TRPM8 and Nav1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

<p>Abstract</p> <p>Background</p> <p>Familial amyloidotic polyneuropathy (FAP) is a peripheral neuropathy caused by the extracellular accumulation and deposition of insoluble transthyretin (TTR) aggregates. However the molecular mechanism that underlies TTR toxicity in peripheral nerves is unclear. Previous studies have suggested that amyloidogenic proteins can aggregate into oligomers which disrupt intracellular calcium homeostasis by increasing the permeability of the plasma membrane to extracellular calcium. The aim of the present study was to examine the effect of TTR on calcium influx in dorsal root ganglion neurons.</p> <p>Results</p> <p>Levels of intracellular cytosolic calcium were monitored in dorsal root ganglion (DRG) neurons isolated from embryonic rats using the calcium-sensitive fluorescent indicator Fluo4. An amyloidogenic mutant form of TTR, L55P, induced calcium influx into the growth cones of DRG neurons, whereas wild-type TTR had no significant effect. Atomic force microscopy and dynamic light scattering studies confirmed that the L55P TTR contained oligomeric species of TTR. The effect of L55P TTR was decreased by blockers of voltage-gated calcium channels (VGCC), as well as by blockers of Na_v1.8 voltage-gated sodium channels and transient receptor potential M8 (TRPM8) channels. siRNA knockdown of TRPM8 channels using three different TRPM8 siRNAs strongly inhibited calcium influx in DRG growth cones.</p> <p>Conclusions</p> <p>These data suggest that activation of TRPM8 channels triggers the activation of Na_v1.8 channels which leads to calcium influx through VGCC. We suggest that TTR-induced calcium influx into DRG neurons may contribute to the pathophysiology of FAP. Furthermore, we speculate that similar mechanisms may mediate the toxic effects of other amyloidogenic proteins such as the β-amyloid protein of Alzheimer's disease.</p

Perilipin 5 Deletion Unmasks an Endoplasmic Reticulum Stress-Fibroblast Growth Factor 21 Axis in Skeletal Muscle.

Lipid droplets (LDs) are critical for the regulation of lipid metabolism, and dysregulated lipid metabolism contributes to the pathogenesis of several diseases, including type 2 diabetes. We generated mice with muscle-specific deletion of the LD-associated protein perilipin 5 (PLIN5, Plin5MKO ) and investigated PLIN5's role in regulating skeletal muscle lipid metabolism, intracellular signaling, and whole-body metabolic homeostasis. High-fat feeding induced changes in muscle lipid metabolism of Plin5MKO mice, which included increased fatty acid oxidation and oxidative stress but, surprisingly, a reduction in inflammation and endoplasmic reticulum (ER) stress. These muscle-specific effects were accompanied by whole-body glucose intolerance, adipose tissue insulin resistance, and reduced circulating insulin and C-peptide levels in Plin5MKO mice. This coincided with reduced secretion of fibroblast growth factor 21 (FGF21) from skeletal muscle and liver, resulting in reduced circulating FGF21. Intriguingly, muscle-secreted factors from Plin5MKO , but not wild-type mice, reduced hepatocyte FGF21 secretion. Exogenous correction of FGF21 levels restored glycemic control and insulin secretion in Plin5MKO mice. These results show that changes in lipid metabolism resulting from PLIN5 deletion reduce ER stress in muscle, decrease FGF21 production by muscle and liver, and impair glycemic control. Further, these studies highlight the importance for muscle-liver cross talk in metabolic regulation

Uteroplacental insufficiency temporally exacerbates salt-induced hypertension associated with a reduced natriuretic response in male rat offspring

Intrauterine growth restriction increases the risk of developing chronic diseases in adulthood. Lifestyle factors, such as poor dietary choices, may elevate this risk. We determined whether being born small increases the sensitivity to a dietary salt challenge, in the context of hypertension, kidney disease and arterial stiffness. Bilateral uterine vessel ligation or sham surgery (offspring termed Restricted and Control, respectively) was performed on 18-day pregnant WKY rats. Male offspring were allocated to receive a diet high in salt (8% sodium chloride) or remain on standard rat chow (0.52% sodium chloride) from 20-26\ua0weeks of age for 6\ua0weeks. Systolic blood pressure (tail-cuff), renal function (24\ua0h urine excretions) and vascular stiffness (pressure myography) were assessed. Restricted males were born 15% lighter than Controls and remained smaller throughout the study. Salt-induced hypertension was exacerbated in Restricted offspring, reaching a peak systolic pressure of ∼175\ua0mmHg earlier than normal weight counterparts. The natriuretic response to high dietary salt in Restricted animals was less than in Controls and may explain the early rise in arterial pressure. Growth restricted males allocated to high salt diet also had increased passive arterial stiffness of mesenteric resistance arteries. Other aspects of renal function, including salt-induced hyperfiltration, albuminuria and glomerular damage were not exacerbated by uteroplacental insufficiency. This study demonstrates that male offspring exposed to uteroplacental insufficiency and born small have an increased sensitivity to salt-induced hypertension and arterial remodelling. This article is protected by copyright. All rights reserved

Comparative Study on the Therapeutic Potential of Neurally Differentiated Stem Cells in a Mouse Model of Multiple Sclerosis

Background: Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ). Methodology/Principal Findings: The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cellderived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS

In vivo and in vitro cardiovascular effects of Papuan taipan (Oxyuranus scutellatus) venom: exploring "sudden collapse"

‘Sudden collapse’ following envenoming by some Australasian elapids is a poorly understood cause of mortality. We have previously shown that Oxyuranus scutellatus venom causes cardiovascular collapse in anaesthetized rats. Prior administration of a sub lethal dose of venom attenuated the response to subsequent administration of higher (lethal) venom doses. In this study, we investigated the possible mechanisms mediating this ‘protective effect’. Papuan taipan venom (5 μg/kg, i.v.) produced a small transient hypotension in anaesthetized rats, while 10 μg/kg resulted in a 73 ± 12% decrease in arterial pressure. Venom (20 μg/kg or 50 μg/kg) produced cardiovascular collapse in all animals tested (n = 12). Cardiovascular collapse by 50 μg/kg venom was prevented by prior administration of ‘priming’ doses of venom (5, 10 and 20 μg/kg). Also, prior administration of indomethacin (30 mg/kg, i.v.) or heparin (300 units/kg, i.v.) prevented sudden collapse induced by venom (20 μg/kg). Venom was without effect in isolated hearts indicating that a direct cardiac effect was unlikely to be responsible for ‘sudden collapse’. Venom induced endothelium-dependent and -independent relaxation in pre-contracted rat mesenteric artery rings which was inhibited by indomethacin, IbTx and Rp-8-CPT-cAMPs. This relaxation was markedly reduced upon second exposure. Our results indicate that cardiovascular collapse induced by O. scutellatus venom may be due to a combination of release of dilator autacoids and to direct relaxation of vascular smooth muscle involving the cAMP/protein kinase A cascade. Further work will involve identification of the venom component(s) responsible for this action and may provide insight into the management of envenomed patients

Somatostatin decreases voltage-gated Ca2+ currents in GH3 cells through activation of somatostatin receptor 2

The secretion of growth hormone ( GH) is inhibited by hypothalamic somatostatin ( SRIF) in somatotropes through five subtypes of the somatostatin receptor ( SSTR1 SSTR5). We aimed to characterize the subtype( s) of SSTRs involved in the Ca2+ current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin- perforated patch clamp to record voltage- gated Ca2+ currents, using a holding potential of -80 mV in the presence of K+ and Na+ channel blockers. We first established the presence of T-, L-, N-, and P/ Q- type Ca2+ currents in GH3 cells using a variety of channel blockers ( Ni+, nifedipine, omega- conotoxin GVIA, and omega-agatoxin IVA). SRIF ( 200 nM) reduced Land N- type but not T- or P/ Q- type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+ currents to find the maximal effective concentration. Activation of SSTR2 with 10(-7) and 10(-8) M L- 797,976 decreased the voltage- gated Ca2+ current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10(-7) M did not modify the voltage- gated Ca2+ current and did not affect the Ca2+ current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage- gated Ca2+ currents by SRIF, which contributes to the decrease in intracellular Ca2+ concentration and GH secretion by SRIF

Hypotensive and vascular relaxant effects of phospholipase A₂ toxins from Papuan taipan (Oxyuranus scutellatus) venom

Phospholipase A₂ (PLA₂) toxins are common and abundant components of Australasian elapid venoms. These toxins are associated with a range of activities including neurotoxicity, myotoxicity and coagulation disturbances. We have recently reported that sudden cardiovascular collapse induced by Papuan taipan (Oxyuranus scutellatus) venom involves a combination of the release of dilator autacoids and a direct effect on the smooth muscle. In this study, we aimed to isolate PLA₂ components from Papuan taipan venom and investigate their contribution to the hypotensive action of this venom. O. scutellatus venom was fractionated using size-exclusion high performance liquid chromatography (HPLC), and fractions screened for activity in anaesthetized rats. Fraction three from O. scutellatus venom (i.e. OSC3, 14.2 ± 1.0% of whole venom) produced a 64% decrease in mean arterial pressure. Reverse-phase HPLC indicated that OSC3 consisted of two major components (i.e. OSC3a and OSC3b). OSC3a and OSC3b produced a significant hypotensive response in anaesthetized rats which were attenuated by prior administration of indomethacin or the combination of mepyramine and heparin. N-terminal analysis indicated that OSC3a and b displayed sequence homology to PLA₂ toxins isolated from coastal taipan (O. scutellatus scutellatus) venom. These findings indicate that PLA₂ components may play an important role in the development of hypotension and vascular relaxation which may contribute to the effects observed after envenoming by these Australasian elapids