Impact of Type II LRRK2 inhibitors on signalling and mitophagy

<strong>Abstract</strong> Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (Ponatinib and GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open- vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggestings this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors. <strong>FILE DESCRIPTIONS</strong> The blots shown in the Figures were developed using the two fluorescent channels available in LI-COR (800 nM, green – 700 nM, red). Different contrasts of the same membranes have been uploaded. <strong>Figure 2 </strong> <strong>Figure_2A-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 2A <strong>Figure_2A_700_1-LRRK2_Rab10_totals_GAPDH.png </strong> Top to bottom 1: total LRRK2 blot shown in Figure 2A 2: GPDH loading control for blots in Figure 2A and total Rab10 shown in Figure 2A. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa <strong>Figure_2A_700_2-Rab12_total.png</strong> Total Rab12 shown in Figure 2A <strong>Figure_2A_700_3</strong><strong>-tubulin.png</strong> Top to bottom 3: alpha-tubulin loading control for blots in Figure 2A <strong>Figure_2A_800_1-LRRK2pS935_Rab10pT73.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 2A 2: Rab10 pThr73 blot shown in Figure 2A <strong>Figure_2A_800_2-Rab12pS105.png</strong> Phospho-Rab12 pSer105 shown in Figure 2A <strong>Figure_2B-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 2B <strong>Figure_2B_700_1-LRRK2_Rab10_totals_GAPDH_tubulin.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 2B 2: GPDH loading control for blots in Figure 2B and total Rab10 shown in Figure 2B. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa 3: alpha-tubulin loading control for blots in Figure 2B <strong>Figure_2B_700_2-Rab12_total.png</strong> Total Rab12 shown in Figure 2B <strong>Figure-2B_800_1-LRRK2pS935_Rab10pT73.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 2B 2: Rab10 pThr73 blot shown in Figure 2B <strong>Figure-2B_800_2-Rab12pS105.png</strong> Phospho-Rab12 pSer105 shown in Figure 2B <strong>Figure_2C-quantitations</strong>: raw data for charts in Figure 2C <strong>Figure_2C_700_1-LRRK2_Rab10_totals_GAPDH.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 2C 2: GPDH loading control for blots in Figure 2C and total Rab10 shown in Figure 2C. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa 3: alpha-tubulin loading control for blots in Figure 2C – higher contrast of Figure-2C 700-3-alpha_tubulin – low contrast) <strong>Figure_2C_700_2-Rab12_total.png</strong> Total Rab12 shown in Figure 2C <strong>Figure_2C_700_3-tubulin.png</strong> Top to bottom 1-2: lower contrast of Figure-2C 680-1 LRRK2_Rab10_totals_GAPDH 3: alpha-tubulin loading control for blots in Figure 2C <strong>Figure_2C_800_1-LRRK2pS935_Rab10pT73.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 2C 2: Rab10 pThr73 blot shown in Figure 2C <strong>Figure_2C_800_2-Rab12pS105.png</strong> Phospho-Rab12 pSer105 shown in Figure 2C <strong>Figure_2D-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 2D <strong>Figure_2D_700_1-LRRK2_total_tubulin.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 2D 2: lower contrast of total Rab10 blot show in Figure 2D 3: alpha-tubulin loading control for blots in Figure 2D <strong>Figure_2D_700_2-Rab10_total_1.png</strong> Top to bottom 1-3: higher contrast of Figure_2D_700_1-LRRK2_total_tubulin 2: total Rab10 blot show in Figure 2D <strong>Figure_2D_700_3-Rab12_total_1.png</strong> Total Rab12 shown in Figure 2C <strong>Figure_2D_700_4-Rab12_total_2.png</strong> Total Rab12 shown in Figure 2C, membrane incubated with the total Rab12 rabbit antibody from Proteintech <strong>Figure_2D_800_1_LRRK2pS935.png</strong> LRRK2 pSer935 blot shown in Figure 2D <strong>Figure_2D_800_2_Rab10pT73.png</strong> Rab10 pThr73 blot shown in Figure 2D <strong>Figure_2D_800_3_Rab10_total_2.png</strong> Total Rab10 shown in Figure 2D, membrane incubated with the rabbit anti-total Rab10 antibody from Abcam <strong>Figure_2D_800_4-Rab12pS105.png</strong> Phospho-Rab12 pSer105 shown in Figure 2D <strong>Figure 3</strong> <strong>Figure_3_700_1-LRRK2_Rab10_totals_GAPDH.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 3. This total was multiplex with anti LRRK2 phospho-Ser935 antibody. 2: GPDH loading control for blots in Figure 3 and total Rab10 shown in Figure 3. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa. <strong>Figure_3_700_2-Rab10_total_2.png</strong> Total Rab10 shown in Figure 3, membrane incubated with the rabbit anti-total Rab10 antibody from Abcam. <strong>Figure_3_700_3-LRRK2_total_for_LRRK2pS910.png</strong> Total LRRK2 blot, not shown in the paper, multiplexed with the anti LRRK2 phospho-Ser910 antibody shown in Figure 3 <strong>Figure_3_700_4-LRRK2_totals_for_LRRK2pS955_pS973.png</strong> Total LRRK2 blots, not shown in the paper, multiplexed with the anti LRRK2 phospho-Ser955 and phospho-Ser973 antibodies shown in Figure 3 <strong>Figure_3_800_1-LRRK2pS935.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 3 2: lower contrast of Rab10 pThr73 blot shown in Figure 3 <strong>Figure_3_800_2-Rab10pT73.png</strong> Top to bottom 1: higher contrast of LRRK2 pSer935 blot shown in Figure 3 2: Rab10 pThr73 blot shown in Figure 3 <strong>Figure_3_800_3-LRRK2pS910.png</strong> LRRK2 pSer910 blot shown in Figure 3 <strong>Figure_3_800_4-LRRK2pS955.png</strong> LRRK2 pSer955 blot shown in Figure 3 <strong>Figure_3_800_5-LRRK2pS973.png</strong> LRRK2 pSer973 blot shown in Figure 3 <strong>Figure_3-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 3 <strong>Figure 4</strong> <strong>Figure_4A_4B_700_1-LRRK2_Rab10_totals_tubulin.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 4A 2: total Rab10 shown in Figure 4A 3: alpha-tubulin loading control for blots in Figure 4A 4: total LRRK2 blot shown in Figure 4B 5: total Rab10 shown in Figure 4B 6: alpha-tubulin loading control for blots in Figure 4B <strong>Figure_4A_4B_800_1-LRRK2pS935_Rab10pT73.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 4A 2: Rab10 pThr73 blot shown in Figure 4A 4: LRRK2 pSer935 blot shown in Figure 4B 5: Rab10 pThr73 blot shown in Figure 4B <strong>Figure_4A_4B-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 4A_4B <strong>Figure_4C_700_1-LRRK2_Rab10_totals_GAPDH_tubulin.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 4C. This total was multiplex with anti LRRK2 phospho-Ser935 antibody. 2: GPDH loading control for blots in Figure 4C and total Rab10 shown in Figure 4C. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa <strong>Figure_4C_800_1-LRRK2pS935_Rab10pT73.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 4C 2: higher contrast of Rab10 pThr73 blot shown in Figure 4C <strong>Figure_4C_800_2-LRRK2pS935_Rab10pT73.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 4C 2: Rab10 pThr73 blot shown in Figure 4C <strong>Figure_4C-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 4C <strong>Figure_4D-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 4D <strong>Figure_4D_700_1-LRRK2_total_tubulin.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 4D 2: lower contrast of GAPDH and total Rab10 blot show in Figure 4D 3: alpha-tubulin loading control for blots in Figure 4D <strong>Figure_4D_700_2-Rab10_total_GAPDH.png </strong> Top to bottom 1: higher contrast of total LRRK2 blot shown in Figure 4D 2: GAPDH loading control for blots in Figure 4D and total Rab10 blot show in Figure 4D 3: higher contrast of alpha-tubulin loading control for blots in Figure 4D <strong>Figure_4D_800_1-LRRK2pS935.png</strong> 1: LRRK2 pSer935 blot shown in Figure 4D 2: higher contrast of Rab10 pThr73 blot shown in Figure 4D <strong>Figure_4D_800_2-Rab10pT73.png</strong> 1: lower contrast of LRRK2 pSer935 blot shown in Figure 4D 2: Rab10 pThr73 blot shown in Figure 4D <strong>Figure 5</strong> <strong>Figure_5A-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 5A <strong>Figure_5A_700_1-LRRK2_total.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 5A 2: higher contrast of total Rab10 blot show in Figure 5A 3: higher contrast of alpha-tubulin loading control for blots in Figure 5A <strong>Figure_5A_700_2-Rab10_total.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 5A 2: total Rab10 blot show in Figure 5A 3: higher contrast of alpha-tubulin loading control for blots in Figure 5A <strong>Figure_5A_700_3-tubulin.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 5A 2: lower contrast of total Rab10 blot show in Figure 5A 3: alpha-tubulin loading control for blots in Figure 5A <strong>Figure_5A_800_1-LRRK2pS935.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 5A 2: higher contrast of Rab10 pThr73 blot shown in Figure 5A <strong>Figure_5A_800_2-Rab10pT73.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 5A 2: Rab10 pThr73 blot shown in Figure 5A <strong>Figure_5B_700_1-LRRK2_total.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 5B 2: higher contrast of total Rab10 blot show in Figure 5B 3: higher contrast of alpha-tubulin loading control for blots in Figure 5B <strong>Figure_5B_700_2-Rab10_total.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 5B 2: total Rab10 blot show in Figure 5B 3: higher contrast of alpha-tubulin loading control for blots in Figure 5B <strong>Figure_5B_700_3-tubulin.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 5B 2: lower contrast of total Rab10 blot show in Figure 5B 3: alpha-tubulin loading control for blots in Figure 5B <strong>Figure_5B_800_1-LRRK2pS935.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 5B 2: higher contrast of Rab10 pThr73 blot shown in Figure 5B <strong>Figure_5B_800_2-Rab10pT73.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 5B 2: Rab10 pThr73 blot shown in Figure 5B <strong>Figure_5B-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 5B <strong>Figure 6</strong> <strong>Figure_6A-quantitations.xls</strong>: raw data for charts in Figure 6A <strong>Figure-6A_700_1-LRRK2_Rab10_totals.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 6A 2: total Rab10 shown in Figure 6A 3: higher contrast of alpha-tubulin loading control for blots in Figure 6A <strong>Figure_6A_700_2-tubulin.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 6A 2: lower contrast of total Rab10 shown in Figure 6A 3: alpha-tubulin loading control for blots in Figure 6A <strong>Figure_6A_800_1-LRRK2pS935.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 6A 2: higher contrast of Rab10 pThr73 blot shown in Figure 6A <strong>Figure_6A_800_2-Rab10pT73.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 6A 2: Rab10 pThr73 blot shown in Figure 6A <strong>Figure_6B-quantitations.xls</strong>: raw data for charts in Figure 6B <strong>Figure_6B_700_1-LRRK2_Rab10_totals.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 6B 2: total Rab10 shown in Figure 6B 3: higher contrast of alpha-tubulin loading control for blots in Figure 6B <strong>Figure_6B_700_2-tubulin.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 6B 2: lower contrast of total Rab10 shown in Figure 6B 3: alpha-tubulin loading control for blots in Figure 6B <strong>Figure_6B_800_1-LRRK2pS935.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 6B 2: higher contrast of Rab10 pThr73 blot shown in Figure 6B <strong>Figure_6B_800_2-Rab10pT73.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 6B 2: Rab10 pThr73 blot shown in Figure 6B <strong>Figure_6C-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 6C <strong>Figure_6C_700_1-LRRK2_Rab10_totals.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 6C 2: total Rab10 blot shown in Figure 6C 3: higher contrast of alpha-tubulin loading control for blots in Figure 6C <strong>Figure_6C_700_2-tubulin.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 6C 2: lower contrast of Rab10 shown in Figure 6C 3: alpha-tubulin loading control for blots in Figure 6C <strong>Figure_6C_800_1-LRRK2pS935.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 6C 2: higher contrast of Rab10 pThr73 blot shown in Figure 6C <strong>Figure_6C_800_2-Rab10pT73.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 6C 2: Rab10 pThr73 blot shown in Figure 6C <strong>Figure_6D-quantitations.xls</strong>:<strong> </strong>raw data for charts in Figure 6D <strong>Figure_6D_700_1-LRRK2_Rab10_totals.png</strong> Top to bottom 1: total LRRK2 blot shown in Figure 6D 2: total Rab10 blot shown in Figure 6D 3: higher contrast of alpha-tubulin loading control for blots in Figure 6D <strong>Figure_6D_700_2-tubulin.png</strong> Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 6D 2: lower contrast of total Rab10 shown in Figure 6D 3: alpha-tubulin loading control for blots in Figure 6D <strong>Figure_6D_800_1-LRRK2pS935.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 6D 2: higher contrast of Rab10 pThr73 blot shown in Figure 6D <strong>Figure_6D_800_2-Rab10pT73.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 6D 2: Rab10 pThr73 blot shown in Figure 6D <strong>Figure 7</strong> <strong>Figure_7A-quantitations.xls</strong>: raw data for charts in Figure 7A <strong>Figure_7A_700_1_LRRK2_total_CoIp.png</strong> Immunoprecipitated total LRRK2 shown in Figure 7A <strong>Figure_7A_700_2_LRRK2_total_Lysates.png</strong> (Input for the CoIp) Top to bottom 1: total LRRK2 blot shown in Figure 7A 2: higher contrast of total Rab10 blot shown in Figure 7A 3: higher contrast of alpha-tubulin loading control for blots in Figure 7A <strong>Figure_7A_700_3_Rab10_total_tubulin_Lysates.png </strong>(Input for the CoIp) Top to bottom 1: lower contrast of total LRRK2 blot shown in Figure 7A 2: total Rab10 shown in Figure 7A 3: alpha-tubulin loading control for blots in Figure 7A <strong>Figure_7A_800_1_14-3-3_CoIp.png</strong> Blot of 14-3-3 co-immunoprecipitated with LRRK2 shown in Figure 7A <strong>Figure_7A_800_2_LRRK2pS935_CoIp.png</strong> Immunoprecipitated LRRK2pSer935 shown in Figure 7A <strong>Figure_7A_800_3_LRRK2pS935_Lysates.png</strong> Top to bottom 1: LRRK2 pSer935 blot shown in Figure 7A 2: higher contrast of Rab10 pThr73 blot shown in Figure 7A <strong>Figure_7A_800_4_Rab10pT73_Lysates.png</strong> Top to bottom 1: lower contrast of LRRK2 pSer935 blot shown in Figure 7A 2: Rab10 pThr73 blot shown in Figure 7A <strong>Figure_7A_800_5_14-3-3_Lysates.png</strong> 14-3-3 blot shown in Figure 7A (Input for the CoIp) <strong>Figure_7A_merge-700_800_LRRK2_total_LRRK2pS935.png</strong> Merged picture (in red LRRK2 total and in green LRRK2pS935) shown in Figure 7A <strong>Figure 7B: </strong>LSM710 laser scanning confocal microscope (Carl Zeiss) output files .lsm used to prepare Figure 7B <strong>Figure_7B_LRRK2_HA-empty_DMSO.lsm</strong> <strong>Figure_7B_LRRK2_HA-empty_GSK3357679A.lsm</strong> <strong>Figure_7B_LRRK2_HA-empty_GZD-824.lsm</strong> <strong>Figure_7B_LRRK2_HA-empty_MLi-2.lsm</strong>