Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells.

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.All authors were supported by EU FP7 grant DDPDGENES. S.L. was supported by European Research Council grant 261063 (BRAINCELL), Knut and Alice Wallenberg Foundation grant 2015.0041, Swedish Research Council (STARGET), and the Swedish Foundation for Strategic Research (RIF14-0057). A.Z. was supported by the Human Frontier Science Program. E.A. was supported by Swedish Research Council (VR projects: 2011-3116 and 2011-3318), Swedish Foundation for Strategic Research (SRL program), and Karolinska Institutet (SFO Thematic Center in Stem cells and Regenerative Medicine). E.A. and R.A.B. were supported by the EU FP7 grant NeuroStemcellRepair. R.A.B. was also supported by an NIHR Biomedical Research Centre award to the University of Cambridge/Addenbrookes Hospital. iCell dopaminergic neurons were a generous gift from Cellular Dynamics International. Single-cell RNA-seq servic0es were provided by the Eukaryotic Single-cell Genomics facility and the National Genomics Infrastructure at Science for Life Laboratory.This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.cell.2016.09.02

Gene Expression Profiling of Two Distinct Neuronal Populations in the Rodent Spinal Cord

BACKGROUND: In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. METHODOLOGY/PRINCIPAL FINDINGS: We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50-250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. CONCLUSIONS/SIGNIFICANCE: We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord

Genetic identification of brain cell types underlying schizophrenia

With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. Applying knowledge of the cellular taxonomy of the brain from single-cell RNA-sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. We found that the common variant genomic results consistently mapped to pyramidal cells, medium spiny neurons, and certain interneurons but far less consistently to embryonic, progenitor, or glial cells. These enrichments were due to sets of genes specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (synaptic genes, FMRP interactors, antipsychotic targets, etc.) generally implicate the same brain cell types. Our results suggest a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. The genetic risk associated with medium spiny neurons did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia

Gene expresion in rodent spinal neuronal populations and their response to injury

Motor neurons are the centre of convergence for all neural activity relating to movements. The activity integrated in the motor neurons is transmitted to appropriate muscles generating coordinated muscle contractions. Motor neurons, long considered passive integrators of the motor signal, have been shown to actively participate in shaping the output to the muscles during different behaviors, where active synaptic components resulting in plateau potentials and persistent inward currents can be activated during motor neuron recruitment. In the present thesis the functional significance of motor neurons during normal and injury states have been examined using a combination of electrophysiology and gene expression profiling. First the transmitter phenotype of motor neurons was examined. Motor neurons have long been thought to release only acetylcholine at their terminals thus following the central dogma proposed by Dale, stating that a neuron releases the same neurotransmitter from all its terminals. We find that motor neurons release not only acetylcholine but also glutamate at central synapses, whereas we did not discover any sign of glutamate release at the neuromuscular junction. This finding jeopardizes the central dogma, indicating a new level of possible modulation by motor neurons in shaping the motor output through a differentiated release of two fast neurotransmitters at distinct axon terminals. To further elucidate the functional role of motor neurons in relation to other spinal neuronal populations, the expression profiles of motor neurons and descending commissural interneurons (dCIN) were compared. This task required development of a method, which can be used for reliable gene expression profiling with RNA extracted from as few as 50 fluorescently identified and laser dissected cells. Based on this methodology, we find 49 significantly differentially expressed genes that may relate to the functional differences between motor neurons and dCINs in transmitting and shaping the motor output. Our method was subsequently used to measure the transcriptional response of motor neurons following spinal cord injury. Injury causes long-term changes in spinal networks located caudal to the injury resulting in maladaptive pathophysiological states including spasticity. In normal animals the expression of plateau potentials caused by persistent inward calcium and sodium currents (PICs) is conditional and depends on the presence of monoamines released from descending pathways. Motor neurons therefore lose the ability to express plateaus immediately after a spinal cord injury as the descending fibers are severed. The ability of motor neurons to express PICs reappears after a few weeks and has been implicated in injury-induced spasticity. We use the expression profiles of motor neurons to examine the molecular underpinnings of this return of plateaus in the late phase of the injury response, 21 and 60 days post injury. We find that the ancillary subunits of the channel complexes conducting the PICs, rather than the pore forming subunits, are subject to extensive regulation. Genes coding for receptors and intracellular pathways relating to the expression of plateau potentials also undergo regulation. Lastly, we examined the general transcriptional response of motor neurons throughout the injury response; 0, 2, 7, 21 and 60 days post injury and the underlying regulatory control of gene expression. We find that motor neurons are involved in the general injury response with a transient up-regulation of inflammatory and immunologically related processes in the early phase, while developmental pathways are up-regulated in late phases of the injury response. Promoter analysis conducted on expression clusters revealed general targets of regulation for identified transcription factors that participate in the injury response of the motor neurons. We conclude that the motor neurons engage an extensive molecular machinery to regulate and modulate their electrophysiological properties as a response to injury. This suggests that electrophysiological properties are subject to dynamic regulation that also could be at play in normal states of the spinal cord, thus modulating the functional response of the motor neurons and shaping the motor output. Together, the results presented in this thesis have provided new knowledge about the normal function of motor neurons and a novel insight into the development of spasticity that can help define new therapies for spinal cord injury