This preprint is under consideration at Molecular Neurodegeneration. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Preclinical Modeling of Chronic Inhibition of the Parkinson’s Disease Associated Kinase LRRK2 Reveals Altered Function of the Endolysosomal System in Vivo
Mark R. Cookson
National Institute on Aging
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1058-3831
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson’s Disease (PD) and renders the encoded protein kinase hyperactive. To date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo.
Methods
We evaluated the utility of newly available phospho-antibodies for Rab substrates pT73 Rab10, pS106 Rab12, pT71 Rab29 and LRRK2 autophosphorylation to examine the pharmacodynamic response to acute treatment paradigms with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice to define the relative target engagement between brain and LRRK2-enriched peripheral tissues. The molecular effects of 10 days and 10 weeks of chronic in-diet dosing were also evaluated using TMTpro reagents for LC-MS/MS total and phospho- proteomics in brain and kidney tissues.
Results
We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and that we validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that was reverted by kinase inhibition.
Conclusions
This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.
Keywords
Parkinson’s Disease, LRRK2 chronic inhibition, proteomics, phospho-proteomics
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 25 Sep, 2020
No community comments so far
Editorial decision: Major revision
On 17 Nov, 2020
Review #4 received
Received 13 Nov, 2020
Review #2 received
Received 04 Nov, 2020
Review #3 received
Received 02 Nov, 2020
Reviewer #5 agreed
On 31 Oct, 2020
Reviewer #4 agreed
On 29 Oct, 2020
Reviewer #3 agreed
On 29 Oct, 2020
Reviewer #2 agreed
On 28 Oct, 2020
Review #1 received
Received 30 Sep, 2020
Reviewers invited
Invitations sent on 23 Sep, 2020
Reviewer #1 agreed
On 23 Sep, 2020
Editor assigned
On 21 Sep, 2020
Submission checks complete
On 20 Sep, 2020
Editor invited
On 20 Sep, 2020
First submitted
On 16 Sep, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
Learn more about our company.
This preprint is under consideration at Molecular Neurodegeneration. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Preclinical Modeling of Chronic Inhibition of the Parkinson’s Disease Associated Kinase LRRK2 Reveals Altered Function of the Endolysosomal System in Vivo
Mark R. Cookson
National Institute on Aging
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1058-3831
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 25 Sep, 2020
No community comments so far
Editorial decision: Major revision
On 17 Nov, 2020
Review #4 received
Received 13 Nov, 2020
Review #2 received
Received 04 Nov, 2020
Review #3 received
Received 02 Nov, 2020
Reviewer #5 agreed
On 31 Oct, 2020
Reviewer #4 agreed
On 29 Oct, 2020
Reviewer #3 agreed
On 29 Oct, 2020
Reviewer #2 agreed
On 28 Oct, 2020
Review #1 received
Received 30 Sep, 2020
Reviewers invited
Invitations sent on 23 Sep, 2020
Reviewer #1 agreed
On 23 Sep, 2020
Editor assigned
On 21 Sep, 2020
Submission checks complete
On 20 Sep, 2020
Editor invited
On 20 Sep, 2020
First submitted
On 16 Sep, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson’s Disease (PD) and renders the encoded protein kinase hyperactive. To date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo.
Methods
We evaluated the utility of newly available phospho-antibodies for Rab substrates pT73 Rab10, pS106 Rab12, pT71 Rab29 and LRRK2 autophosphorylation to examine the pharmacodynamic response to acute treatment paradigms with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice to define the relative target engagement between brain and LRRK2-enriched peripheral tissues. The molecular effects of 10 days and 10 weeks of chronic in-diet dosing were also evaluated using TMTpro reagents for LC-MS/MS total and phospho- proteomics in brain and kidney tissues.
Results
We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and that we validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that was reverted by kinase inhibition.
Conclusions
This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8