Using Patient-Derived Induced Pluripotent Stem Cells to Identify Parkinson’s Disease-Relevant Phenotypes

Sison, Samantha L.; Vermilyea, Scott C.; Emborg, Marina E.; Ebert, Allison D.

doi:10.1007/s11910-018-0893-8

Cited by 39 publications

(37 citation statements)

References 119 publications

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“…As LRRK2 G2019S iPSC patient-derived dopaminergic neurons show changes in neurite arborization (reviewed by Sison et al 19 ), we evaluated the morphology of the differentiated cell lines. Midbrain dopaminergic neurons from both Cj-ESC and , targeted glycine (yellow), exon junctions (red) and amino acid substitutions compared to the human LRRK2 kinase amino acid sequence (human amino acids in purple below the marmoset residues).…”

Section: Morphology Of Differentiated Dopaminergic Marmoset Neuronsmentioning

confidence: 99%

In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson’s Disease in Common Marmosets

Vermilyea

Babinski

Tran

et al. 2020

Sci Rep

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Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common cause of Parkinson's disease (PD) 1 . Located in the kinase domain of LRRK2, G2019S is hypothesized to slow transition of the kinase from active to inactive forms, thus effectively increasing Vmax of the enzyme by ~2 fold. This increased kinase activity has been identified as the basis of many PD-associated dysregulated intracellular mechanisms 2-7 . Relative to other common PD-associated gene mutations (e.g. SNCA, Parkin, PINK1, GBA), LRRK2 G2019S has a relatively high penetrance 1,8,9 . The prevalence, penetrance, and sequence conservation of this mutation makes it an excellent target for genetic modeling of PD in a nonhuman species.Because of its genetic similarity to the human genome and shorter lifespan compared to rhesus monkeys 10 , the common marmoset (Callithrix jacchus) has emerged as a candidate nonhuman primate species for modeling age-related disorders, including PD. Genetic approaches for modeling PD (or other diseases) in monkeys has been achieved by introducing the mutant gene using viral vector technologies, via direct intracerebral delivery or microinjection delivery in the oocyte 11,12 . In both cases, the mutant protein is over-expressed at supraphysiological levels. In addition, intracerebral delivery requires brain surgery, and its effects are limited to the target region, which contradicts the current understanding of neurodegeneration as a multisystem disease.Cj-iPSC parental wild type and LRRK2 G2019S clones were fixed and immunostained for the dopaminergic and neuronal markers...

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Section: Morphology Of Differentiated Dopaminergic Marmoset Neuronsmentioning

confidence: 99%

In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson’s Disease in Common Marmosets

Vermilyea

Babinski

Tran

et al. 2020

Sci Rep

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“…Although it is known that death of dopaminergic neurons is the underlying cause of motor symptoms in PD, the exact biochemical processes driving this cell death remain elusive. Mounting evidence from human, animal and cell-based model systems have implicated mitochondrial dysfunction and increased oxidative stress as major drivers in the disease process [1][2][3][4]. Previous studies within our lab utilizing induced pluripotent stem cells (iPSCs) harboring the familial PD mutation G2019S in the leucine rich repeat kinase (LRRK) 2 gene have found pronounced mitochondrial and sirtuin dysfunction in differentiated dopaminergic neurons [4].…”

Section: Introductionmentioning

confidence: 99%

Nicotinamide mononucleotide treatment increases NAD+ levels in an iPSC Model of Parkinson’s Disease but does not impact sirtuin activity

Fulleylove-Krause¹,

Sison²,

Ebert³

2020

Preprint

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Objectives: Parkinson’s disease (PD) is a common neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. Although the underlying mechanisms of dopaminergic neuron loss is not fully understood, evidence suggests mitochondrial malfunction as a key contributor to disease pathogenesis. We previously found that human PD patient stem cell-derived dopaminergic neurons exhibit reduced nicotinamide adenine dinucleotide (NAD+) levels and reduce activity of sirtuins, a group of NAD+-dependent deacetylase enzymes that participate in the regulation of mitochondrial function, energy production, and cell survival. Thus, here we tested whether treatment of PD stem cell-derived dopaminergic neurons with nicotinamide mononucleotide (NMN), an NAD+ precursor, could increase NAD+ levels and improve sirtuin activity. Results: We treated PD iPSC-derived dopaminergic neurons with NMN and found that NAD+ levels did increase. The deacetylase activity of sirtuin (SIRT) 2 was improved with NMN treatment, but NMN had no impact on deacetylase activity of SIRT 1 or 3. These results suggest that NMN can restore NAD+ levels and SIRT 2 activity, but that additional mechanisms are involved SIRT 1 and 3 dysregulation in PD dopaminergic neurons.

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“…Wang et al, 2012). Accordingly, a number of different cell types, including fibroblasts and iPSC-derived neurons from PD patients harboring mutations in LRRK2 exhibit increased oxidative stress and reactive oxygen species and defects in mitochondrial network integrity (Sison et al, 2018;Smith et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

Weindel

Bell

Huntington

et al. 2019

Preprint

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Despite many connections between mutations in leucine-rich repeat kinase 2 (LRRK2) and susceptibility to mycobacterial infection, we know little about its function outside of the brain, where it is studied in the context of Parkinson's Disease (PD). Here, we report that LRRK2 controls peripheral macrophages and brain-resident glial cells' ability to respond to and express inflammatory molecules. LRRK2 KO macrophages express elevated basal levels of type I interferons, resulting from defective purine metabolism, mitochondrial damage, and engagement of mitochondrial DNA with the cGAS DNA sensing pathway. While LRRK2 KO mice can control Mycobacterium tuberculosis (Mtb) infection, they exhibit exacerbated lung inflammation and altered activation of glial cells in PD-relevant regions of the brain.These results directly implicate LRRK2 in peripheral immunity and support the "multiple-hit hypothesis" of neurodegenerative disease, whereby infection coupled with genetic defects in LRRK2 create an immune milieu that alters activation of glial cells and may trigger PD.

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Using Patient-Derived Induced Pluripotent Stem Cells to Identify Parkinson’s Disease-Relevant Phenotypes

Cited by 39 publications

References 119 publications

In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson’s Disease in Common Marmosets

In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson’s Disease in Common Marmosets

Nicotinamide mononucleotide treatment increases NAD+ levels in an iPSC Model of Parkinson’s Disease but does not impact sirtuin activity

LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

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