The Translation Post Vol.3 Issue 1

Featured Article - Kinases in Neurodegenerative Diseases

Kinases in Brain

The kinase family is one of the largest target families in the human genome. Altogether, it is estimated that there are more than 500 members of the major classes of protein serine/threonine, tyrosine, and dual specificity kinases within the human genome [1,2]. Protein phosphorylation is one of the most significant signal transduction mechanisms by which intercellular signals regulate crucial intracellular processes such as ion transport, cellular proliferation, and hormone responses. Consistent with the complex role of this post-translational modification in the cell, protein kinases can be regulated by activator proteins, inhibitor proteins, ligand binding to regulatory subunits, cofactors, and phosphorylation by other proteins or by themselves (autophosphorylation). For discovering reversible protein phosphorylation as a biological regulatory mechanism, Edmond H. Fischer and Edwin G. Krebs were awarded the 1992 Nobel Prize for Physiology and Medicine. The kinase family™s key function in signal transduction for all organisms makes it a very attractive target class for therapeutic interventions in many disease states such as cancer, diabetes, inflammation, and arthritis. In this regard, protein kinases represent as much as thirty percent of all protein targets under investigation by pharmaceutical companies. Recent successful launches of drugs with kinase inhibition as the mode of action demonstrate the ability to deliver kinase inhibitors as drugs with the appropriate selectivity, potency, and pharmacokinetic properties [3,4]. To date, kinase brain research, however, has not delivered many candidates for treating neurodegenerative disorders such as Alzheimer™s disease. The latest reports of potential treatments of brain disorders by using kinase inhibitors are reviewed in this article.

Alzheimer™s Disease

Alzheimer™s disease (AD) is an age-related progressive neurodegenerative disorder with devastating symptoms in a growing aged population. It is the most common form of dementia affecting about 5% of adults over 65 years. Currently available medications appear to be able to produce moderate symptomatic benefits but not to stop disease progression. The search for novel therapeutic approaches targeting the presumed underlying pathogenic mechanisms has been a major focus of research and it is expected that novel medications with disease-modifying properties will emerge from these efforts in the future, in particular those targeting amyloid b protein and tau pathologies.

The characteristic neuropathological hallmarks of AD include neuritic plaques and neurofibrillary tangles. Neuritic plaques are extracellular lesions composed of a central core of aggregated amyloid-β peptide surrounded by dystrophic neuritis, activated microglia and reactive astrocytes [5]. Neurofibrillary tangles are intracellular bundles of paired helical and straight filaments. They are composed of tau protein in an abnormally hyperphosphorylated form. It appears that the formation of these two protein aggregates is at the root of the pathogenesis of AD, and consequently it is believed that targeting the underlying mechanisms leading to plaques and tangles will ultimately generate novel therapeutics with disease-modifying properties [6].
Hyperphosphorylation of tau deregulates its ability to promote microtubule assembly resulting in its detachment from microtubules, breakdown of the microtubule network, disturbance of axonal transport and ultimately neurodegeneration [7]. More than 30 phosphorylation sites on tau protein have been described and numerous kinases are able to phosphorylate tau protein in vitro. These include glycogen synthase kinase 3-beta (GSK3-b), cdc2-like kinase (cdk5), extracellular signal-regulating kinase-2 (ERK2), microtubule-affinity-regulating kinase (MARK), protein kinase A (PKA), members of the stress-activated protein kinase (SAPK) family, Ca²⁺/calmodulin-dependent kinase II and casein kinases I and II [8,9].
Cdk5/p25 over-expression in transgenic mice leads to tau hyperphosphorylation and aggregation as well as to neuronal loss [10]. Cdk5 is one of the few Cdk family members that have cellular functions outside the cell cycle. Although Cdk5 protein is present in several tissues, its activity is detected almost exclusively in brain extracts. Cdk5 hyperactivity is toxic to cultured neurons that can be explained by hyperphosphorylation of tau protein and β-catenin [11]. GSK3-β is activated during tau aggregation, suggesting that more than one kinase is involved in tau hyperphosphorylation. Several animal models have been developed, that reproduce characteristic features of tau-related neurofibrillary degeneration. Chronic inhibition of GSK3-b by lithium reduces tau hyperphosphorylation at several sites in these models and decreases tau aggregation [12]. In one of these animal models, oral treatment with a synthetic kinase inhibitor with limited kinase selectivity has been shown to delay the onset of the typical motor deficits accompanied by a reduction of tau hyperphosphorylation [13]. These observations strongly support the use of inhibitors of aberrant phosphorylation of tau as an approach to developing a disease-modifying treatment for AD and other tau-related neurodegenerative diseases.
Lithium is considered a first-line pharmacotherapy in geriatric manic behavior, also called bipolar disorder in the elderly. Although specific for GSK3 compared with other protein kinases, lithium also affects other enzymes, and a relatively high dose is required to inhibit GSK3 activity. Lithium competes for Mg²⁺ preventing polyglutamine toxicity in Huntington™s disease (HD) but has limited protection against neuronal death in AD [14].
Besides tau phosphorylation, glutamate transporter regulation by the protein kinase C (PKC) family has emerged as a therapeutic area for the prevention of neurodegenerative diseases [15]. Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and is critical for essentially all physiological processes ranging from control of motor and somatosensory function to information processing and storage. Glutamate activates ionotropic and metabotropic receptors. Ionotropic receptors are ion chanels mainly permeable to Na⁺, Ca²⁺, or K⁺. Metabotropic receptors are coupled to the activation of G proteins that subsequently regulate signaling pathways, such as adenylate cyclase or phospholipase C, and ion channels. Excess activation of glutamate receptors contributes to the loss of neurons observed in several chronic neurodegenerative diseases, such as AD, amyotrophic lateral sclerosis (ALS), and HD. PKC enzymes alter glutamate transporter function by either changing the number of transporters expressed at the cell membrane or by changing the intrinsic activity of the transporters already located at the cell surface. Although the activation of glutamate receptors by PKC enzymes seems to be a ubiquitous mechanism, the different subtypes of PKC have opposite effects depending on cell type and transmitter concentrations. Known inhibitors of PKC isoforms have been efficient in activating one type of response from the glutamate receptors over another, which suggests that inhibiting specific members of the PKC family can regulate glutamate transporter activity on neuron death.

Parkinson™s Disease

Parkinson™s disease (PD) is the second most common neurodegenerative disorder after AD. The age-adjusted prevalence for PD raises from 0.15% in the 50 to 59 year old population to 1.75% in the 80 year old and more population. The pathological hallmarks are dopaminergic cell loss in the substantia nigra and the presence of Lewy bodies and Lewy neuritis [16]. Lewy bodies and dystrophic Lewy neuritis are cytoplasmic accumulations of aggregated proteins. There are about a dozen of identified PD genes. The PTEN-induced kinase 1 (PINK1/PARK6) and leucine-rich repeat kinase 2 (LRRK2/PARK8) are the only two kinases identified in this mixed pool of enzymes closely related to the ubiquitin proteasome pathway. PINK1 is localized in the mitochondria and protects against stress induced mitochondrial dysfunction and apoptosis [17]. Autosomal recessively inherited mutations in PINK1 are reported in PD patients from consanguineous families and in sporadic patients with early onset PD. LRRK2 was identified recently as the causative gene in families linked to the autosomal dominantly inherited LRRK2 locus. Mutations in LRRK2 protein are associated with abnormalities consistent with Lewy body PD as well as other neurodegenerative pathologies. Among different mutations found in LRRK2, the Gly2019Ser mutation has been identified in both familial and sporadic forms of PD in several distinct populations, making this site a major target to develop a diagnostic genetic test [18].
The mixed lineage kinase (MLK) family members are key participants in the activation of c-Jun N-terminal kinase (JNK), which is thought to underlie neuronal dysfunction and subsequent death. In 2002, Cephalon launched a phase II/III clinical trial with CEP-1347 compound, a potent inhibitor of the MLK family members, which enhances the survival of neurons that produce dopamine in the specific area of the brain affected by PD. Unfortunately, the study was stopped in 2005 for lack of efficacy of CEP-1347 in patients with PD [19].
More recently, G-protein-coupled receptor kinase 5 (GRK5) has been found to accumulate in Lewy bodies and to colocalize with a-synuclein in the pathological structures of the brain of sporadic PD patients [20]. Genetic association study revealed haplotypic association of the GRK5 gene with susceptibility to sporadic PD. These results suggest that phosphorylation of α-synuclein by GRK5 plays a crucial role in the pathogenesis of sporadic PD.
The discovery of the different gene defects described above highlighted the relevance of the ubiquitin proteasome and cell signaling pathways. Like treatment of rats with proteasome inhibitors closely mimics PD in rodents, modification of kinase activity with specific inhibitors provides a very attractive treatment strategy of both familial and sporadic disease. Little is know about PINK1 and LRRK2 substrates and potential inhibitors, we should expect however in a close future a myriad of projects targeting these kinases.

Genetic studies of Parkinson™s disease have identified key associated mutations in several genes, including PARK7, PINK1, parkin, a-synuclein and UCHL1 among others. PARK7 participates in the oxidative stress response. PINK1 is a mitochondrial protein implicated in protection against mitochondrial dysfunction. Polymeric α-synuclein is a major constituent of Lewy bodies. Parkin and UCHL1 participate in protein degradation via the ubiquitin proteasome pathway. Mutations or altered gene expression of these proteins lead to faulty protein trafficking, mitochondrial dysfunction and oxidative stress that contribute in a multi-factorial way to the Parkinson™s disease phenotype.

Huntington™s Disease and Other Neurodegenerative Disorders

Polyglutamine diseases such as Huntington™s disease, Kennedy™s disease, dentatorubro-pallidoluysian atrophy (DRPLA), and some of the autosomal dominantly inherited spinocerebellar ataxias result from an increased number of CAG nucleotide repeats that encode polyglutamine tracts within the corresponding gene products [18]. The altered proteins show no other homology whether in cellular function or localization but the polyglutamine tract, which can vary in length. A change of approximately 10-20% in repeat length differentiates between normal and accelerated neuronal degeneration. Longer expansions correlate with earlier onset and more severe disease cases. Proteins with elongated polyglutamine tracts misfold and aggregate as antiparallel b strands termed polar zippers and form intracellular inclusions. These inclusions are typically but not exclusively found in the brain regions or neurons that are affected. Furthermore, they can be found in non-neuronal tissues [21]. Formation of inclusions is the cell response to the toxicity of the polyglutamine tracts, they subsequently activate cell death signaling via caspases 1 and 8 for instance in HD.
As seen previously for AD, glutamate receptor regulation by PKC is related to neuron loss in HD and ALS [15]. GSK3 also regulates activity of heat shock proteins such as HSF1, which can reduce neuron death and polyglutamine toxicity in HD [22]. Double-stranded RNA-dependent protein kinase (PKR) has been associated with HD in a couple of studies that described activated PKR is increased in HD patients [23]. Phosphorylation of the repetitive KSP region in neurofilaments by Cdk5, a mutated region in ALS patients, has led to new research grounds [11]. Although the molecular mechanisms are slowly deciphered, kinase-targeted therapies remain currently elusive for these diseases.

Concluding Remarks

Although kinases have been a major target for cancer treatment in the past years (see The Translation Post vol.1.1 for review), it appears that they are not yet primary targets in developing drugs for brain disorders. The understanding of the cellular mechanism in the brain pathologies such as Alzheimer and Parkinson™s diseases has tremendously advanced in the past decade. Kinases have been identified within the path leading to protein aggregation, but not one in particular could be specifically inhibited, nor more that one is responsible for protein activation. The most promising candidates are in Parkinson™s disease for which PINK1 and LRRK2 have stirred a lot of research interest these past two years.

Abgent and Neurosciences

Abgent offers a comprehensive collection of antibodies against AD, PD and HD including kinases, ubiquitin ligases, and amyloid-related proteins, including: GSK3, Cdk5, PKC isoforms, GRK5, PARK1-11, APP and Tau protein. Additionally, Abgent has set up collaborations with experts in Parkinson™s disease for evaluating our numerous LRRK2 products.

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