Joy A. Umbach

Directed differentiation of human induced pluripotent stem cells generates active motor neurons

The potential for directed differentiation of human‐induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS‐derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS‐derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS‐derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. STEM CELLS 2009;27:806–811

Presynaptic Dysfunction in Drosophila csp Mutants

Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.

Suppression Cloning of the cDNA for a Candidate Subunit of a Presynaptic Calcium Channel

A novel strategy, termed suppression cloning, was used to identify a 7.4 kb cDNA encoding a putative subunit of the calcium channels that regulate transmitter release at nerve endings of Torpedo californica. The 585 nt open reading frame of this cDNA encodes a polypeptide of about 21.7 kd that is essential for the expression in frog oocytes of omega-conotoxin-sensitive, dihydropyridine-resistant, calcium channels. Sequence analysis reveals that this protein is closely related to two cloned cysteine string proteins of undertermined function that were recently localized to Drosophila nerve terminals using monoclonal antibodies.

Functional Neuromuscular Junctions Formed by Embryonic Stem Cell-Derived Motor Neurons

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

Human embryonic stem cell-derived motor neurons expressing SOD1 mutants exhibit typical signs of motor neuron degeneration linked to ALS

SUMMARY Human embryonic stem cell (hESC)-derived neurons have the potential to model neurodegenerative disorders. Here, we demonstrate the expression of a mutant gene, superoxide dismutase 1(SOD1), linked to familial amyotrophic lateral sclerosis (ALS) in hESC-derived motor neurons. Green fluorescent protein (GFP) expression under the control of the HB9 enhancer was used to identify SOD1-transfected motor neurons that express human wild-type SOD1 or one of three different mutants (G93A, A4V and I113T) of SOD1. Neurons transfected with mutant SOD1 exhibited reduced cell survival and shortened axonal processes as compared with control-transfected cells, which could survive for 3 weeks or more. The results indicate that hESC-derived cell populations can be directed to express disease-relevant genes and to display characteristics of the disease-specific cell type. These genetically manipulated hESC-derived motor neurons can facilitate and advance the study of disease-specific cellular pathways, and serve as a model system to test new therapeutic approaches.

Nerve Growth Factor Signals via Preexisting TrkA Receptor Oligomers

Nerve growth factor (NGF) promotes neuronal survival and differentiation by activating TrkA receptors. Similar to other receptor tyrosine kinases, ligand-induced dimerization is thought to be required for TrkA receptor activation. To study this process, we expressed TrkA receptors in Xenopus laevis oocytes and analyzed their response to NGF by using a combination of functional, biochemical, and structural approaches. TrkA receptor protein was detected in the membrane fraction of oocytes injected with TrkA receptor cRNA, but not in uninjected or mock-injected oocytes. Application of NGF to TrkA receptor-expressing oocytes promoted tyrosine phosphorylation and activated an oscillating transmembrane inward current, indicating that the TrkA receptors were functional. Freeze-fracture electron microscopic analysis demonstrated novel transmembrane particles in the P-face (protoplasmic face) of oocytes injected with TrkA cRNA, but not in uninjected or mock injected oocytes. Incubating TrkA cRNA-injected oocytes with the transcriptional inhibitor actinomycin D did not prevent the appearance of these P-face particles or electrophysiological responses to NGF, demonstrating that they did not arise from de novo transcription of an endogenous Xenopus oocyte gene. The appearance of these particles in the plasma membrane correlated with responsiveness to NGF as detected by electrophysiological analysis and receptor phosphorylation, indicating that these novel P-face particles were TrkA receptors. The dimensions of these particles (8.6 x 10 nm) were too large to be accounted for by TrkA monomers, suggesting the formation of TrkA receptor oligomers. Application of NGF did not lead to a discernible change in the size or shape of these TrkA receptor particles during an active response. These results indicate that in Xenopus oocytes, NGF activates signaling via pre-formed TrkA receptor oligomers.

Lithium Ions Enhance Cysteine String Protein Gene Expression In Vivo and In Vitro

Abstract: Lithium is a well established pharmacotherapy for the treatment of recurrent manic‐depressive illness. However, the mechanism by which lithium exerts its therapeutic action remains elusive. Here we report that lithium at 1 mM significantly increased the expression of cysteine string proteins (CSPs) in a pheochromocytoma cell line (PC12 cells) differentiated by nerve growth factor. These cells concomitantly exhibited increased expression of CSPs in their cell bodies and boutons. Enhanced CSP expression was also observed in the brain of rats fed a lithium‐containing diet, which elevated serum lithium to a therapeutically relevant concentration of ∼1.0 mM. However, both in vitro and in vivo, the expression of another synaptic vesicle protein, synaptophysin, and the t‐SNARE, synaptosomal‐associated protein of 25 kDa (SNAP‐25), was not significantly altered by lithium. These observations indicate that lithium‐induced changes of CSP gene expression may contribute to the therapeutic efficacy of this monovalent cation.

Lithium ions modulate the expression of VMAT2 in rat brain

Recent work has indicated that lithium (at 1 mM, a concentration that is efficacious in the treatment of manic-depressive disorders) modulates the level of vesicular monoamine transporter 1 (VMAT1) mRNA in PC12 cells as a function of the differentiation status of these cells. To ascertain whether VMAT expression in neurons is sensitive to lithium, in vivo, rats were fed a lithium-supplemented diet for 21 days (which raised serum lithium to 0.98+/-0.1 mM). Northern analysis revealed an overall increase (199+/-27%) of the neuronal VMAT isoform (VMAT2) in rat brain after lithium. However, in situ hybridization analysis revealed regional differences in the effects of lithium. Thus, VMAT2 mRNA increased by 50-100% over control in the raphe nuclei, ventral tegmental area, and substantia nigra of rats fed the lithium diet. Concomitantly, VMAT2 mRNA declined by about 50% in the locus coeruleus. Because VMAT2 is expressed in neurons that are strongly implicated in regulating mood and behavior, these data support the hypothesis that alterations of VMAT2 expression contribute to the therapeutic effects of lithium in psychiatric disorders.

Convergent effects of lithium and valproate on the expression of proteins associated with large dense core vesicles in NGF-differentiated PC12 cells.

Lithium and valproate are chemically unrelated compounds that are used to treat manic-depressive illness. Previously, we reported that lithium ions upregulate genes encoding proteins primarily associated with large dense core vesicles (LDCV) in nerve growth factor (NGF)-differentiated PC12 cells, but not in undifferentiated PC12 cells. Moreover, lithium did not alter the expression of proteins associated with small-clear, synaptic-like vesicles (SSV) in these cells. Based on these observations, we investigated whether valproate had actions similar to those of lithium in PC12 cells. Thus, undifferentiated or NGF-differentiated PC12 cells were exposed to lithium (1 mM) or valproate (1 mM) for 48 h. Extracts from these cells were submitted to semiquantitative Northern and Western analyses. In NGF-differentiated cells, both agents increased the expression of proteins associated with LDCV, the vesicular monoamine transporter 1 (VMAT1), and cysteine string protein (CSP). These same treatments did not alter the expression of proteins primarily associated with SSV, the vesicular acetylcholine transporter (VAChT), and synaptophysin (SY). Furthermore, neither drug affected the expression of these proteins in undifferentiated cells. Interestingly, secretion of 3H-dopamine was increased in cells exhibiting the increase of VMAT1 and csp. Taken together, the convergent effects of these chemically diverse compounds suggest that altered dynamics of LDCV may play a vital role in the biochemical pathway, leading to the relief of the symptoms of manic depression.

Glia of the Cholinergic Electromotor Nucleus of Torpedo Are the Source of the cDNA Encoding a GAT‐1‐Like GABA Transporter

Abstract: A PCR‐based strategy was used to clone DNAs encoding Na+‐ and Cl−‐dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter‐1 (GAT‐1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT‐1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT‐1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.