Short‐term hyperglycemia produces oxidative damage and apoptosis in neurons

Dorsal root ganglia neurons in culture die through programmed cell death when exposed to elevated glucose, providing an in vitro model system for the investigation of the mechanisms leading to diabetic neuropathy. This study examines the time course of programmed cell death induction, regulation of cellular antioxidant capacity, and the protective effects of antioxidants in neurons exposed to hyperglycemia. We demonstrate that the first 2 h of hyperglycemia are sufficient to induce oxidative stress and programmed cell death. Using fluorimetric analysis of reactive oxygen species (ROS) production, in vitro assays of antioxidant enzymes, and immunocytochemical assays of cell death, we demonstrate superoxide formation, inhibition of aconitase, and lipid peroxidation within 1 h of hyperglycemia. These are followed by caspase‐3 activation and DNA fragmentation. Antioxidant potential increases by 3–6 h but is insufficient to protect these neurons. Application of the antioxidant α‐lipoic acid potently prevents glucose‐induced oxidative stress and cell death. This study identifies cellular therapeutic targets to prevent diabetic neuropathy. Since oxidative stress is a common feature of the micro‐ and macrovascular complications of diabetes, the present findings have broad application to the treatment of diabetic patients.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154304/1/fsb2fj042513fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154304/2/fsb2fj042513fje-sup-0001.pd

Cell Culture Modeling to Test Therapies Against Hyperglycemia-Mediated Oxidative Stress and Injury

The concept that oxidative stress is a key mediator of nerve injury in diabetes has led us to design therapies that target oxidative stress mechanisms. Using an in vitro model of glucose-treated dorsal root ganglion (DRG) neurons in culture, we can examine both free radical generation, using fluorimetric probes for reactive oxygen species, and cell death via the TUNEL assay. The cell culture system is scaled down to a 96-well plate format, and so is well suited to high-throughput screening. In the present study, we test the ability of three drugs, nicotinamide, allopurinol, and α-lipoic acid, alone and in combination to prevent DRG neuron oxidative stress and cell death. This combination of drugs is currently in clinical trial in type 1 diabetic patients. We demonstrate independent effects on oxidative stress and neuronal survival for the three drugs, and neuronal protection using the three drugs in combination. The data strengthen the rationale for the current clinical trial. In addition, we describe an effective tool for rapid preclinical testing of novel therapies against diabetic neuropathy. Antioxid. Redox Signal. 7, 1494–1506.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63115/1/ars.2005.7.1494.pd

Phosphatidylinositol 3‐kinase and Akt effectors mediate insulin‐like growth factor‐I neuroprotection in dorsal root ganglia neurons

Insulin‐like growth factor‐I (IGF‐I) protects neurons of the peripheral nervous system from apoptosis, but the underlying signaling pathways are not well understood. We studied IGF‐I mediated signaling in embryonic dorsal root ganglia (DRG) neurons. DRG neurons express IGF‐I receptors (IGF‐IR), and IGF‐I activates the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. High glucose exposure induces apoptosis, which is inhibited by IGF‐I through the PI3K/Akt pathway. IGF‐I stimulation of the PI3K/Akt pathway phosphorylates three known Akt effectors: the survival transcription factor cyclic AMP response element binding protein (CREB) and the pro‐apoptotic effector proteins glycogen synthase kinase‐3β (GSK‐3β) and forkhead (FKHR). IGF‐I regulates survival at the nuclear level through accumulation of phospho‐Akt in DRG neuronal nuclei, increased CREB‐mediated transcription, and nuclear exclusion of FKHR. High glucose increases expression of the pro‐apoptotic Bcl protein Bim (a transcriptional target of FKHR). However, IGF‐I does not regulate Bim or anti‐apoptotic Bcl‐xL protein expression levels, which suggests that IGF‐I neuroprotection is not through regulation of their expression. High glucose also induces loss of the initiator caspase‐9 and increases caspase‐3 cleavage, effects blocked by IGF‐I. These data suggest that IGF‐I prevents apoptosis in DRG neurons by regulating PI3K/Akt pathway effectors, including GSK‐3β, CREB, and FKHR, and by blocking caspase activation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154325/1/fsb2fj041581fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154325/2/fsb2fj041581fje-sup-0001.pd

NT-3, like NGF, Is Required for Survival of Sympathetic Neurons, but Not Their Precursors

AbstractSuperior cervical ganglia of postnatal mice with a targeted disruption of the gene for neurotrophin-3 have 50% fewer neurons than those of wild-type mice. In culture, neurotrophin-3 increases the survival of proliferating sympathetic precursors. Both precursor death (W. ElShamy et al., 1996, Development 122, 491–500) and, more recently, neuronal death (S. Wyatt et al., 1997, EMBO J. 16, 3115–3123) have been described in mice lacking NT-3. Consistent with the second report, we found that, in vivo, neurogenesis and precursor survival were unaffected by the absence of neurotrophin-3 but neuronal survival was compromised so that only 50% of the normal number of neurons survived to birth. At the time of neuron loss, neurotrophin-3 expression, assayed with a lacZ reporter, was detected in sympathetic target tissues and blood vessels, including those along which sympathetic axons grow, suggesting it may act as a retrograde neurotrophic factor, similar to nerve growth factor. To explore this possibility, we compared neuron loss in neurotrophin-3-deficient mice with that in nerve growth factor-deficient mice and found that neuronal losses occurred at approximately the same time in both mutants, but were less severe in mice lacking neurotrophin-3. Eliminating one or both neurotrophin-3 alleles in mice that lack nerve growth factor does not further reduce sympathetic neuron number in the superior cervical ganglion at E17.5 but does alter axon outgrowth and decrease salivary gland innervation. Taken together these results suggest that neurotrophin-3 is required for survival of some sympathetic neurons that also require nerve growth factor

Cortical Neurons Develop Insulin Resistance and Blunted Akt Signaling: A Potential Mechanism Contributing to Enhanced Ischemic Injury in Diabetes

Patients with diabetes are at higher risk of stroke and experience increased morbidity and mortality after stroke. We hypothesized that cortical neurons develop insulin resistance, which decreases neuroprotection via circulating insulin and insulin-like growth factor-I (IGF-I). Acute insulin treatment of primary embryonic cortical neurons activated insulin signaling including phosphorylation of the insulin receptor, extracellular signal-regulated kinase (ERK), Akt, p70S6K, and glycogen synthase kinase-3- (GSK-3-). To mimic insulin resistance, cortical neurons were chronically treated with 25-mM glucose, 0.2-mM palmitic acid (PA), or 20-nM insulin before acute exposure to 20-nM insulin. Cortical neurons pretreated with insulin, but not glucose or PA, exhibited blunted phosphorylation of Akt, p70S6K, and GSK-3- with no change detected in ERK. Inhibition of the phosphatidylinositol 3-kinase (PI3-K) pathway during insulin pretreatment restored acute insulin-mediated Akt phosphorylation. Cortical neurons in adult BKS-db/db mice exhibited higher basal Akt phosphorylation than BKS-db+ mice and did not respond to insulin. Our results indicate that prolonged hyperinsulinemia leads to insulin resistance in cortical neurons. Decreased sensitivity to neuroprotective ligands may explain the increased neuronal damage reported in both experimental models of diabetes and diabetic patients after ischemia-reperfusion injury. Antioxid. Redox Signal. 14, 1829-1839.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90430/1/ars-2E2010-2E3816.pd

Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin α8β1 in the embryonic kidney

The epithelial–mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin α8β1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin α8β1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, α8β1-AP detects a novel ligand of 70–90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin α8β1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas α8β1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by α8β1-AP and forms a complex with α8β1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating α8β1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects

Human neural stem cell transplantation into the corpus callosum of Alzheimer’s mice

The hippocampus has been the target of stem cell transplantations in preclinical studies focused on Alzheimer’s disease, with results showing improvements in histological and behavioral outcomes. The corpus callosum is another structure that is affected early in Alzheimer’s disease. Therefore, we hypothesize that this structure is a novel target for human neural stem cell transplantation in transgenic Alzheimer’s disease mouse models. This study demonstrates the feasibility of targeting the corpus callosum and identifies an effective immunosuppression regimen for transplanted neural stem cell survival. These results support further preclinical development of the corpus callosum as a therapeutic target in Alzheimer’s disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138852/1/acn3443_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138852/2/acn3443.pd

Dual CCR2/CCR5 antagonist treatment attenuates adipose inflammation, but not microvascular complications in ob/ob mice

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138252/1/dom12950.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138252/2/dom12950_am.pd

Lack of Neurotrophin-3 Results in Death of Spinal Sensory Neurons and Premature Differentiation of Their Precursors

AbstractTo understand mechanisms resulting in the absence of two-thirds of spinal sensory neurons in mice lacking NT-3, we have compared dorsal root ganglia development in normal and mutant embryos. The reduction in neurons, achieved by E13, results from several deficits: first, elevated neuronal apoptosis significantly reduces neuronal numbers; second, elevated neurogenesis between E11 and E12, without changes in rates of precursor proliferation or apoptosis, depletes the precursor pool; consequently, the reduced precursor pool prevents increases in neuronal numbers between E12 and E13, when most neurons are born in normal animals. Although deficits occur before final target innervation, we show that NT-3 is expressed at all stages in regions accessible to these neurons or their axons and is only restricted to final targets after innervation