In this study, changes in blood oxygenation and volume were monitored while monolingual right-handed subjects read English sentences. Our results confirm the role of the left peri-sylvian cortex in language processing. Interestingly, individual subject analyses reveal a pattern of activation characterized by several small, limited patches rather than a few large, anatomically well-circumscribed centers. Between-subject analyses confirm a lateralized pattern of activation and reveal active classical language areas including Broca's area, Wernicke's area, and the angular gyms. In addition they point to areas only more recently considered as language-relevant including the anterior portion of the superior temporal sulcus. This area has not been reliably observed in imaging studies of isolated word processing. This raises the hypothesis that activation in this area is dependent on processes specific to sentence reading. 0

Mutations enhancing the kinase activity of LRRK2 cause Parkinson's disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:CORdimer interface [CORB (R1728H/L)]. Most activating-variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared to a biochemical kinase assay, as only a minority of activating variants [CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)], enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppressed microtubule association in the presence of a Type I kinase inhibitor [ARM(M712V), LRR(R1320S), ROC (A1442P, K1468E, S1508R), CORA(A1589S), CORB (Y1699C, R1728H/L) and WD40(R2143M, S2350I, G2385R)]. Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion/exclusion in ongoing and future LRRK2 inhibitor clinical trials.

The prevalence of Parkinson's disease (PD) is increasing but the development of novel treatment strategies and therapeutics altering the course of the disease would benefit from specific, sensitive, and non‐invasive biomarkers to detect PD early. Here, we describe a scalable and sensitive mass spectrometry (MS)‐based proteomic workflow for urinary proteome profiling. Our workflow enabled the reproducible quantification of more than 2,000 proteins in more than 200 urine samples using minimal volumes from two independent patient cohorts. The urinary proteome was significantly different between PD patients and healthy controls, as well as between LRRK2 G2019S carriers and non‐carriers in both cohorts. Interestingly, our data revealed lysosomal dysregulation in individuals with the LRRK2 G2019S mutation. When combined with machine learning, the urinary proteome data alone were sufficient to classify mutation status and disease manifestation in mutation carriers remarkably well, identifying VGF, ENPEP, and other PD‐associated proteins as the most discriminating features. Taken together, our results validate urinary proteomics as a valuable strategy for biomarker discovery and patient stratification in PD.

Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1–2% of all cases of Parkinson’s disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2—either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors.

The phosphorylated form of LRRK2, pS935 LRRK2, has been proposed as a target modulation biomarker for LRRK2 inhibitors. The primary aim of the study was to characterize and qualify this biomarker for therapeutic trials of LRRK2 inhibitors in Parkinson's disease (PD). To this end, analytically validated assays were used to monitor levels of pS935 LRRK2 and total LRRK2 in peripheral blood mononuclear cells (PBMCs) from the following donor groups: healthy controls, idiopathic PD, and G2019S carriers with and without PD. Neither analyte correlated with age, gender, or disease severity. While total LRRK2 levels were similar across the four groups, there was a significant reduction in pS935 LRRK2 levels in disease-manifesting G2019S carriers compared to idiopathic PD. In aggregate, these data indicate that phosphorylation of LRRK2 at S935 may reflect a state marker for G2019S LRRK2-driven PD, the underlying biology for which requires investigation in future studies. This study also provides critical foundational data to inform the integration of pS935 and total LRRK2 levels as biomarkers in therapeutic trials of LRRK2 kinase inhibitors.