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.

Synaptic spines are dynamic structures that regulate neuronal responsiveness and plasticity. Here we describe a role for the schizophrenia risk factor, Disrupted-in-Schizophrenia 1 (DISC1), in the maintenance of spine morphology and function. We show that DISC1 anchors Kalirin-7 (Kal-7) thereby regulating access of Kal-7 to Rac1 and so controlling the duration and intensity of Rac1 activation in response to NMDA receptor activation in cortical culture as well as in vivo brain. This offers explanation for why Rac1 and its activator (Kal-7) serve as key mediators of spine enlargement and that constitutive Rac1 activation decreases spine size. This novel mechanism likely underlies disturbances in glutamatergic neurotransmission frequently reported in schizophrenia that can lead to alteration of dendritic spines with consequential major pathological changes in brain function. Furthermore, the concept of a “signalosome” involving disease-associated factors, such as DISC1 and glutamate, may well contribute to the multifactorial and polygenetic characteristics of schizophrenia.

Parkinson's disease (PD) is a common neurodegenerative disease caused by genetic and environmental factors. We analyzed induced pluripotent stem cell (iPSC)-derived neural cells from PD patients and presymptomatic individuals carrying mutations in the PINK1 and LRRK2 genes, and healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial function in iPSC-derived neural cells from PD patients and at-risk individuals could be rescued with coenzyme Q10, rapamycin or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insights into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in PD.

Activity-dependent rapid structural and functional modifications of central excitatory synapses contribute to synapse maturation, experience-dependent plasticity, and learning and memory and are associated with neurodevelopmental and psychiatric disorders. However, the signal transduction mechanisms that link glutamate receptor activation to intracellular effectors that accomplish structural and functional plasticity are not well understood. Here we report that NMDA receptor activation in pyramidal neurons causes CaMKII-dependent phosphorylation of the guanine-nucleotide exchange factor (GEF) kalirin-7 at residue threonine 95, regulating its GEF activity, leading to activation of small GTPase Rac1 and rapid enlargement of existing spines. Kalirin-7 also interacts with AMPA receptors and controls their synaptic expression. By demonstrating that kalirin expression and spine localization are required for activity-dependent spine enlargement and enhancement of AMPAR-mediated synaptic transmission, our study identifies a signaling pathway that controls structural and functional spine plasticity.

Dynamic remodeling of spiny synapses is crucial for cortical circuit development, refinement, and plasticity, while abnormal morphogenesis is associated with neuropsychiatric disorders. Here we show in cultured rat cortical neurons that activation of Epac2, a PKA-independent cAMP target and Rap guanine-nucleotide exchange factor (GEF), induces spine shrinkage, increases spine motility, removes synaptic GluR2/3-containing AMPA receptors, and depresses excitatory transmission, while its inhibition promotes spine enlargement and stabilization. Epac2 is required for dopamine D1-like receptor-dependent spine shrinkage and GluR2 removal from spines. Epac2 interaction with neuroligin promotes its membrane recruitment and enhances its GEF activity. Rare missense mutations in the EPAC2 gene, previously found in individuals with autism, affect basal and neuroligin-stimulated GEF activity, dendritic Rap signaling, synaptic protein distribution, and spine morphology. Thus, we identify a novel mechanism that promotes dynamic remodeling and depression of spiny synapses, whose mutations may contribute to some aspects of disease.