A muscarinic receptor antagonist reverses multiple indices of diabetic peripheral neuropathy: preclinical and clinical studies using oxybutynin.

Preclinical studies indicate that diverse muscarinic receptor antagonists, acting via the M1 sub-type, promote neuritogenesis from sensory neurons in vitro and prevent and/or reverse both structural and functional indices of neuropathy in rodent models of diabetes. We sought to translate this as a potential therapeutic approach against structural and functional indices of diabetic neuropathy using oxybutynin, a muscarinic antagonist approved for clinical use against overactive bladder. Studies were performed using sensory neurons maintained in vitro, rodent models of type 1 or type 2 diabetes and human subjects with type 2 diabetes and confirmed neuropathy. Oxybutynin promoted significant neurite outgrowth in sensory neuron cultures derived from adult normal rats and STZ-diabetic mice, with maximal efficacy in the 1-100 nmol/l range. This was accompanied by a significantly enhanced mitochondrial energetic profile as reflected by increased basal and maximal respiration and spare respiratory capacity. Systemic (3-10 mg/kg/day s.c.) and topical (3% gel daily) oxybutynin reversed paw heat hypoalgesia in the STZ and db/db mouse models of diabetes and reversed paw tactile allodynia in STZ-diabetic rats. Loss of nerve profiles in the skin and cornea of db/db mice was also prevented by daily topical delivery of 3% oxybutynin for 8 weeks. A randomized, double-blind, placebo-controlled interventional trial was performed in subjects with type 2 diabetes and established peripheral neuropathy. Subjects received daily topical treatment with 3% oxybutynin gel or placebo for 6 months. The a priori designated primary endpoint, significant change in intra-epidermal nerve fibre density (IENFD) in skin biopsies taken before and after 20 weeks of treatments, was met by oxybutynin but not placebo. Secondary endpoints showing significant improvement with oxybutynin treatment included scores on clinical neuropathy, pain and quality of life scales. This proof-of-concept study indicates that muscarinic antagonists suitable for long-term use may offer a novel therapeutic opportunity for treatment of diabetic neuropathy. Trial registry number: NCT03050827.

Antagonism of the Muscarinic Acetylcholine Type 1 Receptor Enhances Mitochondrial Membrane Potential and Expression of Respiratory Chain Components via AMPK in Human Neuroblastoma SH-SY5Y Cells and Primary Neurons.

Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M1R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M1R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M1R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M1R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M1R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain, and enhance nerve repair in peripheral neuropathy.

Development of iPSC-based clinical trial selection platform for patients with ultrarare diseases.

A "Leap-of-Faith" approach is used to treat patients with previously unknown ultrarare pathogenic mutations, often based on evidence from patients having dissimilar but more prevalent mutations. This uncertainty reflects the need to develop personalized prescreening platforms for these patients to assess drug efficacy before considering clinical trial enrollment. In this study, we report an 18-year-old patient with ultrarare Leigh-like syndrome. This patient had previously participated in two clinical trials with unfavorable responses. We established an induced pluripotent stem cell (iPSC)-based platform for this patient, and assessed the efficacy of a panel of drugs. The iPSC platform validated the safety and efficacy of the screened drugs. The efficacy of three of the screened drugs was also investigated in the patient. After 3 years of treatment, the drugs were effective in shifting the metabolic profile of this patient toward healthy control. Therefore, this personalized iPSC-based platform can act as a prescreening tool to help in decision-making with respect to patient's participation in future clinical trials.

CEBPß regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth.

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPß, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPß overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPß can be a promising therapeutic approach.

Oxidative Stress and Mitochondrial Dysfunction Associated with Peripheral Neuropathy in Type 1 Diabetes.

Significance: This review highlights the many intracellular processes generating reactive oxygen species (ROS) in the peripheral nervous system in the context of type 1 diabetes. The major sources of superoxide and hydrogen peroxide (H2O2) are described, and scavenging systems are explained. Important roles of ROS in regulating normal redox signaling and in a disease setting, such as diabetes, contributing to oxidative stress and cellular damage are outlined. The primary focus is the role of hyperglycemia in driving elevated ROS production and oxidative stress contributing to neurodegeneration in diabetic neuropathy (within the dorsal root ganglia [DRG] and peripheral nerve). Recent Advances: Contributors to ROS production under high intracellular glucose concentration such as mitochondria and the polyol pathway are discussed. The primarily damaging impact of ROS on multiple pathways including mitochondrial function, endoplasmic reticulum (ER) stress, autophagy, and epigenetic signaling is covered. Critical Issues: There is a strong focus on mechanisms of diabetes-induced mitochondrial dysfunction and how this may drive ROS production (in particular superoxide). The mitochondrial sites of superoxide/H2O2 production via mitochondrial metabolism and aerobic respiration are reviewed. Future Directions: Areas for future development are highlighted, including the need to clarify diabetes-induced changes in autophagy and ER function in neurons and Schwann cells. In addition, more clarity is needed regarding the sources of ROS production at mitochondrial sites under high glucose concentration (and lack of insulin signaling). New areas of study should be introduced to investigate the role of ROS, nuclear lamina function, and epigenetic signaling under diabetic conditions in peripheral nerve.

Sensory neurons derived from diabetic rats exhibit deficits in functional glycolysis and ATP that are ameliorated by IGF-1.

OBJECTIVE: The distal dying-back of the longest nerve fibres is a hallmark of diabetic neuropathy, and impaired provision of energy in the form of adenosine triphosphate (ATP) may contribute to this neurodegenerative process. We hypothesised that energy supplementation via glycolysis and/or mitochondrial oxidative phosphorylation is compromised in cultured dorsal root ganglion (DRG) sensory neurons from diabetic rodents, thus contributing to axonal degeneration. Functional analysis of glycolysis and mitochondrial respiration and real-time measurement of ATP levels in live cells were our specific means to test this hypothesis. METHODS: DRG neuron cultures from age-matched control or streptozotocin (STZ)-induced type 1 diabetic rats were used for in vitro studies. Three plasmids containing ATP biosensors of varying affinities were transfected into neurons to study endogenous ATP levels in real time. The Seahorse XF analyser was used for glycolysis and mitochondrial respiration measurements. RESULTS: Fluorescence resonance energy transfer (FRET) efficiency (YFP/CFP ratio) of the ATP biosensors AT1.03 (low affinity) and AT1.03YEMK (medium affinity) were significantly higher than that measured using the ATP-insensitive construct AT1.03R122/6K in both cell bodies and neurites of DRG neurons (p < 0.0001). The ATP level was homogenous along the axons but higher in cell bodies in cultured DRG neurons from both control and diabetic rats. Treatment with oligomycin (an ATP synthase inhibitor in mitochondria) decreased the ATP levels in cultured DRG neurons. Likewise, blockade of glycolysis using 2-deoxy-d-glucose (2-DG: a glucose analogue) reduced ATP levels (p < 0.001). Cultured DRG neurons derived from diabetic rats showed a diminishment of ATP levels (p < 0.01), glycolytic capacity, glycolytic reserve and non-glycolytic acidification. Application of insulin-like growth factor-1 (IGF-1) significantly elevated all the above parameters in DRG neurons from diabetic rats. Oligomycin pre-treatment of DRG neurons, to block oxidative phosphorylation, depleted the glycolytic reserve and lowered basal respiration in sensory neurons derived from control and diabetic rats. Depletion was much higher in sensory neurons from diabetic rats compared to control rats. In addition, an acute increase in glucose concentration, in the presence or absence of oligomycin, elevated parameters of glycolysis by 1.5- to 2-fold while having no impact on mitochondrial respiration. CONCLUSION: We provide the first functional evidence for decreased glycolytic capacity in DRG neurons derived from type 1 diabetic rats. IGF-1 protected against the loss of ATP supplies in DRG cell bodies and axons in neurons derived from diabetic rats by augmenting various parameters of glycolysis and mitochondrial respiration.

Oxidized phosphatidylcholines trigger ferroptosis in cardiomyocytes during ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) injury increases the generation of oxidized phosphatidylcholines (OxPCs), which results in cell death. However, the mechanism by which OxPCs mediate cell death and cardiac dysfunction is largely unknown. The aim of this study was to determine the mechanisms by which OxPC triggers cardiomyocyte cell death during reperfusion injury. Adult rat ventricular cardiomyocytes were treated with increasing concentrations of various purified fragmented OxPCs. Cardiomyocyte viability, bioenergetic response, and calcium transients were determined in the presence of OxPCs. Five different fragmented OxPCs resulted in a decrease in cell viability, with 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) having the most potent cardiotoxic effect in both a concentration and time dependent manner (P < 0.05). POVPC and PONPC also caused a significant decrease in Ca2+ transients and net contraction in isolated cardiomyocytes compared to vehicle treated control cells (P < 0.05). PONPC depressed maximal respiration rate (P < 0.01; 54%) and spare respiratory capacity (P < 0.01; 54.5%). Notably, neither caspase 3 activation or TUNEL staining was observed in cells treated with either POVPC or PONPC. Further, cardiac myocytes treated with OxPCs were indistinguishable from vehicle-treated control cells with respect to nuclear high-mobility group box protein 1 (HMGBP1) activity. However, glutathione peroxidase 4 activity was markedly suppressed in cardiomyocytes treated with POVPC and PONPC coincident with increased ferroptosis. Importantly, cell death induced by OxPCs could be suppressed by E06 Ab, directed against OxPCs or by ferrostatin-1, which bound the sn-2 aldehyde of POVPC during I/R. The findings of the present study demonstrate that oxidation of phosphatidylcholines during I/R generate bioactive phospholipid intermediates that disrupt mitochondrial bioenergetics and calcium transients and provoke wide spread cell death through ferroptosis. Neutralization of OxPC with E06 or with ferrostatin-1 prevents cell death during reperfusion. Our study demonstrates a novel signaling pathway that operationally links generation of OxPC during cardiac I/R to ferroptosis. Interventions designed to target OxPCs may prove beneficial in mitigating ferroptosis during I/R injury in individuals with ischemic heart disease.NEW & NOTEWORTHY Oxidized phosphatidylcholines (OxPC) generated during reperfusion injury are potent inducers of cardiomyocyte death. Our studies have shown that OxPCs exert this effect through a ferroptotic process that can be attenuated. A better understanding of the OxPC cell death pathway can prove a novel strategy for prevention of cell death during myocardial reperfusion injury.

Metformin as a potential therapeutic for neurological disease: mobilizing AMPK to repair the nervous system.

Introduction: Metformin is currently first line therapy for type 2 diabetes (T2D). The mechanism of action of metformin involves activation of AMP-activated protein kinase (AMPK) to enhance mitochondrial function (for example, biogenesis, refurbishment and dynamics) and autophagy. Many neurodegenerative diseases of the central and peripheral nervous systems arise from metabolic failure and toxic protein aggregation where activated AMPK could prove protective. Areas covered: The authors review literature on metformin treatment in Parkinson's disease, Huntington's disease and other neurological diseases of the CNS along with neuroprotective effects of AMPK activation and suppression of the mammalian target of rapamycin (mTOR) pathway on peripheral neuropathy and neuropathic pain. The authors compare the efficacy of metformin with the actions of resveratrol. Expert opinion: Metformin, through activation of AMPK and autophagy, can enhance neuronal bioenergetics, promote nerve repair and reduce toxic protein aggregates in neurological diseases. A long history of safe use in humans should encourage development of metformin and other AMPK activators in preclinical and clinical research. Future studies in animal models of neurological disease should strive to further dissect in a mechanistic manner the pathways downstream from metformin-dependent AMPK activation, and to further investigate mTOR dependent and independent signaling pathways driving neuroprotection.

Sex-Specific Effects of Chronic Creatine Supplementation on Hippocampal-Mediated Spatial Cognition in the 3xTg Mouse Model of Alzheimer's Disease.

The creatine (Cr) energy system has been implicated in Alzheimer's disease (AD), including reductions in brain phosphoCr and Cr kinase, yet no studies have examined the neurobehavioral effects of Cr supplementation in AD, including the 3xTg mouse model. This studied investigated the effects of Cr supplementation on spatial cognition, plasticity- and disease-related protein levels, and mitochondrial function in the 3xTg hippocampus. Here, 3xTg mice were fed a control or Cr-supplemented (3% Cr (w/w)) diet for 8-9 weeks and tested in the Morris water maze. Mitochondrial oxygen consumption (Seahorse) and protein levels (Western blots) were measured in the hippocampus in subsets of mice. Overall, 3xTg females exhibited impaired memory as compared to males. In females, Cr supplementation decreased escape latency and was associated with increased spatial search strategy use. In males, Cr supplementation decreased the use of spatial search strategies. Pilot data indicated mitochondrial enhancements with Cr supplementation in both sexes. In females, Cr supplementation increased CREB phosphorylation and levels of IκB (NF-κB suppressor), CaMKII, PSD-95, and high-molecular-weight amyloid ß (Aß) species, whereas Aß trimers were reduced. These data suggest a beneficial preventative effect of Cr supplementation in females and warrant caution against Cr supplementation in males in the AD-like brain.

Early Onset of Sex-Dependent Mitochondrial Deficits in the Cortex of 3xTg Alzheimer's Mice.

Alzheimer's disease (AD) is a major public health concern worldwide. Advanced age and female sex are two of the most prominent risk factors for AD. AD is characterized by progressive neuronal loss, especially in the cortex and hippocampus, and mitochondrial dysfunction has been proposed to be an early event in the onset and progression of the disease. Our results showed early perturbations in mitochondrial function in 3xTg mouse brain, with the cortex being more susceptible to mitochondrial changes than the hippocampus. In the cortex of 3xTg females, decreased coupled and uncoupled respiration were evident early (at 2 months of age), while in males it appeared later at 6 months of age. We observed increased coupled respiration in the hippocampus of 2-month-old 3xTg females, but no changes were detected later in life. Changes in mitochondrial dynamics were indicated by decreased mitofusin (Mfn2) and increased dynamin related protein 1 (Drp1) (only in females) in the hippocampus and cortex of 3xTg mice. Our findings highlight the importance of controlling and accounting for sex, brain region, and age in studies examining brain bioenergetics using this common AD model in order to more accurately evaluate potential therapies and improve the sex-specific translatability of preclinical findings.

Topical Delivery of Muscarinic Receptor Antagonists Prevents and Reverses Peripheral Neuropathy in Female Diabetic Mice.

Muscarinic antagonists promote sensory neurite outgrowth in vitro and prevent and/or reverse multiple indices of peripheral neuropathy in rodent models of diabetes, chemotherapy-induced peripheral neuropathy, and HIV protein-induced neuropathy when delivered systemically. We measured plasma concentrations of the M1 receptor-selective muscarinic antagonist pirenzepine when delivered by subcutaneous injection, oral gavage, or topical application to the skin and investigated efficacy of topically delivered pirenzepine against indices of peripheral neuropathy in diabetic mice. Topical application of 2% pirenzepine to the paw resulted in plasma concentrations 6 hours postdelivery that approximated those previously shown to promote neurite outgrowth in vitro. Topical delivery of pirenzepine to the paw of mice with streptozotocin-induced diabetes dose-dependently (0.1%-10.0%) prevented tactile allodynia, thermal hypoalgesia, and loss of epidermal nerve fibers in the treated paw and attenuated large fiber motor nerve conduction slowing in the ipsilateral limb. Efficacy against some indices of neuropathy was also noted in the contralateral limb, indicating systemic effects following local treatment. Topical pirenzepine also reversed established paw heat hypoalgesia, whereas withdrawal of treatment resulted in a gradual decline in efficacy over 2-4 weeks. Efficacy of topical pirenzepine was muted when treatment was reduced from 5 to 3 or 1 day/wk. Similar local effects were noted with the nonselective muscarinic receptor antagonist atropine when applied either to the paw or to the eye. Topical delivery of muscarinic antagonists may serve as a practical therapeutic approach to treating diabetic and other peripheral neuropathies. SIGNIFICANCE STATEMENT: Muscarinic antagonist pirenzepine alleviates diabetic peripheral neuropathy when applied topically in mice.

Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase ß to Augment Mitochondrial Function and Prevent Neurodegeneration.

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKß to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 µM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 µM) or short hairpin RNA to CaMKKß. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 µM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M1R-modulated, CaMKKß-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Mitochondrial Respiration Correlates with Prognostic Markers in Chronic Lymphocytic Leukemia and Is Normalized by Ibrutinib Treatment.

Mitochondrial bioenergetics profiling, a measure of oxygen consumption rates, correlates with prognostic markers and can be used to assess response to therapy in chronic lymphocytic leukemia (CLL) cells. In this study, we measured mitochondrial respiration rates in primary CLL cells using respirometry to evaluate mitochondrial function. We found significant increases in mitochondrial respiration rates in CLL versus control B lymphocytes. We also observed amongst CLL patients that advanced age, female sex, zeta-chain-associated protein of 70 kD (ZAP-70+), cluster of differentiation 38 (CD38+), and elevated ß2-microglobulin (ß2-M) predicted increased maximal respiration rates. ZAP-70+ CLL cells exhibited significantly higher bioenergetics than B lymphocytes or ZAP-70- CLL cells and were more sensitive to the uncoupler, carbonyl cyanide-p-trifluoro-methoxyphenylhydrazone (FCCP). Univariable and multivariable linear regression analysis demonstrated that ZAP-70+ predicted increased maximal respiration. ZAP-70+ is a surrogate for B cell receptor (BCR) activation and can be targeted by ibrutinib, which is a clinically approved Bruton's tyrosine kinase (BTK) inhibitor. Therefore, we evaluated the oxygen consumption rates (OCR) of CLL cells and plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4) levels from ibrutinib-treated patients and demonstrated decreased OCR similar to control B lymphocytes, suggesting that ibrutinib treatment resets the mitochondrial bioenergetics, while diminished CCL3/CCL4 levels indicate the down regulation of the BCR signaling pathway in CLL. Our data support evaluation of mitochondrial respiration as a preclinical tool for the response assessment of CLL cells.

Activation of Cannabinoid Receptors Attenuates Endothelin-1-Induced Mitochondrial Dysfunction in Rat Ventricular Myocytes.

Evidence suggests that the activation of the endocannabinoid system offers cardioprotection. Aberrant energy production by impaired mitochondria purportedly contributes to various aspects of cardiovascular disease. We investigated whether cannabinoid (CB) receptor activation would attenuate mitochondrial dysfunction induced by endothelin-1 (ET1). Acute exposure to ET1 (4 hours) in the presence of palmitate as primary energy substrate induced mitochondrial membrane depolarization and decreased mitochondrial bioenergetics and expression of genes related to fatty acid oxidation (ie, peroxisome proliferator-activated receptor-gamma coactivator-1α, a driver of mitochondrial biogenesis, and carnitine palmitoyltransferase-1ß, facilitator of fatty acid uptake). A CB1/CB2 dual agonist with limited brain penetration, CB-13, corrected these parameters. AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, mediated the ability of CB-13 to rescue mitochondrial function. In fact, the ability of CB-13 to rescue fatty acid oxidation-related bioenergetics, as well as expression of proliferator-activated receptor-gamma coactivator-1α and carnitine palmitoyltransferase-1ß, was abolished by pharmacological inhibition of AMPK using compound C and shRNA knockdown of AMPKα1/α2, respectively. Interventions that target CB/AMPK signaling might represent a novel therapeutic approach to address the multifactorial problem of cardiovascular disease.

Amelioration of Both Central and Peripheral Neuropathy in Mouse Models of Type 1 and Type 2 Diabetes by the Neurogenic Molecule NSI-189.

While peripheral neuropathy is the most common complication of long-term diabetes, cognitive deficits associated with encephalopathy and myelopathy also occur. Diabetes is a risk factor for Alzheimer disease (AD) and increases the risk of progression from mild cognitive impairment to AD. The only current recommendation for preventing or slowing the progression of peripheral neuropathy is to maintain close glycemic control, while there is no recommendation for central nervous system disorders. NSI-189 is a new chemical entity that when orally administered promotes neurogenesis in the adult hippocampus, increases hippocampal volume, enhances synaptic plasticity, and reduces cognitive dysfunction. To establish the potential for impact on peripheral neuropathy, we first showed that NSI-189 enhances neurite outgrowth and mitochondrial functions in cultured adult rat primary sensory neurons. Oral delivery of NSI-189 to murine models of type 1 (female) and type 2 (male) diabetes prevented multiple functional and structural indices of small and large fiber peripheral neuropathy, increased hippocampal neurogenesis, synaptic markers and volume, and protected long-term memory. NSI-189 also halted progression of established peripheral and central neuropathy. NSI-189, which is currently in clinical trials for treatment of major depressive disorder, offers the opportunity for the development of a single therapeutic agent against multiple indices of central and peripheral neuropathy.

Poly(ADP-ribose) polymerase-1 inhibits mitochondrial respiration by suppressing PGC-1α activity in neurons.

Poly(ADP-ribose) polymerase-1 (PARP1) is a ubiquitous nuclear enzyme that regulates DNA repair and genomic stability. In oxidative genotoxic conditions, PARP1 activity is enhanced significantly, leading to excessive depletion of nicotinamide adenine dinucleotide (NAD+) and mitochondrial dysfunction. We hypothesized that PARP1-induced NAD+ depletion inhibits NAD+-dependent sirtuin deacetylase activity, thereby interfering with the mitochondrial regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). The DNA alkylator, N'-Nitro-N-nitroso-N-methylguanidine (MNNG), induced NAD+ depletion, inhibited sirtuin deacetylase activity and enhanced acetylation of PGC-1α. This was associated with reduced interaction between PGC-1α and nuclear respiratory factor 1 (NRF-1), which is a nuclear transcription factor that drives mitochondrial replication by regulating mitochondrial transcription factor A (TFAM). MNNG also reduced binding of NRF-1 to the tfam upstream promoter region and reduced TFAM mRNA, mitochondrial DNA copy number and respiratory function. MNNG effects were mitigated by PARP1 inhibition and genetic loss of function, by enhancing intracellular NAD+ levels, and with sirtuin (SIRT1) gain of function, supporting a mechanism dependent on PARP1 activity, NAD+-depletion and SIRT1 inhibition. This and other work from our group supports a destructive sequelae of events related to PARP1-induced sirtuin inhibition and sirtuin-mediated regulation of transcription.

Insulin-like growth factor-1 activates AMPK to augment mitochondrial function and correct neuronal metabolism in sensory neurons in type 1 diabetes.

OBJECTIVE: Diabetic sensorimotor polyneuropathy (DSPN) affects approximately half of diabetic patients leading to significant morbidity. There is impaired neurotrophic growth factor signaling, AMP-activated protein kinase (AMPK) activity and mitochondrial function in dorsal root ganglia (DRG) of animal models of type 1 and type 2 diabetes. We hypothesized that sub-optimal insulin-like growth factor 1 (IGF-1) signaling in diabetes drives loss of AMPK activity and mitochondrial function, both contributing to development of DSPN. METHODS: Age-matched control Sprague-Dawley rats and streptozotocin (STZ)-induced type 1 diabetic rats with/without IGF-1 therapy were used for in vivo studies. For in vitro studies, DRG neurons from control and STZ-diabetic rats were cultured and treated with/without IGF-1 in the presence or absence of inhibitors or siRNAs. RESULTS: Dysregulation of mRNAs for IGF-1, AMPKα2, ATP5a1 (subunit of ATPase), and PGC-1ß occurred in DRG of diabetic vs. control rats. IGF-1 up-regulated mRNA levels of these genes in cultured DRGs from control or diabetic rats. IGF-1 treatment of DRG cultures significantly (P < 0.05) increased phosphorylation of Akt, P70S6K, AMPK and acetyl-CoA carboxylase (ACC). Mitochondrial gene expression and oxygen consumption rate (spare respiratory capacity), ATP production, mtDNA/nDNA ratio and neurite outgrowth were augmented (P < 0.05). AMPK inhibitor, Compound C, or AMPKα1-specific siRNA suppressed IGF-1 elevation of mitochondrial function, mtDNA and neurite outgrowth. Diabetic rats treated with IGF-1 exhibited reversal of thermal hypoalgesia and, in a separate study, reversed the deficit in corneal nerve profiles. In diabetic rats, IGF-1 elevated the levels of AMPK and P70S6K phosphorylation, raised Complex IV-MTCO1 and Complex V-ATP5a protein expression, and restored the enzyme activities of Complex IV and I in the DRG. IGF-1 prevented TCA metabolite build-up in nerve. CONCLUSIONS: In DRG neuron cultures IGF-1 signals via AMPK to elevate mitochondrial function and drive axonal outgrowth. We propose that this signaling axis mediates IGF-1-dependent protection from distal dying-back of fibers in diabetic neuropathy.

Muscarinic receptor antagonists activate ERK-CREB signaling to augment neurite outgrowth of adult sensory neurons.

A major cellular effector activated by G protein coupled receptors is extracellular signal-regulated kinase (ERK). The ERK signaling cascade regulates a variety of cellular processes including growth and proliferation. Both G protein and ß-arrestin-mediated signaling lead to ERK activation by phosphorylation through different kinases. Recently, we have shown muscarinic acetylcholine type 1 receptor (M1R) antagonists, muscarinic toxin 7 (MT7) and pirenzepine, elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models. Thus, we tested the novel hypothesis that muscarinic antagonists could drive neurite outgrowth through altered M1R-ERK signaling. We have used two dimensional isoelectric focusing/SDS-PAGE combined with analysis using multiple phospho-epitope specific antibodies to study ERK1/2 phosphorylation and activation of its downstream nuclear effector cyclic response element binding protein (CREB). Activated CREB is known to exhibit neuroprotective and growth promoting effects. One hour of treatment with MT7 and pirenzepine activated ERK through M1R and induced a significant increase in levels of pCREB(S133) in cultured sensory neurons. Further, pharmacological blockade or siRNA based knockdown of ERK abolished the MT7 and pirenzepine mediated neuritogenic effect. In addition, we have shown drug-induced alterations of charged protein fractions that may possess additional post-translationally modified forms of ERK and CREB. For the first time we show that long-term treatment, e.g. 1 h, with muscarinic antagonists selective or specific for M1R can activate a biased ß-arrestin dependent ERK-CREB signal cascade. Our study gives novel insight into muscarinic antagonist-mediated modulation of M1R-ERK-CREB signaling which could be exploited for therapy in neuropathic diseases.

High glucose concentration suppresses a SIRT2 regulated pathway that enhances neurite outgrowth in cultured adult sensory neurons.

In peripheral nerve under hyperglycemic conditions high flux of d-glucose through the polyol pathway drives an aberrant redox state contributing to neurodegeneration in diabetic sensorimotor polyneuropathy (DSPN). Sirtuins, including SIRT2, detect the redox state via the NAD+/NADH ratio to regulate mitochondrial function via, in part, AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). In adult dorsal root ganglia (DRG) sensory neurons mitochondrial dysfunction has been proposed as an etiological factor in dying-back neuropathy in diabetes. We tested the hypothesis that a high concentration of d-glucose depleted SIRT2 expression via enhancement of polyol pathway activity. We posited that this would lead to impaired mitochondrial function and suppression of neurite outgrowth in cultured sensory neurons. The use of dominant negative mutants or neurons from SIRT2 knockout (KO) mice to block SIRT2 signaling revealed that neurons derived from control or type 1 diabetic rodents required SIRT2 for optimal neurite outgrowth. Over-expression of WT-SIRT2 elevated neurite outgrowth in normal and diabetic cultures. SIRT2 protein isoforms 2.1 and 2.2 were reduced by 20-30% in DRG of type 1 diabetic mice (p < .05). After 72 h exposure to high d-glucose (25 mM vs 5 mM) cultured sensory neurons showed a significant 2-fold (p < .05) decrease in SIRT2 expression, P-AMPK, levels of respiratory Complexes II/III and respiratory capacity. DRG neurons expressed aldose reductase and the aforementioned deficits were prevented by treatment with aldose reductase inhibitors (lidorestat or sorbinil) or sorbitol dehydrogenase inhibitor (SDI-158). In cultures derived from type 1 diabetic rats treatment with SDI-158 elevated expression of SIRT2, P-AMPK/PGC-1α and neurite outgrowth (p < .05). SIRT2 KO neurons exhibited deficits in the LKB-1/AMPK/PGC-1α pathway and mitochondrial function. In cultured neurons the SIRT2 pathway enhances axonal outgrowth and this signaling axis encompassing activation of AMPK/PGC-1α is impaired in DSPN, in part, due to enhanced polyol pathway activity caused by hyperglycemia.