Illuminating Parkinson’s therapy with optogenetics

Abstract

Controlling transplanted dopamine neurons with light helps define their mechanism of action.

In the 1990s, transplants of fetal neural cells into the brain of Parkinson’s patients were shown to alleviate symptoms of the disease. These promising results were later replicated in animal disease models using grafts of dopamine neuron–like cells derived from human pluripotent stem cells. Parkinson’s is caused by the degeneration of midbrain dopamine neurons, which erodes the neural circuitry that coordinates movement, and the efficacy of cell therapy in humans and animals is generally believed to stem from an increased supply of dopamine to striatal γ-amino butyric acid (GABA) neurons. In fact, however, the mechanistic basis of this therapy has yet to be defined because suitable tools have been lacking. In this issue, Steinbeck et al.¹ apply optogenetics to study the function of transplanted dopamine neurons in a mouse model of Parkinson’s disease. This work demonstrates for the first time the utility of optogenetics for investigating how cell therapy promotes recovery in animal models of neurological disease.

In optogenetics, a light-sensitive molecule, such as the ion channel channelrhodopsin 2, is expressed in a cell or an organism in order to control a biological process with high temporal precision by applying light². The approach is particularly powerful for elucidating the function of neurons, which interact on a time scale of milliseconds. Neurons derived from human stem cells are often immature and heterogeneous, and only mature neurons expressing the optogenetic molecule are precisely controllable³. When the latter are integrated into a neural circuitry in vitro or in vivo, their electrochemical activity can be switched on and off at will, thereby modulating the behavior of downstream neurons⁴.

Previous efforts to define the mechanism of action of transplanted dopamine neurons usually involved destruction of the grafted cells using toxins. The problem with such approaches is that irreversible ablation of the grafted cells makes it difficult to discriminate between different possible activities of the cells, such as dopamine release, immunomodulation or secretion of paracrine factors, and to define cause-effect relationships. Steinbeck et al.¹ exploit optogenetics as an on-off switch for neuronal activity, including dopamine release, allowing this function of the cells to be tested independently of their other possible functions.

They begin by inserting halorhodopsin (HALO), an inhibitory chloride pump, into human embryonic stem cells (hESCs) by lentiviral infection and then differentiate the cells into dopamine neuron–like cells. Light activation of HALO triggers an influx of chloride ions and reduces neuronal activities, including transmitter release and synaptic transmission. The authors demonstrate precise regulation of neuronal activity in culture, as measured in the calcium response and especially in the dopamine release elicited by glutamate or depolarization. To determine whether the human neurons can be controlled after transplantation into Parkinson’s disease mice, they implant the cells in the striatum along with a fiber optic cannula next to the graft to deliver light.

Parkinson’s disease mice are generated by injecting a neurotoxin, 6-hydroxydopamine, into one side of the brain to destroy dopamine neurons. The animals usually turn to the side of the lesion upon stimulation with amphetamine and pick up food with the ipsilateral paw. Steinbeck et al.¹ find that when a HALO-expressing graft is deactivated by shining light on it, the original motor deficits that were cured by the graft immediately return. This result demonstrates that the correction of motor deficits by the grafted cells depends on their neuronal activity, including dopamine release. However, if the animals are first treated with apomorphine, which activates dopamine receptors on striatal GABA neurons, bypassing the need for dopamine, optical deactivation of the grafted neurons no longer causes the symptoms to reappear. This result provides strong evidence that the beneficial effects of the graft depend on its releasing dopamine.

One of the critical functions of the nigrastriatal dopamine pathway is to modulate the excitatory (glutamate) inputs from the cerebral cortex and thalamus. By electrophysiological recording from brain slices, the authors find that stimulation of neighboring tissues (corpus callosum) elicits dopamine release from the graft and excitatory postsynaptic potentials (EPSPs) in striatal GABA neurons, and that the latter can be blocked by an antagonist of D1 receptors, one subtype of the G protein–coupled receptors (GPCRs) that mediate the function of dopamine. Optogenetic silencing results in significant reversible reduction in evoked EPSP amplitudes, suggesting that the grafted neurons enhance EPSPs on host striatal GABA neurons through the activation of D1 receptors (Fig. 1). This finding is very interesting as it suggests that grafted dopamine neurons modulate synaptic transmission from cortical and thalamic glutamatergic neurons onto striatal GABA neurons, very much like what endogenous dopamine neurons do⁵.

Figure 1.

Dopamine neurons engineered to express HALO release dopamine, which binds to D1 receptors and regulates glutamatergic inputs to GABA neurons, thereby restoring the motor function of grafted mice. Activation of HALO by light inhibits the release of dopamine, causing the motor deficits to reappear.

By applying optogenetics, this study reveals quite directly how transplantation of dopamine neurons works to restore motor functions. Nevertheless, because the HALO transgene is expressed by all neuronal types under the synapsin promoter and the hESC-derived population may include neurons other than dopamine neurons, one cannot exclude the possibility of functional contribution by other neuronal cell types.

In future studies, the same framework could be applied to interrogate graft function and graft-to-host connectivity in many other neurological models of cell therapy. Equally exciting would be the study of hESC-derived neurons that express the excitatory channel channelrhodopsin 2, which could be used not only as a complementary tool for exploring graft-to-host connectivity but also as a means of regulating neuronal circuitries or restoring specific innervation pathways⁶.

Optogenetic control of transplanted human neurons may turn out to have clinical relevance. For example, in Parkinson’s disease therapy one can envision a switch that turns down dopamine neuron activity to prevent graft-induced dyskinesias or turns it up to enhance therapeutic benefits. However, such applications face various technological hurdles in the design of suitable lasers and implantable fiber optics. A more attractive approach may be drug-based regulation of transplanted cells with chemogenetic tools, such as the DREADD (designer receptors exclusively activated by designer drugs) system⁷. DREADDs are a family of engineered GPCRs that can precisely control three major GPCR signaling pathways (Gq, Gi and Gs). These receptors can be activated by the pharmacologically inert compound clozapine-N-oxide, an orally bioavailable drug that easily penetrates the blood-brain barrier, but not by acetylcholine, the ligand of the native receptor⁸. Human cells can be engineered to carry the excitatory (hM3Dq receptor)⁹ and/ or the inhibitory (hM4Di receptor)¹⁰ form so that cellular activity may be turned up or down remotely. Ultimately, equipping regenerative cell-based medicines with optical or chemogenetic switches may enable more subtle modulation of therapeutic activity than what is possible with pharmacological approaches.