A Bioinformatic Investigation of Proteasome And Autophagy Expression in The Central Nervous System

The ubiquitin proteasome system (UPS) and autophagy lysosome pathway (ALP) play major roles in protein quality control. However, data regarding the relative significance of UPS and ALP in the central nervous system (CNS) remain limited. In this study, we reckon the quantitative expression status of UPS- and ALP-related genes and their products in the CNS compared to that in other tissues. We collected human and mouse gene expression datasets from the reference expression dataset (RefEx) and Genevestigator (a tool for handling curated transcriptomic data from public repositories) and human proteomics data from the proteomics database (ProteomicsDB). The expression levels of genes and proteins in four categories—ubiquitin, proteasome, autophagy, and lysosome in cells and tissues were extracted. Perturbation of expression by drugs was also analyzed based on the four categories. Compared to that for the other three categories, proteasome gene expression was consistently low in the CNS of mice, and was more pronounced in humans. Neural stem cells and neurons showed low proteasome gene expressions when compared to non-neuronal stem cells. Proteomic analyses, however, did not show trends similar to those observed in the gene expression analyses. Perturbation analyses revealed that agents such as azithromycin and vitamin D3 upregulated the expression of both the UPS and ALP. Disproportional expression of the UPS and ALP might play a role in the pathophysiology of CNS disorders and this imbalance might be redressed by several therapeutic candidates.

Shaping the size of a neuronal lineage: the role of Imp and Syp RBPs in the precise elimination of neurons by apoptosis.

Neuronal stem cells produce a finite and stereotyped number of neuronal progenies. This process must be finely regulated during development and adult stages to ensure proper brain function. In Drosophila, stem cells, called Neuroblasts, produce an invariant number of neurons. Two RNA binding proteins, Imp and Syp, play a central role in controlling the speed of division and the end of the proliferative phase of individual NBs, two parameters that influences the final number of neurons produced. Here, we have discovered a novel function for Imp and Syp, where both RBPs shape the number of neurons produced by a stem cell by controlling program cell death (PCD) in immature neurons. By studying a neuroblast lineage, called Lin A/15, which produces motoneurons (MNs) and glia, we have demonstrated that Lin A/15 stem cell spends 40% of its time producing immature MNs which are eliminated by apoptosis. We have revealed that only the first born MNs (Imp +) survive while the last born MNs (Imp- Syp+) are eliminated by apoptosis. Both RBPs play a central role in neuronal survival, Imp promotes neuronal survival while Syp promotes cell death in immature motoneurons. Interestingly their opposite temporal gradient in Lin A/15 stem cell also determines the end of Lin A/15 stem cell neurogenesis by PCD. Both RNA binding proteins are conserved in vertebrates and seem to play a central role in the number of neurons produce during development. The Drosophila model and its genetic tools offer a unique chance to decipher their function in neural stem cell versus immature neurons.

Effects of Rosemary Oil (Rosmarinus Officinalis) Supplementation on the Fate of the Transplanted Human Olfactory Bulb Neural Stem Cells Against Ibotenic Acid-Induced Neurotoxicity (Alzheimer Model) in Rat

Rosemary oil (ROO) is known to have multiple pharmacological effects: it is an antioxidant, an anti-inflammatory, and cytoprotective. In the present study, we examined the effects of ROO on Human olfactory bulb neuronal stem cells (hOBNSCs) after their transplantation into rats, with the ibotenic (IBO) acid-induced cognitive deficit model. After 7 weeks, cognitive functions were assessed using the Morris water maze (MWM). After two months blood and the hippocampus were collected for biochemical, gene expression, and histomorphometric analyses. Learning ability and memory function were significantly enhanced after hOBNSCs transplantation and were nearly returned to normal in the treated group. The IBO acid injection was associated with a significant decline of total leukocyte count (TLC) and a significant increase in total and toxic neutrophils. As well, the level of IL-1β, TNF-α CRP in serum and levels of MDA and NO in hippocampus tissue were significantly elevated, while antioxidant markers (CAT, GSH, and SOD) were reduced in treated tissue compared to controls. The administration of ROO before or with cell transplantation attenuated all these parameters. In particular, the level of NO nearly returned to normal when rosemary was administrated before cell transplantation. Gene expression analysis revealed the potential protective effect of ROO and hOBNSCs via down-expression of R-βAmyl and R- CAS 3 and R-GFAP genes.

A comprehensive series of temporal transcription factors in the fly visual system

The brain consists of thousands of different neuronal types that are generated through multiple divisions of neuronal stem cells. These stem cells have the capacity to generate different neuronal types at different stages of their development. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs). While a number of tTFs are known in different animals and across various parts of the nervous system, these have been mostly identified by informed guesses and antibody availability. We used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila medulla neurons in the optic lobe. We tested the genetic interactions among these tTFs. While we verify the general principle that tTFs regulate the progression of the series by activating the next tTFs in the series and repressing the previous ones, we also identify more complex regulations. Two of the tTFs, Eyeless and Dichaete, act as hubs integrating the input of several upstream tTFs before allowing the series to progress and in turn regulating the expression of several downstream tTFs. Moreover, we show that tTFs not only specify neuronal identity by controlling the expression of cell type-specific genes. Finally, we describe the very first steps of neuronal differentiation and find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons days before they are being used in functioning neurons; we show that these mechanisms are conserved in humans. Our results offer a comprehensive description of a temporal series of tTFs in a neuronal system, offering mechanistic insights into the regulation of the progression of the series and the regulation of neuronal diversity. This represents a proof-of-principle for the use of single-cell mRNA sequencing for the comparison of temporal patterning across phyla that can lead to an understanding of how the human brain develops and how it has evolved.

Cryptic developmental events determine medulloblastoma radiosensitivity and cellular heterogeneity without altering transcriptomic profile

It is unclear why medulloblastoma patients receiving similar treatments experience different outcomes. Transcriptomic profiling identified subgroups with different prognoses, but in each subgroup, individuals remain at risk of incurable recurrence. To investigate why similar-appearing tumors produce variable outcomes, we analyzed medulloblastomas triggered in transgenic mice by a common driver mutation expressed at different points in brain development. We genetically engineered mice to express oncogenic SmoM2, starting in multipotent glio-neuronal stem cells, or committed neural progenitors. Both groups developed medulloblastomas with similar transcriptomic profiles. We compared medulloblastoma progression, radiosensitivity, and cellular heterogeneity, determined by single-cell transcriptomic analysis (scRNA-seq). Stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing stem-like cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, down-regulated stem-like cells and were curable with radiation. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, with more Ccr2+ macrophages and fewer Igf1+ microglia, indicating that developmental events affected the subsequent tumor microenvironment. Reduced mTORC1 activity in M-Smo tumors suggests that differential Igf1 contributed to differences in phenotype. Developmental events in tumorigenesis that were obscure in transcriptomic profiles thus remained cryptic determinants of tumor composition and outcome. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic/methylomic studies with analyses that resolve cellular heterogeneity.

Bioinformatic Analysis of Transcriptional Regulation by Nur77 in Central Nervous System and Immune System

ObjectiveThe objectives of this work were to find genes regulated by Nur77 in neurons and to evaluate the possible common role of this transcription factor in neurons and lymphatic cells using published experimentally generated databases of ChIP-Seq and a microarray. We also characterized Nur77 binding throughout the genome. ResultsWe identified 113 Nur77 target genes in neuronal stem cells and 116 in neuronal cells. Cell adhesion and anchoring processes emerged as regulated by Nur77 in neurons and lymphatic cells. We found 9 common genes regulated by Nur77. Finally, we described a significant distribution of Nur77 binding sites in strong enhancers and active promoters. This work is a first step to understand the role of Nur77 and its common targets in neurons and immune cells.

A New Tool for Safety Evaluation and a Combination of Measures for Efficacy Assessment of Cotransplanting Human Allogenic Neuronal Stem Cells and Mesenchymal Stem Cells for the Treatment of Parkinson Disease: Protocol for an Interventional Study (Preprint)

BACKGROUND Parkinson disease (PD) is a neurodegenerative disorder associated with a broad spectrum of motor and nonmotor symptoms. Any proposed cure needs to address the many aspects of the disease. Stem cell therapy may have potential in this regard as indicated in recent preclinical and clinical studies. OBJECTIVE This protocol aims to examine the safety and therapeutic benefit of human Wharton jelly-derived mesenchymal stem cells (WJ-MScs) and their derivatives, neuronal stem cells (NSCs) in PD. METHODS This clinical trial is a double-arm, single-blinded, phase I-II interventional study. Participants have been allocated to 1 of 2 groups: one receiving allogeneic WJ-MSCs alone, the other receiving NSCs and WJ-MScs. Participants are being followed-up and assessed over a period of 6 months. To assess safety, an incidence of treatment-emergent adverse events (TEAEs) tool tailored for PD is being used immediately and up to 6 months after treatment. For efficacy assessment, a number of factors are being used, including the gold standard severity test and the Unified Parkinson Disease Rating Scale. In addition, the following standardized assessments for different common symptoms in PD are being included: motor (both subjectively and objectively assessed with wearable sensors), sensory, quality of life and psychological well-being, cognition, and sleep quality. Furthermore, immune-modulatory cytokines and neuronal damage versus regeneration markers in PD, including the neuronal protein linked to PD, α-synuclein, are being monitored. RESULTS Ten patients have been enrolled in this study and thus participant recruitment has been completed. The study status is active and beyond the recruiting stage. Study chart implementation, data collection, and analysis are ongoing. CONCLUSIONS The combination of NSCs and MSCs in PD may be useful for harnessing the best of the immunomodulation and neural repair characteristics of these cell types. The tailored comprehensive and scaled TEAEs and the variety of evaluation tools used enables a comprehensive assessment of this cellular therapy treatment protocol. A consideration of this expanded tool set is important in the design of future clinical studies for PD. CLINICALTRIAL ClinicalTrials.gov NCT03684122; https://clinicaltrials.gov/ct2/show/NCT03684122 INTERNATIONAL REGISTERED REPORT DERR1-10.2196/29695

NEUROPROTECTIVE EFFECT OF THYROID HORMONES IN CEREBRAL HYPOPERFUSION

Clinical studies demonstrate that a decrease in the blood concentration of thyroid hormones within the euthyroid range is associated with higher mortality rates, damage, severity and poor prognosis for lost function recovery in patients who survived a stroke during one year. Experimental studies prove the neuroprotective role of thyroid hormones in animal models of cerebral hypoperfusion: bilateral ligation of carotid arteries and occlusion of the middle cerebral artery. Thyroid hormones can penetrate through the blood-brain barrier. Their nuclear effects in the nervous tissue are mediated by the TRα and TRβ receptors. Thyroid hormones provide astrocytic differentiation, reduce proliferation and astrogliosis, increase the glutamate uptake by astrocytes, reduce excitotoxicity and stimulate ATP synthesis, suppress aquaporin-4 (AQP4) expression, and reduce cerebral edema risk. They also reduce tonic GABA signaling in the peri-infarction area, increase the synthesis of BDNF and GDNF neurotrophic factors. Moreover, they have an anti-apoptotic effect. Due to TRα1 receptors on neuronal stem cells of the subgranular hippocampus zone and subventricular zone, thyroid hormones shift the balance between neurogenesis and oligodendrogenesis towards neurogenesis both in intact animals and in those with cerebral hypoperfusion. The non-genomic actions of thyroid hormones are initiated at receptors in the plasma membrane, in the cytoplasm, or in mitochondria, resulting in endothelial cell migration and angiogenesis. Thus, within the normal reaction range the structural and functional state of the thyroid gland is associated with neuroplasticity. So, the level of thyroid hormones can predict the severity of a disease accompanied by cerebral hypoperfusion. Thyroid hormones can also be considered as prototypes of neuroprotective drugs. Keywords: stroke, thyroid hormones, cerebral hypoperfusion, thyroid gland, neurons, neuroglia, angiogenesis, neurotransmitters. Клинические исследования демонстрируют, что снижение концентрации тиреоидных гормонов в крови в пределах эутиреоза ассоциировано с большими показателями летальности, объема повреждения, тяжести и худшим прогнозом восстановления утраченных функций как минимум на протяжении одного года после инсульта головного мозга. Это подтверждается экспериментальными работами, демонстрирующими нейропротекторную роль тиреоидных гормонов в животных моделях церебральной гипоперфузии: двусторонней перевязки общих сонных артерий и окклюзии средней мозговой артерии. Показано, что тиреоидные гормоны способны проникать через гематоэнцефалический барьер. Их ядерные эффекты в нервной ткани опосредованы рецепторами TRα и TRβ. Тиреоидные гормоны обеспечивают астроцитарную дифференцировку, снижают пролиферацию и астроглиоз, повышают поглощение глутамата астроцитами, снижая эксайтотоксичность и стимулируя синтез АТФ, подавляют экспрессию aquaporin-4 (AQP4), снижая риск отека головного мозга. Также они снижают тоническую ГАМК-передачу сигналов в периинфарктной области и увеличивают синтез нейротрофических факторов BDNF и GDNF; обладают антиапоптотическим эффектом. Благодаря наличию TRα1-рецепторов на нейрональных стволовых клетках субгранулярной зоны гиппокампа и субвентрикулярной зоны тиреоидные гормоны обладают способностью смещать баланс между нейрогенезом и олигодендрогенезом в сторону нейрогенеза как у интактных животных, так и при церебральной гипоперфузии. Негеномные эффекты тиреоидных гормонов инициируются на рецепторах в плазматической мембране, в цитоплазме или в митохондриях, в результате активизируется миграция эндотелиальных клеток и ангиогенез. Таким образом, структурно-функциональное состояние щитовидной железы в пределах нормы реакции ассоциировано с нейропластичностью, что позволяет использовать уровень тиреоидных гормонов в качестве предиктора тяжести заболеваний, сопровождающихся церебральной гипоперфузией. Также тиреоидные гормоны можно рассматривать в качестве прототипов нейропротекторных лекарственных средств. Ключевые слова: инсульт, тиреоидные гормоны, церебральная гипоперфузия, щитовидная железа, нейроны, нейроглия, ангиогенез, нейромедиаторы.

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6,438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.