SUMMARY Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons1 or midbrain dopamine (DA) neurons2 has been achieved. However, the effective use of hPSCs for cell therapy has lagged behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease (PD)3, 4, DA neurons from human PSCs generally display poor in vivo performance5. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth6, 7 Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in 11 days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD models using three host species. Long-term engraftment in 6-OHDA-lesioned mice and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell based therapies in PD.
Dopamine (DA) is important to a wide range of striatal functions, including associative learning and habit formation. At synapses between cortical pyramidal neurons and principal striatal medium spiny neurons (MSNs), postsynaptic D1 and D2 DA receptors are postulated to be necessary for the induction of long-term potentiation and depression, respectively. Because these receptors are restricted to two distinct MSN populations, this postulate demands that synaptic plasticity be unidirectional in each cell type. Using brain slices from DA receptor transgenic mice, we show that this is not the case. Rather, DA plays complementary roles in these two types of MSN to ensure that synaptic plasticity is bidirectional and Hebbian. In models of Parkinson's disease, this system is thrown out of balance, leading to unidirectional changes in plasticity that could underlie network pathology and symptoms.
The basal ganglia are a chain of subcortical nuclei that facilitate action selection. Striatal direct and indirect pathways form the functional backbone of the basal ganglia circuit. Twenty years ago it was proposed that the ability of the striatum to use the rise and fall of dopamine (DA) to control action selection was due to the segregation of D1 and D2 DA receptors in direct and indirect pathway spiny projection neurons. Although this sparked a debate, the evidence accumulated since clearly supports this model. In particular, recent advances in the means of marking neural circuits with optical or molecular reporters has revealed a clear-cut dichotomy between these two cell types at the molecular, anatomical and physiological levels. The contrast provided by these studies has provided new insights into how the striatum responds to fluctuations in DA signaling and how diseases that alter this signaling change striatal function.
Summary The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. We have now developed a new ‘BACarray’ methodology, based on affinity purification of polysomal mRNAs from genetically defined cell populations. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. Even two morphologically indistinguishable subclasses of MSNs display vastly different translational profiles. Striatopallidal neurons are characterized by a strong and cell-specific release of intracellular Ca2+ in response to sphingosine 1-phosphate, consistent with their selective expression of Gpr6. In contrast, striatonigral neurons demonstrate a selective cell-specific increase in GABAA receptor subunits in response to chronic cocaine treatment. BACarray translational profiling is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations.
Why dopamine-containing neurons of the brain's substantia nigra pars compacta die in Parkinson's disease has been an enduring mystery. Our studies suggest that the unusual reliance of these neurons on L-type Ca(v)1.3 Ca2+ channels to drive their maintained, rhythmic pacemaking renders them vulnerable to stressors thought to contribute to disease progression. The reliance on these channels increases with age, as juvenile dopamine-containing neurons in the substantia nigra pars compacta use pacemaking mechanisms common to neurons not affected in Parkinson's disease. These mechanisms remain latent in adulthood, and blocking Ca(v)1.3 Ca2+ channels in adult neurons induces a reversion to the juvenile form of pacemaking. Such blocking ('rejuvenation') protects these neurons in both in vitro and in vivo models of Parkinson's disease, pointing to a new strategy that could slow or stop the progression of the disease.
Intracellular α-synuclein (α-syn)-rich protein aggregates called Lewy pathology (LP) and neuronal death are commonly found in the brains of patients with clinical Parkinson disease (cPD). It is widely believed that LP appears early in the disease and spreads in synaptically coupled brain networks, driving neuronal dysfunction and death. However, post-mortem analysis of human brains and connectome-mapping studies show that the pattern of LP in cPD is not consistent with this simple model, arguing that, if LP propagates in cPD, it must be gated by cell- or region-autonomous mechanisms. Moreover, the correlation between LP and neuronal death is weak. In this Review, we briefly discuss the evidence for and against the spreading LP model, as well as evidence that cell-autonomous factors govern both α-syn pathology and neuronal death.
Parkinson's disease (PD) is a pervasive, aging-related neurodegenerative disease whose cardinal motor symptoms reflect the loss of a small group of neurons – dopaminergic neurons in the substantia nigra pars compacta (SNc)1. Mitochondrial oxidant stress is widely viewed as responsible for this loss2, but why these particular neurons should be stressed is a mystery. Using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, it was discovered that the unusual engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. This stress engaged defenses that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, antagonists of cloned uncoupling proteins. Knocking out DJ-1, a gene associated with an early onset form of PD, down-regulated the expression of two uncoupling proteins (UCP4, 5), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of PD.
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