Andreas Heuer

Human ESC-Derived Dopamine Neurons Show Similar Preclinical Efficacy and Potency to Fetal Neurons when Grafted in a Rat Model of Parkinson’s Disease

Summary Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs). Before these cells can be used for cell replacement therapy in Parkinson’s disease (PD), it is important to verify their functional properties and efficacy in animal models. Here we provide a comprehensive preclinical assessment of hESC-derived midbrain dopamine neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons. Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for clinical translation of hESC-derived dopamine neurons using approaches similar to those established with fetal cells for the treatment of Parkinson’s disease.

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.

Activin A directs striatal projection neuron differentiation of human pluripotent stem cells

The efficient generation of striatal neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) is fundamental for realising their promise in disease modelling, pharmaceutical drug screening and cell therapy for Huntingtons disease. GABAergic medium-sized spiny neurons (MSNs) are the principal projection neurons of the striatum and specifically degenerate in the early phase of Huntingtons disease. Here we report that activin A induces lateral ganglionic eminence (LGE) characteristics in nascent neural progenitors derived from hESCs and hiPSCs in a sonic hedgehog-independent manner. Correct specification of striatal phenotype was further demonstrated by the induction of the striatal transcription factors CTIP2, GSX2 and FOXP2. Crucially, these human LGE progenitors readily differentiate into postmitotic neurons expressing the striatal projection neuron signature marker DARPP32, both in culture and following transplantation in the adult striatum in a rat model of Huntingtons disease. Activin-induced neurons also exhibit appropriate striatal-like electrophysiology in vitro. Together, our findings demonstrate a novel route for efficient differentiation of GABAergic striatal MSNs from human pluripotent stem cells. Highlighted article: Striatal projection neurons, which degenerate in Huntingtons disease, can be generated in vitro using activin A treatment, in a manner independent of sonic hedgehog signalling.

Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson’s Disease

Summary Stem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long-term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson’s disease through gene expression analysis of >30 batches of grafted human embryonic stem cell (hESC)-derived progenitors. We found that many of the commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a good manufacturing practice (GMP) differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.

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.

Derangement of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and extracellular signal-regulated kinase (ERK) dependent striatal plasticity in L-DOPA-induced dyskinesia.

BACKGROUND Bidirectional long-term plasticity at the corticostriatal synapse has been proposed as a central cellular mechanism governing dopamine-mediated behavioral adaptations in the basal ganglia system. Balanced activity of medium spiny neurons (MSNs) in the direct and the indirect pathways is essential for normal striatal function. This balance is disrupted in Parkinsons disease and in l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID), a common motor complication of current pharmacotherapy of Parkinsons disease. METHODS Electrophysiological recordings were performed in mouse cortico-striatal slice preparation. Synaptic plasticity, such as long-term potentiation (LTP) and depotentiation, was investigated. Specific pharmacological inhibitors or genetic manipulations were used to modulate the Ras-extracellular signal-regulated kinase (Ras-ERK) pathway, a signal transduction cascade implicated in behavioral plasticity, and synaptic activity in different subpopulations of striatal neurons was measured. RESULTS We found that the Ras-ERK pathway, is not only essential for long-term potentiation induced with a high frequency stimulation protocol (HFS-LTP) in the dorsal striatum, but also for its reversal, synaptic depotentiation. Ablation of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal activator of Ras proteins, causes a specific loss of HFS-LTP in the medium spiny neurons in the direct pathway without affecting LTP in the indirect pathway. Analysis of LTP in animals with unilateral 6-hydroxydopamine lesions (6-OHDA) rendered dyskinetic with chronic L-DOPA treatment reveals a complex, Ras-GRF1 and pathway-independent, apparently stochastic involvement of ERK. CONCLUSIONS These data not only demonstrate a central role for Ras-ERK signaling in striatal LTP, depotentiation, and LTP restored after L-DOPA treatment but also disclose multifaceted synaptic adaptations occurring in response to dopaminergic denervation and pulsatile administration of L-DOPA.

Selective cognitive impairment in the YAC128 Huntington's disease mouse

People with HD have a demonstrated early extra-dimensional set-shifting deficit. In the present study, we use a novel water T-maze set-shifting procedure and demonstrate its validity as a set-shifting task in a mouse model of Huntingtons disease. Three groups of YAC128 mice of different ages (27, 69 and 117 weeks) were run on the task, which incorporated six distinct stages in which the mice must learn a rule and then switch to a different rule. The six stages were: directional learning, directional learning reversal, light discrimination, light discrimination reversal, return to place learning and a maze rotation spatial learning test. Rule changes from place learning to light discrimination and back constitute extra-dimensional shifts. The results of the study demonstrate robust light/dark discrimination reversal learning deficits in transgenic mice from 27 weeks of age, and a directional learning to light discrimination extra-dimensional set-shifting deficit from 69 weeks of age. The extra-dimensional shift deficit was confirmed with control trials demonstrating the validity of the deficit and the task. The onset of reversal learning and extra-dimensional shift deficits corresponded with the development of mutant huntingtin N-terminal fragment aggregates in neurons of relevant forebrain regions.

Monosynaptic Tracing using Modified Rabies Virus Reveals Early and Extensive Circuit Integration of Human Embryonic Stem Cell-Derived Neurons.

Summary Human embryonic stem cell (hESC)-derived dopamine neurons are currently moving toward clinical use for Parkinson’s disease (PD). However, the timing and extent at which stem cell-derived neurons functionally integrate into existing host neural circuitry after transplantation remain largely unknown. In this study, we use modified rabies virus to trace afferent and efferent connectivity of transplanted hESC-derived neurons in a rat model of PD and report that grafted human neurons integrate into the host neural circuitry in an unexpectedly rapid and extensive manner. The pattern of connectivity resembled that of local endogenous neurons, while ectopic connections were not detected. Revealing circuit integration of human dopamine neurons substantiates their potential use in clinical trials. Additionally, our data present rabies-based tracing as a valuable and widely applicable tool for analyzing graft connectivity that can easily be adapted to analyze connectivity of a variety of different neuronal sources and subtypes in different disease models.

DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor

Summary Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson’s disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still unknown. Here we have used a new model where the activity of the transplanted DA neurons can be selectively modulated using a bimodal chemogenetic (DREADD) approach, allowing either enhancement or reduction of the therapeutic effect. We show that exclusive activation of a cAMP-linked (Gs-coupled) DREADD or serotonin 5-HT6 receptor, located on the grafted DA neurons, is sufficient to induce GIDs. These findings establish a mechanistic link between the 5-HT6 receptor, intracellular cAMP, and GIDs in transplanted PD patients. This effect is thought to be mediated through counteraction of the D2 autoreceptor feedback inhibition, resulting in a dysplastic DA release from the transplant.

Dopamine-rich grafts alleviate deficits in contralateral response space induced by extensive dopamine depletion in rats.

Unilateral infusion of 6-hydroxydopamine into the nigro-striatal pathway in the rat is the most common dopamine lesion model of Parkinsons disease. In the present study, we explore the impact of near complete unilateral loss of dopamine along the nigro-striatal pathway and subsequent cell replacement therapy in a choice reaction time task in rats, with assessment of spatial responding towards either side of the body (ipsilateral or contralateral to the lesion) on alternate days. Results indicated a stable contralateral deficit in response accuracy, reaction times and motor function for 50 consecutive days of testing, with no signs of recovery or compensation. All lesioned rats developed a near-hole bias and displayed prolonged movement and reaction times when responses had to be directed towards a distal response location on the side of the body contralateral to the lesion, as well as a smaller ipsilateral impairment in response accuracy and movement times. Grafts of dopamine-rich tissue into the denervated striatum improved some, but not all, of the deficits induced by the lesion. Specifically, grafted rats performed at a similar level to control animals when assessed on the ipsilateral side, they demonstrated a partial restitution of their ability to respond to far contralateral stimuli, and they exhibited a marked reduction in the time to complete all lateralised responses on both sides. The present characterisation of the task and the effects of cell replacement via primary fetal mesencephalic tissue demonstrate restorative properties in alleviating the marked spatial response bias induced by unilateral loss of dopamine.