Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.
Induced pluripotent stem cells (iPSCs) are being developed as a source for autologous cell therapies, many of which aim to treat aged patients1–5. To explore the impact of age on iPSC quality, we produced iPSCs from blood cells of 16 donors aged 21–100. We find that while reprogramming resets most of the epigenome, iPSCs retain an epigenetic signature of age that diminishes with passaging. Reprogramming via clonal expansion also exposes somatic mutations present in individual donor cells, which are missed by other methods. We find that exomic mutations in iPSCs increase linearly with age and each iPSC line analyzed carries at least one gene-disrupting mutation, of which several have previously been linked to cancer or dysfunction. Unexpectedly, elderly donors (>90 yrs) harbor fewer mutations than predicted and their distribution suggests that blood in elderly donors derives from a contracted progenitor pool. These studies show that harnessing clonal expansion during reprogramming can uncover age-associated processes relevant to the clinical use of iPSCs.
SUMMARY The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ~10%−15% of disease in non-African populations. The ~60 kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Here, we produce induced pluripotent stem cells (iPSCs) from risk and non-risk individuals, delete each haplo-type using genome editing, and generate vascular smooth muscle cells (VSMCs). Risk VSMCs exhibit globally altered transcriptional networks that intersect with previously identified CAD risk genes and pathways, concomitant with aberrant adhesion, contraction, and proliferation. Unexpectedly, deleting the risk haplotype rescues VSMC stability, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This study shows that the risk haplotype selectively predisposes VSMCs to adopt a cell state associated with CAD phenotypes, defines new VSMC-based networks of CAD risk genes, and es-tablishes haplotype-edited iPSCs as powerful tools for functionally annotating the human genome.
The Huntington's disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htt’s ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.
Huntingtin is a completely soluble 3,144 amino acid (aa) protein characterized by the presence of an amino-terminal polymorphic polyglutamine (polyQ) tract, whose aberrant expansion causes the progressively neurodegenerative Huntington's disease (HD). Biological evidence indicates that huntingtin (htt) is beneficial to cells (particularly to brain neurons) and that loss of its neuronal function may contribute to HD. The exact protein domains involved in its neuroprotective function are unknown. Evolutionary analyses of htt primary aa have so far been limited to a few species, but its thorough assessment may help to clarify the functions emerging during evolution. We made an extensive comparative analysis of the available htt protein homologues from different organisms along the metazoan phylogenetic tree and defined the presence of 3 different conservative blocks corresponding to human htt aa 1-386 (htt1), 683-1,586 (htt2), and 2,437-3,078 (htt3), in which HEAT (Huntingtin, Elongator factor3, the regulatory A subunit of protein phosphatase 2A, and TOR1) repeats are well conserved. We also describe the cloning and sequencing of sea urchin htt mRNA, the oldest deuterostome homologue so far available. Multiple alignment shows the first appearance of a primitive polyQ in sea urchin, which predates an ancestral polyQ sequence in a nonchordate environment and defines the polyQ characteristic as being typical of the deuterostome branch. The fact that glutamines have conserved positions in deuterostomes and the polyQ size increases during evolution suggests that the protein has a possibly Q-dependent role. Finally, we report an evident relaxing constraint of the N-terminal block in Ciona and drosophilids that correlates with the absence of polyQ and which may indicate that the N-terminal portion of htt has evolved different functions in Ciona and protostomes.
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