+ in ageing and healthy longevity has been revealed from laboratory animal studies and early-stage clinical testing. While basic researchers and clinicians have investigated the molecular mechanisms and translation potential of NAD+, there are still major gaps in applying laboratory science to design the most effective trials. This mini-review was based on the programme and discussions of the 3rd NO-Age Symposium held at the Akershus University Hospital, Norway on the 28th October 2019. This symposium brought together leading basic researchers on NAD+ and clinicians who are leading or are going to perform NAD+ augmentation-related clinical studies. This meeting covered talks about NAD+ synthetic pathways, subcellular homeostasis of NAD+, the benefits of NAD+ augmentation from maternal milk to offspring, current clinical trials of the NAD+ precursor nicotinamide riboside (NR) on Ataxia-Telangiectasia (A-T), Parkinson's disease (PD), post-sepsis fatigue, as well as other potential NR-based clinical trials. Importantly, a consensus is emerging with respect to the design of clinical trials in order to measure meaningful parameters and ensure safety.

The NAD+-mitophagy axis in healthy longevity and in artificial intelligence-based clinical applications.

Nicotinamide adenine dinucleotide (NAD+) is an important natural molecule involved in fundamental biological processes, including the TCA cycle, OXPHOS, ß-oxidation, and is a co-factor for proteins promoting healthy longevity. NAD+ depletion is associated with the hallmarks of ageing and may contribute to a wide range of age-related diseases including metabolic disorders, cancer, and neurodegenerative diseases. One of the central pathways by which NAD+ promotes healthy ageing is through regulation of mitochondrial homeostasis via mitochondrial biogenesis and the clearance of damaged mitochondria via mitophagy. Here, we highlight the contribution of the NAD+-mitophagy axis to ageing and age-related diseases, and evaluate how boosting NAD+ levels may emerge as a promising therapeutic strategy to counter ageing as well as neurodegenerative diseases including Alzheimer's disease. The potential use of artificial intelligence to understand the roles and molecular mechanisms of the NAD+-mitophagy axis in ageing is discussed, including possible applications in drug target identification and validation, compound screening and lead compound discovery, biomarker development, as well as efficacy and safety assessment. Advances in our understanding of the molecular and cellular roles of NAD+ in mitophagy will lead to novel approaches for facilitating healthy mitochondrial homoeostasis that may serve as a promising therapeutic strategy to counter ageing-associated pathologies and/or accelerated ageing.

High Intensity Interval Training Ameliorates Mitochondrial Dysfunction in the Left Ventricle of Mice with Type 2 Diabetes.

Both human and animal studies have shown mitochondrial and contractile dysfunction in hearts of type 2 diabetes mellitus (T2DM). Exercise training has shown positive effects on cardiac function, but its effect on the mitochondria have been insufficiently explored. The aim of this study was to assess the effect of exercise training on mitochondrial function in T2DM hearts. We divided T2DM mice (db/db) into a sedentary and an interval training group at 8 weeks of age and used heterozygote db/+ as controls. After 8 weeks of training, we evaluated mitochondrial structure and function, as well as the levels of mRNA and proteins involved in key metabolic processes from the left ventricle. db/db animals showed decreased oxidative phosphorylation capacity and fragmented mitochondria. Mitochondrial respiration showed a blunted response to Ca2+ along with reduced protein levels of the mitochondrial calcium uniporter. Exercise training ameliorated the reduced oxidative phosphorylation in complex (C) I + II, CII and CIV, but not CI or Ca2+ response. Mitochondrial fragmentation was partially restored. mRNA levels of isocitrate, succinate and oxoglutarate dehydrogenase were increased in db/db mice and normalized by exercise training. Exercise training induced an upregulation of two transcripts of peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1α1 and PGC1α4) previously linked to endurance training adaptations and strength training adaptations, respectively. The T2DM heart showed mitochondrial dysfunction at multiple levels and exercise training ameliorated some, but not all mitochondrial dysfunctions.

Lactate-Mediated Protection of Retinal Ganglion Cells.

Loss of retinal ganglion cells (RGCs) is a leading cause of blinding conditions. The purpose of this study was to evaluate the effect of extracellular l-lactate on RGC survival facilitated through lactate metabolism and ATP production. We identified lactate as a preferred energy substrate over glucose in murine RGCs and showed that lactate metabolism and consequently increased ATP production are crucial components in promoting RGC survival during energetic crisis. Lactate was released to the extracellular environment in the presence of glucose and detained intracellularly during glucose deprivation. Lactate uptake and metabolism was unaltered in the presence and absence of glucose. However, the ATP production declined significantly for 24 h of glucose deprivation and increased significantly in the presence of lactate. Finally, lactate exposure for 2 and 24 h resulted in increased RGC survival during glucose deprivation. In conclusion, the metabolic pathway of lactate in RGCs may be of great future interest to unravel potential pharmaceutical targets, ultimately leading to novel therapies in the prevention of blinding neurodegenerative diseases, for example, glaucoma.

Dual Properties of Lactate in Müller Cells: The Effect of GPR81 Activation.

Purpose: Besides being actively metabolized, lactate may also function as a signaling molecule by activation of the G-protein-coupled receptor 81 (GPR81). Thus, we aimed to characterize the metabolic effects of GPR81 activation in Müller cells. Method: Primary Müller cells from mice were treated with and without 10 mM L-lactate in the presence or absence of 6 mM glucose. The effects of lactate receptor GPR81 activation were evaluated by the addition of 5 mM 3,5-DHBA (3,5-dihydroxybenzoic acid), a GPR81 agonist. Western blot analyses were used to determine protein expression of GPR81. Cell survival was assessed through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assays. Lactate release was quantified by commercially available lactate kits. 13C-labeling studies via mass spectroscopy and Seahorse analyses were performed to evaluate metabolism of lactate and glucose, and mitochondrial function. Finally, Müller cell function was evaluated by measuring glutamate uptake. Results: The lactate receptor, GPR81, was upregulated during glucose deprivation. Treatment with a GPR81 agonist did not affect Müller cell survival. However, GPR81 activation diminished lactate release allowing lactate to be metabolized intracellularly. Furthermore, GPR81 activation increased metabolism of glucose and mitochondrial function. Finally, maximal glutamate uptake decreased in response to GPR81 activation during glucose deprivation. Conclusions: The present study revealed dual properties of lactate via functioning as an active metabolic energy substrate and a regulatory molecule by activation of the GPR81 receptor in primary Müller cells. Thus, combinational therapy of lactate and GPR81 agonists may be of future interest in maintaining Müller cell survival, ultimately leading to increased resistance toward retinal neurodegeneration.

Are the neuroprotective effects of exercise training systemically mediated?

To date there is no cure available for dementia, and the field calls for novel therapeutic targets. A rapidly growing body of literature suggests that regular endurance training and high cardiorespiratory fitness attenuate cognitive impairment and reduce dementia risk. Such benefits have recently been linked to systemic neurotrophic factors induced by exercise. These circulating biomolecules may cross the blood-brain barrier and potentially protect against neurodegenerative disorders such as Alzheimer's disease. Identifying exercise-induced systemic neurotrophic factors with beneficial effects on the brain may lead to novel molecular targets for maintaining cognitive function and preventing neurodegeneration. Here we review the recent literature on potential systemic mediators of neuroprotection induced by exercise. We focus on the body of translational research in the field, integrating knowledge from the molecular level, animal models, clinical and epidemiological studies. Taken together, the current literature provides initial evidence that exercise-induced, blood-borne biomolecules, such as BDNF and FNDC5/irisin, may be powerful agents mediating the benefits of exercise on cognitive function and may form the basis for new therapeutic strategies to better prevent and treat dementia.

A Ketogenic Diet Improves Mitochondrial Biogenesis and Bioenergetics via the PGC1α-SIRT3-UCP2 Axis.

A ketogenic diet (KD; high-fat, low-carbohydrate) can benefit refractory epilepsy, but underlying mechanisms are unknown. We used mice inducibly expressing a mutated form of the mitochondrial DNA repair enzyme UNG1 (mutUNG1) to cause progressive mitochondrial dysfunction selectively in forebrain neurons. We examined the levels of mRNAs and proteins crucial for mitochondrial biogenesis and dynamics. We show that hippocampal pyramidal neurons in mutUNG1 mice, as well as cultured rat hippocampal neurons and human fibroblasts with H2O2 induced oxidative stress, improve markers of mitochondrial biogenesis, dynamics and function when fed on a KD, and when exposed to the ketone body ß-hydroxybutyrate, respectively, by upregulating PGC1α, SIRT3 and UCP2, and (in cultured cells) increasing the oxygen consumption rate (OCR) and the NAD+/NADH ratio. The mitochondrial level of UCP2 was significantly higher in the perikarya and axon terminals of hippocampus CA1 pyramidal neurons in KD treated mutUNG1 mice compared with mutUNG1 mice fed a standard diet. The ß-hydroxybutyrate receptor GPR109a (HCAR2), but not the structurally closely related lactate receptor GPR81 (HCAR1), was upregulated in mutUNG1 mice on a KD, suggesting a selective influence of KD on ketone body receptor mechanisms. We conclude that progressive mitochondrial dysfunction in mutUNG1 expressing mice causes oxidative stress, and that exposure of animals to KD, or of cells to ketone body in vitro, elicits compensatory mechanisms acting to augment mitochondrial mass and bioenergetics via the PGC1α-SIRT3-UCP2 axis (The compensatory processes are overwhelmed in the mutUNG1 mice by all the newly formed mitochondria being dysfunctional).

Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP.

Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism.

Essential Roles of Lactate in Müller Cell Survival and Function.

Müller cells are pivotal in sustaining retinal ganglion cells, and an intact energy metabolism is essential for upholding Müller cell functions. The present study aimed to investigate the impact of lactate on Müller cell survival and function. Primary mice Müller cells and human Müller cell lines (MIO-M1) were treated with or without lactate (10 or 20 mM) for 2 and 24 hours. Simultaneously, Müller cells were incubated with or without 6 mM of glucose. L-lactate exposure increased Müller cell survival independently of the presence of glucose. This effect was abolished by the addition of the monocarboxylate inhibitor 4-cinnamic acid to the treatment media, whereas survival continued to increase in response to addition of D-lactate during glucose restriction. ATP levels decreased over time in MIO-M1 cells and remained stable over time in primary Müller cells. Lactate was preferably metabolized in MIO-M1 cells compared to glucose, and 10 mM of L-Lactate exposure prevented complete glycogen depletion in MIO-M1 cells. Glutamate uptake increased after 2 hours and decreased after 24 hours in glucose-restricted Müller cells compared to cells with glucose supplement. The addition of 10 mM of lactate to the treatment media increased glutamate uptake in glucose supplemented and restricted cells. In conclusion, lactate is a key component in maintaining Müller cell survival and function. Hence, lactate administration may be of great future interest, ultimately leading to novel therapies to rescue retinal ganglion cells.

Optimization of Storage Temperature for Retention of Undifferentiated Cell Character of Cultured Human Epidermal Cell Sheets.

Cultured epidermal cell sheets (CES) containing undifferentiated cells are useful for treating skin burns and have potential for regenerative treatment of other types of epithelial injuries. The undifferentiated phenotype is therefore important for success in both applications. This study aimed to optimize a method for one-week storage of CES for their widespread distribution and use in regenerative medicine. The effect of storage temperatures 4 °C, 8 °C, 12 °C, 16 °C, and 24 °C on CES was evaluated. Analyses included assessment of viability, mitochondrial reactive oxygen species (ROS), membrane damage, mitochondrial DNA (mtDNA) integrity, morphology, phenotype and cytokine secretion into storage buffer. Lowest cell viability was seen at 4 °C. Compared to non-stored cells, ABCG2 expression increased between temperatures 8-16 °C. At 24 °C, reduced ABCG2 expression coincided with increased mitochondrial ROS, as well as increased differentiation, cell death and mtDNA damage. P63, C/EBPδ, CK10 and involucrin fluorescence combined with morphology observations supported retention of undifferentiated cell phenotype at 12 °C, transition to differentiation at 16 °C, and increased differentiation at 24 °C. Several cytokines relevant to healing were upregulated during storage. Importantly, cells stored at 12 °C showed similar viability and undifferentiated phenotype as the non-stored control suggesting that this temperature may be ideal for storage of CES.

Exercise induces cerebral VEGF and angiogenesis via the lactate receptor HCAR1.

Physical exercise can improve brain function and delay neurodegeneration; however, the initial signal from muscle to brain is unknown. Here we show that the lactate receptor (HCAR1) is highly enriched in pial fibroblast-like cells that line the vessels supplying blood to the brain, and in pericyte-like cells along intracerebral microvessels. Activation of HCAR1 enhances cerebral vascular endothelial growth factor A (VEGFA) and cerebral angiogenesis. High-intensity interval exercise (5 days weekly for 7 weeks), as well as L-lactate subcutaneous injection that leads to an increase in blood lactate levels similar to exercise, increases brain VEGFA protein and capillary density in wild-type mice, but not in knockout mice lacking HCAR1. In contrast, skeletal muscle shows no vascular HCAR1 expression and no HCAR1-dependent change in vascularization induced by exercise or lactate. Thus, we demonstrate that a substance released by exercising skeletal muscle induces supportive effects in brain through an identified receptor.

Endogenous GABA controls oligodendrocyte lineage cell number, myelination, and CNS internode length.

Adjusting the thickness and internodal length of the myelin sheath is a mechanism for tuning the conduction velocity of axons to match computational needs. Interactions between oligodendrocyte precursor cells (OPCs) and developing axons regulate the formation of myelin around axons. We now show, using organotypic cerebral cortex slices from mice expressing eGFP in Sox10-positive oligodendrocytes, that endogenously released GABA, acting on GABAA receptors, greatly reduces the number of oligodendrocyte lineage cells. The decrease in oligodendrocyte number correlates with a reduction in the amount of myelination but also an increase in internode length, a parameter previously thought to be set by the axon diameter or to be a property intrinsic to oligodendrocytes. Importantly, while TTX block of neuronal activity had no effect on oligodendrocyte lineage cell number when applied alone, it was able to completely abolish the effect of blocking GABAA receptors, suggesting that control of myelination by endogenous GABA may require a permissive factor to be released from axons. In contrast, block of AMPA/KA receptors had no effect on oligodendrocyte lineage cell number or myelination. These results imply that, during development, GABA can act as a local environmental cue to control myelination and thus influence the conduction velocity of action potentials within the CNS. GLIA 2017;65:309-321.

A ketogenic diet accelerates neurodegeneration in mice with induced mitochondrial DNA toxicity in the forebrain.

Mitochondrial genome maintenance plays a central role in preserving brain health. We previously demonstrated accumulation of mitochondrial DNA damage and severe neurodegeneration in transgenic mice inducibly expressing a mutated mitochondrial DNA repair enzyme (mutUNG1) selectively in forebrain neurons. Here, we examine whether severe neurodegeneration in mutUNG1-expressing mice could be rescued by feeding the mice a ketogenic diet, which is known to have beneficial effects in several neurological disorders. The diet increased the levels of superoxide dismutase 2, and mitochondrial mass, enzymes, and regulators such as SIRT1 and FIS1, and appeared to downregulate N-methyl-D-aspartic acid (NMDA) receptor subunits NR2A/B and upregulate γ-aminobutyric acid A (GABAA) receptor subunits α1. However, unexpectedly, the ketogenic diet aggravated neurodegeneration and mitochondrial deterioration. Electron microscopy showed structurally impaired mitochondria accumulating in neuronal perikarya. We propose that aggravation is caused by increased mitochondrial biogenesis of generally dysfunctional mitochondria. This study thereby questions the dogma that a ketogenic diet is unambiguously beneficial in mitochondrial disorders.