Loss of synaptic ribbons is an early cause in ROS-induced acquired sensorineural hearing loss.

Considerable evidence of reactive oxygen species (ROS) involvement in cochlear hair cell (HC) loss, leading to acquired sensorineural hearing loss (SNHL), were reported. Cochlear synaptopathy between HCs and spiral ganglion neurons has been gathering attention as a cochlear HC loss precursor not detectable by normal auditory evaluation. However, the molecular mechanisms linking ROS with HC loss, as well as the relationship between ROS and cochlear synaptopathy have not been elucidated. Here, we examined these linkages using NOX4-TG mice, which constitutively produce ROS without stimulation. mRNA levels of Piccolo 1, a major component of the synaptic ribbon (a specialized structure surrounded by synaptic vesicles in HCs), were decreased in postnatal day 6 NOX4-TG mice cochleae compared to those in WT mice; they were also decreased by noise exposure in 2-week-old WT cochleae. As noise exposure induces ROS production, this suggests that the synaptic ribbon is a target of ROS. The level of CtBP2, another synaptic ribbon component, was significantly lower in NOX4-TG cochleae of 1-month-old and 4-month-old mice compared to that in WT mice, although no significant differences were noted at 1.5- and 2-months. The decrease in CtBP2 plateaued in 4-month-old NOX4-TG, while it gradually decreased from 1 to 6 months in WT mice. Furthermore, CtBP2 level in 2-month-old NOX4-TG mice decreased significantly after exposure to cisplatin and noise compared to that in WT mice. These findings suggest that ROS lead to developmental delays and early degeneration of synaptic ribbons, which could be potential targets for novel therapeutics for ROS-induced SNHL.

Congenital hearing impairment associated with peripheral cochlear nerve dysmyelination in glycosylation-deficient muscular dystrophy.

Hearing loss (HL) is one of the most common sensory impairments and etiologically and genetically heterogeneous disorders in humans. Muscular dystrophies (MDs) are neuromuscular disorders characterized by progressive degeneration of skeletal muscle accompanied by non-muscular symptoms. Aberrant glycosylation of α-dystroglycan causes at least eighteen subtypes of MD, now categorized as MD-dystroglycanopathy (MD-DG), with a wide spectrum of non-muscular symptoms. Despite a growing number of MD-DG subtypes and increasing evidence regarding their molecular pathogeneses, no comprehensive study has investigated sensorineural HL (SNHL) in MD-DG. Here, we found that two mouse models of MD-DG, Largemyd/myd and POMGnT1-KO mice, exhibited congenital, non-progressive, and mild-to-moderate SNHL in auditory brainstem response (ABR) accompanied by extended latency of wave I. Profoundly abnormal myelination was found at the peripheral segment of the cochlear nerve, which is rich in the glycosylated α-dystroglycan-laminin complex and demarcated by "the glial dome." In addition, patients with Fukuyama congenital MD, a type of MD-DG, also had latent SNHL with extended latency of wave I in ABR. Collectively, these findings indicate that hearing impairment associated with impaired Schwann cell-mediated myelination at the peripheral segment of the cochlear nerve is a notable symptom of MD-DG.

Nox3-Derived Superoxide in Cochleae Induces Sensorineural Hearing Loss.

Reactive oxygen species (ROS) produced by NADPH oxidases (Nox) contribute to the development of different types of sensorineural hearing loss (SNHL), a common impairment in humans with no established treatment. Although the essential role of Nox3 in otoconia biosynthesis and its possible involvement in hearing have been reported in rodents, immunohistological methods targeted at detecting Nox3 expression in inner ear cells reveal ambiguous results. Therefore, the mechanism underlying Nox3-dependent SNHL remains unclear and warrants further investigation. We generated Nox3-Cre knock-in mice, in which Nox3 was replaced with Cre recombinase (Cre). Using Nox3-Cre;tdTomato mice of either sex, in which tdTomato is expressed under the control of the Nox3 promoter, we determined Nox3-expressing regions and cell types in the inner ear. Nox3-expressing cells in the cochlea included various types of supporting cells, outer hair cells, inner hair cells, and spiral ganglion neurons. Nox3 expression increased with cisplatin, age, and noise insults. Moreover, increased Nox3 expression in supporting cells and outer hair cells, especially at the basal turn of the cochlea, played essential roles in ROS-related SNHL. The extent of Nox3 involvement in SNHL follows the following order: cisplatin-induced hearing loss > age-related hearing loss > noise-induced hearing loss. Here, on the basis of Nox3-Cre;tdTomato, which can be used as a reporter system (Nox3-Cre+/-;tdTomato+/+ and Nox3-Cre+/+;tdTomato+/+), and Nox3-KO (Nox3-Cre+/+;tdTomato+/+) mice, we demonstrate that Nox3 inhibition in the cochlea is a promising strategy for ROS-related SNHL, such as cisplatin-induced HL, age-related HL, and noise-induced HL.SIGNIFICANCE STATEMENT We found Nox3-expressing regions and cell types in the inner ear, especially in the cochlea, using Nox3-Cre;tdTomato mice, a reporter system generated in this study. Nox3 expression increased with cisplatin, age, and noise insults in specific cell types in the cochlea and resulted in the loss (apoptosis) of outer hair cells. Thus, Nox3 might serve as a molecular target for the development of therapeutics for sensorineural hearing loss, particularly cisplatin-induced, age-related, and noise-induced hearing loss.

Late-onset hearing loss case associated with a heterozygous truncating variant of DIAPH1.

Diaphanous-related formin 1 (DIAPH1) is a formin homology F-actin elongating protein encoded by DIAPH1. Homozygous recessive variants resulting in the loss of DIAPH1 function cause seizures, cortical blindness, and microcephaly syndrome (SCBMS), but hearing loss has not been reported. In contrast, dominant variants of human DIAPH1 are associated with DFNA1 non-syndromic sensorineural hearing loss. The deafness phenotype is due partly to abnormal F-actin elongation activity caused by disruption of the DIAPH1 autoinhibitory mechanism. We report an elderly female heterozygous for the c.3145C>T: p.R1049X variant who showed late-onset sensorineural hearing loss in her fifth decade. p.R1049X lacks F-actin elongation activity because this variant truncates one-third of the FH2 domain, which is vital for DIAPH1 dimerization and processive F-actin elongation activity. Concordantly, no increase of F-actin or processive F-actin elongation activity was observed after overexpression of p.R1049X DIAPH1 in HeLa cells or by single-molecule microscopy using Xenopus XTC cells. However, overexpression of the p.R1049X variant impairs formation of cell-cell junctions and mitosis. We speculate that late-onset hearing loss is a long-term consequence of heterozygosity for the recessive p.R1049X variant, a phenotype that may have been overlooked among carriers of other recessive alleles of DIAPH1.

Effects of flurbiprofen on the functional regulation of serotonin transporter and its misfolded mutant.

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

Novel role of Rac-Mid1 signaling in medial cerebellar development.

Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

Differential disruption of autoinhibition and defect in assembly of cytoskeleton during cell division decide the fate of human DIAPH1-related cytoskeletopathy.

BACKGROUND: Diaphanous-related formin 1 (DIA1), which assembles the unbranched actin microfilament and microtubule cytoskeleton, is encoded by DIAPH1. Constitutive activation by the disruption of autoinhibitory interactions between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD) dysregulates DIA1, resulting in both hearing loss and blood cell abnormalities. METHODS AND RESULTS: Here, we report the first constitutively active mutant in the DID (p.A265S) of humans with only hearing loss and not blood cell abnormality through whole exome sequencing. The previously reported DAD mutants and our DID mutant (p.A265S) shared the finding of diminished autoinhibitory interaction, abnormally upregulated actin polymerisation activity and increased localisations at the plasma membrane. However, the obvious defect in the DIA1-driven assembly of cytoskeleton 'during cell division' was only from the DAD mutants, not from p.A265S, which did not show any blood cell abnormality. We also evaluated the five DID mutants in the hydrophobic pocket since four of these five additional mutants were predicted to critically disrupt interaction between the DID and DAD. These additional pathogenic DID mutants revealed varying degrees of defect in the DIA1-driven cytoskeleton assembly, including nearly normal phenotype during cell division as well as obvious impaired autoinhibition, again coinciding with our key observation in DIA1 mutant (p.A265S) in the DID. CONCLUSION: Here, we report the first mutant in the DID of humans with only hearing loss. The differential cell biological phenotypes of DIA1 during cell division appear to be potential determinants of the clinical severity of DIAPH1-related cytoskeletopathy in humans.

Spinocerebellar ataxia type 14 caused by a nonsense mutation in the PRKCG gene.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disorder characterized by cerebellar ataxia with myoclonus, dystonia, spasticity, and rigidity. Although missense mutations and a deletion mutation have been found in the protein kinase C gamma (PRKCG) gene encoding protein kinase C γ (PKCγ) in SCA14 families, a nonsense mutation has not been reported. The patho-mechanisms underlying SCA14 remain poorly understood. However, gain-of-function mechanisms and loss-of-function mechanisms, but not dominant negative mechanisms, were reported the patho-mechanism of SCA14. We identified the c.226C>T mutation of PRKCG, which caused the p.R76X in PKCγ by whole-exome sequencing in patients presenting cerebellar atrophy with cognitive and hearing impairment. To investigate the patho-mechanism of our case, we studied aggregation formation, cell death, and PKC inhibitory effect by confocal microscopy, western blotting with cleaved caspase 3, and pSer PKC motif antibodies, respectively. PKCγ(R76X)-GFP have aggregations the same as wild-type (WT) PKCγ-GFP. The PKCγ(R76X)-GFP inhibited PKC phosphorylation activity more than GFP alone. It also induced more apoptosis in COS7 and SH-SY5Y cells compared to WT-PKCγ-GFP and GFP. We first reported SCA14 patients with p.R76X in PKCγ who have cerebellar atrophy with cognitive and hearing impairment. Our results suggest that a dominant negative mechanism due to truncated peptides produced by p.R76X may be at least partially responsible for the cerebellar atrophy.

The Role of Cysteine String Protein α Phosphorylation at Serine 10 and 34 by Protein Kinase Cγ for Presynaptic Maintenance.

Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinson's disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ-CSPα-HSC70/HSP70-SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD.SIGNIFICANCE STATEMENT Cysteine string protein α (CSPα) is a protein belonging to the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles, which maintain the presynaptic terminal. However, the function of CSPα phosphorylation by protein kinase C (PKC) for neuronal cell survival remains unclear. The experiments presented here demonstrate that PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. CSPα phosphorylation at Ser10 and Ser34 by PKCγ protects the presynaptic terminal by promoting HSP70 chaperone activity. This report suggests that CSPα phosphorylation, because of its role in modulating HSP70 chaperone activity, may be a target for the treatment of neurodegeneration.

Pharmacological induction of heat shock proteins ameliorates toxicity of mutant PKCγ in spinocerebellar ataxia type 14.

Amyloid and amyloid-like protein aggregations are hallmarks of multiple, varied neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. We previously reported that spinocerebellar ataxia type 14 (SCA14), a dominant-inherited neurodegenerative disease that affects cerebellar Purkinje cells, is characterized by the intracellular formation of neurotoxic amyloid-like aggregates of genetic variants of protein kinase Cγ (PKCγ). A number of protein chaperones, including heat shock protein 70 (Hsp70), promote the degradation and/or refolding of misfolded proteins and thereby prevent their aggregation. Here, we report that, in various SCA14-associated, aggregating PKCγ variants, endogenous Hsp70 is incorporated into aggregates and that expression of these PKCγ mutants up-regulates Hsp70 expression. We observed that PKCγ binds Hsp70 and that this interaction is enhanced in the SCA14-associated variants, mediated by the kinase domain that is involved in amyloid-like fibril formation as well as the C2 domain of PKCγ. Pharmacological up-regulation of Hsp70 by the Hsp90 inhibitors celastrol and herbimycin A attenuated the aggregation of mutant PKCγ in primary cultured Purkinje cells. Up-regulation of Hsp70 diminished net PKCγ aggregation by preventing aggregate formation, resulting in decreased levels of apoptotic cell death among primary cultured Purkinje cells expressing the PKCγ variant. Of note, herbimycin A also ameliorated abnormal dendritic development. Extending our in vitro observations, administration of celastrol to mice up-regulated cerebellar Hsp70. Our findings identify heat shock proteins as important endogenous regulators of pathophysiological PKCγ aggregation and point to Hsp90 inhibition as a potential therapeutic strategy in the treatment of SCA14.

Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.

Protein kinase C-ε (PKC-ε) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (εPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for εPS is unknown. Liposome assays revealed that the εPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2 Wortmannin, but not LY294002, inhibits PKC-ε concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ε concentration at cups and the rate of phagocytosis. PKC-ε colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ε-expressing macrophages revealed a loss of Golgi-associated PKC-ε during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ε at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgi-associated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ε is tethered to the TGN via an εPS-PI4P interaction. The TGN-associated pool of PKC-ε concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that εPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ε localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.

A Novel Rac1-GSPT1 Signaling Pathway Controls Astrogliosis Following Central Nervous System Injury.

Astrogliosis (i.e. glial scar), which is comprised primarily of proliferated astrocytes at the lesion site and migrated astrocytes from neighboring regions, is one of the key reactions in determining outcomes after CNS injury. In an effort to identify potential molecules/pathways that regulate astrogliosis, we sought to determine whether Rac/Rac-mediated signaling in astrocytes represents a novel candidate for therapeutic intervention following CNS injury. For these studies, we generated mice with Rac1 deletion under the control of the GFAP (glial fibrillary acidic protein) promoter (GFAP-Cre;Rac1flox/flox). GFAP-Cre;Rac1flox/flox (Rac1-KO) mice exhibited better recovery after spinal cord injury and exhibited reduced astrogliosis at the lesion site relative to control. Reduced astrogliosis was also observed in Rac1-KO mice following microbeam irradiation-induced injury. Moreover, knockdown (KD) or KO of Rac1 in astrocytes (LN229 cells, primary astrocytes, or primary astrocytes from Rac1-KO mice) led to delayed cell cycle progression and reduced cell migration. Rac1-KD or Rac1-KO astrocytes additionally had decreased levels of GSPT1 (G1 to S phase transition 1) expression and reduced responses of IL-1ß and GSPT1 to LPS treatment, indicating that IL-1ß and GSPT1 are downstream molecules of Rac1 associated with inflammatory condition. Furthermore, GSPT1-KD astrocytes had cell cycle delay, with no effect on cell migration. The cell cycle delay induced by Rac1-KD was rescued by overexpression of GSPT1. Based on these results, we propose that Rac1-GSPT1 represents a novel signaling axis in astrocytes that accelerates proliferation in response to inflammation, which is one important factor in the development of astrogliosis/glial scar following CNS injury.

Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction.

Previous studies have convincingly argued that reactive oxygen species (ROS) contribute to the development of several major types of sensorineural hearing loss, such as noise-induced hearing loss (NIHL), drug-induced hearing loss, and age-related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG) mouse line expressing the human NADPH oxidase 4 (NOX4, NOX4-TG mice), which is a constitutively active ROS-producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX4-TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high-frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHCs). The vulnerability to loss of hearing function and OHCs was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat-shock protein 47 (HSP47) in models using HEK293 cells, including H2 O2 treatment and cells with stable and transient expression of NOX4. Furthermore, the up-regulated levels of Hsp47 were observed in both the cochlea and heart of NOX4-TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL. Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo, and counteracting ROS-related hearing loss.

Identification and characterization of PKCγ, a kinase associated with SCA14, as an amyloidogenic protein.

Amyloid assemblies are associated with a wide range of human disorders, including Alzheimer's and Parkinson's diseases. Here, we identify protein kinase C (PKC) γ, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKCγ in cultured cells, as well as in vitro incubation of PKCγ without heat or chemical denaturants, causes amyloid-like fibril formation of this protein. We also observed that SCA14-associated mutations in PKCγ accelerate the amyloid-like fibril formation both in cultured cells and in vitro. We show that the C1A and kinase domains of PKCγ are involved in its soluble dimer and aggregate formation and that SCA14-associated mutations in the C1 domain cause its misfolding and aggregation. Furthermore, long-term time-lapse imaging indicates that aggregates of mutant PKCγ are highly toxic to neuronal cells. Based on these findings, we propose that PKCγ could form amyloid-like fibrils in physiological and/or pathophysiological conditions such as SCA14. More generally, our results provide novel insights into the mechanism of amyloid-like fibril formation by multi-domain proteins.

The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release.

NADPH oxidase (Nox) family proteins produce superoxide (O2 (⨪)) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2 (⨪) intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2 (⨪) dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2 (⨪) leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2 (⨪) even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2 (⨪) in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2 (⨪) leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2 (⨪). Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2 (⨪) release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2 (⨪) leakage. Although we identified Duox1 A-loop residues (His(1071), His(1072), and Gly(1074)) important for reducing O2 (⨪) release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2 (⨪)-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex.

Maintenance of stereocilia and apical junctional complexes by Cdc42 in cochlear hair cells.

Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.

The role of Pak-interacting exchange factor-ß phosphorylation at serines 340 and 583 by PKCγ in dopamine release.

Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKCγ, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKCγ-knock-out (KO) and that PKCγ-KO mice showed parkinsonian syndrome. However, the PKCγ substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKCγ substrates involved in dopamine release, we used PKCγ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKCγ-KO mice. We focused on Pak-interacting exchange factor-ß (ßPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates ßPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated ßPIX in vivo. Classical PKC inhibitors and ßPIX knock-down (KD) significantly suppressed Ca(2+)-evoked dopamine release in PC12 cells. Wild-type ßPIX, and not the ßPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by ßPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of ßPIX at Ser340 and Ser583 has pivotal roles in Ca(2+)-evoked dopamine release in the striatum. Therefore, we propose that PKCγ positively modulates dopamine release through ß2PIX phosphorylation. The PKCγ-ßPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

Negative charges in the flexible N-terminal domain of Rho GDP-dissociation inhibitors (RhoGDIs) regulate the targeting of the RhoGDI-Rac1 complex to membranes.

In its resting state, Rho GDP-dissociation inhibitor (RhoGDI) α forms a soluble cytoplasmic heterodimer with the GDP-bound form of Rac. Upon stimulation, the dissociation of RhoGDIα from the RhoGDIα-Rac complex is a mandatory step for Rac activation; however, this mechanism is poorly understood. In this study, we examined how the cytoplasm/membrane cycles of the RhoGDI-Rac complex are regulated, as well as where RhoGDI dissociates from the RhoGDI-Rac complex, during FcγR-mediated phagocytosis. The negatively charged and flexible N terminus (25 residues) of RhoGDIα, particularly its second negative amino acid cluster possessing five negatively charged amino acids, was a pivotal regulator in the cytoplasm/membrane cycles of the RhoGDI-Rac complex. We also found that RhoGDIα translocated to the phagosomes as a RhoGDIα-Rac1 complex, and this translocation was mediated by an interaction between the polybasic motif in the C terminus of Rac1 and anionic phospholipids produced on phagosomes, such as phosphatidic acid, that is, by a phagosome-targeting mechanism of Rac1. Thus, we demonstrated that the targeting/accumulation of the RhoGDIα-Rac1 complex to phagosomes is regulated by a balance between three factors: 1) the negatively charged and flexible N-terminal of RhoGDIα, 2) the binding affinity of RhoGDIα for Rac1, and 3) anionic phospholipids produced on phagosomes. Moreover, we demonstrated that the mechanism of targeting/accumulation of the RhoGDIα-Rac1 complex is also applicable for the RhoGDIß-Rac1 complex.

Diacylglycerol kinases terminate diacylglycerol signaling during the respiratory burst leading to heterogeneous phagosomal NADPH oxidase activation.

It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase ß abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase ß mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms.

Diacylglycerol kinase γ regulates antigen-induced mast cell degranulation by mediating Ca(2+) influxes.

Diacylglycerol (DAG) is an important lipid that acts as a signaling messenger during mast cell degranulation after allergen cross-linking of immunoglobulin (Ig) E-bound FcεRI receptors. In this study, we determined the role of diacylglycerol kinase (DGK), which negatively regulates DAG-dependent signaling by converting DAG to phosphatidic acid (PA), in the regulation of mast cell degranulation. Treating RBL (rat basophilic leukemia)-2H3 mast cells with a type I DGK inhibitor significantly reduced antigen-induced degranulation and PA production. Among type I DGK isoforms, we observed that DGKα and DGKγ mRNAs were expressed in RBL-2H3 mast cells using reverse transcription polymerase chain reaction. DGKγ knockdown, but not DGKα, by isoform-specific short hairpin RNAs reduced mast cell degranulation and Ca(2+) influxes from the extracellular environment. These results suggest that DGKγ regulates mast cell degranulation after FcεRI cross-linking through mobilization of intracellular Ca(2+) through Ca(2+) influxes.