casein-kinase-ii and Parkinson-Disease

Phosphorylation is a key post-translational modification necessary for normal cellular signaling and, therefore, lies at the heart of cellular function. In neurodegenerative disorders, abnormal hyperphosphorylation of pathogenic proteins is a common phenomenon that contributes in important ways to the disease process. A prototypical protein that is hyperphosphorylated in the brain is α-synuclein (α-syn) - found in Lewy bodies and Lewy neurites - the pathological hallmarks of Parkinson's disease (PD) and other α-synucleinopathies. The genetic linkage of α-syn to PD as well as its pathological association in both genetic and sporadic cases have made it the primary protein of interest. In understanding how α-syn dysfunction occurs, increasing focus is being placed on its abnormal aggregation and the contribution of phosphorylation to this process. Studies of both the kinases and phosphatases that regulate α-syn phosphorylation are beginning to reveal the roles of this post-translational modification in disease pathogenesis. Modulation of α-syn phosphorylation may ultimately prove to be a viable strategy for disease-modifying therapeutic interventions. In this review, we explore mechanisms related to α-syn phosphorylation, its biophysical and functional consequences, and its role in neurodegeneration.

Topics: alpha-Synuclein; Animals; Casein Kinase I; Casein Kinase II; Cell Cycle Proteins; Drosophila; G-Protein-Coupled Receptor Kinases; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Lewy Bodies; Mice; Mice, Transgenic; Molecular Targeted Therapy; Neurons; Parkinson Disease; Phosphoprotein Phosphatases; Phosphorylation; Polo-Like Kinase 1; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rats; Serine

Following the discovery of the human kinome, protein kinases have become the second most important group of drug targets as they can be modulated by small ligand molecules. Moreover, orally active protein kinase inhibitors have recently reached the market and there are many more in clinical trials. The lack of treatments for neurodegenerative diseases has increased human and financial efforts in the search for new therapeutic targets that could provide new effective drug candidates. The importance of kinases in the molecular pathway of neuronal survival is under study, but different key pathways have been described. New roles for the old casein kinases 1 and 2, currently known as protein kinases CK1 and CK2, have recently been discovered in the molecular pathology of different neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases and amyotrophic lateral sclerosis. The search for specific inhibitors of these enzymes has become an important challenge for the treatment of these devastating diseases. The role of these two kinases in the molecular pathology of different neurodegenerative diseases together with different chemical families that are able to more or less specifically inhibit CK1 and CK2 are discussed in this review.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Animals; Binding, Competitive; Casein Kinase I; Casein Kinase II; Drug Design; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Mice; Neurodegenerative Diseases; Parkinson Disease; Phosphorylation; Rats

Parkinson disease (PD) is no longer considered a complex motor disorder characterized by parkinsonism but rather a systemic disease with variegated non-motor deficits and neurological symptoms, including impaired olfaction, sleep disorders, gastrointestinal and urinary abnormalities and cardiovascular dysfunction, in addition to other symptoms and signs such as pain, depression and mood disorders. Many of these alterations appear before or in parallel with motor deficits and then worsen with disease progression. Although there is a close relation between motor symptoms and the presence of Lewy bodies (LBs) and neurites filled with abnormal α-synuclein, other neurological alterations are independent of LBs, thereby indicating that different mechanisms probably converge in the degenerative process. This review presents cardinal observations at very early stages of PD and provides personal experience based on the study of a consecutive series of brains with PD-related pathology and without parkinsonism, mainly cases categorized as stages 2-3 of Braak. Alterations in the substantia nigra, striatum and frontal cortex in pPD are here revised in detail. Early modifications in the substantia nigra at pre-motor stages of PD (preclinical PD: pPD) include abnormal small aggregates of α-synuclein which is phosphorylated, nitrated and oxidized, and which exhibits abnormal solubility and truncation. This occurs in association with a plethora of altered molecular events including increased oxidative stress, altered oxidative stress responses, altered balance of L-ferritin and H-ferritin, reduced expression of neuronal globin α and β chains in neurons with α-synuclein deposits, increased expression of endoplasmic reticulum stress markers, increased p62 and ubiquitin immunoreactivity in relation to α-synuclein deposits, and altered distribution of LC3 and other autophagosome/lysosome markers. In spite of the relatively small decrease in the number of dopaminergic neurons in the substantia nigra, which does not reach thresholds causative of parkinsonism, levels of tyrosine hydroxylase and cannabinoid 1 receptor are reduced, whereas levels of adenosine receptor 2A are increased in the caudate in pPD. Moreover, biochemical alterations are also present in the cerebral cortex (at least in the frontal cortex) in pPD including increased oxidative stress and oxidative damage to proteins α-synuclein, β-synuclein, superoxide dismutase 2, aldolase A, enolase 1, and glyceraldehyde de

Topics: alpha-Synuclein; Casein Kinase II; Disease Progression; Endoplasmic Reticulum; Guanine; Humans; Iron; Mitochondrial Diseases; Nerve Tissue Proteins; Neurons; Oxidative Stress; Parkinson Disease; Substantia Nigra; Superoxide Dismutase; Ubiquitination

Parkinson's disease (PD) is a common neurodegenerative disorder which is mostly sporadic but familial-linked PD (FPD) cases have also been found. The first reported gene mutation that linked to PD is α-synuclein (α-syn). Studies have shown that mutations, increased expression or abnormal processing of α-syn can contribute to PD, but it is believed that multiple mechanisms are involved. One of the contributing factors is post-translational modification (PTM), such as phosphorylation of α-syn at serine 129 by G-protein-coupled receptor kinases (GRKs) and casein kinase 2α (CK2α). Another known important contributing factor to PD pathogenesis is oxidative and nitrosative stress. In this study, we found that GRK6 and CK2α can be S-nitrosylated by nitric oxide (NO) both in vitro and in vivo. S-nitrosylation of GRK6 and CK2α enhanced their kinase activity towards the phosphorylation of α-syn at S129. In an A53T α-syn transgenic mouse model of PD, we found that increased GRK6 and CK2α S-nitrosylation were observed in an age dependent manner and it was associated with an increased level of pSer129 α-syn. Treatment of A53T α-syn transgenic mice with Nω-Nitro-L-arginine (L-NNA) significantly reduced the S-nitrosylation of GRK6 and CK2α in the brain. Finally, deletion of neuronal nitric oxide synthase (nNOS) in A53T α-syn transgenic mice reduced the levels of pSer129 α-syn and α-syn in an age dependent manner. Our results provide a novel mechanism of how NO through S-nitrosylation of GRK6 and CK2α can enhance the phosphorylation of pSer129 α-syn in an animal model of PD.

Topics: Age Factors; alpha-Synuclein; Animals; Casein Kinase II; Disease Models, Animal; G-Protein-Coupled Receptor Kinases; Gene Deletion; HEK293 Cells; Humans; Mice; Mice, Transgenic; Mutation; Nitric Oxide; Nitric Oxide Synthase Type I; Nitroarginine; Nitrosative Stress; Parkinson Disease; Phosphorylation; Serine

Reducing the burden of α-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target α-synuclein oligomers has been a limitation to drug discovery programs.. Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein-synuclein interactions. We have developed two robust models to monitor α-synuclein oligomerization by generating novel stable cell lines expressing α-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system.. A pilot screen was performed resulting in the identification of two potential hits, a p38 MAPK inhibitor and a casein kinase 2 inhibitor, thereby demonstrating the suitability of our protein-fragment complementation assay for the measurement of α-synuclein oligomerization in living cells at high throughput.. The application of the strategy described herein to monitor α-synuclein oligomer formation in living cells with high throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

Topics: alpha-Synuclein; Casein Kinase II; Cell Line; Drug Design; Drug Discovery; High-Throughput Screening Assays; Humans; Lewy Body Disease; Models, Biological; Molecular Targeted Therapy; p38 Mitogen-Activated Protein Kinases; Parkinson Disease; Pilot Projects; Protein Multimerization

PTEN-induced putative kinase 1 (PINK1), a Parkinson's disease (PD)-related protein, has two isoforms, the mitochondria-localized full-length isoform PINK1FL and the cytoplasm-localized short isoform PINK1-cyto. Studies have suggested that PINK1FL can selectively accumulate at the surface of damaged mitochondria and cooperate with another Parkinson's Disease-related protein PARKIN to trigger the degradation of MIRO1, a mitochondria trafficking regulator. The functions of PINK1-cyto are, however, not yet clear. To investigate the functions of PINK1-cyto, we expressed different proteins in cultured HEK293 cells by transfecting it with different plasmids, and detected the protein levels by Western blot after expressing for 24 h. We found that in cultured HEK293 cells, PINK1-cyto could also cooperate with PARKIN degrade MIRO1 in the presence of CK23, and the regulatory subunit of Casein Kinase II. Interestingly, this function of CK2P was not dependent on CK2alpha, the catalytic subunit of Casein Kinase II. We also found that CK2P could promote the direct interaction between PINK1-cyto and MIRO1 by immunocoprecipitation analysis. This result suggested that in addition to CK2alpha, CK2beta could also form a kinase complex.

Topics: Casein Kinase II; HEK293 Cells; Humans; Mitochondrial Proteins; Parkinson Disease; Protein Kinases; Protein Transport; rho GTP-Binding Proteins; Ubiquitin-Protein Ligases

Topics: Aged; Case-Control Studies; Casein Kinase II; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Parkinson Disease; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Receptors, G-Protein-Coupled; Risk Factors; Spain

Aggregated alpha-synuclein and the point mutations Ala30Pro and Ala53Thr of alpha-synuclein are associated with Parkinson's disease. The physiological roles of alpha-synuclein and methionine oxidation of the alpha-synuclein protein structure and function are not fully understood. Methionine sulfoxide reductase A (MsrA) reduces methionine sulfoxide residues and functions as an antioxidant. To monitor the effect of methionine oxidation to alpha-synuclein on basic cellular processes, alpha-synucleins were expressed in msrA null mutant and wild-type yeast cells. Protein degradation was inhibited in the alpha-synuclein-expressing msrA null mutant cells compared to alpha-synuclein-expressing wild-type cells. Increased inhibition of degradation and elevated accumulations of fibrillated proteins were observed in SynA30P-expressing msrA null mutant cells. Additionally, methionine oxidation inhibited alpha-synuclein phosphorylation in yeast cells and in vitro by casein kinase 2. Thus, a compromised MsrA function combined with alpha-synuclein overexpression may promote processes leading to synucleinopathies.

Topics: alpha-Synuclein; Amino Acid Sequence; Casein Kinase II; Gene Expression Regulation, Enzymologic; Gene Knockout Techniques; Methionine; Methionine Sulfoxide Reductases; Mutation; Neurofibrils; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Stress; Parkinson Disease; Saccharomyces cerevisiae; Up-Regulation

We reported previously that phosphorylation by casein kinase II (CKII) regulates the interaction between alpha-synuclein and its binding partner synphilin-1, and that both CKII alpha and beta subunits co-localize with alpha-synuclein in cytoplasmic inclusions in transfected cells. In this study, we extended these observations to the brains of patients with Parkinson's disease (PD) and examined whether CKII subunits are present in Lewy bodies. Immunohistochemical studies on PD brains harboring Lewy bodies revealed a positive stain for CKII beta but not for CKII alpha. In addition, CKII beta subunits co-localized with alpha-synuclein in most Lewy bodies. These findings suggest that CKII beta subunits may play a role in the formation of intracytoplasmic inclusions in human alpha-synucleinopathies either through phosphorylation events or through a separate mechanism linked to the beta subunit itself.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Casein Kinase II; Female; Humans; Immunohistochemistry; Inclusion Bodies; Lewy Bodies; Male; Parkinson Disease; Phosphorylation; Substantia Nigra

alpha-Synuclein (alpha-syn) is the major component of pathologic inclusions that characterize neurodegenerative disorders such as Parkinson disease, dementia with Lewy body disease, and multiple system atrophy. The present study uses novel phospho-specific antibodies to assess the presence and regulation of phosphorylated Ser87 and Ser129 in alpha-syn in human brain samples and in a transgenic mouse model of alpha-synucleinopathies. By immunohistochemistry, alpha-syn phosphorylated at Ser129, but not at Ser87, was abundant in alpha-syn inclusions. Under normal conditions, Ser129 phosphorylation, but not Ser87 phosphorylation, was detected at low levels in the soluble biochemical fractions in human alpha-syn transgenic mice and stably transfected cultured cells. Therefore, a role for Ser87 phosphorylation in alpha-synucleinopathies is unlikely, and in vitro assays showed that phosphorylation at this site would inhibit polymerization. In vitro studies also indicated that hyperphosphorylation of Ser129 alpha-syn in pathologic inclusions may be due in part to the intrinsic properties of aggregated alpha-syn to act as substrates for kinases but not phosphatases. Further studies in transgenic mice and cultured cells suggest that cellular toxicity, including proteasomal dysfunction, increases casein kinase 2 activity, which results in elevated Ser129 alpha-syn phosphorylation. These data provide novel explanations for the presence of hyperphosphorylated Ser129 alpha-syn in pathologic inclusions.

Topics: Adult; Aged; Aged, 80 and over; alpha-Synuclein; Animals; Binding Sites; Casein Kinase II; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Immunohistochemistry; Inclusion Bodies; Male; Mice; Mice, Transgenic; Middle Aged; Neurons; Parkinson Disease; Phosphorylation; Proteasome Endopeptidase Complex; Serine; Substantia Nigra; Substrate Specificity; Up-Regulation

Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of alpha-synuclein. Although certain post-translational modifications of alpha-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of alpha-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50-60% of the LBs that show an intense halo pattern resembling that of alpha-synuclein. By utilizing a cellular model of alpha-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of alpha-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of alpha-synuclein. Finally, we show that Pin1 binds synphilin-1, an alpha-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with alpha-synuclein, thus likely facilitating the formation of alpha-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for alpha-synuclein aggregation and in the resultant Lewy body formations in PD.

Topics: alpha-Synuclein; Amino Acid Sequence; Animals; Binding Sites; Carrier Proteins; Casein Kinase II; Chlorocebus aethiops; COS Cells; Half-Life; Humans; Lewy Bodies; Molecular Sequence Data; Nerve Tissue Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Parkinson Disease; Peptidylprolyl Isomerase; Phosphorylation; Protein Processing, Post-Translational; Solubility

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.

Topics: alpha-Synuclein; Amino Acid Sequence; Animals; Antibody Specificity; Brain Chemistry; Casein Kinase II; Casein Kinases; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Parkinson Disease; PC12 Cells; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Rats; Serine; Synucleins