Naoko Adachi

Enzymological Analysis of Mutant Protein Kinase Cγ Causing Spinocerebellar Ataxia Type 14 and Dysfunction in Ca2+ Homeostasis

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cγ (PKCγ). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCγ kinase activity. To determine the effect of these mutations on PKCγ function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCγs. We found that wild-type PKCγ, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCγ. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCγ. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCγs was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

Aggregate formation of mutant protein kinase C gamma found in spinocerebellar ataxia type 14 impairs ubiquitin-proteasome system and induces endoplasmic reticulum stress

Several causal missense mutations in protein kinase Cγ (γPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant γPKC found in SCA14 is susceptible to two types of aggregation, cytoplasmic dot‐like and perinuclear massive aggregation, and causes cell death in Chinese hamster ovary cells. Long‐term time‐lapse imaging revealed that firstly accumulated dot‐like aggregation of mutant γPKC‐green fluorescent protein (GFP) gradually formed perinuclear massive aggregations, followed by cell death. However, it remains unclear how aggregate formation of mutant γPKC causes cell death. In the present study, we examined whether these mutant aggregations affect the ubiquitin‐proteasome system (UPS) and endoplasmic reticular (ER) stress. Two mutant γPKC‐GFPs (S119P and G128D) were strongly ubiquitinated, and dot‐like aggregations of these mutants were ubiquitin‐positive and colocalized with proteasome 20S. Furthermore, proteasome activity in cells with aggregates, especially massive ones, was significantly decreased. Aggregate formation of mutant γPKC‐GFP induced phosphorylation of PERK (PKR‐like ER kinase) and nuclear expression of CHOP (C/EBP homologous protein), hallmarks of ER stress and subsequently activated caspase‐3. These results indicate that aggregate formation of mutant γPKC found in SCA14 impairs UPS and induces ER stress, leading to apoptotic cell death.

Mutant γPKC found in spinocerebellar ataxia type 14 induces aggregate-independent maldevelopment of dendrites in primary cultured Purkinje cells

Missense mutations in protein kinase Cgamma (gammaPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant gammaPKC found in SCA14 is susceptible to aggregation and induces apoptosis in cultured cell lines. In the present study, we investigated whether mutant gammaPKC formed aggregates and how mutant gammaPKC affects the morphology and survival of cerebellar Purkinje cells (PCs), which are degenerated in SCA14 patients. Adenovirus-transfected primary cultured PCs expressing mutant gammaPKC-GFP also had aggregates and underwent apoptosis. Long-term time-lapse observation revealed that PCs have a potential to eliminate aggregates of mutant gammaPKC-GFP. Mutant gammaPKC-GFP disturbed the development of PC dendrites and reduced synapse formation, regardless of the presence or absence of its aggregates. In PCs without aggregates, mutant gammaPKC-GFP formed soluble oligomers, resulting in reduced mobility and attenuated translocation of mutant gammaPKC-GFP upon stimulation. These molecular properties of mutant gammaPKC might affect the dendritic morphology in PCs, and be involved in the pathogenesis of SCA14.

Importance of chroman ring and tyrosine phosphorylation in the subtype-specific translocation and activation of diacylglycerol kinase α by d-α-tocopherol

Diacylglycerol kinase (DGK) has been suggested to be a pharmacological target of d‐α‐tocopherol for diabetic renal dysfunctions. However, the DGK subtypes involved in the d‐α‐tocopherol‐induced improvement of diabetic renal dysfunctions and the activation mechanisms of DGK by d‐α‐tocopherol are still unknown. Therefore, using GFP‐tagged DGKα, β, γ, ɛ and ζ, we analyzed their response to d‐α‐tocopherol and its derivatives. Only DGKα was translocated from the cytoplasm to the plasma membrane with elevation of kinase activity. In addition, troglitazone and trolox possessing ‘chroman ring’ similarly to d‐α‐tocopherol, induced the subtype‐specific translocation of DGKα. Furthermore, the translocation of DGKα was abolished by pretreatment with tyrosine kinase inhibitors or by mutation on Tyr‐334 of the kinase (YF mutant). d‐α‐tocopheryl succinate enhanced the Src‐mediated tyrosine phosphorylation of wild‐type DGKα but the same reagent did not enhance that of the YF mutant. These results demonstrate that tyrosine phosphorylation on Tyr‐334 and chroman ring are important for the d‐α‐tocopherol‐induced translocation of DGKα.

Protein kinase Cε: function in neurons

Protein kinase Cε is expressed at higher levels in the brain compared to other tissues such as the heart and kidney, suggesting that it plays an important role in the nervous system. Several neural functions of PKCε, including neurotransmitter release and ion channel regulation, have been identified using PKCε knockout mice. In this review, we focus on the involvement of protein kinase Cε in neurite outgrowth, presynaptic regulation, alcohol actions, ischemic preconditioning and pain.

Protein kinase Cepsilon: function in neurons.

Protein kinase Cε is expressed at higher levels in the brain compared to other tissues such as the heart and kidney, suggesting that it plays an important role in the nervous system. Several neural functions of PKCε, including neurotransmitter release and ion channel regulation, have been identified using PKCε knockout mice. In this review, we focus on the involvement of protein kinase Cε in neurite outgrowth, presynaptic regulation, alcohol actions, ischemic preconditioning and pain.

Diacylglycerol kinase β accumulates on the perisynaptic site of medium spiny neurons in the striatum

Following activation of Gq protein‐coupled receptors, phospholipase C yields a pair of second messengers, i.e. diacylglycerol (DAG) and inositol 1,4,5‐trisphosphate. The former activates protein kinase C and the latter mobilizes Ca2+ from intracellular store. DAG kinase (DGK) then phosphorylates DAG to produce another second messenger (phosphatidic acid). Of 10 mammalian DGK isozymes, DGKβ is expressed in dopaminergic projection fields with the highest level in the striatum and its particular splice variant is differentially expressed in patients with bipolar disorder. To gain molecular anatomical evidence for its signaling role, we investigated the cellular expression and subcellular localization of DGKβ in the striatum of rat brain. DGKβ was expressed in medium spiny neurons constituting the striatonigral and striatopallidal pathways, whereas striatal interneurons were below the detection threshold. DGKβ was distributed in somatodendritic elements of medium spiny neurons and localized in association with the smooth endoplasmic reticulum and plasma membrane or in the narrow cytoplasmic space between them. In particular, DGKβ exhibited dense accumulation at perisynaptic sites on dendritic spines forming asymmetrical synapses. The characteristic anatomical localization was consistent with exclusive enrichment of DGKβ in the microsomal and postsynaptic density fractions. Intriguingly, DGKβ was very similar in immunohistochemical and immunochemical distribution to Gq‐coupled receptors, such as metabotropic glutamate receptors 1 and 5, and also to other downstream molecules involving DAG metabolism, such as phospholipase C β and DAG lipase. These findings suggest that abundant DGKβ is provided to perisynaptic sites of medium spiny neurons so that it can effectively produce phosphatidic acid upon activation of Gq‐coupled receptors and modulate the cellular state of striatal output neurons.

Effect of Trehalose on the Properties of Mutant γPKC, Which Causes Spinocerebellar Ataxia Type 14, in Neuronal Cell Lines and Cultured Purkinje Cells

Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation, which induces apoptotic cell death. The disaccharide trehalose has been reported to inhibit aggregate formation and to alleviate symptoms in cellular and animal models of Huntington disease, Alzheimer disease, and prion disease. Here, we show that trehalose can be incorporated into SH-SY5Y cells and reduces the aggregation of mutant γPKC-GFP, thereby inhibiting apoptotic cell death in SH-SY5Y cells and primary cultured Purkinje cells (PCs). Trehalose acts by directly stabilizing the conformation of mutant γPKC without affecting protein turnover. Trehalose was also found to alleviate the improper development of dendrites in PCs expressing mutant γPKC-GFP without aggregates but not in PCs with aggregates. In PCs without aggregates, trehalose improves the mobility and translocation of mutant γPKC-GFP, probably by inhibiting oligomerization and thereby alleviating the improper development of dendrites. These results suggest that trehalose counteracts various cellular dysfunctions that are triggered by mutant γPKC in both neuronal cell lines and primary cultured PCs by inhibiting oligomerization and aggregation of mutant γPKC.

Mutant protein kinase C gamma that causes spinocerebellar ataxia type 14 (SCA14) is selectively degraded by autophagy

Several causal missense mutations in the protein kinase Cγ (γPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously showed that mutant γPKC found in SCA14 is susceptible to aggregation and causes apoptosis. Aggregation of misfolded proteins is generally involved in the pathogenesis of many neurodegenerative diseases. Growing evidence indicates that macroautophagy (autophagy) is important for the degradation of misfolded proteins and the prevention of neurodegenerative diseases. In the present study, we examined whether autophagy is involved in the degradation of the mutant γPKC that causes SCA14. Mutant γPKC‐GFP was transiently expressed in SH‐SY5Y cells by using an adenoviral tetracycline‐regulated system. Subsequently, temporal changes in clearance of aggregates and degradation of γPKC‐GFP were evaluated. Rapamycin, an autophagic inducer, accelerated clearance of aggregates and promoted degradation of mutant γPKC‐GFP, but it did not affect degradation of wild‐type γPKC‐GFP. These effects of rapamycin were not observed in embryonic fibroblast cells from Atg5‐deficient mice, which are not able to perform autophagy. Furthermore, lithium, another type of autophagic inducer, also promoted the clearance of mutant γPKC aggregates. These results indicate that autophagy contributes to the degradation of mutant γPKC, suggesting that autophagic inducers could provide therapeutic potential for SCA14.

Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca2+ and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate), a type of phorbol ester. Wild-type (WT) γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in pathogenesis of SCA14.