Gordon W. Glazner

Complete Sequence of a Novel Protein Containing a Femtomolar‐Activity‐Dependent Neuroprotective Peptide

Abstract : The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pl 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity‐dependent neurotrophic factor ; (2) a glutaredoxin active site ; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuron—astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the β‐amyloid peptide (the Alzheimers disease neurotoxin), N‐methyl‐D‐aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E‐deficient mice accelerated the acquisition of developmental reflexes and prevented short‐term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E‐deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.

Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy

Insulin deficiency in type I diabetes may lead to cognitive impairment, cerebral atrophy and white matter abnormalities. We studied the impact of a novel delivery system using intranasal insulin (I-I) in a mouse model of type I diabetes (streptozotocin-induced) for direct targeting of pathological and cognitive deficits while avoiding potential adverse systemic effects. Daily I-I, subcutaneous insulin (S-I) or placebo in separate cohorts of diabetic and non-diabetic CD1 mice were delivered over 8 months of life. Radio-labelled insulin delivery revealed that I-I delivered more rapid and substantial insulin levels within the cerebrum with less systemic insulin detection when compared with S-I. I-I delivery slowed development of cognitive decline within weekly cognitive/behavioural testing, ameliorated monthly magnetic resonance imaging abnormalities, prevented quantitative morphological abnormalities in cerebrum, improved mouse mortality and reversed diabetes-mediated declines in mRNA and protein for phosphoinositide 3-kinase (PI3K)/Akt and for protein levels of the transcription factors cyclic AMP response element binding protein (CREB) and glycogen synthase kinase 3beta (GSK-3beta) within different cerebral regions. Although the murine diabetic brain was not subject to cellular loss, a diabetes-mediated loss of protein and mRNA for the synaptic elements synaptophysin and choline acetyltransferase was prevented with I-I delivery. As a mechanism of delivery, I-I accesses the brain readily and slows the development of diabetes-induced brain changes as compared to S-I delivery. This therapy and delivery mode, available in humans, may be of clinical utility for the prevention of pathological changes in the diabetic human brain.

Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system.

Glial cell line‐derived neurotrophic factor (GDNF) is a neurotrophic factor that influences the survival and function of several neuronal populations in the central (CNS) and peripheral nervous systems. The actions of GDNF are mediated by a multicomponent receptor complex composed of the tyrosine kinase product of c‐ret and the ligand‐binding protein GDNF receptor alpha (GDNFR‐α). In the present study, we used in situ hybridization to localize cells expressing the mRNA for these GDNF receptor subunits in rat CNS. As reported previously, GDNFR‐α and c‐ret mRNA are present in the substantia nigra and ventral tegmental area, regions containing GDNF‐responsive dopamine neurons. However, both mRNA were found in motor neurons of spinal cord and brainstem nuclei that innervate skeletal muscle. These areas include alpha motor neurons in the ventral horn of spinal cord and neurons in hypoglossal, facial, trigeminal, and abducens nuclei. In areas rostral to the substantia nigra, c‐ret mRNA is not detected, whereas GDNFR‐α is found in numerous brain structures, including the hippocampus, cortex, medial geniculate, and the medial habenula, the latter area expressing the highest levels of GDNFR‐α mRNA in brain. These results provide evidence that c‐ret and GDNFR‐α mRNA are expressed in neuronal populations involved in motor function and provides further support for GDNF as a target‐derived neurotrophic for these motor neurons. The observation that GDNFR‐α mRNA is localized in several brain structures that do not contain detectable levels of c‐ret mRNA indicates that either GDNFR‐α utilizes signal transduction molecules other than c‐ret in these areas or that other GDNF‐like ligands that utilize GDNFR‐α as a receptor may be present. J. Comp. Neurol. 391:42–49, 1998.

Differential Effects of BDNF, ADNF9, and TNFα on Levels of NMDA Receptor Subunits, Calcium Homeostasis, and Neuronal Vulnerability to Excitotoxicity

Calcium influx through N-methyl-d-aspartate (NMDA) receptors can result in neuronal apoptosis or necrosis and may play a pivotal role in neuronal death in many different neurodegenerative diseases. In the present study we employed primary neuronal cultures and three different excitoprotective factors, brain-derived neurotrophic factor (BDNF), activity-dependent neurotrophic factor (ADNF9), and tumor necrosis factor alpha (TNFalpha), to elucidate the mechanisms whereby trophic factors modify the excitotoxic process. Neurons pretreated with BDNF exhibited increased levels of the NMDA receptor subunits NR1 and NR2A, which was associated with increased calcium responses to NMDA and vulnerability to excitotoxic necrosis and reduced vulnerability to apoptosis. ADNF9 and TNFalpha suppressed calcium responses to glutamate and protected neurons against both excitotoxic necrosis and apoptosis, but had no effect on levels of NMDA receptor subunits. Inhibition of phosphorylation and DNA binding of NF-kappaB, by H7 and kappaB decoy DNA, respectively, suggest that the excitotoxic-modulating actions of BDNF are mediated by kinases, while those of ADNF9 and TNFalpha are mediated by both kinases and the transcription factor NF-kappaB. Our data show that, whereas BDNF increases neuronal responses to glutamate while ADNF9 and TNFalpha decrease the same, all three protect against excitotoxic apoptosis.

Distal Degenerative Sensory Neuropathy in a Long-Term Type 2 Diabetes Rat Model

OBJECTIVE—Peripheral neuropathy associated with type 2 diabetes (DPN) is not widely modeled. We describe unique features of DPN in type 2 diabetic Zucker diabetic fatty (ZDF) rats. RESEARCH DESIGN AND METHODS—We evaluated the structural, electrophysiological, behavioral, and molecular features of DPN in ZDF rats and littermates over 4 months of hyperglycemia. The status of insulin signaling transduction molecules that might be interrupted in type 2 diabetes and selected survival-, stress-, and pain-related molecules was emphasized in dorsal root ganglia (DRG) sensory neurons. RESULTS—ZDF rats developed slowing of motor sciatic-tibial and sensory sciatic digital conduction velocity and selective mechanical allodynia with preserved thermal algesia. Diabetic sural axons, preserved in number, developed atrophy, but there was loss of large-calibre dermal and small-calibre epidermal axons. In diabetic rats, insulin signal transduction pathways in lumbar DRGs were preserved or had trends toward upregulation: mRNA levels of insulin receptor β-subunit (IRβ), insulin receptor substrate (IRS)-1, and IRS-2. The numbers of neurons expressing IRβ protein were also preserved. There were trends toward early rises of mRNA levels of heat shock protein 27 (HSP27), the α2δ1 calcium channel subunit, and phosphatidylinositol 3-kinase in diabetes. Others were unchanged, including nuclear factor-κB (NF-κB; p50/p105) and receptor for advanced glycosylation endproducts (RAGE) as was the proportion of neurons expressing HSP27, NF-κB, and RAGE protein. CONCLUSIONS—ZDF type 2 diabetic rats develop a distal degenerative sensory neuropathy accompanied by a selective long-term pain syndrome. Neuronal insulin signal transduction molecules are preserved.

Activation of Nuclear Factor-κB via Endogenous Tumor Necrosis Factor α Regulates Survival of Axotomized Adult Sensory Neurons

Embryonic dorsal root ganglion (DRG) neurons die after axonal damage in vivo, and cultured embryonic DRG neurons require exogenous neurotrophic factors that activate the neuroprotective transcription factor nuclear factor-κB (NF-κB) for survival. In contrast, adult DRG neurons survive permanent axotomy in vivo and in defined culture media devoid of exogenous neurotrophic factors in vitro. Peripheral axotomy in adult rats induces local accumulation of the cytokine tumor necrosis factor α (TNFα), a potent activator of NF-κB activity. We tested the hypothesis that activation of NF-κB stimulated by endogenous TNFα was required for survival of axotomized adult sensory neurons. Peripheral axotomy of lumbar DRG neurons by sciatic nerve crush induced a very rapid (within 2 h) and significant elevation in NF-κB-binding activity. This phenomenon was mimicked in cultured neurons in which there was substantial NF-κB nuclear translocation and a significant rise in NF-κB DNA-binding activity after plating. Inhibitors of NF-κB (SN50 or NF-κB decoy DNA) resulted in necrotic cell death of medium to large neurons (≥40 μm) within 24 h (60 and 75%, respectively), whereas inhibition of p38 and mitogen-activated protein/extracellular signal-regulated kinase did not effect survival. ELISA revealed that these cultures contained TNFα, and exposure to an anti-TNFα antibody inhibited NF-κB DNA-binding activity by ∼35% and killed ∼40% of medium to large neurons within 24 h. The results show for the first time that cytokine-mediated activation of NF-κB is a component of the signaling pathway responsible for maintenance of adult sensory neuron survival after axon damage.

Nuclear factor-κB mediates the cell survival-promoting action of activity-dependent neurotrophic factor peptide-9

Abstract : Activity‐dependent neurotrophic factor (ADNF) is produced by astrocytes in response to neuronal depolarization and, in turn, promotes neuronal survival. A nineamino acid ADNF peptide (ADNF9) exhibits full neurotrophic activity and potently protects cultured embryonic rat hippocampal neurons from oxidative injury and apoptosis. Picomolar concentrations of ADNF9 induced an increase in nuclear factor‐κB (NF‐κB) DNA‐binding activity within 1 h of exposure, with a maximum increase of ~10‐fold by 6 h. Activation of NF‐κB was correlated with increased resistance of neurons to apoptosis induced by exposure to Fe2+. The antiapoptotic action of ADNF9 was abolished when NF‐κB activation was specifically blocked with κB decoy DNA. Oxidative stress was attenuated in neurons pretreated with ADNF9, and this effect of ADNF9 was blocked by κB decoy DNA, suggesting that ADNF9 suppresses apoptosis by reducing oxidative stress. ADNF9 also prevented neuronal apoptosis following trophic factor withdrawal via an NF‐κB‐mediated mechanism. Thus, NF‐κB mediates the neuron survival‐promoting effects of ADNF9 in experimental models relevant to developmental neuronal death and neurodegenerative disorders.

Interleukin-1 alpha and vasoactive interstinal peptide: Enigmatic regulation of neuronal survival

A neurotrophic role for interleukin‐1 alpha (IL‐1α) was investigated in dissociated spinal cord‐dorsal root ganglion cultures. Three observations suggested a survival‐promoting action for IL‐1α in nine‐day‐old cultures: (1) neutralizing antiserum to murine IL‐1α decreased neuronal survival; (2) treatment with IL‐1α in electrically blocked cultures increased neuronal survival; and (3) antiserum to the type I IL‐1 receptor decreased neuronal survival. Treatment with VIP prevented neuronal cell death associated with the antiserum to IL‐1α. In contrast, treatment of one‐month‐old cultures with IL‐1α produced neuronal cell death and neutralizing antiserum to the IL‐1 receptor had no effect on neuronal survival in these cultures. These experiments suggested that an IL‐1‐like substance was necessary for neuronal survival during a specific stage in development and that a relationship between VIP and IL‐1α might account in part for the neurotrophic properties of VIP.

Maternal vasoactive intestinal peptide and the regulation of embryonic growth in the rodent.

Vasoactive intestinal peptide (VIP) has been shown to regulate early postimplantation growth in rodents through central nervous system receptors. However, the source of VIP mediating these effects is unknown. Although VIP binding sites are present prenatally, VIP mRNA was not detected in the rat central nervous system before birth and was detected in the periphery only during the last third of pregnancy. In the present study, the embryonic day (E11) rat embryo/trophoblast was shown to have four times the VIP concentration of the E17 fetus and to have VIP receptors in the central nervous system. However, no VIP mRNA was detected in the E11 rat embryo or embryonic membranes by in situ hybridization or reverse transcriptase-PCR. RIA of rat maternal serum revealed a peak in VIP concentration at days E10-E12 of pregnancy, with VIP rising to levels 6-10-fold higher than during the final third of pregnancy. After intravenous administration of radiolabeled VIP to pregnant female mice, undegraded VIP was found in the E10 embryo. These results suggest that maternal tissues may provide neuroendocrine support for embryonic growth through a surge of VIP during early postimplantation development in the rodent.

Activity-Dependent Neurotrophic Factor Peptide (ADNF9) Protects Neurons Against Oxidative Stress-Induced Death

Abstract : Activity‐dependent neurotrophic factor (ADNF) and a 14‐amino acid fragment of this peptide (sequence VLGGGSALLRSIPA) protect neurons from death associated with an array of toxic conditions, including amyloid β‐peptide, N‐methyl‐d‐aspartate, tetrodotoxin, and the neurotoxic HIV envelope coat protein gp120. We report that an even smaller, nine‐amino acid fragment (ADNF9) with the sequence SALLRSIPA potently protects cultured embryonic day 18 rat hippocampal neurons from oxidative injury and neuronal apoptosis induced by FeSO4 and trophic factor withdrawal. Among the characteristics of this protection are maintenance of mitochondrial function and a reduction in accumulation of intracellular reactive oxygen species.