Early fate decision for mitochondrially encoded proteins by a molecular triage.

Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems either promote the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. Although well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially encoded proteins, key subunits of the oxidative phosphorylation (OXPHOS) system. Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial protein biogenesis and quality control. Conserved prohibitin (PHB)/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in the vicinity of the mitoribosomal tunnel exit allows for a prompt decision on whether newly synthesized proteins are fed into OXPHOS assembly or are degraded.

Early steps in the biogenesis of mitochondrially encoded oxidative phosphorylation subunits.

The complexes mediating oxidative phosphorylation (OXPHOS) in the inner mitochondrial membrane consist of proteins encoded in the nuclear or the mitochondrial DNA. The mitochondrially encoded membrane proteins (mito-MPs) represent the catalytic core of these complexes and follow complicated pathways for biogenesis. Owing to their overall hydrophobicity, mito-MPs are co-translationally inserted into the inner membrane by the Oxa1 insertase. After insertion, OXPHOS biogenesis factors mediate the assembly of mito-MPs into complexes and participate in the regulation of mitochondrial translation, while protein quality control factors recognize and degrade faulty or excess proteins. This review summarizes the current understanding of these early steps occurring during the assembly of mito-MPs by concentrating on results obtained in the model organism baker's yeast.

Prevention of Chemotherapy-Induced Peripheral Neuropathy by Inhibiting C-X-C Motif Chemokine Receptor 2.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major drawback in the use of chemotherapeutic agents for patients with cancer. Although studies have investigated a broad number of molecules that might be related to CIPN, the differences in the chemokine pathways between various chemotherapeutic agents, such as vincristine and oxaliplatin, which are some of the most widely used treatments, have not been fully elucidated. We confirmed that the administration (intraperitoneal injections for seven days) of vincristine (0.1 mg/kg) and oxaliplatin (3 mg/kg) induced pain by using the von Frey behavioral test. Subsequent applications with vincristine and oxaliplatin led to mechanical allodynia that lasted more than one week from the fifth day. After the induction of mechanical allodynia, the mRNA expression of CXCR2, CXCL1, CXCL3, and CXCL5 was examined in the dorsal root ganglia (DRG) and spinal cord of the CIPN models. As a result, the mRNA expression of CXCR2 robustly increased in the lumbar spinal cord in the oxaliplatin-treated mice. Next, to evaluate the involvement of CXCR2 in CIPN, reparixin, a CXCR1/2 inhibitor, was administered intrathecally or intraperitoneally with vincristine or oxaliplatin and was further verified by treatment with ruxolitinib, which inhibits Janus kinase 2 downstream of the CXCR1/2 pathway. Reparixin and ruxolitinib blocked oxaliplatin-induced allodynia but not vincristine-induced allodynia, which suggests that CXCR2-related pathways are associated with the development of oxaliplatin-induced neuropathy. Together with the above results, this suggests that the prevention of oxaliplatin-induced neuropathy by CXCR2 inhibition can lead to successful chemotherapy, and it is important to provide appropriate countermeasures against CIPN development for each specific chemotherapeutic agent.

Transient receptor potential melastatin 2 governs stress-induced depressive-like behaviors.

Major depressive disorder (MDD) is a devastating disease that arises in a background of environmental risk factors, such as chronic stress, that produce reactive oxygen species (ROS) in the brain. The chronic stress-induced ROS production involves Ca2+ signals; however, the mechanism is poorly understood. Transient receptor potential melastatin type 2 (TRPM2) is a Ca2+-permeable cation channel that is highly expressed in the brain. Here we show that in animal models of chronic unpredictable stress (CUS), deletion of TRPM2 (Trpm2-/- ) produces antidepressant-like behaviors in mice. This phenotype correlates with reduced ROS, ROS-induced calpain activation, and enhanced phosphorylation of two Cdk5 targets including synapsin 1 and histone deacetylase 5 that are linked to synaptic function and gene expression, respectively. Moreover, TRPM2 mRNA expression is increased in hippocampal tissue samples from patients with MDD. Our findings suggest that TRPM2 is a key agent in stress-induced depression and a possible target for treating depression.

Emerging View on the Molecular Functions of Sec62 and Sec63 in Protein Translocation.

Most secreted and membrane proteins are targeted to and translocated across the endoplasmic reticulum (ER) membrane through the Sec61 protein-conducting channel. Evolutionarily conserved Sec62 and Sec63 associate with the Sec61 channel, forming the Sec complex and mediating translocation of a subset of proteins. For the last three decades, it has been thought that ER protein targeting and translocation occur via two distinct pathways: signal recognition particle (SRP)-dependent co-translational or SRP-independent, Sec62/Sec63 dependent post-translational translocation pathway. However, recent studies have suggested that ER protein targeting and translocation through the Sec translocon are more intricate than previously thought. This review summarizes the current understanding of the molecular functions of Sec62/Sec63 in ER protein translocation.

The Role of Prostaglandin E1 as a Pain Mediator through Facilitation of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 via the EP2 Receptor in Trigeminal Ganglion Neurons of Mice.

Cyclooxygenase metabolizes dihomo-γ-linolenic acid and arachidonic acid to form prostaglandin (PG) E, including PGE1 and PGE2, respectively. Although PGE2 is well known to play an important role in the development and maintenance of hyperalgesia and allodynia, the role of PGE1 in pain is unknown. We confirm whether PGE1 induced pain using orofacial pain behavioral test in mice and determine the target molecule of PGE1 in TG neurons with whole-cell patch-clamp and immunohistochemistry. Intradermal injection of PGE1 to the whisker pads of mice induced a reduced threshold, enhancing the excitability of HCN channel-expressing trigeminal ganglion (TG) neurons. The HCN channel-generated inward current (Ih) was increased by 135.3 ± 4.8% at 100 nM of PGE1 in small- or medium-sized TG, and the action of PGE1 on Ih showed a concentration-dependent effect, with a median effective dose (ED50) of 29.3 nM. Adenylyl cyclase inhibitor (MDL12330A), 8-bromo-cAMP, and the EP2 receptor antagonist AH6809 inhibited PGE1-induced Ih. Additionally, PGE1-induced mechanical allodynia was blocked by CsCl and AH6809. PGE1 plays a role in mechanical allodynia through HCN2 channel facilitation via the EP2 receptor in nociceptive neurons, suggesting a potential therapeutic target in that PGE1 could be involved in pain as endogenous substances under inflammatory conditions.

Association of TRPV1 and TLR4 through the TIR domain potentiates TRPV1 activity by blocking activation-induced desensitization.

BACKGROUND: We have previously reported that histamine-induced pruritus was attenuated in toll-like receptor 4 (TLR4) knockout mice due to decreased transient receptor potential V1 (TRPV1) sensitivity. Our results implied that TLR4 potentiated TRPV1 activation in sensory neurons; however, the molecular mechanism has yet to be elucidated. In this study, we investigated the molecular mechanisms of TLR4-mediated TRPV1 potentiation using TLR4-deficient sensory neurons and a heterologous expression system. METHODS: Primary sensory neurons were obtained from wild-type or TLR4 knockout mice, and HEK293T cells expressing TRPV1 and TLR4 were prepared by transient transfection. TRPV1 activity was analyzed by calcium imaging, fluorophotometry, and patch-clamp recording. Subcellular protein distribution was tested by immunocytochemistry and cell surface biotinylation assay. Protein interaction was assessed by western blot and immunoprecipitation assay. RESULTS: Direct association between TRPV1 and TLR4 was detected in HEK293T cells upon heterologous TRPV1 and TLR4 expression. In an immunoprecipitation assay using TLR4-deletion mutants and soluble toll/interleukin-1 receptor (TIR) protein, the cytoplasmic TIR domain of TLR4 was required for TLR4-TRPV1 association and TRPV1 potentiation. In TLR4-deficient sensory neurons, the activation-induced desensitization of TRPV1 increased, accompanied by enhanced TRPV1 clearance from the cell membrane upon activation compared to wild-type neurons. In addition, heterologous TLR4 expression inhibited activation-induced TRPV1 endocytosis and lysosomal degradation in HEK293T cells. CONCLUSION: Our data show that direct association between TRPV1 and TLR4 through the TIR domain enhances TRPV1 activity by blocking activation-induced TRPV1 desensitization.

Fast Cyclic Square-Wave Voltammetry To Enhance Neurotransmitter Selectivity and Sensitivity.

Although fast-scan cyclic voltammetry (FSCV) has been widely used for in vivo neurochemical detection, the sensitivity and selectivity of the technique can be further improved. In this study, we develop fast cyclic square-wave voltammetry (FCSWV) as a novel voltammetric technique that combines large-amplitude cyclic square-wave voltammetry (CSWV) with background subtraction. A large-amplitude, square-shaped potential was applied to induce cycling through multiple redox reactions within a square pulse to increase sensitivity and selectivity when combined with a two-dimensional voltammogram. As a result, FCSWV was significantly more sensitive than FSCV ( n = 5 electrodes, two-way ANOVA, p = 0.0002). In addition, FCSWV could differentiate dopamine from other catecholamines (e.g., epinephrine and norepinephrine) and serotonin better than conventional FSCV. With the confirmation that FCSWV did not influence local neuronal activity, despite the large amplitude of the square waveform, it could monitor electrically induced phasic changes in dopamine release in rat striatum before and after injecting nomifensine, a dopamine reuptake inhibitor.

Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs.

Generation of functional dopamine (DA) neurons is an essential step for the development of effective cell therapy for Parkinson's disease (PD). The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and therefore is considered safe to use in the development of cell-based therapy. Thus, we used mRNA-based gene delivery method to generate safe DA neurons. In this study, we generated transformation-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2. The delivery of mRNA encoding dopaminergic fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. Additionally, the generation efficiency of DA neurons was improved by the addition of small molecules, db-cAMP, and the adjustment of transfection timing. The successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.

Antipruritic effect of curcumin on histamine-induced itching in mice.

Itching is a common clinical symptom of skin disease that significantly affects a patient's quality of life. Transient receptor potential vanilloid 1 (TRPV1) receptors of keratinocytes and peripheral nerve fibers in skin are involved in the regulation of itching as well as pain. In this study, we investigated whether curcumin, which acts on TRPV1 receptors, affects histamine-induced itching in mice, using behavioral tests and electrophysiological approaches. We found that histamine-induced itching was blocked by topical application of curcumin in a concentration-dependent manner. In ex-vivo recordings, histamine-induced discharges of peripheral nerves were reduced by the application of curcumin, indicating that curcumin acts directly on peripheral nerves. Additionally, curcumin blocked the histamine-induced inward current via activation of TRPV1 (curcumin IC50=523 nM). However, it did not alter chloroquine-induced itching behavior in mice, which is associated with transient receptor potential ankyrin 1 (TRPA1). Taken together, our results suggest that histamine-induced itching can be blocked by topical application of curcumin through the inhibitory action of curcumin on TRPV1 receptors in peripheral nerves.

TRPM2 contributes to LPC-induced intracellular Ca2+ influx and microglial activation.

Microglia are the resident immune cells which become activated in some pathological conditions in central nervous system (CNS). Lysophosphatidylcholine (LPC), an endogenous inflammatory phospholipid, is implicated in immunomodulatory function of glial cells in the CNS. Although several studies uncovered that LPC induces intracellular Ca2+ influx and morphologic change in microglia, there is still no direct evidence showing change of phosphorylation of mitogen-activated protein kinase (MAPK) p38 (p-p38), a widely used microglia activation marker, by LPC. Furthermore, the cellular mechanism of LPC-induced microglia activation remains unknown. In this study, we found that LPC induced intracellular Ca2+ increase in primary cultured microglia, which was blocked in the presence of Gd3+, non-selective transient receptor potential (TRP) channel blocker. RT-PCR and whole cell patch clamp recordings revealed molecular and functional expression of TRP melastatin 2 (TRPM2) in microglia. Using western blotting, we also observed that LPC increased phosphorylation of p38 MAPK, and the increase of p-p38 expression is also reversed in TRPM2-knockout (KO) microglia. Moreover, LPC induced membrane trafficking of TRPM2 and intrathecal injection of LPC increased Iba-1 immunoreactivity in the spinal cord, which were significantly reduced in KO mice. In addition, LPC-induced intracellular Ca2+ increase and inward currents were abolished in TRPM2-KO microglia. Taken together, our results suggest that LPC induces intracellular Ca2+ influx and increases phosphorylation of p38 MAPK via TRPM2, which in turn activates microglia.

Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage.

Recent groundbreaking work has demonstrated that combined expression of the transcription factors Brn2, Ascl1, and Myt1L (BAM; also known as Wernig factors) convert mouse fibroblasts into postmitotic neuronal cells. However, questions remain regarding whether trans-conversion is achieved directly or involves an intermediary precursor stage. Trans-conversion toward expandable neural precursor cells (NPCs) is more useful than direct one-step neuron formation with respect to yielding a sufficient number of cells and the feasibility of manipulating NPC differentiation toward certain neuron subtypes. Here, we show that co-expression of Wernig factors and Bcl-xL induces fibroblast conversion into NPCs (induced NPCs (iNPCs)) that are highly expandable for >100 passages. Gene expression analyses showed that the iNPCs exhibited high expression of common NPC genes but not genes specific to defined embryonic brain regions. This finding indicated that a regional identity of iNPCs was not established. Upon induction, iNPCs predominantly differentiated into astrocytes. However, the differentiation potential was not fixed and could be efficiently manipulated into general or specific subtypes of neurons by expression of additional genes. Specifically, overexpression of Nurr1 and Foxa2, transcription factors specific for midbrain dopamine neuron development, drove iNPCs to yield mature midbrain dopamine neurons equipped with presynaptic DA neuronal functions. We further assessed the therapeutic potential of iNPCs in Parkinson disease model rats.

The Sec62-Sec63 translocon facilitates translocation of the C-terminus of membrane proteins.

The Sec62-Sec63 complex mediates post-translational translocation of a subset of primarily secretory proteins into the endoplasmic reticulum (ER) in yeast. Therefore, it has been thought that membrane proteins, which are mainly co-translationally targeted into the ER, are not handled by the Sec62-Sec63 translocon. By systematic analysis of single and multi-spanning membrane proteins with broad sequence context [with differing hydrophobicity, flanking charged residues and orientation of transmembrane (TM) segments], we show that mutations in the N-terminal cytosolic domain of yeast Sec62 impair its interaction with Sec63 and lead to defects in membrane insertion and translocation of the C-terminus of membrane proteins. These results suggest that there is an unappreciated function of the Sec62-Sec63 translocon in regulating topogenesis of membrane proteins in the eukaryotic cell.

DAMGO modulates two-pore domain K(+) channels in the substantia gelatinosa neurons of rat spinal cord.

The analgesic mechanism of opioids is known to decrease the excitability of substantia gelatinosa (SG) neurons receiving the synaptic inputs from primary nociceptive afferent fiber by increasing inwardly rectifying K(+) current. In this study, we examined whether a µ-opioid agonist, [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), affects the two-pore domain K(+) channel (K2P) current in rat SG neurons using a slice whole-cell patch clamp technique. Also we confirmed which subtypes of K2P channels were associated with DAMGO-induced currents, measuring the expression of K2P channel in whole spinal cord and SG region. DAMGO caused a robust hyperpolarization and outward current in the SG neurons, which developed almost instantaneously and did not show any time-dependent inactivation. Half of the SG neurons exhibited a linear I~V relationship of the DAMGO-induced current, whereas rest of the neurons displayed inward rectification. In SG neurons with a linear I~V relationship of DAMGO-induced current, the reversal potential was close to the K(+) equilibrium potentials. The mRNA expression of TWIK (tandem of pore domains in a weak inwardly rectifying K(+) channel) related acid-sensitive K(+) channel (TASK) 1 and 3 was found in the SG region and a low pH (6.4) significantly blocked the DAMGO-induced K(+) current. Taken together, the DAMGO-induced hyperpolarization at resting membrane potential and subsequent decrease in excitability of SG neurons can be carried by the two-pore domain K(+) channel (TASK1 and 3) in addition to inwardly rectifying K(+) channel.

Trans-activation of TRPV1 by D1R in mouse dorsal root ganglion neurons.

TRPV1, a ligand-gated ion channel expressed in nociceptive sensory neurons is modulated by a variety of intracellular signaling pathways. Dopamine is a neurotransmitter that plays important roles in motor control, cognition, and pain modulation in the CNS, and acts via a variety of dopamine receptors (D1R-D5R), a class of GPCRs. Although nociceptive sensory neurons express D1-like receptors, very little is known about the effect of dopamine on TRPV1 in the peripheral nervous system. Therefore, in this study, we examined the effects of D1R activation on TRPV1 in mouse DRG neurons using Ca(2+) imaging and immunohistochemical analysis. The D1R agonist SKF-38393 induced reproducible Ca(2+) responses via Ca(2+) influx through TRPV1 rather than Ca(2+) mobilization from intracellular Ca(2+) stores. Immunohistochemical analysis revealed co-expression of D1R and TRPV1 in mouse DRG neurons. The PLC-specific inhibitor blocked the SKF-38393-induced Ca(2+) response, whereas the PKC, DAG lipase, AC, and PKA inhibitors had no effect on the SKF-38393-induced Ca(2+) response. Taken together, our results suggest that the SKF-38393-induced Ca(2+) response results from the direct activation of TRPV1 by a PLC/DAG-mediated membrane-delimited pathway. These results provide evidence that the trans-activation of TRPV1 following D1R activation may contribute to the modulation of pain signaling in nociceptive sensory neurons.

In vitro generation of mature dopamine neurons by decreasing and delaying the expression of exogenous Nurr1.

Neural stem/progenitor cell (NSC/NPC) cultures can be a source of dopamine (DA) neurons for experimental and transplantation purposes. Nurr1, a steroid receptor transcription factor, can overcome the limitations associated with differentiation of cultured NPCs into DA neurons. However, forced Nurr1 expression in NPC cultures generates non-neuronal and/or immature DA cells. We show here that the Nurr1 level and period of expression crucially affect the differentiation and maturation of Nurr1-induced DA neurons. Mature DA neurons were generated by manipulating Nurr1 expression patterns to resemble those in the developing midbrain.

Neuritin produces antidepressant actions and blocks the neuronal and behavioral deficits caused by chronic stress.

Decreased neuronal dendrite branching and plasticity of the hippocampus, a limbic structure implicated in mood disorders, is thought to contribute to the symptoms of depression. However, the mechanisms underlying this effect, as well as the actions of antidepressant treatment, remain poorly characterized. Here, we show that hippocampal expression of neuritin, an activity-dependent gene that regulates neuronal plasticity, is decreased by chronic unpredictable stress (CUS) and that antidepressant treatment reverses this effect. We also show that viral-mediated expression of neuritin in the hippocampus produces antidepressant actions and prevents the atrophy of dendrites and spines, as well as depressive and anxiety behaviors caused by CUS. Conversely, neuritin knockdown produces depressive-like behaviors, similar to CUS exposure. The ability of neuritin to increase neuroplasticity is confirmed in models of learning and memory. Our results reveal a unique action of neuritin in models of stress and depression, and demonstrate a role for neuroplasticity in antidepressant treatment response and related behaviors.

Imiquimod induces a Toll-like receptor 7-independent increase in intracellular calcium via IP(3) receptor activation.

Imiquimod is an itch-promoting, small, synthetic compound that is generally used to treat genital warts and basal cell carcinoma. The pruritogenic effect of imiquimod is considered to be due to TLR7 activation; however that idea has been challenged by our studies showing intact pruritogenic effects of imiquimod in TLR7 KO mice. Thus, the signaling pathways of imiquimod have not been completely elucidated. Here we investigated the novel effects of imiquimod on intracellular calcium ([Ca(2+)]i) signaling. We found that imiquimod induces [Ca(2+)]i increases in PC12 and F11 cells, and even in NIH-3T3 and HEK293T cells, which do not express TLR7. This [Ca(2+)]i increase was due to Ca(2+) release from the internal store without extracellular Ca(2+) influx. Neither FCCP, a mitochondrial Ca(2+) reuptake inhibitor, nor dantrolene, a ryanodine receptor inhibitor, affected the imiquimod-induced [Ca(2+)]i increase. However, 2APB, an IP3 receptor blocker, inhibited the imiquimod-induced [Ca(2+)]i increase. U73122, a PLCß inhibitor, failed to block the imiquimod-induced [Ca(2+)]i increase. These data indicate that imiquimod triggers IP3 receptor-dependent Ca(2+) signaling independently of TLR7.

TRPC4 deletion elicits behavioral defects in sociability by dysregulating expression of microRNA-138-2.

To investigate whether the defects in transient receptor potential canonical 4 (TRPC4), which is strongly expressed in the hippocampus, are implicated in ASD, we examined the social behaviors of mice in which Trpc4 was deleted (Trpc4-/-). Trpc4-/- mice displayed the core symptoms of ASD, namely, social disability and repetitive behaviors. In microarray analysis of the hippocampus, microRNA (miR)-138-2, the precursor of miR-138, was upregulated in Trpc4-/- mice. We also found that binding of Matrin3 (MATR3), a selective miR-138-2 binding nuclear protein, to miR-138-2 was prominently enhanced, resulting in the downregulation of miR-138 in Trpc4-/- mice. Some parameters of the social defects and repetitive behaviors in the Trpc4-/- mice were rescued by increased miR-138 levels following miR-138-2 infusion in the hippocampus. Together, these results suggest that Trpc4 regulates some signaling components that oppose the development of social behavioral deficits through miR-138 and provide a potential therapeutic strategy for ASD.