Katie E. Frizzi

Ciliary neurotrophic factor activates NF-κB to enhance mitochondrial bioenergetics and prevent neuropathy in sensory neurons of streptozotocin-induced diabetic rodents

Diabetes causes mitochondrial dysfunction in sensory neurons that may contribute to peripheral neuropathy. Ciliary neurotrophic factor (CNTF) promotes sensory neuron survival and axon regeneration and prevents axonal dwindling, nerve conduction deficits and thermal hypoalgesia in diabetic rats. In this study, we tested the hypothesis that CNTF protects sensory neuron function during diabetes through normalization of impaired mitochondrial bioenergetics. In addition, we investigated whether the NF-κB signal transduction pathway was mobilized by CNTF. Neurite outgrowth of sensory neurons derived from streptozotocin (STZ)-induced diabetic rats was reduced compared to neurons from control rats and exposure to CNTF for 24 h enhanced neurite outgrowth. CNTF also activated NF-κB, as assessed by Western blotting for the NF-κB p50 subunit and reporter assays for NF-κB promoter activity. Conversely, blockade of NF-κB signaling using SN50 peptide inhibited CNTF-mediated neurite outgrowth. Studies in mice with STZ-induced diabetes demonstrated that systemic therapy with CNTF prevented functional indices of peripheral neuropathy along with deficiencies in dorsal root ganglion (DRG) NF-κB p50 expression and DNA binding activity. DRG neurons derived from STZ-diabetic mice also exhibited deficiencies in maximal oxygen consumption rate and associated spare respiratory capacity that were corrected by exposure to CNTF for 24 h in an NF-κB-dependent manner. We propose that the ability of CNTF to enhance axon regeneration and protect peripheral nerve from structural and functional indices of diabetic peripheral neuropathy is associated with targeting of mitochondrial function, in part via NF-κB activation, and improvement of cellular bioenergetics.

Repeated monitoring of corneal nerves by confocal microscopy as an index of peripheral neuropathy in type-1 diabetic rodents and the effects of topical insulin

We developed a reliable imaging and quantitative analysis method for in vivo corneal confocal microscopy (CCM) in rodents and used it to determine whether models of type 1 diabetes replicate the depletion of corneal nerves reported in diabetic patients. Quantification was reproducible between observers and stable across repeated time points in two rat strains. Longitudinal studies were performed in normal and streptozotocin (STZ)‐diabetic rats, with innervation of plantar paw skin quantified using standard histological methods after 40 weeks of diabetes. Diabetic rats showed an initial increase, then a gradual reduction in occupancy of nerves in the sub‐basal plexus so that values were significantly lower at week 40 (68 ± 6%) than age‐matched controls (80 ± 2%). No significant loss of stromal or intra‐epidermal nerves was detected. In a separate study, insulin was applied daily to the eye of control and STZ‐diabetic mice and this treatment prevented depletion of nerves of the sub‐basal plexus. Longitudinal studies are viable in rodents using CCM and depletion of distal corneal nerves precedes detectable loss of epidermal nerves in the foot, suggesting that diabetic neuropathy is not length dependent. Loss of insulin‐derived neurotrophic support may contribute to the pathogenesis of corneal nerve depletion in type 1 diabetes.

Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation.

PURPOSE Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis. We investigated the potential for HSPC transplantation to treat corneal defects in cystinosis. METHODS We isolated HSPCs from transgenic DsRed mice and systemically transplanted irradiated Ctns-/- mice. A year posttransplantation, we investigated the fate and function of HSPCs by in vivo confocal and fluorescence microscopy (IVCM), quantitative RT-PCR (RT-qPCR), mass spectrometry, histology, and by measuring the IOP. To determine the mechanism by which HSPCs may rescue disease cells, we transplanted Ctns-/- mice with Ctns-/- DsRed HSPCs virally transduced to express functional CTNS-eGFP fusion protein. RESULTS We found that a single systemic transplantation of wild-type HSPCs prevented ocular pathology in the Ctns-/- mice. Engraftment-derived HSPCs were detected within the cornea, and also in the sclera, ciliary body, retina, choroid, and lens. Transplantation of HSPC led to substantial decreases in corneal cystine crystals, restoration of normal corneal thickness, and lowered IOP in mice with high levels of donor-derived cell engraftment. Finally, we found that HSPC-derived progeny differentiated into macrophages, which displayed tunneling nanotubes capable of transferring cystinosin-bearing lysosomes to diseased cells. CONCLUSIONS To our knowledge, this is the first demonstration that HSPCs can rescue hereditary corneal defects, and supports a new potential therapeutic strategy for treating ocular pathologies.

Peripheral Neuropathy in Mouse Models of Diabetes.

Peripheral neuropathy is a frequent complication of chronic diabetes that most commonly presents as a distal degenerative polyneuropathy with sensory loss. Around 20% to 30% of such patients may also experience neuropathic pain. The underlying pathogenic mechanisms are uncertain, and therapeutic options are limited. Rodent models of diabetes have been used for more than 40 years to study neuropathy and evaluate potential therapies. For much of this period, streptozotocin‐diabetic rats were the model of choice. The emergence of new technologies that allow relatively cheap and routine manipulations of the mouse genome has prompted increased use of mouse models of diabetes to study neuropathy. In this article, we describe the commonly used mouse models of type 1 and type 2 diabetes, and provide protocols to phenotype the structural, functional, and behavioral indices of peripheral neuropathy, with a particular emphasis on assays pertinent to the human condition.

Spinal Disinhibition in Experimental and Clinical Painful Diabetic Neuropathy

Impaired rate-dependent depression (RDD) of the Hoffman reflex is associated with reduced dorsal spinal cord potassium chloride cotransporter expression and impaired spinal γ-aminobutyric acid type A receptor function, indicative of spinal inhibitory dysfunction. We have investigated the pathogenesis of impaired RDD in diabetic rodents exhibiting features of painful neuropathy and the translational potential of this marker of spinal inhibitory dysfunction in human painful diabetic neuropathy. Impaired RDD and allodynia were present in type 1 and type 2 diabetic rats but not in rats with type 1 diabetes receiving insulin supplementation that did not restore normoglycemia. Impaired RDD in diabetic rats was rapidly normalized by spinal delivery of duloxetine acting via 5-hydroxytryptamine type 2A receptors and temporally coincident with the alleviation of allodynia. Deficits in RDD and corneal nerve density were demonstrated in patients with painful diabetic neuropathy compared with healthy control subjects and patients with painless diabetic neuropathy. Spinal inhibitory dysfunction and peripheral small fiber pathology may contribute to the clinical phenotype in painful diabetic neuropathy. Deficits in RDD may help identify patients with spinally mediated painful diabetic neuropathy who may respond optimally to therapies such as duloxetine.

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.

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.

Internode length is reduced during myelination and remyelination by neurofilament medium phosphorylation in motor axons

ABSTRACT The distance between nodes of Ranvier, referred to as internode length, positively correlates with axon diameter, and is optimized during development to ensure maximal neuronal conduction velocity. Following myelin loss, internode length is reestablished through remyelination. However, remyelination results in short internode lengths and reduced conduction rates. We analyzed the potential role of neurofilament phosphorylation in regulating internode length during remyelination and myelination. Following ethidium bromide induced demyelination, levels of neurofilament medium (NF‐M) and heavy (NF‐H) phosphorylation were unaffected. Preventing NF‐M lysine‐serine‐proline (KSP) repeat phosphorylation increased internode length by 30% after remyelination. To further analyze the role of NF‐M phosphorylation in regulating internode length, gene replacement was used to produce mice in which all KSP serine residues were replaced with glutamate to mimic constitutive phosphorylation. Mimicking constitutive KSP phosphorylation reduced internode length by 16% during myelination and motor nerve conduction velocity by ˜27% without altering sensory nerve structure or function. Our results suggest that NF‐M KSP phosphorylation is part of a cooperative mechanism between axons and Schwann cells that together determine internode length, and suggest motor and sensory axons utilize different mechanisms to establish internode length. HIGHLIGHTSAxonal NF‐M remains phosphorylated after demyelination and through remyelination.NF‐M phosphorylation is part of a cooperative mechanism that determines internode length.NF‐M phosphorylation influences internode length establishment in motor but not in sensory axons.

708. Treatment of Inherited Eye Defects By Systemic Hematopoietic Stem Cell Transplantation

Corneal disease is a significant cause of blindness worldwide, largely due to the low availability of donor corneas and the risks associated with transplantation, such as immune rejection, cataract, and glaucoma. Stem cell transplantation represents an attractive alternative for corneal repair because of their capacity to differentiate into corneal cells. Limbal epithelial stem (LEST) cells, which reside within the corneo-limbus, have been used for more than a decade to repair corneal epithelium, and mesenchymal stem cells (MSCs) have been shown to migrate and promote corneal healing and even differentiate into epithelial/keratocyte-like cells. However, risks of immune rejection with LEST cells, and heterogeneity in repair mechanisms with MSCs have led to discrepancies in treatment outcomes. Alternatively, very little is known about the potential for hematopoietic stem cells (HSCs) in eye repair. HSCs have attractive features; they are multipotent cells capable of self-renewal and easily mobilized from bone marrow into the circulatory system. Here we report that engraftment of HSPCs can treat corneal defects in a mouse model of a degenerative hereditary metabolic disorder, cystinosis. Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin, resulting in cystine crystal accumulation in tissues. In eyes, a primary affected organ, crystals accumulate within the cornea causing photophobia and eventually blindness, if untreated. Using the mouse model of cystinosis, Ctns-/- mice, which develops similar eye anomalies than patients, we showed that systemic HSC transplantation led to the long-term preservation of the eyes. Effective therapy, however, was dependent on achieving relatively high level of donor-derived blood cell engraftment (more than 50%). Abundant GFP-expressing bone marrow-derived cells were detected within the cornea but also in the sclera, ciliary body, retina, choroid, and lens. In addition, HSCs engraftment led to substantial decrease in corneal cystine crystals, restoration of normal corneal thickness and intraocular pressure, and improved vision after one-year post-transplantation. Finally, we showed that HSCs mainly differentiated into MHC class II+ macrophages, which transferred cystinosin-bearing lysosomes via tunneling nanotubes to disease cells. This work is the first demonstration that HSCs can rescue corneal defects, which brings forth a new potential therapeutic strategy for treating ocular pathologies.