Painful peripheral neuropathy is a severe and difficult‐to‐treat neurological complication associated with cancer chemotherapy. Although chemotherapeutic drugs such as paclitaxel are known to cause tonic activation of presynaptic NMDA receptors (NMDARs) to potentiate nociceptive input, the molecular mechanism involved in this effect is unclear. α2δ‐1, commonly known as a voltage‐activated calcium channel subunit, is a newly discovered NMDAR‐interacting protein and plays a critical role in NMDAR‐mediated synaptic plasticity. Here we show that paclitaxel treatment in rats increases the α2δ‐1 expression level in the dorsal root ganglion and spinal cord and the mRNA levels of GluN1, GluN2A, and GluN2B in the spinal cord. Paclitaxel treatment also potentiates the α2δ‐1–NMDAR interaction and synaptic trafficking in the spinal cord. Strikingly, inhibiting α2δ‐1 trafficking with pregabalin, disrupting the α2δ‐1–NMDAR interaction with an α2δ‐1 C‐terminus–interfering peptide, or α2δ‐1 genetic ablation fully reverses paclitaxel treatment‐induced presynaptic NMDAR‐mediated glutamate release from primary afferent terminals to spinal dorsal horn neurons. In addition, intrathecal injection of pregabalin or α2δ‐1 C‐terminus–interfering peptide and α2δ‐1 knockout in mice markedly attenuate paclitaxel‐induced pain hypersensitivity. Our findings indicate that α2δ‐1 is required for paclitaxel‐induced tonic activation of presynaptic NMDARs at the spinal cord level. Targeting α2δ‐1–bound NMDARs, not the physiological α2δ‐1–free NMDARs, may be a new strategy for treating chemotherapy‐induced neuropathic pain. Open science badges This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
Background and Purpose: Glutamate N-methyl-D-aspartate receptors (NMDARs) play a major role in the initiation of ischemic brain damage. However, NMDAR antagonists have no protective effects in stroke patients, possibly because they impair physiological functions of NMDARs. α2δ−1 (encoded by Cacna2d1) is strongly expressed in many brain regions. We determined the contribution of α2δ−1 to NMDAR hyperactivity and brain injury induced by ischemia and reperfusion. Methods: Mice were subjected to 90 min of middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Neurological deficits, brain infarct volumes, and calpain/caspase-3 activity in brain tissues were measured. NMDAR activity of hippocampal CA1 neurons was measured in an in vitro ischemic model. Results: MCAO increased α2δ−1 protein glycosylation in the cerebral cortex, hippocampus, and striatum. Coimmunoprecipitation showed that ischemia rapidly enhanced the α2δ−1–NMDAR physical interaction in the mouse brain tissue. Inhibiting α2δ−1 with gabapentin, uncoupling the α2δ−1–NMDAR interaction with an α2δ−1 C-terminus–interfering peptide, or genetically ablating Cacna2d1 had no effect on basal NMDAR currents but strikingly abolished oxygen-glucose deprivation-induced NMDAR hyperactivity in hippocampal CA1 neurons. Systemic treatment with gabapentin or α2δ−1 C-terminus–interfering peptide or Cacna2d1 genetic knockout reduced MCAO-induced infarct volumes, neurological deficit scores, and calpain/caspase-3 activation in brain tissues. Conclusions: α2δ−1 is essential for brain ischemia-induced neuronal NMDAR hyperactivity, and α2δ−1–bound NMDARs mediate brain damage caused by cerebral ischemia. Targeting α2δ−1–bound NMDARs, without impairing physiological α2δ−1–free NMDARs, may be a promising strategy for treating ischemic stroke.
NMDAR activity in the hypothalamic paraventricular nucleus (PVN) is increased and critically involved in heightened sympathetic vasomotor tone in hypertension. Calcium/calmodulin-dependent protein kinase II (CaMKII) binds to and modulates NMDAR activity. In this study, we determined the role of CaMKII in regulating NMDAR activity of PVN presympathetic neurons in male spontaneously hypertensive rats (SHRs). NMDAR-mediated EPSCs and puff NMDA-elicited currents were recorded in spinally projecting PVN neurons in SHRs and male Wistar-Kyoto (WKY) rats. The basal amplitude of evoked NMDAR-EPSCs and puff NMDA currents in retrogradely labeled PVN neurons were significantly higher in SHRs than in WKY rats. The CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP) normalized the increased amplitude of NMDAR-EPSCs and puff NMDA currents in labeled PVN neurons in SHRs but had no effect in WKY rats. Treatment with AIP also normalized the higher frequency of NMDAR-mediated miniature EPSCs of PVN neurons in SHRs. CaMKII-mediated phosphorylation level of GluN2B serine 1303 (S1303) in the PVN, but not in the hippocampus and frontal cortex, was significantly higher in SHRs than in WKY rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter the increased level of phosphorylated GluN2B S1303 in the PVN. In addition, microinjection of AIP into the PVN significantly reduced arterial blood pressure and lumbar sympathetic nerve discharges in SHRs. Our findings suggest that CaMKII activity is increased in the PVN and contributes to potentiated presynaptic and postsynaptic NMDAR activity to elevate sympathetic vasomotor tone in hypertension. Heightened sympathetic vasomotor tone is a major contributor to the development of hypertension. Although glutamate NMDA receptor (NMDAR)-mediated excitatory drive in the hypothalamus plays a critical role in increased sympathetic output in hypertension, the molecular mechanism involved in potentiated NMDAR activity of hypothalamic presympathetic neurons remains unclear. Here we show that the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) is increased and plays a key role in the potentiated presynaptic and postsynaptic NMDAR activity of hypothalamic presympathetic neurons in hypertension. Also, the inhibition of CaMKII in the hypothalamus reduces elevated blood pressure and sympathetic nerve discharges in hypertension. This new knowledge extends our understanding of the mechanism of synaptic plasticity in the hypothalamus and suggests new strategies to treat neurogenic hypertension.
ORCID IDs: 0000-0002-1641-8650 (J.Z.); 0000-0002-6719-9814 (S.X.); 0000-0001-5134-731X (J.-K.Z.).DNA polymerase d plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase d, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase a. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression.
While targeting oxidative phosphorylation (OXPHOS) is a rational anticancer strategy, patient bene t with OXPHOS inhibitors in the clinic has yet to be achieved. Based on promising preclinical data, we advanced IACS-010759, a highly potent and selective small-molecule inhibitor of mitochondrial complex I, into two phase I trials in patients with acute myeloid leukemia (NCT02882321) or advanced solid tumors (NCT03291938). Clinical ndings revealed that IACS-010759 had a narrow therapeutic index with emergent dose-limiting toxicities that included elevated blood lactate and neurotoxicity, obstructing efforts to maintain target plasma exposure. Consequently, only modest on-target inhibition and limited antitumor activity were observed. Follow-up reverse translational studies uncovered that IACS-010759 reduced oxygen consumption rates in neurons and damaged myelin. Further, IACS-010759-treated mice displayed behaviors indictive of neuropathy, which were minimized with the co-administration of a histone deacetylase 6 inhibitor. Our ndings urge caution in the continued development of complex I inhibitors as antitumor agents.
Edited by Xiao-Fan Wang Neuropathic pain is associated with persistent changes in gene expression in primary sensory neurons, but the underlying epigenetic mechanisms that cause these changes remain unclear. The muscarinic cholinergic receptors (mAChRs), particularly the M2 subtype (encoded by the cholinergic receptor muscarinic 2 (Chrm2) gene), are critically involved in the regulation of spinal nociceptive transmission. However, little is known about how Chrm2 expression is transcriptionally regulated. Here we show that nerve injury persistently increased the expression of RE1-silencing transcription factor (REST, also known as neuron-restrictive silencing factor [NRSF]), a genesilencing transcription factor, in the dorsal root ganglion (DRG). Remarkably, nerve injury-induced chronic but not acute pain hypersensitivity was attenuated in mice with Rest knockout in DRG neurons. Also, siRNA-mediated Rest knockdown reversed nerve injury-induced chronic pain hypersensitivity in rats. Nerve injury persistently reduced Chrm2 expression in the DRG and diminished the analgesic effect of muscarine. The RE1 binding site on the Chrm2 promoter is required for REST-mediated Chrm2 repression, and nerve injury increased the enrichment of REST in the Chrm2 promoter in the DRG. Furthermore, Rest knockdown or genetic ablation in DRG neurons normalized Chrm2 expression and augmented muscarine's analgesic effect on neuropathic pain and fully reversed the nerve injuryinduced reduction in the inhibitory effect of muscarine on glutamatergic input to spinal dorsal horn neurons. Our findings indicate that nerve injury-induced REST up-regulation in DRG neurons plays an important role in the acute-to-chronic pain transition and is essential for the transcriptional repression of Chrm2 in neuropathic pain.
N-methyl-D-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and a2d-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and a2d-1 in the control of NMDAR activity. We found that a2d-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and a2d-1. Remarkably, PKC inhibition with Gӧ6983 abolished a2d-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/ GluN2A or GluN1/GluN2B. Treatment with PMA increased the a2d-1-GluN1 interaction and promoted a2d-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of a2d-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in a2d-1 knockout mice. Furthermore, inhibiting a2d-1 with pregabalin or disrupting the a2d-1-NMDAR interaction with the a2d-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and a2d-1. Together, our findings demonstrate the interdependence of a2d-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic a2d-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.
SUMMARY As a central component in the maturation of Okazaki fragments, flap endonuclease 1 (FEN1) removes the 5′-flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. The Arabidopsis fen1-1 mutant is hypersensitive to methyl methanesulfonate and shows reduced telomere length. Interestingly, genome-wide chromatin immunoprecipitation and RNA sequencing results demonstrate that FEN1 mutation leads to a decrease in the level of H3K27me3 and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS as well as maintaining genome stability in Arabidopsis.
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