Sally K. Mak

α-Synuclein Suppression by Targeted Small Interfering RNA in the Primate Substantia Nigra

The protein α-synuclein is involved in the pathogenesis of Parkinsons disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal α-synuclein burden. Here, feasibility and safety of α-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against α-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of α-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40–50% suppression of α-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in α-synuclein. Infusion with α-synuclein siRNA, while lowering α-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-α-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

Pathological Modifications of α-Synuclein in 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP)-Treated Squirrel Monkeys

&agr;-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic accumulation of &agr;-synuclein and protein modifications (i.e. nitration, phosphorylation, and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of &agr;-synuclein but not of &bgr;-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy also revealed large numbers of dystrophic axons labeled with &agr;-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) &agr;-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. After toxicant exposure, &agr;-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for &agr;-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce &agr;-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of &agr;-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.

Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage.

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinsons disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.

Higher Vulnerability and Stress Sensitivity of Neuronal Precursor Cells Carrying an Alpha-Synuclein Gene Triplication

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson’s disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this “stem cell pathology” could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.

Increased α-synuclein phosphorylation and nitration in the aging primate substantia nigra.

Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson’s disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes.

Decreased α-synuclein expression in the aging mouse substantia nigra

Because of its normal function in synaptic plasticity and pathologic involvement in age-related neurodegenerative diseases, the protein alpha-synuclein could play an important role in aging processes. Here we compared alpha-synuclein expression in the substantia nigra and other brain regions of young (2-month-old), middle-aged (10-month-old), and old (20-month-old) mice. Levels of nigral alpha-synuclein mRNA, as assessed by both in situ hybridization and qPCR, were high in young mice and progressively declined in middle-aged and old animals. This age-dependent mRNA loss was paralleled by a marked reduction of alpha-synuclein protein; immunoreactivity of midbrain sections stained with an anti-alpha-synuclein antibody was most robust in 2-month-old mice and weakest in 20-month-old animals. Lowering of nigral alpha-synuclein could not be explained by a loss of dopaminergic neurons and was relatively specific since no change in beta-synuclein mRNA and protein occurred with advancing age. Finally, age-related decreases in alpha-synuclein were widespread throughout the mouse brain, affecting other regions (e.g., hippocampus) besides the substantia nigra. The data suggest that loss of alpha-synuclein could contribute to or be a marker of synaptic dysfunction in the aging brain. They also emphasize important differences in alpha-synuclein expression between rodents and primates since earlier reports have shown a marked elevation of alpha-synuclein protein in the substantia nigra of older humans and non-human primates.

Mitochondrial Dysfunction in Skin Fibroblasts from a Parkinson's Disease Patient with an alpha-Synuclein Triplication

Mitochondrial dysfunction has been frequently implicated in the neurodegenerative process that underlies Parkinsons disease (PD), but the basis for this impairment is not fully understood. The goal of this study was to investigate the effects of α-synuclein (α-syn) gene multiplication on mitochondrial function in human tissue. To investigate this question, human fibroblasts were taken from a patient with parkinsonism carrying a triplication in the α-syn gene. Unexpectedly, the cells showed a significant decrease in cell growth compared to matched healthy controls. With regard to mitochondrial function, α-syn triplication fibroblasts exhibited a 39% decrease in ATP production, a 40% reduction in mitochondrial membrane potential, and a 49% reduction in complex I activity. Furthermore, they proved to be more sensitive to the effects of the nigrostrial toxicant paraquat compared to controls. Finally, siRNA knockdown of α-syn resulted in a partial rescue of mitochondrial impairment and reduction of paraquat-induced cell toxicity, suggesting that α-syn plays a causative role for mitochondrial dysfunction in these patient-derived peripheral skin fibroblasts.

Mitochondrial bioenergetics in stem cell models of neurodegeneration

5. SNCA-Tri NPCs are more sensitive to Apoptosis 1754 Parkinson disease (PD) is a multi-factorial progressive neuro-degenerative disorder affecting primarily dopaminergic neurons in the substantia nigra (1). Genetic predisposition, such as a triplication of the α-synuclein gene (SNCA-Tri), can accelerate disease progression and result in earlier onset of the disorder (2). Environmental factors, such as toxicants and free radical generators targeting cellular bioenergetics have been associated with PD epidemiology (3). Dopaminergic neurons are an extreme high energy demanding cell type , and mitochondria are essential for their function. Consequently, mitochondrial dysfunction and a higher susceptibility to programmed cell death of these neurons have been suggested to play a significant role in disease progression (4). Induced pluripotent stem cells (iPSCs) derived neuronal precursor cells (NPCs) generated from PD patients have opened new possibilities for in vitro modeling of PD disease mechanisms and progression (5). Neuronal degeneration of adult dopaminergic neurons is a primary target of research, but equally interesting are the stability and developmental potential of neuronal stem cells.

PD in a petri-dish: overcoming a major bottleneck in modeling Parkinson's disease

Malini Vangipuram1, Anne Huang1, Sally Mak1, Prachi Gujar2, Ha Nam Nguyen2, Alex de la Cruz2, Branden Cord2,4, Adrian Flierl1, Blake Byers2,5, Ramya Sundararajan1, James Byrne2, Robert Diaz1, Kehkooi Kee2, Chhavy Tep-Cullison2, Patrick Lee2, Smruti Phandis2, Loan Ngyuen1, Sam Kim1, Anne Chang6, Theo Palmer2,4, Bill Langston1, Renee Reijo Pera2,3, Birgitt Schüle1 1 Parkinsons Institute, Sunnyvale, CA; 2 Inst. Stem Cell Biology & Reg. Medicine, Stanford University; 3 Dept. Obstetrics & Gynecology, Stanford Univ.; 4 Dept. Neurosurgery, Stanford Univ.; 5 Dept. Bioengineering, Stanford Univ.; 6 Dept. Dermatology Stanford Univ. School of Medicine

High efficiency of neural induction in Parkinson's disease-specific induced pluripotent stem cells

1. Chung, S., et al., Stem Cells, 2006. 24(6): p. 1583-93. 2. Swistowski, A., et al., PLoS One, 2009. 4(7): p. e6233. 3. Kim, D.S., et al., Stem Cell Rev, 2010. 6(2): p. 270-81. 4. Morizane, A., et al., J Neurosci Res, 2011. 89(2): p. 117-26. ACKNOWLEDGMENTS Special thanks to all patients, family members, spouses, and healthy volunteers participating in this study and “putting their skin in the game”. Sponsors: California Institute of Regenerative Medicine, Parkinson Alliance, Blume foundation, Brin Wojcicki Foundation High Efficiency of Neural Induction in Parkinson’s Disease-Specific Induced Pluripotent Stem Cells