Knowledge about age-related eye diseases in the general population in Germany.

BACKGROUND: With a rising prevalence of age-related eye diseases, prevention and early diagnosis of these conditions are key goals of public eye health. Disease-related knowledge in the general public supports these goals but there is little data available. Thus, we have assessed knowledge of cataract, glaucoma, age-related macular degeneration (AMD) and diabetic eye disease in the German adult general population in a cross-sectional study and identified target groups for health education interventions. METHODS: Knowledge assessment content was identified based on a literature review, expert input, and a list of items was generated after a qualitative selection process. The resulting 16-item instrument (4 items per condition) was administered to 1,008 participants from a survey panel, demographically representative of the adult German population. Test properties were evaluated based on a Rasch model and multiple correspondence analysis (MCA). Binary-logistic regression analysis was performed to investigate associations with age, sex, education level, employment status, marital status, income, reported health status, visual difficulties, and recent general practitioner (GP) and ophthalmologist consultations. RESULTS: Replies were correct for a median of 9 out of 16 (range 2 - 16) items, which differed between conditions (p < 0.0001). Most responses were correct for cataract items (median: 3 / 4) and least were correct for AMD items (median: 2 / 4). 27%, 9%, 1% and 19% of respondents replied correctly to all cataract, glaucoma, AMD and diabetic eye disease-related items, respectively. Rasch analysis suggested an adequate targeting of items and in MCA, no evidence of multidimensionality was present. Older age, being retired, decreased general health and recent GP or ophthalmology consultations were significantly associated with more knowledge about common eye conditions (p ≤ 0.005). GP or ophthalmology consultations remained significant in a multivariable model (p ≤ 0.011). CONCLUSIONS: Knowledge gaps regarding eye health are considerable in the German general population and should therefore be addressed in educational interventions targeting the public. Special attention when designing such campaigns needs to be paid to infrequent users of the healthcare system. Knowledge of AMD seems to be poorer compared to other eye conditions.

Deuteron-to-Proton Mass Ratio from Simultaneous Measurement of the Cyclotron Frequencies of H_{2}

By simultaneously measuring the cyclotron frequencies of an H_{2}^{+} ion and a deuteron in a coupled magnetron orbit we have made an extended series of measurements of their cyclotron frequency ratio. From the observed changes in H_{2}^{+} mass energy we have followed the decay of three H_{2}^{+} ions to the vibrational ground state. We are able to assign some of our measured ratios to specific rovibrational levels, hence reducing uncertainty due to H_{2}^{+} rotational energy. Assuming the most probable assignment, we obtain a deuteron-to-proton mass ratio, m_{d}/m_{p}=1.999 007 501 272(9). Combined with the atomic mass of the deuteron [S. Rau et al., Nature (London) 585, 43 (2020).NATUAS0028-083610.1038/s41586-020-2628-7] we also obtain a new value for the atomic mass of the proton, m_{p}=1.007 276 466 574(10) u.

Deuteron-to-Proton Mass Ratio from the Cyclotron Frequency Ratio of H_{2}

We have measured cyclotron frequency ratios of H_{2}^{+} to D^{+} with sufficient precision to resolve the mass increase of H_{2}^{+} due to vibrational energy. Additional discrimination against excited vibrational levels was provided by increasing the rate of vibrational decay through Stark quenching. From our results we obtain a value for the deuteron-to-proton mass ratio, m_{d}/m_{p}=1.999 007 501 274(38), which has an uncertainty three times smaller than the current CODATA value.

HspB1 silences translation of PDZ-RhoGEF by enhancing miR-20a and miR-128 expression to promote neurite extension.

HspB1 is a small heat shock protein implicated in neuronal survival and neurite growth; mutations in HspB1 have been identified in hereditary motor neuronopathies and Charcot Marie Tooth Type 2 neuropathies. In cortical neurons we found that expression of HspB1 decreased RhoA activity and RhoA-GTP protein, and reversed the inhibition of neurite extension induced by NogoA. HspB1 decreased PDZ-RhoGEF, a RhoA specific guanine nucleotide exchange factor, while other regulators of RhoA activity were unchanged. The decrease in PDZ-RhoGEF was independent of proteasomal or lysosomal degradation pathways and was not associated with changes in PDZ-RhoGEF mRNA. We sequenced the 3'UTR of rat PDZ-RhoGEF and found binding sites for miRNAs miR-20a, miR-128 and miR-132. Expression of these microRNAs was substantially increased in cortical neurons transfected with HspB1. Co-transfection of HspB1 with specific inhibitors of miR-20a or miR-128 prevented the decrease in PDZ-RhoGEF and blocked the neurite growth promoting effects of HspB1. Using the 3'UTR of PDZ-RhoGEF mRNA in a luciferase reporter construct we observed that HspB1, miR-20a and miR-128 each inhibited luciferase expression. We conclude that HspB1 regulates RhoA activity through modulation of PDZ-RhoGEF levels achieved by translational control through enhanced expression of specific miRNAs (miR-20a and miR-128). Regulation of RhoA activity by translational silencing of PDZ-RhoGEF may be the mechanism through which HspB1 is involved in regulation of neurite growth. As RhoA-GTPase plays a regulatory role in the organization and stability of cytoskeletal networks through its downstream effectors, the results suggest a possible mechanism linking HspB1 mutations and axonal cytoskeletal pathology.

Test-Retest Reliability of the Impact of Vision Impairment-Very Low Vision Questionnaire.

Purpose: Most patient-reported outcome measures used in ophthalmology show floor effects in a very low vision population, which limits their use in vision restoration trials. The Impact of Vision Impairment-Very Low Vision scale (IVI-VLV) was developed to specifically target a very low vision population, but its test-retest reliability has not been investigated yet. Methods: The German version of the IVI-VLV was administered twice to patients with stable disease of a low vision clinic. Test and retest person measures of the IVI-VLV subscales were obtained from Rasch analysis. Test-retest reliability was investigated by intraclass correlation coefficients and Bland-Altman plots. Results: We included 134 patients (72 women, 62 men) at a mean age of 62 ± 15 years. The intraclass correlation coefficients were 0.920 (95% confidence interval, 0.888-0.944) for the activities of daily living and mobility subscale of the IVI-VLV and 0.929 (95% confidence interval, 0.899-0.949) for the emotional well-being subscale. Bland-Altman plots did not indicate any systematic bias. In linear regression analysis, test-retest differences were not significantly associated with visual acuity or administration interval. Conclusions: Both subscales of the IVI-VLV showed excellent repeatability independent of visual acuity and length of repeat interval. Further validation steps including an assessment of the patient-reported outcome measure's responsiveness are required for use in vision restoration trials. Translational Relevance: The results support repeated use of the IVI-VLV as a patient-reported end point in future studies in very low and ultralow vision populations.

Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy.

BACKGROUND: Painful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms Na(V)1.7 and Na(V)1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in Na(V)1.7 protein levels in DRG in vivo. To further evaluate the role of Na(V)α subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against Na(V)α subunits. RESULTS: Subcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in Na(V)α subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia. CONCLUSIONS: These data support the role of increased Na(V)α protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.

Gene therapy for pain: results of a phase I clinical trial.

OBJECTIVE: Preclinical evidence indicates that gene transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)-based vectors can reduce pain-related behavior in animal models of pain. This clinical trial was carried out to assess the safety and explore the potential efficacy of this approach in humans. METHODS: We conducted a multicenter, dose-escalation, phase I clinical trial of NP2, a replication-defective HSV-based vector expressing human preproenkephalin (PENK) in subjects with intractable focal pain caused by cancer. NP2 was injected intradermally into the dermatome(s) corresponding to the radicular distribution of pain. The primary outcome was safety. As secondary measures, efficacy of pain relief was assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ), and concurrent opiate usage. RESULTS: Ten subjects with moderate to severe intractable pain despite treatment with >200mg/day of morphine (or equivalent) were enrolled into the study. Treatment was well tolerated with no study agent-related serious adverse events observed at any point in the study. Subjects receiving the low dose of NP2 reported no substantive change in pain. Subjects in the middle- and high-dose cohorts reported pain relief as assessed by NRS and SF-MPQ. INTERPRETATION: Treatment of intractable pain with NP2 was well tolerated. There were no placebo controls in this relatively small study, but the dose-responsive analgesic effects suggest that NP2 may be effective in reducing pain and warrants further clinical investigation.

Prevention of diabetic neuropathy by regulatable expression of HSV-mediated erythropoietin.

Previous studies have demonstrated that gene transfer of genes coding for neurotrophic factors to the dorsal root ganglion (DRG) using nonreplicating herpes simplex virus (HSV)-based vectors injected subcutaneously can prevent the progression of diabetic neuropathy. Because prolonged expression of neurotrophic factors could potentially have unwanted adverse effects, we constructed a nonreplicating HSV vector, vHrtEPO, to express erythropoietin (EPO) under the control of a tetracycline response element (TRE)-minimal cytomegalovirus (CMV) fusion promoter. Primary DRG neurons in culture infected with vHrtEPO express and release EPO in response to exposure to doxycycline (DOX). Animals infected with vHrtEPO by footpad inoculation demonstrated regulated expression of EPO in DRG under the control of DOX administered by gavage. Mice rendered diabetic by injection of streptozotocin (STZ), inoculated with vHrtEPO, and treated with DOX 4 days out of 7 each week for 4 weeks were protected against the development of diabetic neuropathy as assessed by electrophysiologic and behavioral measures. These studies indicate that intermittent expression of EPO in DRG achieved from a regulatable vector is sufficient to protect against the progression of neuropathy in diabetic animals, and provides proof-of-principle preclinical evidence for the development of such vectors for clinical trial.

Rho GTPase regulation of α-synuclein and VMAT2: implications for pathogenesis of Parkinson's disease.

Accumulation of α-synuclein (Asyn) in neuronal perikarya and dystrophic neurites is characteristic of idiopathic and familial Parkinson's disease. In this study, we investigated the relationship between α-synuclein expression and neurite outgrowth-maturation using MN9D dopaminergic cells and demonstrated key features of Asyn regulation in hippocampal neurons. Neurite elongation elicited by inhibition of Rho GTPase activity with C3 transferase or by db-cAMP treatment was associated with marked reduction of α-synuclein mRNA and protein expression. Rho inhibition resulted in reduction of transcription factor SRF in the nuclear fraction and retention of MKL-1 - the SRF co-transactivator of SRE - in cytosol, indicating that these effects of Rho inhibition may be mediated though reduction of SRF-SRE transcription. Inhibition of Rho GTPase activity led to decreased nuclear localization of GATA2, a key regulator of α-synuclein promoter activity. Rho inhibition-induced neurite extension was associated with increased VMAT2 and SNARE proteins synaptophysin and synapsin I. These results indicate that in the MN9D dopaminergic cell line, α-synuclein transcription and levels of synaptic vesicle associated proteins are inversely correlated with neurite growth. We confirm that in mature hippocampal neurons inhibition of RhoA and knock down of SRF by siRNA also lead to decrease GATA2 and Asyn. The results suggest that RhoA signaling may be potential therapeutic target for the treatment of synucleinopathies.

Instrumental Activities of Daily Living Tools in Very-Low Vision: Ready for Use in Trials?

Traditional endpoints assessing visual function are limited by their responsiveness to interventions restoring or maintaining vision. An alternative concept is assessing instrumental activities of daily living (IADL). Herein, we review all available vision-specific IADL instruments relevant for vision restoration trials and report data for the most promising instrument. Six relevant instruments exist: The Low Vision Functional Status Evaluation (LVFSE), Timed IADL (TIADL), Melbourne Low-Vision Activities of Daily Living Index (MLVAI), Assessment of Disability Related to Vision (ADREV), Functional Low-Vision Observer Rated Assessment (FLORA), and Very Low Vision IADL (IADL-VLV). Both internal consistency and test-retest data were available for the LVFSE, MLVAI, and IADL-VLV. In a sample from a low-vision clinic (n = 51; age 57 ± 16 years), we report additional validation data on the IVI-VLV including test-retest reliability (intraclass correlation coefficient 0.981 [0.961; 0.991]). The LVSFE was noticeably less reliable than the MLVAI and the IADL-VLV. Content and construct validity data were available for the LVFSE, TIADL, MLVAI, ADREV, and IADL-VLV, but only the MLVAI and IADL-VLV were developed for an ultra-low vision context. Ceiling effects were present across instruments. Thus, of all appropriate IADL instruments related to vision, the IADL-VLV and MLVAI best meet existing requirements for use in vision restoration trials, e.g., in gene therapies or visual prostheses in inherited retinal diseases, but require further validation.

Soluble Nogo receptor down-regulates expression of neuronal Nogo-A to enhance axonal regeneration.

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.

Neuronal Nogo-A regulates glutamate receptor subunit expression in hippocampal neurons.

Nogo-A and its cognate receptor NogoR1 (NgR1) are both expressed in neurons. To explore the function of these proteins in neurons of the CNS, we carried out a series of studies using postnatal hippocampal neurons in culture. Interfering with the binding of Nogo-A to NgR1 either by adding truncated soluble fragment of NgR1 (NgSR) or by reducing NgR1 protein with a specific siRNA, resulted in a marked reduction in Nogo-A expression. Inhibition of Rho-ROCK or MEK-MAPK signaling resulted in a similar reduction in neuronal Nogo-A mRNA and protein. Reducing Nogo-A protein levels by siRNA resulted in an increase in the post-synaptic scaffolding protein PSD95, as well as increases in GluA1/GluA2 AMPA receptor and GluN1/GluN2A/GluN2B NMDA glutamate receptor subunits. siRNA treatment to reduce Nogo-A resulted in phosphorylation of mTOR; addition of rapamycin to block mTOR signaling prevented the up-regulation in glutamate receptor subunits. siRNA reduction of NgR1 resulted in increased expression of the same glutamate receptor subunits. Taken together the results suggest that transcription and translation of Nogo-A in hippocampal neurons is regulated by a signaling through NgR1, and that interactions between neuronal Nogo-A and NgR1 regulate glutamatergic transmission by altering NMDA and AMPA receptor levels through an rapamycin-sensitive mTOR-dependent translation mechanism.

Glial TNFα in the spinal cord regulates neuropathic pain induced by HIV gp120 application in rats.

BACKGROUND: HIV-associated sensory neuropathy (HIV-SN) is one of the most common forms of peripheral neuropathy, affecting about 30% of people with acquired immune deficiency syndrome (AIDS). The symptoms of HIV-SN are dominated by neuropathic pain. Glia activation in the spinal cord has become an attractive target for attenuating chronic pain. This study will investigate the role of spinal TNFα released from glia in HIV-related neuropathic pain. RESULTS: Peripheral gp120 application into the rat sciatic nerve induced mechanical allodynia for more than 7 weeks, and upregulated the expression of spinal TNFα in the mRNA and the protein levels at 2 weeks after gp120 application. Spinal TNFα was colocalized with GFAP (a marker of astrocytes) and Iba1 (a marker of microglia) in immunostaining, suggesting that glia produce TNFα in the spinal cord in this model. Peripheral gp120 application also increased TNFα in the L4/5 DRG. Furthermore, intrathecal administration of TNFα siRNA or soluble TNF receptor reduced gp120 application-induced mechanical allodynia. CONCLUSIONS: Our results indicate that TNFα in the spinal cord and the DRG are involved in neuropathic pain, following the peripheral HIV gp120 application, and that blockade of the glial product TNFα reverses neuropathic pain induced by HIV gp120 application.

TNFα is involved in neuropathic pain induced by nucleoside reverse transcriptase inhibitor in rats.

In patients with HIV/AIDS, neuropathic pain is a common neurological complication. Infection with the HIV itself may lead to neuropathic pain, and painful symptoms are enhanced when patients are treated with nucleoside reverse transcriptase inhibitors (NRTIs). The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current studies, we tested the role of TNFα in antiretroviral drug-induced neuropathic pain. We administered 2',3'-dideoxycytidine (ddC, one of the NRTIs) systemically to induce mechanical allodynia. We found that ddC induced overexpression of both mRNA and proteins of GFAP and TNFα in the spinal dorsal horn. TNFα was colocalized with GFAP in the spinal dorsal horn and with NeuN in the DRG. Knockdown of TNFα with siRNA blocked the mechanical allodynia induced by ddC. Intrathecal administration of glial inhibitor or recombinant TNF soluble receptor, reversed mechanical allodynia induced by ddC. These results suggest that TNFα is involved in NRTI-induced neuropathic pain.

Expression of the EGFR-RAS Inhibitory Proteins DOK1 and MTMR7 and its Significance in Colorectal Adenoma and Adenoma Recurrence.

BACKGROUND AND AIMS: Colorectal adenomas are precursor lesions for colorectal cancer (CRC), a major cause of cancer-related death. Despite all molecular insights, there are still unknown variables in the development of CRC as well as uncertainties regarding adenoma recurrence after resection. We aimed to characterize the expression of docking protein 1 (DOK1) and myotubularin-related protein 7 (MTMR7), which share inhibiting functions on EGFR-RAS-signalling, a major oncogenic driver in CRC, and their association with clinical variables and adenoma recurrence. METHODS: This observational study is based on clinical data obtained from patients who underwent routine endoscopy and consecutive follow-up examinations. Immunohistochemistry was conducted both in dysplastic tissue and adjacent non-dysplastic mucosa followed by microscopical assessment. Recurrence was differentiated between local, segmental and distant relapse. RESULTS: A total of 56 patients (23 females) gathering 96 adenomas/polyps were included. 36 patients experienced a metachronous lesion, 23 patients had simultaneous lesions in their index endoscopy. Female patients showed lower levels of MTMR7 in adenomas (p=0.0318). Adenomas of young patients showed lower DOK1 than those of older patients (p=0.0469). Big adenomas showed a higher expression of DOK1 than small lesions (p=0.0044). In serrated lesions, DOK1 was reduced (p=0.0026) and correlated with the quantity of lesions (p < 0.001). MTMR7 was significantly reduced in distant (p=0.05) and local segmental recurrence (p=0.0362), while DOK1 showed higher expression in recurrence (p=0.0291). CONCLUSIONS: We found ambivalent results regarding the role of the markers as potential tumor suppressors, implying a context-dependent function of these molecules which might change in the course of time. DOK1 may play an inhibiting role in the serrated pathway. Remarkably, molecular markers have the potential to predict recurrence, since a combined expression analysis of high DOK1 and low MTMR7 correlated with the likelihood of segmental adenoma recurrence.

Transgene-mediated GDNF expression enhances synaptic connectivity and GABA transmission to improve functional outcome after spinal cord contusion.

Glial cell line-derived trophic factor (GDNF) is a peptide with pleiotropic survival and growth-promoting effects on neurons. We found that intraspinal injection of a non-replicating herpes simplex virus-based vector coding for GDNF 2 h after blunt trauma to the thoraco-lumbar spinal cord produced sustained improvement in motor behavioral outcomes up to 5 weeks following injury. The improvement in behavior correlated with an increase in synaptophysin and glutamic acid decarboxylase (GAD) in the spinal cord at the level of injury. Addition of recombinant GDNF protein to primary spinal cord neurons in-vitro resulted in enhanced neurite growth and a marked increase in protein levels of GAD65 and GAD67, synapsin I and synaptophysin. GDNF-mediated increases in GAD and the synaptic markers were blocked by the MEK inhibitor UO126, but not by the phosphoinositide 3-kinase inhibitor LY294002. These results suggest that GDNF, acting through the MEK-ERK pathway enhances axonal sprouting, synaptic connectivity, and GABAergic neurotransmission in the spinal cord, that result in improved behavioral outcomes after spinal cord contusion injury.

Neuroprotective effect of herpes simplex virus-mediated gene transfer of erythropoietin in hyperglycemic dorsal root ganglion neurons.

We examined the efficacy of herpes simplex virus vector-mediated gene transfer of erythropoietin in preventing neuropathy in mouse model of streptozotocin-diabetes. A replication-incompetent herpes simplex virus vector with erythropoietin under the control of the human cytomegalovirus promoter (vector DHEPO) was constructed. DHEPO expressed and released erythropoietin from primary dorsal root ganglion neurons in vitro, and following subcutaneous inoculation in the foot, expressed erythropoietin in dorsal root ganglion neurons in vivo. At 2 weeks after induction of diabetes, subcutaneous inoculation of erythropoietin prevented the reduction in sensory nerve amplitude characteristic of diabetic neuropathy measured 4 weeks later, preserved autonomic function measured by pilocarpine-induced sweating, and prevented the loss of nerve fibres in the skin and reduction of neuropeptide calcitonin gene-related peptide in the dorsal horn of spinal cord of the diabetic mice. We further investigated whether vector-mediated local expression of erythropoietin in dorsal root ganglion neurons can protect in vivo as well as in vitro hyperglycemia-induced axonal degeneration. Our findings show that the AKT/GSK-3beta dependent pathway plays an important role in mediating the protection of erythropoietin against diabetic neuropathy. Herpes simplex virus-mediated transfer of erythropoietin to dorsal root ganglia may prove useful in treatment of diabetic neuropathy.

Herpes vector-mediated gene transfer in the treatment of chronic pain.

Chronic pain is a major health concern with up to 50% of patients finding little if any relief following traditional pharmacotherapy. This review describes the treatment of chronic pain using herpes simplex virus type 1 (HSV)-based vectors. HSV can be effectively used to deliver pain-modulating transgenes to sensory neurons in vivo following intradermal inoculation. The vector genome persists in peripheral nerve bodies in an episomal state and serves as a platform for expression of natural pain-relieving molecules that access endogenous antinociceptive circuitry. The vectors are mutated to prevent reactivation from latency or spread to the central nervous system. Dermatome selection for administration of HSV vectors provides targeted delivery of pain gene therapy to primary afferent neurons. This novel approach alleviates pain without systemic side effects or the induction of tolerance and can be used in combination with standard pain treatments.

HSV-mediated transfer of artemin overcomes myelin inhibition to improve outcome after spinal cord injury.

Artemin is a neurotrophic factor of the glial cell line-derived neurotrophic factor (GDNF) family of ligands that acts through the GDNF family receptor alpha3 (GFRalpha3)/ret receptor found predominantly on sensory and sympathetic neurons. In order to explore the potential utility of artemin to improve functional outcome after spinal cord injury (SCI), we constructed a nonreplicating herpes simplex virus (HSV)-based vector to express artemin (QHArt). We found that QHArt efficiently transfects spinal cord neurons to produce artemin. Transgene-mediated artemin supported the extension of neurites by primary dorsal root ganglion neurons in culture, and allowed those cells to overcome myelin inhibition of neurite extension through activation of protein kinase A (PKA) to phosphorylate cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and increase expression of arginase I. Intraspinal injection of QHArt immediately after thoracic spinal cord dorsal over hemisection produced a statistically significant improvement in motor recovery over the course of four weeks measured by locomotor rating score.

Continuous delta-opioid receptor activation reduces neuronal voltage-gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy.

The Na(V)1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased Na(V)1.7 in dorsal root ganglia (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus-based vector expressing proenkephalin reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG Na(V)1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAPK (mitogen-activated protein kinase) and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mm glucose for 18 h also demonstrated an increase in Na(V)1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on Na(V)1.7 production in vitro was mimicked by exposure to PMA and blocked by the myristolated PKC inhibitor 20-28 or the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]; the effect of vector-mediated enkephalin on Na(V)1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic delta-opioid receptor by enkephalin prevents the increase in neuronal Na(V)1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the delta-opioid receptor and voltage-gated sodium channels.