Impact of 100 LRRK2 variants linked to Parkinson’s Disease on kinase activity and microtubule binding

Abstract

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 98 LRRK2 variants linked to PD, including previously described pathogenic mutations, and identify 22 variants that robustly stimulate LRRK2 kinase activity. These include variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)] as well as variants residing within the GTPase domain, predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORB (S1761R, L1795F)] and CORB:CORB dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer910/pSer935), consistent with the notion that the active kinase conformation blocks their phosphorylation. We find that the R1628P variant that has been linked to PD does not stimulate LRRK2 activity. We observe that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation at Thr73 compared to an in vitro kinase assay, as only a minority of activating variants enhance the activity of immunoprecipitated LRRK2, comprising the CORB (Y1699C, R1728H, S1761R) and kinase (G2019S, I2020T, T2031S) variants. Twelve variants including some that activated LRRK2, suppressed microtubule association in the presence of a Type 1 kinase inhibitor [ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)]. Our findings will aid in understanding the impact of these variants on LRRK2 kinase activation, stimulate work to reveal mechanisms by which variants impact biology, and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 clinical trials.

Structural basis of human LRRK2 membrane recruitment and activation

“…Mutations in the LRRK2 gene, which encodes the leucine-rich repeat kinase 2 (LRRK2) protein, are one of the most frequent genetic causes of PD and account for ~5% of familial and ~1% of sporadic cases (Paisan-Ruiz et al, 2004;Tolosa et al, 2020;Zimprich et al, 2004). More than 250 mutations in LRRK2 have been identified, but pathogenicity has been confirmed only for a small subset, including N1437H, R1441C/G/H, Y1699C, G2019S and I2020T (Blanca Ramirez et al, 2017;Bryant et al, 2021;Kalogeropulou et al, 2022). LRRK2 G2019S , the most common mutation in LRRK2-related PD cases, significantly increased the kinase activity (Jaleel et al, 2007).…”

The emerging role of LRRK2 in tauopathies

“…Accordingly, an altered cytosolic to microtubule-bound tau ratio could increase tau’s propensity to aggregate. In particular, a subset of LRRK2 pathogenic variants can form filaments on microtubules [ 29 , 30 ], which impairs the motility of microtubule-associated motors [ 31 ] and therefore makes it likely to impair the binding of tau. However, LRRK2 microtubule-binding is primarily observed in overexpression systems and when cells have been treated with LRRK2-type I kinase inhibitors [ 31 ].…”

Membrane remodeling properties of the Parkinson’s disease protein LRRK2

“…We next tested the effect of nucleotides on LRRK2 binding to liposomes as its GTPase and kinase domains are critical to its physiological function 25 . Presence of GMPPNP, a nonhydrolyzable GTP analog, did not affect the co-sedimentation of LRRK2 with liposomes (Fig.…”

“…The kinase activity of LRRK2 is critical for its physiological function. Many of the LRRK2 mutations that cause, or increase the risk of PD increase LRRK2 kinase activity 9,25,26 . However, as shown above, ATP had no effect on LRRK2-dependent tubulation (Fig.…”