A mitochondrial blood-based patient stratification candidate biomarker for Parkinson’s disease

Rui, Qi; Sammler, Esther; Gonzalez‐Hunt, Claudia P.; Pena, Nicholas; Rouanet, Jeremy P.; Goodson, Steven D.; Fuzzati, Marie; Blandini, Fabio; Erickson, Kirk I.; Weinstein, Andrea M.; Padmanabhan, Shalini; Tonelli, Francesca; Alessi, Dario R.; Shiva, Sruti; Sanders, Laurie H.

doi:10.1101/2022.02.07.479309

Cited by 4 publications

(7 citation statements)

References 78 publications

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“…Yet, only partial inhibition of LRRK2 kinase activity could be sustained without lung pathology in monkeys [22]. Therefore, future preclinical and clinical studies may consider evaluating these multiple biomarkers, and levels of mtDNA damage in human blood hold promise for guiding this balancing act of efficacy and minimizing safety risk [62].…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…DNA damage in the mitochondrial genome was measured utilizing Mito DNA DX [62], currently the most robust way of measuring damage in mtDNA [66]. The assay to calculate mitochondrial DNA lesion frequency was performed as previously described [62]. Briefly, 15 ng of DNA was used to amplify long or short amplicons of the mitochondrial genome (as determined by primer sets).…”

Section: Methodsmentioning

confidence: 99%

“…The amount of amplification is directly proportional to the number of undamaged DNA templates. Average lesion frequency is calculated as described [62]. Results are then normalized and presented as lesions per 10 kb, with the values in experimental samples relative to control samples.…”

Section: Pcr-based Mitochondrial Dna Damage Assaymentioning

confidence: 99%

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G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pena

Gonzalez‐Hunt

Rui

et al. 2022

Preprint

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Pathogenic mutations in LRRK2 cause Parkinson's disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a nonselective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, a novel system that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. Precision medicine is a common approach in modern day cancer research that is not yet routinely applied to neurodegenerative diseases. Abrogation of mtDNA damage with mutant selective tool inhibitor EB-42168 demonstrates the promise of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Pcr-based Mitochondrial Dna Damage Assaymentioning

confidence: 99%

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G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pena

Gonzalez‐Hunt

Rui

et al. 2022

Preprint

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“…Recent studies indicate that mitocondrial DNA damage may be another potential biomarker for PD which is reversed by LRRK2 kinase inhibitors and detectable in LCLs and PBMCs, even though it does not correlate with increased pT73-Rab10 levels (77)(78)(79)(80).…”

Section: Discussionmentioning

confidence: 99%

A potential patient stratification biomarker for Parkinso’s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

Naaldijk

Fernández

Fasiczka

et al. 2023

Preprint

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Parkinson's disease (PD) is a common neurodegenerative movement disorder and leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for disease intervention. However, the ability to stratify patients who will benefit from such treatment modalities based on shared etiology is critical for the success of disease-modifying therapies. Ciliary and centrosomal alterations are commonly associated with pathogenic LRRK2 kinase activity and can be detected in many cell types. We previously found centrosomal deficits in immortalized lymphocytes from G2019S-LRRK2 PD patients. Here, to investigate whether such deficits may serve as a potential blood biomarker for PD which is susceptible to LRKK2 inhibitor treatment, we characterized patient-derived cells from distinct PD cohorts. We report centrosomal alterations in peripheral cells from a subset of early-stage idiopathic PD patients which is mitigated by LRRK2 kinase inhibition, supporting a role for aberrant LRRK2 activity in idiopathic PD. Centrosomal defects are detected in R1441G-LRRK2 and G2019S-LRRK2 PD patients and in non-manifesting LRRK2 mutation carriers, indicating that they acumulate prior to a clinical PD diagnosis. They are present in immortalized cells as well as in primary lymphocytes from peripheral blood. These findings indicate that analysis of centrosomal defects as a blood-based patient stratification biomarker may help nominate PD patients who will benefit from LRRK2-related therapeutics.

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Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease

Sastre,

Zafar,

Torres

et al. 2023

Sci Rep

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Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.

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A mitochondrial blood-based patient stratification candidate biomarker for Parkinson’s disease

Cited by 4 publications

References 78 publications

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

A potential patient stratification biomarker for Parkinso’s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease

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