Kinase inhibitors arrest neurodegeneration in cell and C. elegans models of LRRK2 toxicity.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in Caenorhabditis elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2 and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C- and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C- and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.

Insulin-like growth factor-binding protein 2-driven glioma progression is prevented by blocking a clinically significant integrin, integrin-linked kinase, and NF-κB network.

Insulin-like growth factor-binding protein 2 (IGFBP2) is increasingly recognized as a glioma oncogene, emerging as a target for therapeutic intervention. In this study, we used an integrative approach to characterizing the IGFBP2 network, combining transcriptional profiling of human glioma with validation in glial cells and the replication-competent ASLV long terminal repeat with a splice acceptor/tv-a glioma mouse system. We demonstrated that IGFBP2 expression is closely linked to genes in the integrin and integrin-linked kinase (ILK) pathways and that these genes are associated with prognosis. We further showed that IGFBP2 activates integrin ß1 and downstream invasion pathways, requires ILK to induce cell motility, and activates NF-κB. Most significantly, the IGFBP2/integrin/ILK/NF-κB network functions as a physiologically active signaling pathway in vivo by driving glioma progression; interfering with any point in the pathway markedly inhibits progression. The results of this study reveal a signaling pathway that is both targetable and highly relevant to improving the survival of glioma patients.

ATF6beta is a host cellular target of the Toxoplasma gondii virulence factor ROP18.

The ROP18 kinase has been identified as a key virulence determinant conferring a high mortality phenotype characteristic of type I Toxoplasma gondii strains. This major effector molecule is secreted by the rhoptries into the host cells during invasion; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. In this study, we show that ROP18 targets the host endoplasmic reticulum-bound transcription factor ATF6ß. Disruption of the ROP18 gene severely impairs acute toxoplasmosis by the type I RH strain. Because another virulence factor ROP16 kinase modulates immune responses through its N-terminal portion, we focus on the role of the N terminus of ROP18 in the subversion of host cellular functions. The N-terminal extension of ROP18 contributes to ATF6ß-dependent pathogenicity by interacting with ATF6ß and destabilizing it. The kinase activity of ROP18 is essential for proteasome-dependent degradation of ATF6ß and for parasite virulence. Consistent with a key role for ATF6ß in resistance against this intracellular pathogen, ATF6ß-deficient mice exhibit a high susceptibility to infection by ROP18-deficient parasites. The results reveal that interference with ATF6ß-dependent immune responses is a novel pathogenic mechanism induced by ROP18.

Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease.

Leucine-rich repeat kinase-2 (LRRK2) mutations are a common cause of Parkinson's disease. Here we identify inhibitors of LRRK2 kinase that are protective in in vitro and in vivo models of LRRK2-induced neurodegeneration. These results establish that LRRK2-induced degeneration of neurons in vivo is kinase dependent and that LRRK2 kinase inhibition provides a potential new neuroprotective paradigm for the treatment of Parkinson's disease.

Mechanisms in dominant parkinsonism: The toxic triangle of LRRK2, alpha-synuclein, and tau.

Parkinson's disease (PD) is generally sporadic but a number of genetic diseases have parkinsonism as a clinical feature. Two dominant genes, alpha-synuclein (SNCA) and leucine-rich repeat kinase 2 (LRRK2), are important for understanding inherited and sporadic PD. SNCA is a major component of pathologic inclusions termed Lewy bodies found in PD. LRRK2 is found in a significant proportion of PD cases. These two proteins may be linked as most LRRK2 PD cases have SNCA-positive Lewy bodies. Mutations in both proteins are associated with toxic effects in model systems although mechanisms are unclear. LRRK2 is an intracellular signaling protein possessing both GTPase and kinase activities that may contribute to pathogenicity. A third protein, tau, is implicated as a risk factor for PD. We discuss the potential relationship between these genes and suggest a model for PD pathogenesis where LRRK2 is upstream of pathogenic effects through SNCA, tau, or both proteins.

The WD40 domain is required for LRRK2 neurotoxicity.

BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD). LRRK2 contains an "enzymatic core" composed of GTPase and kinase domains that is flanked by leucine-rich repeat (LRR) and WD40 protein-protein interaction domains. While kinase activity and GTP-binding have both been implicated in LRRK2 neurotoxicity, the potential role of other LRRK2 domains has not been as extensively explored. PRINCIPAL FINDINGS: We demonstrate that LRRK2 normally exists in a dimeric complex, and that removing the WD40 domain prevents complex formation and autophosphorylation. Moreover, loss of the WD40 domain completely blocks the neurotoxicity of multiple LRRK2 PD mutations. CONCLUSION: These findings suggest that LRRK2 dimerization and autophosphorylation may be required for the neurotoxicity of LRRK2 PD mutations and highlight a potential role for the WD40 domain in the mechanism of LRRK2-mediated cell death.

CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation, and toxicity.

Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.

Zeroing in on LRRK2-linked pathogenic mechanisms in Parkinson's disease.

The frequency and potency of mutations in the LRRK2 gene redefine the role of genetic susceptibility in Parkinson's disease. Dominant missense mutations that fulfill initial criteria for potential gain of function mechanisms coupled with enzymatic activity likely amenable to small molecule inhibition position LRRK2 as a promising therapeutic target. Herein, key observations from the clinic to the test tube are highlighted together with points of contention and outstanding critical issues. Resolution of the critical issues will expedite the development of therapies that exploit LRRK2 activity for neuroprotection strategies.

RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression.

Myotonic muscular dystrophy (DM1) is the most common inherited neuromuscular disorder in adults and is considered the first example of a disease caused by RNA toxicity. Using a reversible transgenic mouse model of RNA toxicity in DM1, we provide evidence that DM1 is associated with induced NKX2-5 expression. Transgene expression resulted in cardiac conduction defects, increased expression of the cardiac-specific transcription factor NKX2-5 and profound disturbances in connexin 40 and connexin 43. Notably, overexpression of the DMPK 3' UTR mRNA in mouse skeletal muscle also induced transcriptional activation of Nkx2-5 and its targets. In human muscles, these changes were specific to DM1 and were not present in other muscular dystrophies. The effects on NKX2-5 and its downstream targets were reversed by silencing toxic RNA expression. Furthermore, using Nkx2-5+/- mice, we show that NKX2-5 is the first genetic modifier of DM1-associated RNA toxicity in the heart.

Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease indistinguishable from idiopathic disease. The mechanisms whereby missense alterations in the LRRK2 gene initiate neurodegeneration remain unknown. Here, we demonstrate that seven of 10 suspected familial-linked mutations result in increased kinase activity. Functional and disease-associated mutations in conserved residues reveal the critical link between intrinsic guanosine triphosphatase (GTPase) activity and downstream kinase activity. LRRK2 kinase activity requires GTPase activity, whereas GTPase activity functions independently of kinase activity. Both LRRK2 kinase and GTPase activity are required for neurotoxicity and potentiate peroxide-induced cell death, although LRRK2 does not function as a canonical MAP-kinase-kinase-kinase. These results suggest a link between LRRK2 kinase activity and pathogenic mechanisms relating to neurodegeneration, further supporting a gain-of-function role for LRRK2 mutations.

Inducible overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette.

We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(delta2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce deltaR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5 deltaR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.

The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane.

Multiple yop mutant strains of Yersinia pseudotuberculosis not expressing several virulence effector Yop proteins (YopH, M, E, K and YpkA) were engineered. When high-copy-number plasmids carrying the ypkA or the yopE gene with their endogenous promoters were introduced into the engineered strains, the corresponding Yop protein was secreted at high levels in vitro. These multiple yop mutant strains, when harbouring the yopE gene in trans, behaved as the wild-type strain with respect to YopB-dependent translocation of YopE through the HeLa cell plasma membrane. Using these multiple yop mutant strains, it was demonstrated that the YpkA Ser/Thr protein kinase mediates morphological alterations of infected cultured HeLa cells different from those mediated by YopE and YopH. Furthermore, YpkA is shown to be translocated by a YopB-dependent translocation mechanism from surface-located bacteria and subsequently targeted to the inner surface of the target-cell plasma membrane. The pattern of YpkA localization after infection suggests that this Yop effector is involved in interference with signal transduction.