Allen H. Li

PINK1 mutants associated with recessive Parkinson's disease are defective in inhibiting mitochondrial release of cytochrome c

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinsons disease (PARK6). We investigated molecular mechanisms underlying PINK1 neuroprotective function and PARK6 mutation-induced loss of PINK1 function. Overexpression of wild-type PINK1 blocked mitochondrial release of apoptogenic cytochrome c, caspase-3 activation and apoptotic cell death induced by proteasome inhibitor MG132. N-terminal truncated PINK1 (NDelta35), which lacks mitochondrial localization sequence, did not block MG132-induced cytochrome c release and cytotoxicity. Despite mitochondrial expression, PARK6 mutant (E240K), (H271Q), (G309D), (L347P), (E417G) and C-terminal truncated (CDelta145) PINK1 failed to inhibit MG132-induced cytochrome c release and caspase-3 activation. Overexpression of wild-type PINK1 blocked cytochrome c release and cell death caused by atractyloside, which opens mitochondrial permeability transition pore (mPTP). PARK6 PINK1 mutants failed to inhibit atractyloside-induced cytochrome c release. These results suggest that PINK1 exerts anti-apoptotic effect by inhibiting the opening of mPTP and that PARK6 mutant PINK1 loses its ability to prevent mPTP opening and cytochrome c release.

Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia

Astroglial glutamate transporters, GLT-1 and GLAST, play an essential role in removing released glutamate from the extracellular space and are essential for maintaining a low concentration of extracellular glutamate in the brain. It was hypothesized that impaired function of glial glutamate transporters induced by transient global ischemia may lead to an elevated level of extracellular glutamate and subsequent excitotoxic neuronal death. To test this hypothesis, in the present study, we performed whole-cell patch-clamp recording of hippocampal CA1 astrocytes in control or postischemic slices, and measured glutamate transporter activity by recording glutamate-evoked transporter currents. Six to 24 h after global ischemia, maximal amplitude of glutamate transporter currents recorded from postischemic CA1 astrocytes was significantly reduced. Western blotting analysis indicated that transient global ischemia decreased the protein level of GLT-1 in the hippocampal CA1 area without affecting GLAST protein level. Further real-time quantitative RT-PCR assays showed that global ischemia resulted in a decrease in GLT-1 mRNA level of hippocampal CA1 region. Global ischemia-induced reduction in GLT-1 expression and glutamate transporter function of CA1 astrocytes precedes the initiation of delayed neuronal death in CA1 pyramidal layer. The present study provides the evidence that transient global ischemia downregulates glutamate transporter function of hippocampal CA1 astrocytes by decreasing mRNA and protein levels of GLT-1.

Functional analysis of connexin-26 mutants associated with hereditary recessive deafness

The physiological importance of connexin‐26 (Cx26) gap junctions in regulating auditory function is indicated by the finding that autosomal recessive DFNB1 deafness is associated with mutations of the Cx26 gene. To investigate the pathogenic role of Cx26 mutation in recessive hearing loss, four putative DFNB1 Cx26 mutants (V84L, V95M, R127H, and R143W) were stably expressed in N2A cells, a communication‐deficient cell line. In N2A cells expressing (R127H) Cx26 gap junctions, macroscopic junctional conductance and ability of transferring neurobiotin between transfected cells were greatly reduced. Despite the formation of defective junctional channels, immunoreactivity of (R127H) Cx26 was mainly localized in the cell membrane and prominent in the region of cell–cell contact. Mutant (V84L), (V95M), or (R143W) Cx26 protein formed gap junctions with a junctional conductance similar to that of wild‐type Cx26 junctional channels. (V84L), (V95M), or (R143W) Cx26 gap junctions also permitted neurobiotin transfer between pairs of transfected N2A cells. The present study suggests that (R127H) mutation associated with hereditary sensorineural deafness results in the formation of defective Cx26 gap junctions, which may lead to the malfunction of cochlear gap junctions and hearing loss. Further studies are required to determine the exact mechanism by which mutant (V84L), (V95M), and (R143W) Cx26 proteins, which are capable of forming functional homotypic junctional channels in N2A cells, cause the cochlear dysfunction and sensorineural deafness.

CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Nav1.8 sodium channels in dorsal root ganglion neurons

BackgroundInflammation or nerve injury-induced upregulation and release of chemokine CC chemokine ligand 2 (CCL2) within the dorsal root ganglion (DRG) is believed to enhance the activity of DRG nociceptive neurons and cause hyperalgesia. Transient receptor potential vanilloid receptor 1 (TRPV1) and tetrodotoxin (TTX)-resistant Nav1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons. We therefore tested the hypothesis that CCL2 causes peripheral sensitization of nociceptive DRG neurons by upregulating the function and expression of TRPV1 and Nav1.8 channels.MethodsDRG neuronal culture was prepared from 3-week-old Sprague–Dawley rats and incubated with various concentrations of CCL2 for 24 to 36 hours. Whole-cell voltage-clamp recordings were performed to record TRPV1 agonist capsaicin-evoked inward currents or TTX-insensitive Na+ currents from control or CCL2-treated small DRG sensory neurons. The CCL2 effect on the mRNA expression of TRPV1 or Nav1.8 was measured by real-time quantitative RT-PCR assay.ResultsPretreatment of CCL2 for 24 to 36 hours dose-dependently (EC50 value = 0.6 ± 0.05 nM) increased the density of capsaicin-induced currents in small putative DRG nociceptive neurons. TRPV1 mRNA expression was greatly upregulated in DRG neurons preincubated with 5 nM CCL2. Pretreating small DRG sensory neurons with CCL2 also increased the density of TTX-resistant Na+ currents with a concentration-dependent manner (EC50 value = 0.7 ± 0.06 nM). The Nav1.8 mRNA level was significantly increased in DRG neurons pretreated with CCL2. In contrast, CCL2 preincubation failed to affect the mRNA level of TTX-resistant Nav1.9. In the presence of the specific phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 or Akt inhibitor IV, CCL2 pretreatment failed to increase the current density of capsaicin-evoked inward currents or TTX-insensitive Na+ currents and the mRNA level of TRPV1 or Nav1.8.ConclusionsOur results showed that CCL2 increased the function and mRNA level of TRPV1 channels and Nav1.8 sodium channels in small DRG sensory neurons via activating the PI3K/Akt signaling pathway. These findings suggest that following tissue inflammation or peripheral nerve injury, upregulation and release of CCL2 within the DRG could facilitate pain transmission mediated by nociceptive DRG neurons and could induce hyperalgesia by upregulating the expression and function of TRPV1 and Nav1.8 channels in DRG nociceptive neurons.

G protein-coupled receptor kinase 2 mediates µ-opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus

Nucleus raphe magnus (NRM) sends the projection to spinal dorsal horn and inhibits nociceptive transmission. Analgesic effect produced by µ‐opioid receptor agonists including morphine partially results from activating the NRM‐spinal cord pathway. It is generally believed that µ‐opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce analgesia by hyperpolarizing GABAergic interneurons. In the present study, whole‐cell patch‐clamp recordings combined with single‐cell RT‐PCR analysis were used to test the hypothesis that DAMGO ([D‐Ala2,N‐methyl‐Phe4,Gly‐ol5]enkephalin), a specific µ‐opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD67). Homologous desensitization of µ‐opioid receptors in NRM neurons could result in the development of morphine‐induced tolerance. G protein‐coupled receptor kinase (GRK) is believed to mediate µ‐opioid receptor desensitization in vivo. Therefore, we also investigated the involvement of GRK in mediating homologous desensitization of DAMΑΜGO‐induced electrophysiological effects on NRM neurons by using two experimental strategies. First, single‐cell RT‐PCR assay was used to study the expression of GRK2 and GRK3 mRNAs in individual DAMGO‐responsive NRM neurons. Whole‐cell recording was also performed with an internal solution containing the synthetic peptide, which corresponds to Gβγ‐binding domain of GRK and inhibits Gβγ activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes NRM GABAergic neurons by opening inwardly rectifying K+ channels and that GRK2 mediates short‐term homologous desensitization of µ‐opioid receptors in NRM GABAergic neurons.

Vasoactive intestinal polypeptide enhances the GABAergic synaptic transmission in cultured hippocampal neurons

The whole-cell mode of patch-clamp techniques was used to investigate the effect of vasoactive intestinal polypeptide (VIP) on spontaneous gamma-aminobutyric acid (GABA)-mediated inhibitory postsynaptic currents (IPSCs) of cultured hippocampal neurons. Application of VIP caused a significant increase in the frequency of spontaneous IPSCs with a reversible and dose-dependent manner. VIP had no effect on the mean amplitude and kinetic parameters of spontaneous IPSCs. In the presence of tetrodotoxin, VIP increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without affecting their mean magnitude. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, mimicked the stimulatory effect of VIP on spontaneous IPSCs and mIPSCs. VIP and forskolin failed to modulate GABAergic IPSCs in the presence of Rp-cAMPs, a cell permeable antagonist of cAMP-dependent protein kinase (PKA). Calcium channel blocker CdCl2 did not prevent VIP and forskolin from increasing the frequency of mIPSCs. These results suggest that the activation of presynaptic VIP receptor enhances the GABAergic synaptic transmission in cultured hippocampal neurons through the cAMP-PKA pathway and that VIP is likely to increase GABA release by directly stimulating the vesicular release apparatus.

Identification of two C-terminal amino acids, Ser355 and Thr357, required for short-term homologous desensitization of μ-opioid receptors

Our recent study suggests that a cluster of Ser/Thr residues (T(354)S(355)S(356)T(357)) at the intracellular carboxyl tail of rat mu-opioid receptor (MOR1) is required for the development of short-term homologous desensitization. To investigate the functional role played by individual serine or threonine residue of this (TSST) cluster in the agonist-induced mu-opioid receptor desensitization, point mutant (T354A), (S355A), (S356A) and (T357A) mu-opioid receptors were prepared and stably expressed in human embryonic kidney 293 cells (HEK 293 cells). Similar to wild-type mu-opioid receptors, mutant (T354A) and (S356A) mu-opioid receptors stably expressed in HEK 293 cells developed homologous desensitization after 30 min pretreatment of DAMGO ([D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin), a specific mu-opioid receptor agonist. Substituting Ser(355)or Thr(357) with alanine resulted in a significant attenuation of agonist-induced mu-opioid receptor desensitization. In HEK 293 cells stably expressing double mutant (S355A/T357A) mu-opioid receptors, DAMGO pretreatment failed to significantly affect the efficacy and potency by which DAMGO inhibits forskolin-stimulated adenylyl cyclase activity. Consistent with the general belief that agonist-induced phosphorylation of guanine nucleotide binding protein (G protein)-coupled receptors is involved in homologous desensitization. Treating HEK 293 cells expressing wild-type mu-opioid receptors with 5 microM DAMGO for 30 min induced the receptor phosphorylation. Mutation of Ser(355) and Thr(357) also greatly impaired DAMGO-induced mu-opioid receptor phosphorylation. These results suggest that two C-terminal amino acids, Ser(355) and Thr(357), are required for short-term homologous desensitization and agonist-induced phosphorylation of mu-opioid receptors expressed in HEK 293 cells.

Neurotensin excitation of serotonergic neurons in the rat nucleus raphe magnus: ionic and molecular mechanisms

To understand the cellular and molecular mechanisms by which neurotensin (NT) induces an analgesic effect in the nucleus raphe magnus (NRM), whole-cell patch-clamp recordings were performed to investigate the electrophysiological effects of NT on acutely dissociated NRM neurons. Two subtypes of neurons, primary serotonergic and secondary non-serotonergic cells, were identified from acutely isolated NRM neurons. During current-clamp recordings, NT depolarized NRM serotonergic neurons and evoked action potentials. Voltage-clamp recordings showed that NT excited serotonergic neurons by enhancing a voltage-insensitive and non-selective cationic conductance. Both SR48692, a selective antagonist of subtype 1 neurotensin receptor (NTR-1), and SR 142948A, a non-selective antagonist of NTR-1 and subtype 2 neurotensin receptor (NTR-2), failed to prevent neurotensin from exciting NRM serotonergic neurons. NT-evoked cationic current was inhibited by the intracellular administration of GDP-beta-S. NT failed to induce cationic currents after dialyzing serotonergic neurons with the anti-G(alphaq/11) antibody. Cellular Ca(2+) imaging study using fura-2 showed that NT induced the calcium release from the intracellular store. NT-evoked current was blocked after the internal perfusion of heparin, an IP(3) receptor antagonist, or BAPTA, a fast Ca(2+) chelator. It is concluded that neurotensin enhancement of the cationic conductance of NRM serotonergic neurons is mediated by a novel subtype of neurotensin receptors. The coupling mechanism via G(alphaq/11) proteins is likely to involve the generation of IP(3), and subsequent IP(3)-evoked Ca(2+) release from intracellular stores results in activating the non-selective cationic conductance.

Point mutation associated with X-linked dominant Charcot-Marie-Tooth disease impairs the P2 promoter activity of human connexin-32 gene.

Many lines of evidence suggest that connexin-32 gap junction is involved in the exchange of information and metabolites in the peripheral nervous system. It has been shown that connexin-32 protein and mRNA are expressed in Schwann cells that function as myelinating cells of the peripheral nervous system. The physiological importance of connexin-32 gap junctions in regulating the normal function of myelinating Schwann cell is indicated by recent findings that X-linked dominant Charcot-Marie-Tooth disease, a hereditary peripheral neuropathy, is associated with the mutations of connexin-32 gene. Recently, we encountered a Taiwanese family affected with X-linked dominant Charcot-Marie-Tooth neuropathy. Therefore, we investigated the possible mutation in the coding and noncoding regions of the connexin-32 gene of affected members of this family. Our results suggest that a G-to-A transition at the position -215 (in relation to the transcription initiation site) of the nerve-specific P2 promoter region is associated with the pathogenesis of X-linked dominant Charcot-Marie-Tooth disease. Further experiments using the promoter assay indicate that G-to-A mutation at the position -215 greatly impairs the transcriptional activity of connexin-32 P2 promoter. These findings propose that a reduced expression of connexin-32 mRNA and protein in the myelin sheath could be responsible for the development of X-linked dominant Charcot-Marie-Tooth neuropathy.

Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons

BackgroundSeveral lines of evidence suggest that CCL2 could initiate the hyperalgesia of neuropathic pain by causing central sensitization of spinal dorsal horn neurons and facilitating nociceptive transmission in the spinal dorsal horn. The cellular and molecular mechanisms by which CCL2 enhances spinal pain transmission and causes hyperalgesia remain unknown. The substantia gelatinosa (lamina II) of the spinal dorsal horn plays a critical role in nociceptive transmission. An activated spinal microglia, which is believed to release pro-inflammatory cytokines including TNF-α, plays an important role in the development of neuropathic pain, and CCL2 is a key mediator for spinal microglia activation. In the present study, we tested the hypothesis that spinal CCL2 causes the central sensitization of substantia gelatinosa neurons and enhances spinal nociceptive transmission by activating the spinal microglia and augmenting glutamatergic transmission in lamina II neurons.MethodsCCL2 was intrathecally administered to 2-month-old male rats. An intrathecal injection of CCL2 induced heat hyperalgesia, which was assessed using the hot plate test. Whole-cell voltage-clamp recordings substantia gelatinosa neurons in spinal cord slices were performed to record glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs).ResultsThe hot plate test showed that 1 day after the intrathecal injection of CCL2 (1 μg), the latency of hind-paw withdrawal caused by a heat stimulus was significantly reduced in rats. One day after the intrathecal administration of CCL2, the amplitude of the evoked glutamatergic EPSCs and the frequency of spontaneous glutamatergic miniature EPSCs (mEPSCs) were significantly increased in outer lamina II neurons. Intrathecal co-injection of minocycline, a specific inhibitor of microglial activation, and CCL2 blocked the CCL2-induced reduction in the latency of hind-paw withdrawal and thermal hyperalgesia. Following intrathecal co-administration of CCL2 and minocycline, CCL2 failed to increase the frequency of glutamatergic mEPSCs and failed to promote glutamine release in lamina II neurons. Intrathecal co-injection of WP9QY, a selective TNF-α antagonist, and CCL2 completely inhibited CCL2-induced heat hyperalgesia and inhibited the increase in the frequency of glutamatergic mEPSCs in substantia gelatinosa neurons.ConclusionIn summary, our results suggest that an intrathecal injection of CCL2 causes thermal hyperalgesia by augmenting the excitatory glutamatergic transmission in substantia gelatinosa neurons through a presynaptic mechanism and facilitating nociceptive transmission in the spinal dorsal horn. Further studies show that intrathecal co-administration of minocycline, a specific inhibitor of microglial activation, or WP9QY, a selective TNF-α antagonist, completely inhibited CCL2 potentiation of glutamatergic transmission in substantia gelatinosa neurons and CCL2-induced heat hyperalgesia. The results of the present study suggest that peripheral nerve injury-induced upregulation of the spinal CCL2 level causes the central sensitization of substantia gelatinosa neurons by activating spinal microglia and that TNF-α mediates CCL2-induced thermal hyperalgesia and augmentation of glutamatergic transmission in lamina II neurons.