Jixiang Zhang, PhD

An HDAC6 inhibitor reverses cisplatin-induced mechanical hypersensitivity.
MRC1 (CD206)-positive spinal cord macrophages play a key role.
The HDAC6 inhibitor increases IL-10 by spinal cord MRC1-positive macrophages.
IL-10 signaling to IL-10 receptors on sensory neurons is required.
Normalization of mitochondrial deficits is independent of macrophages and IL-10.

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… Show more

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. Show less

This chapter focuses on the role of histone modifications in chronic pain development. It starts by introducing epigenetic processes, particularly histone modifications, and then chronic pain, especially nerve injury-induced neuropathic pain. It then reviews recent studies on the role of histone modifications in regulating gene expression in chronic pain, including G9a-mediated H3K9me2, EZH2-mediated H3K27me3, and HDAC-mediated histone acetylation. Lastly, it discusses the possible involvement… Show more

This chapter focuses on the role of histone modifications in chronic pain development. It starts by introducing epigenetic processes, particularly histone modifications, and then chronic pain, especially nerve injury-induced neuropathic pain. It then reviews recent studies on the role of histone modifications in regulating gene expression in chronic pain, including G9a-mediated H3K9me2, EZH2-mediated H3K27me3, and HDAC-mediated histone acetylation. Lastly, it discusses the possible involvement of histone variant turnover in chronic pain development and the potential interaction/overlap of G9a, HDAC, and REST in pain-related gene regulation. Current challenges and prospects for future studies on the epigenetic basis of chronic pain are also discussed. Show less

A replicative plasmid system was developed for Mycoplasma bovis.•
The transformation conditions were optimized to improve transformation frequency.
The plasmids served stably as extra-chromosomal elements during 30 passages.
The heterologous protein β-galactosidase was expressed by this plasmid system.
A FLAG tag-fused endogenous protein was expressed and detectable.

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.

Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long-lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic… Show more

Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long-lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain. Show less

Peripheral neuropathic pain induced by the chemotherapeutic cisplatin can persist for months to years after treatment. Histone deacetylase 6 (HDAC6) inhibitors have therapeutic potential for cisplatin-induced neuropathic pain since they persistently reverse mechanical hypersensitivity and spontaneous pain in rodent models. Here, we investigated the mechanisms underlying reversal of mechanical hypersensitivity in male and female mice by a 2 week treatment with an HDAC6 inhibitor, administered 3… Show more

Peripheral neuropathic pain induced by the chemotherapeutic cisplatin can persist for months to years after treatment. Histone deacetylase 6 (HDAC6) inhibitors have therapeutic potential for cisplatin-induced neuropathic pain since they persistently reverse mechanical hypersensitivity and spontaneous pain in rodent models. Here, we investigated the mechanisms underlying reversal of mechanical hypersensitivity in male and female mice by a 2 week treatment with an HDAC6 inhibitor, administered 3 d after the last dose of cisplatin. Mechanical hypersensitivity in animals of both sexes treated with the HDAC6 inhibitor was temporarily reinstated by a single injection of the neutral opioid receptor antagonist 6β-naltrexol or the peripherally restricted opioid receptor antagonist naloxone methiodide. These results suggest that tonic peripheral opioid ligand-receptor signaling mediates reversal of cisplatin-induced mechanical hypersensitivity after treatment with an HDAC6 inhibitor. Pointing to a specific role for δ opioid receptors (DORs), Oprd1 expression was decreased in DRG neurons following cisplatin administration, but normalized after treatment with an HDAC6 inhibitor. Mechanical hypersensitivity was temporarily reinstated in both sexes by a single injection of the DOR antagonist naltrindole. Consistently, HDAC6 inhibition failed to reverse cisplatin-induced hypersensitivity when DORs were genetically deleted from advillin+ neurons. Mechanical hypersensitivity was also temporarily reinstated in both sexes by a single injection of a neutralizing antibody against the DOR ligand met-enkephalin. In conclusion, we reveal that treatment with an HDAC6 inhibitor induces tonic enkephalin-DOR signaling in peripheral sensory neurons to suppress mechanical hypersensitivity. Show less

Type 1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids. However, the epigenetic mechanism regulating the expression of CB1Rs in neuropathic pain is unknown. G9a (encoded by the Ehmt2 gene), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined G9a's role in regulating CB1R expression in the DRG and in CB1R-mediated analgesic effects… Show more

Type 1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids. However, the epigenetic mechanism regulating the expression of CB1Rs in neuropathic pain is unknown. G9a (encoded by the Ehmt2 gene), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined G9a's role in regulating CB1R expression in the DRG and in CB1R-mediated analgesic effects in an animal model of neuropathic pain. We show that nerve injury profoundly reduced mRNA levels of CB1Rs but increased the expression of CB2 receptors in the rat DRG. ChIP results indicated increased enrichment of histone 3 at lysine 9 dimethylation, a G9a-catalyzed repressive histone mark, at the promoter regions of the CB1R genes. G9a inhibition in nerve-injured rats not only up-regulated the CB1R expression level in the DRG but also potentiated the analgesic effect of a CB1R agonist on nerve injury-induced pain hypersensitivity. Furthermore, in mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce CB1R expression in the DRG and to decrease the analgesic effect of the CB1R agonist. Moreover, nerve injury diminished the inhibitory effect of the CB1R agonist on synaptic glutamate release from primary afferent nerves to spinal cord dorsal horn neurons in WT mice but not in mice lacking Ehmt2 in DRG neurons. Our findings reveal that nerve injury diminishes the analgesic effect of CB1R agonists through G9a-mediated CB1R down-regulation in primary sensory neurons. Show less

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… Show more

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. Show less

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 α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR… Show more

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 α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-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 α2δ-1. Remarkably, PKC inhibition with Gӧ6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1–GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1–NMDAR interaction with the α2δ-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 α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1–NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity. Show less

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… Show more

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 gene-silencing 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 injury–induced 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. Show less

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… Show more

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. Show less

Reports of neurological sequelae related to colon cancer are largely restricted to rare instances of paraneoplastic syndromes, due to autoimmune reactions. Systemic inflammation associated with tumor development influences sensory neuron function in other disease models, though the extent to which this occurs in colorectal cancer is unknown. We induced orthotopic colorectal cancer via orthotopic injection of two colorectal cancer cell lines (MC38 and CT26) in two different mouse strains… Show more

Reports of neurological sequelae related to colon cancer are largely restricted to rare instances of paraneoplastic syndromes, due to autoimmune reactions. Systemic inflammation associated with tumor development influences sensory neuron function in other disease models, though the extent to which this occurs in colorectal cancer is unknown. We induced orthotopic colorectal cancer via orthotopic injection of two colorectal cancer cell lines (MC38 and CT26) in two different mouse strains (C57BL/6 and Balb/c, respectively). Behavioral tests of pain sensitivity and activity did not detect significant alterations in sensory sensitivity or diminished well-being throughout tumor development. However, immunohistochemistry revealed widespread reductions in intraepidermal nerve fiber density in the skin of tumor-bearing mice. Though loss of nerve fiber density was not associated with increased expression of cell injury markers in dorsal root ganglia, lumbar dorsal root ganglia neurons of tumor-bearing animals showed deficits in mitochondrial function. These neurons also had reduced cytosolic calcium levels in live-cell imaging and reduced spontaneous activity in multi-electrode array analysis. Bulk RNA sequencing of DRGs from tumor-bearing mice detected activation of gene expression pathways associated with elevated cytokine and chemokine signaling, including CXCL10. This is consistent with the detection of CXCL10 (and numerous other cytokines, chemokines and growth factors) in MC38 and CT26 cell-conditioned media, and the serum of tumor-bearing mice. Our study demonstrates in a pre-clinical setting that colon cancer is associated with latent sensory neuron dysfunction and implicates cytokine/chemokine signaling in this process. These findings may have implications for determining risk factors and treatment responsiveness related to neuropathy in colorectal cancer. Show less

This study demonstrates that the prevalence of synaptic calcium-permeable AMPA receptors is increased in the spinal dorsal horn, which mediates pain hypersensitivity in diabetic neuropathy. Thus, calcium-permeable AMPA receptors play an important role in glutamatergic synaptic plasticity in the spinal cord in painful diabetic neuropathy. This new knowledge improves our understanding of the mechanisms involved in central sensitization associated with diabetic neuropathic pain and suggests that… Show more

This study demonstrates that the prevalence of synaptic calcium-permeable AMPA receptors is increased in the spinal dorsal horn, which mediates pain hypersensitivity in diabetic neuropathy. Thus, calcium-permeable AMPA receptors play an important role in glutamatergic synaptic plasticity in the spinal cord in painful diabetic neuropathy. This new knowledge improves our understanding of the mechanisms involved in central sensitization associated with diabetic neuropathic pain and suggests that calcium-permeable AMPA receptors are an alternative therapeutic target for treating this chronic pain condition. Show less

DNA polymerase δ 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 δ, 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… Show more

DNA polymerase δ 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 δ, 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 α. 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. Show less