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...
When functioning properly, the immune system protects the body from foreign invaders with the help of T‐cells. An error in the formation of T‐cells in the thymus can lead to autoimmune diseases in which the T‐cells attack normal body tissues. When a T‐cell binds to an antigen, a signal transduction pathway is activated, forming a multimeric signaling complex known as the TCR signalosome. LCK, part of the signalosome, is a lymphocyte‐specific protein tyrosine kinase that phosphorylates a variety of proteins in order to activate the T‐cell receptor pathway. TCR stimulation also leads to the activation of LYP, a lymphoid tyrosine phosphatase that down regulates TCR signaling by removing phosphates from the signaling intermediates of the TCR signalosome. LYP also dephosphorylates Y394 of LCK, thus inactivating the kinase and inhibiting TCR signaling. The LYP R620W mutation prevents LYP from binding to the signalosome, resulting in a gain‐of‐function, leading to increased inhibition of TCR signaling. The LYP R620W mutation has been linked to a wide spectrum of human diseases, including a decreased risk in Crohn’s disease and increased risk to rheumatoid arthritis, and possible links to non‐autoimmune disorders such as cardiovascular disease. The Marshfield SMART (Students Modeling A Research Topic) Team modeled the mutated protein LYP R620W using 3D technology. Grant Funding Source: Supported by grants from NIH‐CTSA and NIH‐SEPA.
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