The neuroprotective E3-ubiquitin ligase CHIP is linked to healthy aging. Here, we present a protocol using a patient-derived iPSC line with a triplication of the α-synuclein gene to produce gene-edited cells isogenic for CHIP. We describe iPSC differentiation into cortical neurons and their identity validation. We then detail mass spectrometry-based approaches (SWATH-MS) to identify dominant changes in the steady state proteome generated by loss of CHIP function. This protocol can be adapted to other proteins that impact proteostasis in neurons. For complete details on the use and execution of this protocol, please refer to Dias et al. (2021).

• Klíčová slova
• CRISPR, Cell Biology, Cell Differentiation, Cell culture, Mass Spectrometry, Neuroscience, Proteomics, Stem Cells,
• MeSH
• hmotnostní spektrometrie MeSH
• indukované pluripotentní kmenové buňky * MeSH
• lidé MeSH
• neurony MeSH
• proteom genetika   MeSH
• proteomika metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH

CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular "health". Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP.

• Klíčová slova
• bioinformatics, cell biology, omics, organizational aspects of cell biology, proteomics,
• Publikační typ
• časopisecké články MeSH

Studying the long-term impact of continuous-flow left ventricular assist device (CF-LVAD) offers an opportunity for a complex understanding of the pathophysiology of vascular changes in aortic tissue in response to a nonphysiological blood flow pattern. Our study aimed to analyze aortic mRNA/miRNA expression changes in response to long-term LVAD support. Paired aortic samples obtained at the time of LVAD implantation and at the time of heart transplantation were examined for mRNA/miRNA profiling. The number of differentially expressed genes (Pcorr < 0.05) shared between samples before and after LVAD support was 277. The whole miRNome profile revealed 69 differentially expressed miRNAs (Pcorr < 0.05). Gene ontology (GO) analysis identified that LVAD predominantly influenced genes involved in the extracellular matrix and collagen fibril organization. Integrated mRNA/miRNA analysis revealed that potential targets of miRNAs dysregulated in explanted samples are mainly involved in GO biological process terms related to dendritic spine organization, neuron projection organization, and cell junction assembly and organization. We found differentially expressed genes participating in vascular tissue engineering as a consequence of LVAD duration. Changes in aortic miRNA levels demonstrated an effect on molecular processes involved in angiogenesis.

• Klíčová slova
• aorta, left ventricular assist device, mRNA, mechanical circulatory support, microRNA,
• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• longitudinální studie MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• onemocnění aortální chlopně etiologie   metabolismus   patologie   MeSH
• podpůrné srdeční systémy škodlivé účinky   MeSH
• regulace genové exprese * MeSH
• senioři MeSH
• srdeční selhání patologie   chirurgie   MeSH
• stanovení celkové genové exprese MeSH
• transplantace srdce škodlivé účinky   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• mikro RNA MeSH

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

• MeSH
• blastocysta fyziologie   MeSH
• buněčná diferenciace MeSH
• buněčný rodokmen MeSH
• DNA vazebné proteiny fyziologie   MeSH
• embryonální vývoj MeSH
• endoderm cytologie   MeSH
• mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory   fyziologie   MeSH
• myši MeSH
• proteiny vázající RNA fyziologie   MeSH
• proteosyntéza * MeSH
• TOR serin-threoninkinasy fyziologie   MeSH
• transkripční faktory fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• mTOR protein, mouse MeSH Prohlížeč
• Mybbp1a protein, mouse MeSH Prohlížeč
• proteiny vázající RNA MeSH
• TOR serin-threoninkinasy MeSH
• transkripční faktory MeSH

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.

• Klíčová slova
• aging, brain barrier, cerebrospinal fluid, choroid plexus, development, single-cell RNA sequencing, single-nucleus RNA sequencing,
• MeSH
• analýza jednotlivých buněk MeSH
• buněčná diferenciace genetika   MeSH
• buněčný rodokmen genetika   MeSH
• epitelové buňky metabolismus   MeSH
• hematoencefalická bariéra metabolismus   MeSH
• mozek metabolismus   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši embryologie   MeSH
• nemoci mozku genetika   patofyziologie   MeSH
• plexus chorioideus embryologie   metabolismus   fyziologie   MeSH
• signální transdukce MeSH
• stárnutí fyziologie   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši embryologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

BACKGROUND: The study of the mechanisms controlling wound healing is an attractive area within the field of biology, with it having a potentially significant impact on the health sector given the current medical burden associated with healing in the elderly population. Healing is a complex process and includes many steps that are regulated by coding and noncoding RNAs, proteins and other molecules. Nitric oxide (NO) is one of these small molecule regulators and its function has already been associated with inflammation and angiogenesis during adult healing. RESULTS: Our results showed that NO is also an essential component during embryonic scarless healing and acts via a previously unknown mechanism. NO is mainly produced during the early phase of healing and it is crucial for the expression of genes associated with healing. However, we also observed a late phase of healing, which occurs for several hours after wound closure and takes place under the epidermis and includes tissue remodelling that is dependent on NO. We also found that the NO is associated with multiple cellular metabolic pathways, in particularly the glucose metabolism pathway. This is particular noteworthy as the use of NO donors have already been found to be beneficial for the treatment of chronic healing defects (including those associated with diabetes) and it is possible that its mechanism of action follows those observed during embryonic wound healing. CONCLUSIONS: Our study describes a new role of NO during healing, which may potentially translate to improved therapeutic treatments, especially for individual suffering with problematic healing.

• Klíčová slova
• AP-1, Leptin, Nitric oxide, RNA-sequencing, Transcriptome, Wound healing, Xenopus laevis,
• MeSH
• embryo nesavčí cytologie   metabolismus   fyziologie   MeSH
• glukosa metabolismus   MeSH
• hojení ran * MeSH
• leptin metabolismus   MeSH
• oxid dusnatý metabolismus   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukosa MeSH
• leptin MeSH
• oxid dusnatý MeSH

The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.

• Klíčová slova
• MPF, aging, lamin A/C, meiosis, oocyte, translation,
• MeSH
• faktor podporující zrání genetika   metabolismus   MeSH
• fosforylace MeSH
• jaderný obal metabolismus   ultrastruktura   MeSH
• meióza * MeSH
• mezotelin MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• stárnutí genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faktor podporující zrání MeSH
• mezotelin MeSH
• Msln protein, mouse MeSH Prohlížeč