Selective antagonism of muscarinic receptors is neuroprotective in peripheral neuropathy.

Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M1R) exhibited enhanced neurite outgrowth, confirming the role of M1R in tonic suppression of axonal plasticity. M1R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M1R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetes-induced mitochondrial dysfunction in vitro and in vivo. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible.

Activation of nuclear factor-kappaB via endogenous tumor necrosis factor alpha 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-kappaB (NF-kappaB) 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 alpha (TNFalpha), a potent activator of NF-kappaB activity. We tested the hypothesis that activation of NF-kappaB stimulated by endogenous TNFalpha 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-kappaB-binding activity. This phenomenon was mimicked in cultured neurons in which there was substantial NF-kappaB nuclear translocation and a significant rise in NF-kappaB DNA-binding activity after plating. Inhibitors of NF-kappaB (SN50 or NF-kappaB decoy DNA) resulted in necrotic cell death of medium to large neurons (> or =40 microm) 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 TNFalpha, and exposure to an anti-TNFalpha antibody inhibited NF-kappaB DNA-binding activity by approximately 35% and killed approximately 40% of medium to large neurons within 24 h. The results show for the first time that cytokine-mediated activation of NF-kappaB is a component of the signaling pathway responsible for maintenance of adult sensory neuron survival after axon damage.

Diabetes impairs an interleukin-1ß-dependent pathway that enhances neurite outgrowth through JAK/STAT3 modulation of mitochondrial bioenergetics in adult sensory neurons.

BACKGROUND: A luminex-based screen of cytokine expression in dorsal root ganglia (DRG) and nerve of type 1 diabetic rodents revealed interleukin-1 (IL-1α) and IL-1ß to be significantly depressed. We, therefore, tested the hypothesis that impaired IL-1α and IL-1ß expression in DRG may contribute to aberrant axon regeneration and plasticity seen in diabetic sensory neuropathy. In addition, we determined if these cytokines could optimize mitochondrial bioenergetics since mitochondrial dysfunction is a key etiological factor in diabetic neuropathy. RESULTS: Cytokines IL-1α and IL-1ß were reduced 2-fold (p<0.05) in DRG and/or nerve of 2 and 5 month streptozotocin (STZ)-diabetic rats. IL-2 and IL-10 were unchanged. IL-1α and IL-1ß induced similar 2 to 3-fold increases in neurite outgrowth in cultures derived from control or diabetic rats (p<0.05). STAT3 phosphorylation on Tyr705 or Ser727 was depressed in DRG from STZ-diabetic mice and treatment of cultures derived from STZ-diabetic rats with IL-1ß for 30 min raised phosphorylation of STAT3 on Tyr705 and Ser727 by 1.5 to 2-fold (p<0.05). shRNA-based or AG490 inhibition of STAT3 activity or shRNA blockade of endogenous IL-1ß expression completely blocked neurite outgrowth. Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1ß and maximal oxygen consumption rate and spare respiratory capacity, both key measures of bioenergetic fidelity that were depressed in diabetic compared with control neurons, were enhanced 2-fold. This effect was blocked by AG490. CONCLUSIONS: Endogenous synthesis of IL-1ß is diminished in nerve tissue in type 1 diabetes and we propose this defect triggers reduced STAT3 signaling and mitochondrial function leading to sup-optimal axonal regeneration and plasticity.

Receptor for advanced glycation end-products (RAGE) activates divergent signaling pathways to augment neurite outgrowth of adult sensory neurons.

BACKGROUND: The receptor for advanced glycation end-products (RAGE) is implicated in neuronal differentiation during embryogenesis and in regulation of peripheral nerve regeneration. However, the role of RAGE ligands and the signaling pathways utilized by activated RAGE in mediating axon regeneration in adult neurons remain unknown. We tested the hypothesis that RAGE signaling modulated neurotrophin-induced neurite outgrowth in cultured adult sensory neurons. RESULTS: Dorsal root ganglia (DRG) neurons from adult rats in vitro were exposed to specific RAGE ligands, signal transduction inhibitors and function blocking anti-RAGE IgG to assess their impact on neurite outgrowth. RAGE ligands including human glycated albumin (HGA), S100 calcium binding protein (S100B) and high mobility group 1 protein (HMGB1; alternatively termed amphoterin) in the presence of neurotrophins elevated neurite outgrowth 2-fold (p<0.05). shRNA to RAGE or anti-RAGE IgG blockade of RAGE inhibited neurite outgrowth by 40-90% (p<0.05). Western blotting and gene reporter analysis showed RAGE ligands activated NF-κB, JAK-STAT and ERK pathways. RAGE ligand induction of neurite outgrowth was blocked by inhibition of NF-κB, JAK-STAT or ERK pathways revealing the necessity for combined activation for optimal growth. RAGE ligands rapidly elevated NF-κB p65 expression in the cytoplasm while triggering translocation of NF-κB p50 to the nucleus. shRNA blockade of p50 demonstrated that translocation of p50 to the nucleus was implicated in driving axonal outgrowth. CONCLUSIONS: RAGE signaling is a complex mediator of neurotrophin-dependent neurite outgrowth, operating through divergent but partly inter-dependent pathways.

Guaifenesin derivatives promote neurite outgrowth and protect diabetic mice from neuropathy.

In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of neurite outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the neurite outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy.

Sensory neurons derived from diabetic rats have diminished internal Ca2+ stores linked to impaired re-uptake by the endoplasmic reticulum.

Distal symmetrical sensory neuropathy in diabetes involves the dying back of axons, and the pathology equates with axonal dystrophy generated under conditions of aberrant Ca2+ signalling. Previous work has described abnormalities in Ca2+ homoeostasis in sensory and dorsal horn neurons acutely isolated from diabetic rodents. We extended this work by testing the hypothesis that sensory neurons exposed to long-term Type 1 diabetes in vivo would exhibit abnormal axonal Ca2+ homoeostasis and focused on the role of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). DRG (dorsal root ganglia) sensory neurons from age-matched normal and 3-5-month-old STZ (streptozotocin)-diabetic rats (an experimental model of Type 1 diabetes) were cultured. At 1-2 days in vitro an array of parameters were measured to investigate Ca2+ homoeostasis including (i) axonal levels of intracellular Ca2+, (ii) Ca2+ uptake by the ER (endoplasmic reticulum), (iii) assessment of Ca2+ signalling following a long-term thapsigargin-induced blockade of SERCA and (iv) determination of expression of ER mass and stress markers using immunocytochemistry and Western blotting. KCl- and caffeine-induced Ca2+ transients in axons were 2-fold lower in cultures of diabetic neurons compared with normal neurons indicative of reduced ER calcium loading. The rate of uptake of Ca2+ into the ER was reduced by 2-fold (P<0.05) in diabetic neurons, while markers for ER mass and ER stress were unchanged. Abnormalities in Ca2+ homoeostasis in diabetic neurons could be mimicked via long-term inhibition of SERCA in normal neurons. In summary, axons of neurons from diabetic rats exhibited aberrant Ca2+ homoeostasis possibly triggered by sub-optimal SERCA activity that could contribute to the distal axonopathy observed in diabetes.

NF-kappaB activated by ER calcium release inhibits Abeta-mediated expression of CHOP protein: enhancement by AD-linked mutant presenilin 1.

Mutations in presenilin which result in early-onset Alzheimer disease (AD) cause both increased calcium release from intracellular stores, primarily endoplasmic reticulum (ER), and changes in NF-kappaB activation. Some studies have also reported that neurons containing AD-linked mutant presenilins (mPS1) show increased vulnerability to various stresses, while others report no differences in neuronal death. The majority of these reports center on potential changes in ER stress, because of the enhanced ER calcium release seen in mPS1 neurons. One of the primary death effectors of ER stress is CHOP, also termed GADD153, which acts to transcriptionally inhibit protective cellular molecules such as Bcl-2 and glutathione. Because both CHOP and NF-kappaB are activated by increased intracellular calcium and stress, yet have diametrically opposite effects on neuronal vulnerability, we sought to examine this interaction in greater detail. We observed that IP3-mediated calcium release from ER, stimulated by Abeta exposure, mediated both CHOP expression and NF-kappaB DNA binding activity. Further, specific inhibition of NF-kappaB resulted in greater expression of CHOP, while activation of NF-kappaB inhibited CHOP expression. The enhanced release of calcium from IP3-mediated ER stores in mPS1 neurons stimulated increased NF-kappaB compared to normal neurons, which inhibited CHOP expression. Upon blockage of NF-kappaB, exposure to Abeta caused significantly greater Abeta-mediated CHOP expression and death in mPS1 neurons compared to normal neurons. Thus, AD-linked PS1 mutations disrupt the balance between stress-induced NF-kappaB and CHOP, resulting in greater dependence on stress-induced NF-kappaB activation in mPS1 neurons.