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Exploring the contribution of LRRK2 GTPase activity to kinase activity and cellular phenotypes of Parkinson's disease-associated LRRK2

Biosa, Alice (2013) Exploring the contribution of LRRK2 GTPase activity to kinase activity and cellular phenotypes of Parkinson's disease-associated LRRK2. Doctoral Thesis.

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Abstract

Parkinson’s disease (PD) is a chronic and progressive motor neurodegenerative disorder. It is among the most common incurable diseases in the world: in the United States, at least 500,000 people suffer from PD, and about 50,000 new cases are reported annually. For these reasons, researchers are still looking for new therapies. Several genetic and environmental factors are demonstrated to be responsible for the onset of the disease.
In this work we have focused on the LRRK2 gene in which mutations are the most common causes of familial and sporadic PD. Since, LRRK2 pathobiology is still not well understood, the aim of this study is to understand the overall contribution of the GTPase domain to the regulation of LRRK2 kinase activity and cellular phenotypes related to Parkinson’s disease.
A collection of synthetic mutations analogous to well-characterized and conserved functional substitutions in members of the Ras and Raf families were developed for expression in mammalian cells. GTP binding, GTP hydrolysis, kinase activity and cellular phenotypes of FLAG-tagged LRRK2 were explored in HEK-293T cells. Since it was previously demonstrated that LRRK2 forms homodimers, the formation of dimers by FLAG-tagged wild-type (WT) LRRK2 and functional variants was verified by fast protein liquid chromatography and by co-immunoprecipitation with MYC-tagged WT LRRK2.
Finally, the effect of altered GTPase activity on neurite length was verified in cortical primary neurons obtained from P0 Sprague-Dawley rats and then transfected with WT and mutated FLAG-tagged LRRK2.
Data shows that both GTP binding and hydrolysis are independent from kinase activity and that both of them can contribute to the positive modulation of LRRK2 kinase activity. Moreover, reduced, but not enhanced, GTPase activity critically alters neurite length in primary cortical neurons.
In summary, we generated a complete library of LRRK2 functional variants to elucidate the interplay between ROC and kinase domains. Our biochemical results seem to indicate that LRRK2 enzymatic regulation is quite complicated and it does not follow the canonical GDP/GTP cycle that is typical of the Ras/Raf/ERK pathway.
Moreover, decreased, but not increased, GTPase activity causes inhibition of axonal length. To date, we do not have any data to demonstrate that increased GTP hydrolysis can rescue GTPase-impaired neurite shortening. Therefore, new experiments are required to investigate the potential protective effects of modulating GTP hydrolysis in vitro and in vivo model systems expressing GTPase-impaired pathological mutations.

Item Type:Doctoral Thesis
ID Code:8383
Contributors:Serra, Pier Andrea and Moore, Darren J.
Publisher:Università degli studi di Sassari
Uncontrolled Keywords:Parkinson's disease, LRRK2, enzymatic activity, neurotoxicity
Subjects:Area 05 - Scienze biologiche > BIO/14 Farmacologia
Divisions:001 Università di Sassari > 01-a Nuovi Dipartimenti dal 2012 > Scienze Biomediche
Cicli, scuole e corsi:Ciclo 25 > Scuole > Scienze biomediche > Neuroscienze
Deposited On:28 Mar 2013 09:43

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