A comparative study of Lrrk2 function in primary neuronal cultures.
OBJECTIVE
To assess the contribution of wild-type, mutant and loss of leucine-rich repeat kinase-2 (LRRK2; Lrrk2) on dendritic neuronal arborization.


BACKGROUND
LRRK2 mutations are recognized as the major genetic determinant of susceptibility to Parkinson's disease for which a cellular assay of Lrrk2 mutant function would facilitate the development of targeted molecular therapeutics.


METHODS
Dendritic neuronal arborization (neurite length, branching and the number of processes per cell) was quantified in primary hippocampal and midbrain cultures derived from five lines of recombinant LRRK2 mice, including human BAC wild-type and mutant overexpressors (Y1699C and G2019S), murine knock-out and G2019S knock-in animals.


RESULTS
Neuronal arborization in cultures from BAC Lrrk2 wild-type animals is comparable to non-transgenic littermate controls, despite high levels of human transgene expression. In contrast, primary neurons from both BAC mutant overexpressors presented with significantly reduced neuritic outgrowth and branching, although the total number of processes per cell remained comparable. The mutant-specific toxic gain-of-function observed in cultures from BAC mutant mice may be partially rescued by staurosporine treatment, a non-specific kinase inhibitor. In contrast, neuronal arborization is far more extensive in neuronal cultures derived from murine knock-out mice that lack endogenous Lrrk2 expression. In Lrrk2 G2019S knock-in mice, arguably the most physiologically relevant system, neuritic arborization is not impaired.


CONCLUSIONS
Impairment of neuritic arborization is an exaggerated, albeit mutant specific, consequence of Lrrk2 over-expression in primary cultures. The phenotype and assay described provides a means to develop therapeutic agents that modulate the toxic gain-of-function conferred by mutant Lrrk2.
Dächsel
Justus C
JC
Division of Neurogenetics, Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA.
Behrouz
Bahareh
B
Yue
Mei
M
Beevers
Joel E
JE
Melrose
Heather L
HL
Farrer
Matthew J
MJ
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D005091:Exons / Q000235:genetics; D055879:Gene Knock-In Techniques; D006706:Homeostasis; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D051379:Mice; D018345:Mice, Knockout; D016501:Neurites / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019311:Staurosporine / Q000494:pharmacology
D047428:Protein Kinase Inhibitors; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine