The nuclear envelope compartmentalizes the eukaryotic genome, provides mechanical resistance, and regulates access to the chromatin. However, recent studies have identified several conditions where ...the nuclear membrane ruptures during interphase, breaking down this compartmentalization leading to DNA damage, chromothripsis, and kataegis. This review discusses three major circumstances that promote nuclear membrane rupture, nuclear deformation, chromatin bridges, and micronucleation, and how each of these nuclear catastrophes results in DNA damage. In addition, we highlight recent studies that demonstrate a single chromosome missegregation can initiate a cascade of events that lead to accumulating damage and even multiple rounds of chromothripsis.
The PI 3‐kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3‐phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P ...dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot‐Marie‐Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34‐Schwann cell knockout (Vps34SCKO) mice show severe hypomyelination in peripheral nerves. Vps34–/– Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34–/– Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34–/– Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic through endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to neuregulin 1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.
Main Points
To myelinate axons, Schwann cells require the PI 3‐kinase Vps34, a regulator of endosomal trafficking.
The ErbB2‐ErbB3 tyrosine kinase receptor, which traffics through endosomes and is key to myelination, is altered when Schwann cells lack Vps34.
Abstract
The form of Charcot–Marie–Tooth type 4B (CMT4B) disease caused by mutations in myotubularin-related 5 (MTMR5; also called SET binding factor 1, SBF1) shows a spectrum of axonal and ...demyelinating nerve phenotypes. This contrasts with the CMT4B subtypes caused by MTMR2 or MTMR13 (SBF2) mutations, which are characterized by myelin outfoldings and classic demyelination. Thus, it is unclear whether MTMR5 plays an analogous or distinct role from that of its homolog, MTMR13, in the peripheral nervous system (PNS). MTMR5 and MTMR13 are pseudophosphatases predicted to regulate endosomal trafficking by activating Rab GTPases and binding to the phosphoinositide 3-phosphatase MTMR2. In the mouse PNS, Mtmr2 was required to maintain wild-type levels of Mtmr5 and Mtmr13, suggesting that these factors function in discrete protein complexes. Genetic elimination of both Mtmr5 and Mtmr13 in mice led to perinatal lethality, indicating that the two proteins have partially redundant functions during embryogenesis. Loss of Mtmr5 in mice did not cause CMT4B-like myelin outfoldings. However, adult Mtmr5−/− mouse nerves contained fewer myelinated axons than control nerves, likely as a result of axon radial sorting defects. Consistently, Mtmr5 levels were highest during axon radial sorting and fell sharply after postnatal day seven. Our findings suggest that Mtmr5 and Mtmr13 ensure proper axon radial sorting and Schwann cell myelination, respectively, perhaps through their direct interactions with Mtmr2. This study enhances our understanding of the non-redundant roles of the endosomal regulators MTMR5 and MTMR13 during normal peripheral nerve development and disease.
For a given gene, different mutations influence organismal phenotypes to varying degrees. However, the expressivity of these variants not only depends on the DNA lesion associated with the mutation, ...but also on factors including the genetic background and rearing environment. The degree to which these factors influence related alleles, genes, or pathways similarly, and whether similar developmental mechanisms underlie variation in the expressivity of a single allele across conditions and among alleles is poorly understood. Besides their fundamental biological significance, these questions have important implications for the interpretation of functional genetic analyses, for example, if these factors alter the ordering of allelic series or patterns of complementation. We examined the impact of genetic background and rearing environment for a series of mutations spanning the range of phenotypic effects for both the scalloped and vestigial genes, which influence wing development in Drosophila melanogaster. Genetic background and rearing environment influenced the phenotypic outcome of mutations, including intra-genic interactions, particularly for mutations of moderate expressivity. We examined whether cellular correlates (such as cell proliferation during development) of these phenotypic effects matched the observed phenotypic outcome. While cell proliferation decreased with mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the degree of background dependence. We discuss these findings and propose a phenomenological model to aid in understanding the biology of genes, and how this influences our interpretation of allelic effects in genetic analysis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here, we demonstrate that two ...characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability and determine the timing of micronucleus rupture. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial protein recruitment during post-mitotic nuclear envelope assembly. Micronucleus size strongly correlates with lamin B1 levels and nuclear pore density in intact micronuclei, but, unexpectedly, lamin B1 levels do not completely predict nuclear lamina organization or membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.
Theoretical work predicts that sexual selection can enhance natural selection, increasing the rate of adaptation to new environments and helping purge harmful mutations. While some experiments ...support these predictions, remarkably little work has addressed the role of sexual selection on compensatory adaptation-populations' ability to compensate for the costs of deleterious alleles that are already present. We tested whether sexual selection, as well as the degree of standing genetic variation, affect the rate of compensatory evolution via phenotypic suppression in experimental populations of
These populations were fixed for a spontaneous mutation causing mild abnormalities in the male sex comb, a structure important for mating success. We fine-mapped this mutation to an ∼85 kb region on the X chromosome containing three candidate genes, showed that the mutation is deleterious, and that its phenotypic expression and penetrance vary by genetic background. We then performed experimental evolution, including a treatment where opportunity for mate choice was limited by experimentally enforced monogamy. Although evolved populations did show some phenotypic suppression of the morphological abnormalities in the sex comb, the amount of suppression did not depend on the opportunity for sexual selection. Sexual selection, therefore, may not always enhance natural selection; instead, the interaction between these two forces may depend on additional factors.
Charcot-Marie-Tooth Disorder Type 4B (CMT4B) is a demyelinating peripheral neuropathy caused by mutations in myotubularin-related (MTMR) proteins 2, 13, or 5 (CMT4B1/2/3), which regulate ...phosphoinositide turnover and endosomal trafficking. Although mouse models of CMT4B2 exist, an in vitro model would make possible pharmacological and reverse genetic experiments needed to clarify the role of MTMR13 in myelination. We have generated such a model using Schwann cell-dorsal root ganglion (SC-DRG) explants from Mtmr13−/− mice. Myelin sheaths in mutant cultures contain outfoldings highly reminiscent of those observed in the nerves of Mtmr13−/− mice and CMT4B2 patients. Mtmr13−/− SC-DRG explants also contain reduced Mtmr2, further supporting a role of Mtmr13 in stabilizing Mtmr2. Elevated PI(3,5)P2 has been implicated as a cause of myelin outfoldings in Mtmr2−/− models. In contrast, the role of elevated PI3P or PI(3,5)P2 in promoting outfoldings in Mtmr13−/− models is unclear. We found that over-expression of MTMR2 in Mtmr13−/− SC-DRGs moderately reduced the prevalence of myelin outfoldings. Thus, a manipulation predicted to lower PI3P and PI(3,5)P2 partially suppressed the phenotype caused by Mtmr13 deficiency. We also explored the relationship between CMT4B2-like myelin outfoldings and kinases that produce PI3P and PI(3,5)P2 by analyzing nerve pathology in mice lacking both Mtmr13 and one of two specific PI 3-kinases. Intriguingly, the loss of vacuolar protein sorting 34 or PI3K-C2β in Mtmr13−/− mice had no impact on the prevalence of myelin outfoldings. In aggregate, our findings suggest that the MTMR13 scaffold protein likely has critical functions other than stabilizing MTMR2 to achieve an adequate level of PI 3-phosphatase activity.
Abstract
Identifying the genetic architecture of complex traits is important to many geneticists, including those interested in human disease, plant and animal breeding, and evolutionary genetics. ...Advances in sequencing technology and statistical methods for genome-wide association studies have allowed for the identification of more variants with smaller effect sizes, however, many of these identified polymorphisms fail to be replicated in subsequent studies. In addition to sampling variation, this failure to replicate reflects the complexities introduced by factors including environmental variation, genetic background, and differences in allele frequencies among populations. Using Drosophila melanogaster wing shape, we ask if we can replicate allelic effects of polymorphisms first identified in a genome-wide association studies in three genes: dachsous, extra-macrochaete, and neuralized, using artificial selection in the lab, and bulk segregant mapping in natural populations. We demonstrate that multivariate wing shape changes associated with these genes are aligned with major axes of phenotypic and genetic variation in natural populations. Following seven generations of artificial selection along the dachsous shape change vector, we observe genetic differentiation of variants in dachsous and genomic regions containing other genes in the hippo signaling pathway. This suggests a shared direction of effects within a developmental network. We also performed artificial selection with the extra-macrochaete shape change vector, which is not a part of the hippo signaling network, but showed a largely shared direction of effects. The response to selection along the emc vector was similar to that of dachsous, suggesting that the available genetic diversity of a population, summarized by the genetic (co)variance matrix (G), influenced alleles captured by selection. Despite the success with artificial selection, bulk segregant analysis using natural populations did not detect these same variants, likely due to the contribution of environmental variation and low minor allele frequencies, coupled with small effect sizes of the contributing variants.
Background
Advances in ultrasensitive detection techniques for blood biomarkers allow the quantification of AD‐specific phosphorylated Tau proteins, including Tau phosphorylated at threonine 181 ...(pTau181) in patient plasma. pTau blood‐based biomarker have shown great promise as inclusion criteria and secondary endpoint evaluation in clinical trials.
Method
The University of British Columbia (UBC) CARD biobank plasma samples from clinically diagnosed AD and non‐AD patients were used to establish clinical and analytical validity of the pTau181 plasma assay per FDA fit‐for‐purpose guildelines (Neurcode USA, Inc.). We assessed the analytical measurement interval, clinical reportable range, linearity, intra‐laboratory precision, specimen stability, interference, and clinical performance.
Result
The pTau181 plasma assay provides a robust and accurate biomarker approach for independent determination of AD, with an AUC of 0.9 in our validation study cohort. The cut‐point (≥ 30 ng/L) had 100% sensitivity and 88% specificity for AD diagnosis in autopsy‐confirmed samples. The plasma pTau181 assay appears to be performing well as additional screening metric for inclusion of mild‐to‐moderate AD subjects in Phase 3 clinical trials (i.e. RETHINK‐ALZ and REFOCUS‐ALZ). 89% of the clinical sites participating in the REFOCUS‐ALZ study and 80% of sites participating in the RETHINK‐ALZ had at least 70% of screened subjects meeting this criterion.
Conclusion
AD plasma biomarker (pTau181) quantified using an RUO assay has great potential both as a diagnostic tool and to streamline clinical trials in AD.
Background
The feasibility of detecting tau phosphorylated at threonine‐181(p‐tau181) in CSF makes it a valuable biomarker for diagnosis in the field of Alzheimer’s Disease (AD). Recently, the novel ...technologies accurately measuring biomarkers directly in blood offer a unique advantage for use in clinical testing and trials. This study describes the performance testing of a novel plasma p‐tau181 Simoa assay to predict clinical AD.
Method
The presented data were obtained after analysis of EDTA plasma from cases with clinical AD who had been referred to the UBC Hospital Clinic with complaint of cognitive impairment and were assessed for dementia and AD between 2008 – 2018.
We also analysed serum of 50 random healthy donors and 50 aged ‐ matched controls with normal cognition. The samples were assayed using an ADx Neurosciences developed p‐tau181 specific Simoa assay. Analytical performance (precision, detection limit, parallelism) and analyte stability was evaluated using healthy‐donor samples. Finally, the p‐tau181 specificity was assessed using synthetic peptides.
Result
254 AD patients (female = 125) and 100 controls (female = 39) were included. The average of plasma p‐tau181 was 72.9 ±36.8 ng/L in AD cases and the average of serum p‐tau181 was 8.1± 10.5 ng/L in controls.
The concentration of plasma p‐tau181 in patients with AD was significantly increased with at‐least 9‐fold versus controls (p=<0.001). ROC analysis demonstrated an AUC of 0.92 and a suitable clinical cut‐off of 34.3 ng/L. Analytical performance testing: 1 of the 254 AD samples tested were below the LLOQ of 1.38 ng/L. The intra‐ and inter‐ assay variability was 8.2% CV and 11.8 % CV, respectively. Sample dilution (df 2‐4‐5‐6) resulted in mean parallelism of 93.5 %. P‐tau181 levels were stable up to 5 Freeze/Thaw cycle (mean 98.9%) and up to 24h at 4°C or RT. The assay was found to be highly specific towards p‐tau181 and nonreactive to p‐tau175.
Conclusion
The plasma p‐tau181 concentration in AD cases were significantly higher than controls with limited overlap and in line with published data. The specific measurement of plasma p‐tau181 shows good promise in offering a non‐biased measurement and help in the clinical assessment of AD patients and clinical trials.
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