Deubiquitylases in developmental ubiquitin signaling and congenital diseases

Metazoan development from a one-cell zygote to a fully formed organism requires complex cellular differentiation and communication pathways. To coordinate these processes, embryos frequently encode signaling information with the small protein modifier ubiquitin, which is typically attached to lysine residues within substrates. During ubiquitin signaling, a three-step enzymatic cascade modifies specific substrates with topologically unique ubiquitin modifications, which mediate changes in the substrate’s stability, activity, localization, or interacting proteins. Ubiquitin signaling is critically regulated by deubiquitylases (DUBs), a class of ~100 human enzymes that oppose the conjugation of ubiquitin. DUBs control many essential cellular functions and various aspects of human physiology and development. Recent genetic studies have identified mutations in several DUBs that cause developmental disorders. Here we review principles controlling DUB activity and substrate recruitment that allow these enzymes to regulate ubiquitin signaling during development. We summarize key mechanisms of how DUBs control embryonic and postnatal differentiation processes, highlight developmental disorders that are caused by mutations in particular DUB members, and describe our current understanding of how these mutations disrupt development. Finally, we discuss how emerging tools from human disease genetics will enable the identification and study of novel congenital disease-causing DUBs.

Tracing and tracking epiallele families in complex DNA populations

DNA methylation is a stable epigenetic modification, extremely polymorphic and driven by stochastic and deterministic events. Most of the current techniques used to analyse methylated sequences identify methylated cytosines (mCpGs) at a single-nucleotide level and compute the average methylation of CpGs in the population of molecules. Stable epialleles, i.e. CpG strings with the same DNA sequence containing a discrete linear succession of phased methylated/non-methylated CpGs in the same DNA molecule, cannot be identified due to the heterogeneity of the 5′–3′ ends of the molecules. Moreover, these are diluted by random unstable methylated CpGs and escape detection. We present here MethCoresProfiler, an R-based tool that provides a simple method to extract and identify combinations of methylated phased CpGs shared by all components of epiallele families in complex DNA populations. The methylated cores are stable over time, evolve by acquiring or losing new methyl sites and, ultimately, display high information content and low stochasticity. We have validated this method by identifying and tracing rare epialleles and their families in synthetic or in vivo complex cell populations derived from mouse brain areas and cells during postnatal differentiation. MethCoresProfiler is written in R language. The software is freely available at https://github.com/84AP/MethCoresProfiler/.

Distribution of nadph-diaphorase positive structures of olfactory bulb of rats in ontogenesis

Aim: to identify peculiarities of distribution of NADPH-diaphorase (NADPH-d) positive structures in olfactory bulbs of rats of different age. Materials and Methods. The study was conducted on 22 white male rats. The object of research was olfactory bulbs of newborn rats – 1-3 days, suckling period – 7, 14, 21 days, infantile – 30 days, juvenile – 60, and mature – 180 days. The study was conducted on cryostat serial sections of olfactory bulbs (20 µm). Nitroxidergic structures were identified by histochemical labelling of NADPH-d (by Hope method). In a standard section, the area of NADPH-diaphorase-positive cells (100 in each case), surface area of glomeruli, the number of positive neurons surrounding a glomerulus, were measured. Results. In result of study it was found that in the olfactory bulb of rats of the studied age only groups of superficial and deep short-axon neurons and periglomerular neurons showed positivity to NADPH-d. The end product of reaction was distributed in somas and extensions of a part of cells with the density of distribution depending on the layer of the olfactory bulb and on the age of animals. Besides, positivity to the enzyme was also found in the central parts of glomeruli with distribution of diaphorase depending not on age, but rather on localization of glomeruli. Conclusion. Age-related transformations of positive subpopulation of neurons of olfactory bulb indicate active participation of NO in the processes of postnatal differentiation, growth and development of olfactory bulb.

Distribution of nadph-diaphorase positive structures of olfactory bulb of rats in ontogenesis

Aim: to identify peculiarities of distribution of NADPH-diaphorase (NADPH-d) positive structures in olfactory bulbs of rats of different age. Materials and Methods. The study was conducted on 22 white male rats. The object of research was olfactory bulbs of newborn rats – 1-3 days, suckling period – 7, 14, 21 days, infantile – 30 days, juvenile – 60, and mature – 180 days. The study was conducted on cryostat serial sections of olfactory bulbs (20 µm). Nitroxidergic structures were identified by histochemical labelling of NADPH-d (by Hope method). In a standard section, the area of NADPH-diaphorase-positive cells (100 in each case), surface area of glomeruli, the number of positive neurons surrounding a glomerulus, were measured. Results. In result of study it was found that in the olfactory bulb of rats of the studied age only groups of superficial and deep short-axon neurons and periglomerular neurons showed positivity to NADPH-d. The end product of reaction was distributed in somas and extensions of a part of cells with the density of distribution depending on the layer of the olfactory bulb and on the age of animals. Besides, positivity to the enzyme was also found in the central parts of glomeruli with distribution of diaphorase depending not on age, but rather on localization of glomeruli. Conclusion. Age-related transformations of positive subpopulation of neurons of olfactory bulb indicate active participation of NO in the processes of postnatal differentiation, growth and development of olfactory bulb.

Improving infant formulas: on the way to approaching breast milk

Breast milk is the gold standard of infant feeding during the first year of life that ensures adequate physical and psychological development. The composition of human milk changes all the time in the process of one feeding as well as throughout the whole period of lactation. The ingredients of breast milk promote the normal growth of the baby, affect postnatal differentiation of tissues, the formation of the central nervous system (CNS), auditory and visual analyzers, development of the intestinal microbiota of the infant. Breastfeeding contributes to a strong psycho-emotional bond between mother and child which provides the best conditions for further development of the mind, intelligence and behavioral characteristics.

The RhoGEF DOCK10 is essential for dendritic spine morphogenesis

By regulating actin cytoskeleton dynamics, Rho GTPases and their activators RhoGEFs are implicated in various aspects of neuronal differentiation, including dendritogenesis and synaptogenesis. Purkinje cells (PCs) of the cerebellum, by developing spectacular dendrites covered with spines, represent an attractive model system in which to decipher the molecular signaling underlying these processes. To identify novel regulators of dendritic spine morphogenesis among members of the poorly characterized DOCK family of RhoGEFs, we performed gene expression profiling of fluorescence-activated cell sorting (FACS)-purified murine PCs at various stages of their postnatal differentiation. We found a strong increase in the expression of the Cdc42-specific GEF DOCK10. Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs. Accordingly, in mouse hippocampal neurons, depletion of DOCK10 or expression of a DOCK10 GEF-dead mutant led to a strong decrease in spine density and size. Conversely, overexpression of DOCK10 led to increased spine formation. We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1. Our global approach thus identifies an unprecedented function for DOCK10 as a novel regulator of dendritic spine morphogenesis via a Cdc42-mediated pathway.