A dominant mutation in a neuronal acetylcholine receptor subunit leads to motor neuron degeneration in Caenorhabditis elegans

Inappropriate or excessive activation of ionotropic receptors can have dramatic consequences for neuronal function and, in many instances, leads to cell death. In Caenorhabditis elegans, nicotinic acetylcholine receptor (nAChR) subunits are highly expressed in a neural circuit that controls movement. Here, we show that heteromeric nAChRs containing the acr-2 subunit are diffusely localized in the processes of excitatory motor neurons and act to modulate motor neuron activity. Excessive signaling through these receptors leads to cell-autonomous degeneration of cholinergic motor neurons and paralysis. C. elegans double mutants lacking calreticulin and calnexin-two genes previously implicated in the cellular events leading to necrotic-like cell death (Xu et al. 2001)-are resistant to nAChR-mediated toxicity and possess normal numbers of motor neuron cell bodies. Nonetheless, excess nAChR activation leads to progressive destabilization of the motor neuron processes and, ultimately, paralysis in these animals. Our results provide new evidence that chronic activation of ionotropic receptors can have devastating degenerative effects in neurons and reveal that ion channel-mediated toxicity may have distinct consequences in neuronal cell bodies and processes

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation

Giant panda fibroblasts, but not red panda or Asiatic lion fibroblasts, are able to generate m-SKPs after passage and cryopreservation.

<p>Fibroblast cultures from red panda (<b>a</b>) and Asiatic lion (<b>b</b>) were unable to generate m-SKPs. Female giant panda fibroblasts cultures were able to generate m-SKPs (<b>c</b>). Buccal mucosa cell cultures from the male giant panda could also generate m-SKPs (<b>d</b>) after cryopreservation of cells, m-SKPs generated from the male giant panda could be passaged to p1 (<b>e</b>) and p2 (<b>f</b>). Scale Bar = 100μm.</p

Monolayer fibroblasts cell cultures express markers associated with SKPs.

<p>DF cells isolated from skin biopsies from giant panda (<b>a</b>), red panda (<b>e</b>) and Asiatic lion (<b>i</b>) could be cultured as monolayers. Giant panda (<b>b-d</b>), red panda (<b>f-h</b>) and Asiatic lion (<b>j-l</b>) monolayer DF cultures all expressed vimentin, fibronectin and versican. Nuclei were stained with DAPI. Scale Bar = 100μm.</p

m-SKP yield does not correlate with α-SMA expression in red panda, Asiatic lion or giant panda buccal mucosa fibroblasts.

<p>Monolayer cultures of red panda (<b>a</b>) and Asiatic lion (<b>b</b>) dermal fibroblasts strongly expressed α-SMA, compared to monolayer cultures of giant panda buccal mucosa fibroblasts (<b>c</b>) where α-SMA expression was greatly reduced. Some, but not all giant panda SKP spheroids displayed α-SMA expression (<b>d</b>). Nuclei were stained with DAPI. Scale Bar = 100μm.</p

Giant panda buccal mucosa m-SKPs are able to differentiate along various cell lineages.

<p>Giant panda cells from passage 3 m-SKPs cultured in adipogenic and osteogenic differentiation media exhibited adipogenic differentiation (<b>a</b>) and osteogenic differentiation (<b>c</b>) as shown by oil Red-O and Von Kossa staining, respectively. Control cultures (<b>b</b>) and (<b>d</b>) did not stain positively for adipogenic or osteogenic differentiation, respectively. Giant panda m-SKPs cultured in neuronal differentiation media differentiated along neuronal lineages as shown by βIII tubulin immunostaining (<b>e</b>). βIII tubulin staining was negative in SKP cells cultured in control media (<b>f</b>). The Schwann cell marker S100β was up-regulated in cells cultured in Schwann cell differentiation media and cell morphology was characteristic of Schwann cells (<b>g</b>) when compared to cells cultured in control media (<b>h</b>). Nuclei were stained with DAPI in (<b>e-h</b>). Scale Bar = 100μm.</p

Expression of SKP protein markers is maintained and ABCG2 expression is up-regulated in giant panda m-SKPs.

<p>Expression of SKP associated markers was analysed in m-SKPs generated and passaged from giant panda male buccal mucosa cell cultures. Fibronectin (<b>a</b>), nestin (<b>b</b>), vimentin (<b>c</b>), and versican (<b>d</b>) are expressed in m-SKPs. P75 similarly is expressed both in monolayer (<b>e</b>) and m-SKP (<b>f</b>) cells. ABCG2 expression is not expressed in giant panda monolayer cultures (<b>g</b>) but is induced in m-SKPs (h). Nuclei were stained with DAPI. Scale bar = 100μm.</p