Degeneration of medium spiny GABA neurons in the basal ganglia underlies motor dysfunction in Huntington’s disease (HD) which presently lacks effective therapy. In this study, we have successfully directed human embryonic stem cells (hESCs) to enriched populations of DARPP32-expressing forebrain GABA neurons. Transplantation of these human forebrain GABA neurons and their progenitors, but not spinal GABA cells, into the striatum of quinolinic acid-lesioned mice results in generation of large populations of DARPP32+ GABA neurons, which project to the substantia nigra as well as receiving glutamatergic and dopaminergic inputs, corresponding to correction of motor deficits. This finding raises hopes for cell therapy for HD.

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-Dopa alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates (NHP), demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD 1 . Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues 1,2 . Generation of induced pluripotent stem cells *

Purpose of Review: Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting older individuals. The specific cause underlying dopaminergic (DA) neuron loss in the substantia nigra, a pathological hallmark of PD, remains elusive. Here we highlight peer-reviewed reports using induced pluripotent stem cells (iPSCs) to model PD in vitro and discuss the potential disease-relevant phenotypes that may lead to a better understanding of PD etiology. Benefits of iPSCs are that they retain the genetic background of the donor individual and can be differentiated into specialized neurons to facilitate disease modeling. Recent Findings: Mitochondrial dysfunction, oxidative stress, ER stress, and alpha-synuclein accumulation are common phenotypes observed in PD iPSC-derived neurons. New culturing technologies, such as directed reprogramming and midbrain organoids, offer innovative ways of investigating intraneuronal mechanisms of PD pathology. Summary: PD patient-derived iPSCs are an evolving resource to understand PD pathology and identify therapeutic targets.

The common marmoset monkey (Callithrix jacchus; Cj) is an advantageous nonhuman primate species for modeling age-related disorders, including Parkinson's disease, due to their shorter life span compared to macaques. Cj-derived induced pluripotent stem cells (Cj-iPSCs) from somatic cells are needed for in vitro disease modeling and testing regenerative medicine approaches. Here we report the development of a novel Cj-iPSC line derived from adult marmoset fibroblasts. The Cj-iPSCs showed potent pluripotency properties, including the development of mesodermal lineages in tumors after injection to immunocompromised mice, as well as ectoderm and endoderm lineages after in vitro differentiation regimens, demonstrating differentiated derivatives of all three embryonic layers. In addition, expression of key pluripotency genes (ZFP42, PODXL, DNMT3B, C-MYC, LIN28, KLF4, NANOG, SOX2, and OCT4) was observed. We then tested the neural differentiation capacity and gene expression profiles of Cj-iPSCs and a marmoset embryonic stem cell line (Cj-ESC) after dual-SMAD inhibition. Exposure to CHIR99021 and sonic hedgehog (SHH) for 12 and 16 days, respectively, patterned the cells toward a ventralized midbrain dopaminergic phenotype, confirmed by expression of FOXA2, OTX2, EN-1, and tyrosine hydroxylase. These results demonstrate that common marmoset stem cells will be able to serve as a platform for investigating regenerative medicine approaches targeting the dopaminergic system.

Accumulation of α-synuclein (α-syn) leading to the formation of insoluble intracellular aggregates named Lewy bodies is proposed to have a significant role in Parkinson’s disease (PD) pathology. Nonhuman primate (NHP) models of PD have proven essential for understanding the neurobiological basis of the disease and for the preclinical evaluation of first-in-class and invasive therapies. In addition to neurotoxin, aging and intracerebral gene transfer models, a new generation of models using inoculations of α-syn formulations, as well as transgenic methods is emerging. Understanding of their advantages and limitations will be essential when choosing a platform to evaluate α-syn-related pathology and interpreting the test results of new treatments targeting α-syn aggregation. In this review we aim to provide insight on this issue by critically analyzing the differences in endogenous α-syn, as well as α-syn pathology in PD and PD NHP models.

Induced pluripotent stem cell (iPSC)-derived neurons represent an opportunity for cell replacement strategies for neurodegenerative disorders such as Parkinson’s disease (PD). Improvement in cell graft targeting, distribution, and density can be key for disease modification. We have previously developed a trajectory guide system for real-time intraoperative magnetic resonance imaging (RT-IMRI) delivery of infusates, such as viral vector suspensions for gene therapy strategies. Intracerebral delivery of iPSC-derived neurons presents different challenges than viral vectors, including limited cell survival if cells are kept at room temperature for prolonged periods of time, precipitation and aggregation of cells in the cannula, and obstruction during injection, which must be solved for successful application of this delivery approach. To develop procedures suitable for RT-IMRI cell delivery, we first performed in vitro studies to tailor the delivery hardware (e.g., cannula) and defined a range of parameters to be applied (e.g., maximal time span allowable between cell loading in the system and intracerebral injection) to ensure cell survival. Then we performed an in vivo study to evaluate the feasibility of applying the system to nonhuman primates. Our results demonstrate that the RT-IMRI delivery system provides valuable guidance, monitoring, and visualization during intracerebral cell delivery that are compatible with cell survival.

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...

Abnormal intraneuronal accumulation of the presynaptic protein α-synuclein (α-syn) is implicated in the etiology of dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD). Recent work revealed that mice expressing human α-syn with the alanine-53-threonine (A53T) mutation have a similar phenotype to the human condition, exhibiting long-term potentiation deficits, learning and memory deficits, and inhibitory hippocampal remodeling, all of which were reversed by genetic ablation of microtubule-associated protein tau. Significantly, memory deficits were associated with histological signs of network hyperactivity/seizures. Electrophysiological abnormalities are often seen in parkinsonian dementias. Baseline electroencephalogram (EEG) slowing is used as a supportive diagnostic feature in DLB and PDD, and patients with these diseases may exhibit indicators of broad network dysfunction such as sleep dysregulation, myoclonus, and seizures. Given the translational significance, we examined whether human A53T α-syn expressing mice exhibit endogenous-tau-dependent EEG abnormalities, as measured with epidural electrodes over the frontal and parietal cortices. Using template-based waveform sorting, we determined that A53T mice have significantly high numbers of epileptiform events as early as 3-4 months of age and throughout life, and this effect is markedly attenuated in the absence of tau. Epileptic myoclonus occurred in half of A53T mice and was markedly reduced by tau ablation. In spectral analysis, tau ablation partially reduced EEG slowing in 6-7 month transgenic mice. We found abnormal sleeping patterns in transgenic mice that were more pronounced in older groups, but did not find evidence that this was influenced by tau genotype. Together, these data support the notion that tau facilitates A53T α-syn-induced hyperexcitability that both precedes and coincides with associated synaptic, cognitive, and behavioral effects. Tau also contributes to some aspects of EEG slowing in A53T mice. Importantly, our work supports tau-based approaches as an effective early intervention in α-synucleinopathies to treat aberrant network activity.