Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100b increased at 90 days when cells were positive for the senescence b-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H 2 O 2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.

Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterolrich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn 934 residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol-and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.Ambient temperature detection is a critical biological process carried out by terminals of primary afferent sensory neurons of the dorsal root (DRG) 2 and trigeminal ganglia in the mammalian sensory system (1). Thermosensitive nerves express a subset of proteins of the transient receptor potential (TRP) ion channel family that are activated at different temperatures, making these cationic ion channels central elements in the temperature sensing machinery of peripheral nerve endings (2). Among thermoTRPs, TRPM8 (transient receptor potential melastatine 8) is characterized by its enhanced activity at low temperatures (threshold, ϳ25°C) and by application of cooling compounds such as menthol and icilin (3, 4). These properties, and its selective expression in a subset of small sensory neurons, make TRPM8 an excellent candidate for transducing cold temperatures at nerve endings, a view supported by behavioral findings in TRPM8 knock-out mice (5-7). In addition to mild cold temperature detection, several recent studies (8, 9) have attributed a role to TRPM8 in noxious cold transduction and nociception.Functional studies in chimeric channels suggest that the C terminus of TRPM8 contains structural elements important in temperature-dependent gating (10). However, the molecular mechanisms underlying temperature sensitivity of TRPM8 are still unknown. It has been hypothesized that TRP channels might sense temperature-mediated changes in lipid bilayer tension (11,12). Whereas at physiological temperatures most of the plasma membrane remains in a l...

Background: TRPM8 channel is N-glycosylated, a post-translational modification affecting trafficking and gating properties of other TRP channels. Results: Preventing N-glycosylation reduces responses of TRPM8 to agonists (cold and menthol) due to a change in its biophysical properties. Conclusion: N-Glycosylation is an important determinant of TRPM8 sensitivity to chemical and thermal stimuli. Significance: N-Glycosylation of TRPM8 could play a modulatory role in cold thermoreceptor activity.

Glial cell line-derived neurotrophic factor (GDNF) was assayed for its neurotrophic effects against the neuronal atrophy that causes cognitive deficits in old age. Aged Fisher 344 rats with impairment in the Morris water maze received intrahippocampal injections at the dorsal CA1 area of either a lentiviral vector encoding human GDNF or the same vector encoding human green fluorescent protein as a control. Recombinant lentiviral vectors constructed with human cytomegalovirus promotor and pseudotyped with lyssavirus Mokola glycoprotein specifically transduced the astrocytes in vivo. Astrocyte-secreted GDNF enhanced neuron function as shown by local increases in synthesis of the neurotransmitters acetylcholine, dopamine and serotonin. This neurotrophic effect led to cognitive improvement of the rats as early as 2 weeks after gene transduction. Spatial learning and memory testing showed a significant gain in cognitive abilities due to GDNF exposure, whereas control-transduced rats kept their performance at the chance level. These results confirm the broad spectrum of the neurotrophic action of GDNF and open new gene therapy possibilities for reducing age-related neurodegeneration.

BackgroundTRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole.ResultsWe show that Y745 at the menthol binding site is critical for inhibition mediated by SKF96365 of cold- and voltage-activated TRPM8 currents. In contrast, the inhibition by other antagonists was unaffected by the mutation (BCTC) or only partially reduced (capsazepine, clotrimazole, econazole), suggesting that additional binding sites exist on the TRPM8 channel from where the inhibitors exert their negative modulation. Indeed, a molecular docking model implies that menthol and SKF96365 interact readily with Y745, while BCTC is unable to bind to this residue.ConclusionIn summary, we identify structural elements on the TRPM8 channel that are critical for the action of channel antagonists, providing valuable information for the future design of new, specific modulator compounds.

Cold allodynia is a common symptom of neuropathic and inflammatory pain following peripheral nerve injury. The mechanisms underlying this disabling sensory alteration are not entirely understood. In primary somatosensory neurons, cold sensitivity is mainly determined by a functional counterbalance between cold-activated TRPM8 channels and Shaker-like Kv1.1-1.2 channels underlying the excitability brake current I Here we studied the role of I in damage-triggered painful hypersensitivity to innocuous cold. We found that cold allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in mice, was related to both an increase in the proportion of cold-sensitive neurons (CSNs) in DRGs contributing to the sciatic nerve, and a decrease in their cold temperature threshold. I density was reduced in high-threshold CSNs from CCI mice compared with sham animals, with no differences in cold-induced TRPM8-dependent current density. The electrophysiological properties and neurochemical profile of CSNs revealed an increase of nociceptive-like phenotype among neurons from CCI animals compared with sham mice. These results were validated using a mathematical model of CSNs, including I and TRPM8, showing that a reduction in I current density shifts the thermal threshold to higher temperatures and that the reduction of this current induces cold sensitivity in former cold-insensitive neurons expressing low levels of TRPM8-like current. Together, our results suggest that cold allodynia is largely due to a functional downregulation of I in both high-threshold CSNs and in a subpopulation of polymodal nociceptors expressing TRPM8, providing a general molecular and neural mechanism for this sensory alteration. This paper unveils the critical role of the brake potassium current I in damage-triggered cold allodynia. Using a well-known form of nerve injury and combining behavioral analysis, calcium imaging, patch clamping, and pharmacological tools, validated by mathematical modeling, we determined that the functional expression of I is reduced in sensory neurons in response to peripheral nerve damage. This downregulation not only enhances cold sensitivity of high-threshold cold thermoreceptors signaling cold discomfort, but it also transforms a subpopulation of polymodal nociceptors signaling pain into neurons activated by mild temperature drops. Our results suggest that cold allodynia is linked to a reduction of I in both high-threshold cold thermoreceptors and nociceptors expressing TRPM8, providing a general model for this form of cold-induced pain.

Background:The functional role of the TRPM8 N terminus remains poorly understood. Results: Truncation of the first 40 residues increases TRPM8 responses to cold and menthol, whereas deletions within the 40 -60-residue region yield nonfunctional channels retained in the ER. Conclusion: Initial N terminus of TRPM8 is important for proper biogenesis and function. Significance: Variations within the N terminus can modulate thermal and chemical sensitivity of TRPM8.

We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain increases Cdk5 activity, a key kinase involved in brain development and function and recently in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TG), we engineered a transgenic mouse model (TNFglo) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNFglo mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca2+ influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, suggesting that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity, therefore this mouse model would be valuable for investigating mechanism involved TNF-α in orofacial pain.