Gaynor A. Smith

Successful Function of Autologous iPSC-Derived Dopamine Neurons following Transplantation in a Non-Human Primate Model of Parkinson’s Disease

Autologous transplantation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons is a potential clinical approach for treatment of neurological disease. Preclinical demonstration of long-term efficacy, feasibility, and safety of iPSC-derived dopamine neurons in non-human primate models will be an important step in clinical development of cell therapy. Here, we analyzed cynomolgus monkey (CM) iPSC-derived midbrain dopamine neurons for up to 2 years following autologous transplantation in a Parkinsons disease (PD) model. In one animal, with the most successful protocol, we found that unilateral engraftment of CM-iPSCs could provide a gradual onset of functional motor improvement contralateral to the side of dopamine neuron transplantation, and increased motor activity, without a need for immunosuppression. Postmortem analyses demonstrated robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the transplanted putamen. Our proof of concept findings support further development of autologous iPSC-derived cell transplantation for treatment of PD.

Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons

The main motor symptoms of Parkinsons disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinsons disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs‐derived midbrain‐type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non‐human primate iPSCs. The use of non‐human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell‐derived DA neurons. The aim of this study was to improve the safety of human‐ and non‐human primate iPSC (PiPSC)‐derived DA neurons. According to our results, NCAM+/CD29low sorting enriched VM DA neurons from pluripotent stem cell‐derived neural cell populations. NCAM+/CD29low DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC‐derived NCAM+/CD29low DA neurons were able to restore motor function of 6‐hydroxydopamine (6‐OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC‐derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH‐positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC‐derived cell transplantation therapies in the future. STEM Cells 2013;31:1548–1562

Unilateral nigrostriatal 6-hydroxydopamine lesions in mice I: Motor impairments identify extent of dopamine depletion at three different lesion sites

The unilateral 6-hydroxydopamine mouse lesion models of Parkinsons disease have received increasing attention in recent years, but comparison of the different lesion models was largely focused at a histological level. An extensive behavioural comparison between different mouse models on tests of motor function has yet to be carried out, to pin point tests that accurately discriminate between different extents of dopaminergic depletion. In the present study we examine the consequences of injection of the toxin at three sites along the nigrostriatal tract (substantia nigra, medial forebrain bundle, and striatum) on a broad range of simple motor tasks, and on the dopaminergic pathology. All lesion groups demonstrated marked behavioural deficits and displayed distinct profiles of degeneration along the nigrostriatal dopamine pathway. Tests that correlated closely with the level of substantia nigra cell loss included the corridor, cylinder and balance beam tests, the rotarod, inverted cage lid and three types of rotational assessment (spontaneous, amphetamine-induced and apomorphine-induced). Specific tasks are identified which are capable of distinguishing a near-complete lesion, with amphetamine rotation, corridor and cylinder tests showing the highest correlations with levels of nigral cell loss. Performance in the different behavioural tests was associated with distinct profiles of cell loss in the SN and VTA. We provide a comprehensive behavioural assessment of lesion-induced deficits in mouse models of PD, which should facilitate selection of the most appropriate lesion model and most sensitive behavioural tests for use in future studies investigating therapeutic interventions.

Unilateral nigrostriatal 6-hydroxydopamine lesions in mice II: Predicting L-DOPA-induced dyskinesia

In the 6-hydroxydopamine (6-OHDA) lesioned rodent the location of the lesion produces significantly different behavioural phenotypes, responses to the dopamine precursor l-3,4-dihydroxyphenylalanine (l-DOPA) and neuropathology. Lesion extent is commonly determined by a series of motor tests, but whether any of these tests have a relationship to the development and predictability of dyskinesia is unknown. We used mice with 6-OHDA lesions of the striatum, medial forebrain bundle and substantia nigra to examine the relationship between a range of tests used to determine motor function in the absence of l-DOPA: rotarod, cylinder, corridor, the balance beam, locomotor activity, psycho-stimulant and spontaneous rotational behaviour. The mice were subsequently treated with l-DOPA in progressively increasing doses and the development of l-DOPA-induced dyskinesia assessed. Most of these tests predict dopamine depletion but only rotarod, spontaneous rotations, apomorphine-induced rotations and locomotor activities were significantly correlated with the development of dyskinesia at 6mg/kg and 25mg/kg l-DOPA. The losses of dopaminergic neurons and serotonergic density in the ventral and dorsal striatum were dependent upon lesion type and were also correlated with l-DOPA-induced dyskinesia. The expression of FosB/ΔFosB was differentially affected in the striatum and nucleus accumbens regions in dyskinetic mice according to lesion type.

Progressive decline of glucocerebrosidase in aging and Parkinson's disease

The principal risk factor for developing most adult onset neurodegenerative diseases is aging, with incidence rising significantly after age 50. Despite research efforts, the causes of Parkinsons disease (PD) remain unknown. As neurons age, they show signs of diminished lysosomal and mitochondrial function, including increased oxidative stress and accumulation of misfolded proteins, and these changes become exacerbated PD. We show that activity of the lysosomal hydrolase glucocerebrosidase gradually diminishes with age in the substantia nigra and putamen of healthy controls. This reduction is comparable to glucocerebrosidase activity in GBA1‐mutation carrier PD patients. These data, demonstrate for the first time that an age‐dependent reduction in glucocerebrosidase activity may lower the threshold for developing PD.

Glucocerebrosidase gene therapy prevents α-synucleinopathy of midbrain dopamine neurons

Diminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinsons disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6 months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein.

Progressive axonal transport and synaptic protein changes correlate with behavioral and neuropathological abnormalities in the heterozygous Q175 KI mouse model of Huntington's disease

A long-term goal of modeling Huntingtons disease (HD) is to recapitulate the cardinal features of the disease in mice that express both mutant and wild-type (WT) huntingtin (Htt), as HD commonly manifests as a heterozygous condition in humans, and loss of WT Htt is associated with loss-of-function. In a new heterozygous Q175 knock-in (KI) mouse model, we performed an extensive evaluation of motor and cognitive functional deficits, neuropathological and biochemical changes and levels of proteins involved in synaptic function, the cytoskeleton and axonal transport, at 1-16 months of age. Motor deficits were apparent at 6 months of age in Q175 KI mice and at that time, postmortem striatal gamma-aminobutyric acid (GABA) levels were elevated and mutant Htt inclusions were present throughout the brain. From 6 months of age, levels of proteins associated with synaptic function, including SNAP-25, Rab3A and PSD-95, and with axonal transport and microtubules, including KIF3A, dynein and dynactin, were altered in the striatum, motor cortex, prefrontal cortex and hippocampus of Q175 KI mice, compared with WT levels. At 12-16 months of age, Q175 KI mice displayed motor and cognitive deficits, which were paralleled at postmortem by striatal atrophy, cortical thinning, degeneration of medium spiny neurons, dense mutant Htt inclusion formation, decreased striatal dopamine levels and loss of striatal brain-derived neurotrophic factor (BDNF). Data from this study indicate that the heterozygous Q175 KI mouse represents a realistic model for HD and also provides new insights into the specific and progressive synaptic, cytoskeletal and axonal transport protein abnormalities that may accompany the disease.

Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection

Adeno-associated viral (AAV) gene transfer holds great promise for treating a wide-range of neurodegenerative disorders. The AAV9 serotype crosses the blood-brain barrier and shows enhanced transduction efficiency compared to other serotypes, thus offering advantageous targeting when global transgene expression is required. Neonatal intravenous or intracerebroventricular (i.c.v.) delivery of recombinant AAV9 (rAAV9) have recently proven effective for modeling and treating several rodent models of neurodegenerative disease, however, the technique is associated with variable cellular tropism, making tailored gene transfer a challenge. In the current study, we employ the human synapsin 1 (hSYN1) gene promoter to drive neuron-specific expression of green fluorescent protein (GFP) after neonatal i.c.v. injection of rAAV9 in mice. We observed widespread GFP expression in neurons throughout the brain, spinal cord, and peripheral nerves and ganglia at 6 weeks-of-age. Region-specific quantification of GFP expression showed high neuronal transduction rates in substantia nigra pars reticulata (43.9±5.4%), motor cortex (43.5±3.3%), hippocampus (43.1±2.7%), cerebellum (29.6±2.3%), cervical spinal cord (24.9±3.9%), and ventromedial striatum (16.9±4.3%), among others. We found that 14.6±2.2% of neuromuscular junctions innervating the gastrocnemius muscle displayed GFP immunoreactivity. GFP expression was identified in several neuronal sub-types, including nigral tyrosine hydroxylase (TH)-positive dopaminergic cells, striatal dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32)-positive neurons, and choline acetyltransferase (ChAT)-positive motor neurons. These results build on contemporary gene transfer techniques, demonstrating that the hSYN1 promoter can be used with rAAV9 to drive robust neuron-specific transgene expression throughout the nervous system.

Sustained Systemic Glucocerebrosidase Inhibition Induces Brain α-Synuclein Aggregation, Microglia and Complement C1q Activation in Mice

Abstract Aims: Loss-of-function mutations in GBA1, which cause the autosomal recessive lysosomal storage disease, Gaucher disease (GD), are also a key genetic risk factor for the α-synucleinopathies, including Parkinsons disease (PD) and dementia with Lewy bodies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase and reductions in this enzyme result in the accumulation of the glycolipid substrates glucosylceramide and glucosylsphingosine. Deficits in autophagy and lysosomal degradation pathways likely contribute to the pathological accumulation of α-synuclein in PD. In this report we used conduritol-β-epoxide (CBE), a potent selective irreversible competitive inhibitor of glucocerebrosidase, to model reduced glucocerebrosidase activity in vivo, and tested whether sustained glucocerebrosidase inhibition in mice could induce neuropathological abnormalities including α-synucleinopathy, and neurodegeneration. Results: Our data demonstrate that daily systemic CBE treatment over 28 days caused accumulation of insoluble α-synuclein aggregates in the substantia nigra, and altered levels of proteins involved in the autophagy lysosomal system. These neuropathological changes were paralleled by widespread neuroinflammation, upregulation of complement C1q, abnormalities in synaptic, axonal transport and cytoskeletal proteins, and neurodegeneration. Innovation: A reduction in brain GCase activity has been linked to sporadic PD and normal aging, and may contribute to the susceptibility of vulnerable neurons to degeneration. This report demonstrates that systemic reduction of GCase activity using chemical inhibition, leads to neuropathological changes in the brain reminiscent of α-synucleinopathy. Conclusions: These data reveal a link between reduced glucocerebrosidase and the development of α-synucleinopathy and pathophysiological abnormalities in mice, and support the development of GCase therapeutics to reduce α-synucleinopathy in PD and related disorders. Antioxid. Redox Signal. 23, 550–564.

Enhanced ubiquitin-dependent degradation by Nedd4 protects against α-synuclein accumulation and toxicity in animal models of Parkinson's disease

Parkinsons disease is a neurodegenerative disorder, characterized by accumulation and misfolding of α-synuclein. Although the level of α-synuclein in neurons is fundamentally linked to the onset of neurodegeneration, multiple pathways have been implicated in its degradation, and it remains unclear which are the critical ubiquitination enzymes that protect against α-synuclein accumulation in vivo. The ubiquitin ligase Nedd4 targets α-synuclein to the endosomal–lysosomal pathway in cultured cells. Here we asked whether Nedd4-mediated degradation protects against α-synuclein-induced toxicity in the Drosophila and rodent models of Parkinsons disease. We show that overexpression of Nedd4 can rescue the degenerative phenotype from ectopic expression of α-synuclein in the Drosophila eye. Overexpressed Nedd4 in the Drosophila brain prevented the α-synuclein-induced locomotor defect whereas reduction in endogenous Nedd4 by RNAi led to worsening motor function and increased loss of dopaminergic neurons. Accordingly, AAV-mediated expression of wild-type but not the catalytically inactive Nedd4 decreased the α-synuclein-induced dopaminergic cell loss in the rat substantia nigra and reduced α-synuclein accumulation. Collectively, our data in two evolutionarily distant model organisms strongly suggest that Nedd4 is a modifier of α-synuclein pathobiology and thus a potential target for neuroprotective therapies.