Search

Levels of expression or of phosphorylation of leucine-rich repeat kinase 2 (LRRK2) and its Rab substrates have the potential for use as disease or pharmacodynamic biomarkers. LRRK2 phosphorylation levels at the S910-S935-S955-S973 phosphosites are reduced for most disease mutant forms of LRRK2, while for phospho-S1292, phospho-T72-Rab8 and phospho-T73-Rab10 levels are increased for most mutants. In addition, all of these 5 phosphosites on LRRK2 are rapidly dephosphorylated upon LRRK2 inhibitor treatment, considered potential therapeutics. The main objective of this study is therefore to characterize leucine-rich repeat kinase 2 (LRRK2) and Rab8/10 in human, non-human primate and rat urine for use as potential disease or pharmacodynamic biomarkers. With urine collected from human subjects as well as animals, we have applied an ultracentrifugation based fractionation protocol to isolate small extracellular vesicles (EVs). We used western blot with antibodies directed against LRRK2, LRRK2 phosphorylation sites as well as Rab8 and Rab10 total and phosphorylated proteins in order to measure these LRRK2 and Rab epitopes in urinary EVs (uEVs). We confirm the presence of LRRK2 and Rab8/10 in human and non-human primate (NHP) uEVs, including total LRRK2 as well phosphorylated forms of LRRK2 pS910, pS935, pS955, pS973, pS1292 and phosphorylated Rab8 and Rab10. We also confirm LRRK2 and Rab expression in rodent uEVs. We quantified LRRK2 and Rab epitopes in human cohorts and found in a first cohort that pS1292-LRRK2 levels were elevated in individuals carrying the LRRK2 G2019S mutation, but did not differ significantly between healthy and PD groups, whether for LRRK2 G2019S carriers or not. In a second cohort, we found that PD was associated to an increase in Rab8 levels and a decrease in S910-LRRK2 and S935-LRRK2 phosphorylation rates. In animals, we found that acute treatment with LRRK2 kinase inhibitor led to decreases in Rab10 phosphorylation rates and to a decrease in pS935-LRRK2 rates in uEVs from NHPs. The identification of changes in LRRK2 phosphorylation at heterologous phosphorylation sites in uEVs of PD patients further reinforces the potential of the LRRK2-Rab pathway as a source of PD biomarkers in uEVs.

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson's disease (PD), and orally bioavailable, brain penetrant and highly potent LRRK2 kinase inhibitors are in early stages of clinical testing. Detection of LRRK2 phosphorylation, as well as phosphorylation of Rab10, a LRRK2 kinase substrate, have been proposed as target engagement biomarkers for LRRK2 inhibitor clinical trials. However, these readouts do not seem able to stratify patients based on enhanced LRRK2 kinase activity. Here, we describe a robust cell biological assay based on centrosomal cohesion alterations which were observed in peripheral blood mononuclear cellderived lymphoblastoid cell lines (LCLs) from patients with G2019S LRRK2 mutations as compared with healthy controls, and could also be detected in a subset of sporadic PD patient samples. We suggest that LCLs may be a valuable resource for LRRK2 research, and that determination of centrosomal cohesion deficits may assist in the stratification of a subset of sporadic PD patients., This work was supported by funding from the Michael J. Fox Foundation for Parkinson's Research (U.S.A.), FEDER, the Spanish Ministry of Science, Innovation and Universities (SAF2017-89402-R), and by the French Ministry of Health's PHRC program (CONVERGENCE 2008-A00219-42), the University of Lille, Inserm and the Lille University Hospital.

The trichomonad species Tritrichomonas fetus and Pentatrichomonas hominis were recently identified in the feces of dogs with diarrhea. However the prevalence and pathogenicity of these parasites in the canine population still remained poorly resolved. Therefore the aim of the present study was (1) to determine the prevalence of trichomonads infecting puppies living in French breeding kennels, (2) to confirm the predominance of P. hominis in dogs, (3) to investigate the genetic diversity of P. hominis isolates identified in the French canine population and (4) to evaluate the risk factors for infection by P. hominis and the influence of the parasite on feces consistency. A total of 215 both diarrheic and non-diarrheic puppies from 25 French breeding kennels were included in this epidemiological survey. Fecal samples from each puppy were examined for 6 gastrointestinal pathogens: parvovirus type 2 (CPV2), coronavirus, Toxocara canis, Cystoisospora ohioensis-complex, Cystoisospora canis, and Giardia intestinalis. A part of each collected stool was also tested for the presence of motile trichomonads by microscopy after culturing. The prevalence of trichomonad infection was 15.8% (34/215) among puppies and 20% (5/25) among breeding kennels. DNA from 26 of the 34 positive samples was successfully amplified using a trichomonad-specific primer pair. Analysis of the sequences of PCR products indicated that P. hominis was the only trichomonad infecting the canine population. All the puppies infected with P. hominis belonged to large breed dogs. Moreover, puppies from large breeding kennels, excreting a high level of G. intestinalis and/or excreting a high level of C. canis oocysts showed a higher probability of being positive for P. hominis infection. Univariate analysis also revealed an increased risk for P. hominis infection in puppies with abnormal feces. However, in a multivariate analysis, CPV2 was the only gastrointestinal pathogen associated with abnormal feces. Since enteropathogens were commonly found in dogs infected by P. hominis, the pathogenic potential of this trichomonad species remained uncertain and has to be further evaluated by experimental infection studies.