Background. Neuropathic pain (NP) is caused by lesion or disease of somatosensory system. It impacts quality of life of 8% of UK population and only 33% of these patients have effective NP management with existing medications1. Protein kinase C delta (PKCδ) is calcium-independent novel PKC isozyme and is involved in numerous cellular functions, including neurotoxicity, neurodegeneration and apoptosis. PKCδ expression correlates with augmented sensitivity and intensity of NP2,3. Rottlerin, a compound isolated from Mallotus Philippensis plant, was reported to act as PKCδ inhibitor4.
Hypothesis. Inhibition of PKCδ activity by rottlerin may represent a novel therapeutic strategy for effective NP management.
Methods. Cell culture. HD33n1 human induced pluripotent stem cells (hiPSC) were used to generate fully functional sensory-like neurons according to previously developed protocol5. To verify the data obtained on hiPSC-derived sensory-like neurons, we used trigeminal ganglia (TG) neurons isolated from 10-week-old Sprague-Dawley rats (n=3); Schedule 1 for tissue harvesting.
Electrophysiology. Patch-clamp conventional whole-cell electrophysiology technique was used to investigate electrical properties of neurons. Electrophysiological parameters were recorded with Axopatch 200A amplifier, Digidata 1440A digitizer and pCLAMP 10.2 software (Molecular Devices) at room temperature. Bath solution was prepared with 144.8mM NaCl, 2.5mM KCl, 0.5mM MgCl2, 1.2mM CaCl2, 10mM glucose, and 5mM HEPES, pH 7.4 with 1M NaOH. Pipette solution contained 140mM KCl, 6mM NaCl, 4mM Na2-ATP, 4.2mM Na-GTP, 3mM MgCl2, 1mM CaCl2, 5mM HEPES, pH 7.2 with 1M KOH.
qPCR. RNA was extracted from HD33n1 hiPSCs-derived sensory-like neurons treated with rottlerin in the presence and absence of “pain cocktail” (10 μM adenosine triphosphate, 1μM noradrenaline, 1μM bradykinin and 1μM substance P). The Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis following standard procedure. Changes in relative gene expression of PKCD in the presence and absence of “pain cocktail” in control and rottlerin treated samples were calculated using the 2−ΔΔCT method, and the data were normalised to GAPDH.
Statistical analysis. All data are expressed as mean ± S.E.M. Statistical comparisons were performed using Student’s t-tests; differences were considered significant at p< 0.05.
Results. Acute application of rottlerin hyperpolarised HD33n1 hiPSCs-derived sensory-like neurons and rat TG neurons in concentration-dependent manner. 10μM rottlerin significantly hyperpolarised HD33n1 hiPSC-derived sensory-like neurons from -45.6±0.1mV (n=3) to -54.2±1.8mV (n=3); p<0.01. Similarly, acute application of 10μM rottlerin significantly hyperpolarised rat TG neurons from -57.7±4.0mV (n=5) to -66.4±5.1mV (n=5); p<0.05. Synergetic application of all components of the “pain cocktail” was ineffective to cause depolarisation in rat TG neurons in the presence of rottlerin, thus suggesting its anti-excitatory effect.
Chronic administration of HD33n1 hiPSC-derived sensory-like neurons with 10μM rottlerin decreased PKCδ expression, whereas administration with “pain cocktail” increased it. Combined chronic administration of rottlerin and “pain cocktail” reduced PKCδ expression compare to “pain cocktail” alone.
Summary. These data show for the first-time the ability of PKCδ inhibitor rottlerin to attenuate excitability of HD33n1 hiPSC-derived sensory-like and rat TG neuronal in vitro NP model. This may suggest a novel strategy to improve pharmacological management of NP as well as associated co-morbidities.