Ubiquitination and deubiquitination of 4E-T regulate neural progenitor cell maintenance and neurogenesis by controlling P-body formation.

During embryogenesis, neural stem/progenitor cells (NPCs) proliferate and differentiate to form brain tissues. Here, we show that in the developing murine cerebral cortex, the balance between the NPC maintenance and differentiation is coordinated by ubiquitin signals that control the formation of processing bodies (P-bodies), cytoplasmic membraneless organelles critical for cell state regulation. We find that the deubiquitinase Otud4 and the E3 ligase Trim56 counter-regulate the ubiquitination status of a core P-body protein 4E-T to orchestrate the assembly of P-bodies in NPCs. Aberrant induction of 4E-T ubiquitination promotes P-body assembly in NPCs and causes a delay in their cell cycle progression and differentiation. In contrast, loss of 4E-T ubiquitination abrogates P-bodies and results in premature neurogenesis. Thus, our results reveal a critical role of ubiquitin-dependent regulation of P-body formation in NPC maintenance and neurogenesis during brain development.

Immunohistochemical analysis of the developing mouse cortex.

The cerebral cortex is the most complex structure in the mammalian brain, whose development requires coordinated proliferation of neural stem/precursor cells (NPCs) and their differentiation into neurons and glia. Perturbations in NPC homeostasis can lead to abnormal cortical development which is frequently seen in neurodevelopmental disorders. In this chapter, we describe the preparation of cortical tissues from mice and step-by-step protocol for immunohistochemistry to study cortical development. With this technique, we employ commonly used molecular markers and thymidine analog methods to analyze NPC populations. We also discuss assay conditions that can be optimized according to the specific needs to improve experimental outcomes.

Development of iPSC-based clinical trial selection platform for patients with ultrarare diseases.

A "Leap-of-Faith" approach is used to treat patients with previously unknown ultrarare pathogenic mutations, often based on evidence from patients having dissimilar but more prevalent mutations. This uncertainty reflects the need to develop personalized prescreening platforms for these patients to assess drug efficacy before considering clinical trial enrollment. In this study, we report an 18-year-old patient with ultrarare Leigh-like syndrome. This patient had previously participated in two clinical trials with unfavorable responses. We established an induced pluripotent stem cell (iPSC)-based platform for this patient, and assessed the efficacy of a panel of drugs. The iPSC platform validated the safety and efficacy of the screened drugs. The efficacy of three of the screened drugs was also investigated in the patient. After 3 years of treatment, the drugs were effective in shifting the metabolic profile of this patient toward healthy control. Therefore, this personalized iPSC-based platform can act as a prescreening tool to help in decision-making with respect to patient's participation in future clinical trials.

CEBPß regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth.

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPß, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPß overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPß can be a promising therapeutic approach.

Nucleocytoplasmic transport of the RNA-binding protein CELF2 regulates neural stem cell fates.

The development of the cerebral cortex requires balanced expansion and differentiation of neural stem/progenitor cells (NPCs), which rely on precise regulation of gene expression. Because NPCs often exhibit transcriptional priming of cell-fate-determination genes, the ultimate output of these genes for fate decisions must be carefully controlled in a timely fashion at the post-transcriptional level, but how that is achieved is poorly understood. Here, we report that de novo missense variants in an RNA-binding protein CELF2 cause human cortical malformations and perturb NPC fate decisions in mice by disrupting CELF2 nucleocytoplasmic transport. In self-renewing NPCs, CELF2 resides in the cytoplasm, where it represses mRNAs encoding cell fate regulators and neurodevelopmental disorder-related factors. The translocation of CELF2 into the nucleus releases mRNA for translation and thereby triggers NPC differentiation. Our results reveal that CELF2 translocation between subcellular compartments orchestrates mRNA at the translational level to instruct cell fates in cortical development.

Sensory neurons derived from diabetic rats exhibit deficits in functional glycolysis and ATP that are ameliorated by IGF-1.

OBJECTIVE: The distal dying-back of the longest nerve fibres is a hallmark of diabetic neuropathy, and impaired provision of energy in the form of adenosine triphosphate (ATP) may contribute to this neurodegenerative process. We hypothesised that energy supplementation via glycolysis and/or mitochondrial oxidative phosphorylation is compromised in cultured dorsal root ganglion (DRG) sensory neurons from diabetic rodents, thus contributing to axonal degeneration. Functional analysis of glycolysis and mitochondrial respiration and real-time measurement of ATP levels in live cells were our specific means to test this hypothesis. METHODS: DRG neuron cultures from age-matched control or streptozotocin (STZ)-induced type 1 diabetic rats were used for in vitro studies. Three plasmids containing ATP biosensors of varying affinities were transfected into neurons to study endogenous ATP levels in real time. The Seahorse XF analyser was used for glycolysis and mitochondrial respiration measurements. RESULTS: Fluorescence resonance energy transfer (FRET) efficiency (YFP/CFP ratio) of the ATP biosensors AT1.03 (low affinity) and AT1.03YEMK (medium affinity) were significantly higher than that measured using the ATP-insensitive construct AT1.03R122/6K in both cell bodies and neurites of DRG neurons (p < 0.0001). The ATP level was homogenous along the axons but higher in cell bodies in cultured DRG neurons from both control and diabetic rats. Treatment with oligomycin (an ATP synthase inhibitor in mitochondria) decreased the ATP levels in cultured DRG neurons. Likewise, blockade of glycolysis using 2-deoxy-d-glucose (2-DG: a glucose analogue) reduced ATP levels (p < 0.001). Cultured DRG neurons derived from diabetic rats showed a diminishment of ATP levels (p < 0.01), glycolytic capacity, glycolytic reserve and non-glycolytic acidification. Application of insulin-like growth factor-1 (IGF-1) significantly elevated all the above parameters in DRG neurons from diabetic rats. Oligomycin pre-treatment of DRG neurons, to block oxidative phosphorylation, depleted the glycolytic reserve and lowered basal respiration in sensory neurons derived from control and diabetic rats. Depletion was much higher in sensory neurons from diabetic rats compared to control rats. In addition, an acute increase in glucose concentration, in the presence or absence of oligomycin, elevated parameters of glycolysis by 1.5- to 2-fold while having no impact on mitochondrial respiration. CONCLUSION: We provide the first functional evidence for decreased glycolytic capacity in DRG neurons derived from type 1 diabetic rats. IGF-1 protected against the loss of ATP supplies in DRG cell bodies and axons in neurons derived from diabetic rats by augmenting various parameters of glycolysis and mitochondrial respiration.

Upregulation of Neural Cell Adhesion Molecule 1 and Excessive Migration of Purkinje Cells in Cerebellar Cortex.

Purkinje cells (PCs) are large GABAergic projection neurons of the cerebellar cortex, endowed with elaborate dendrites that receive a multitude of excitatory inputs. Being the only efferent neuron of the cerebellar cortex, PCs project to cerebellar nuclei and control behaviors ranging from movement to cognition and social interaction. Neural cell adhesion molecule 1 (NCAM1) is widely expressed in the embryonic and postnatal development of the brain and plays essential roles in neuronal migration, axon pathfinding and synapse assembly. However, despite its high expression levels in cerebellum, little is known to date regarding the role(s) of NCAM1 in PCs development. Among other aspects, elucidating how the expression of NCAM1 in PCs could impact their postnatal migration would be a significant achievement. We analyzed the Acp2 mutant mouse (nax: naked and ataxia), which displays excessive PC migration into the molecular layer, and investigated how the excessive migration of PCs along Bergmann glia could correlate to NCAM1 expression pattern in early postnatal days. Our Western blot and RT-qPCR analysis of the whole cerebellum show that the protein and mRNA of NCAM1 in wild type are not different during PC dispersal from the cluster stage to monolayer formation. However, RT-qPCR analysis from FACS-based isolated PCs shows that Ncam1 is significantly upregulated when PCs fail to align and instead overmigrate into the molecular layer. Our results suggest two alternative interpretations: (1) NCAM1 promotes excessive PC migration along Bergmann glia, or (2) NCAM1 upregulation is an attempt to prevent PCs from invading the molecular layer. If the latter scenario proves true, NCAM1 may play a key role in PC monolayer formation.

Mechanisms Targeting the Unfolded Protein Response in Asthma.

Lung cells are constantly exposed to various internal and external stressors that disrupt protein homeostasis. To cope with these stimuli, cells evoke a highly conserved adaptive mechanism called the unfolded protein response (UPR). UPR stressors can impose greater protein secretory demands on the endoplasmic reticulum (ER), resulting in the development, differentiation, and survival of these cell types to meet these increasing functional needs. Dysregulation of the UPR leads to the development of the disease. The UPR and ER stress are involved in several human conditions, such as chronic inflammation, neurodegeneration, metabolic syndrome, and cancer. Furthermore, potent and specific compounds that target the UPR pathway are under development as future therapies. The focus of this review is to thoroughly describe the effects of both internal and external stressors on the ER in asthma. Furthermore, we discuss how the UPR signaling pathway is activated in the lungs to overcome cellular damage. We also present an overview of the pathogenic mechanisms, with a brief focus on potential strategies for pharmacological interventions.

The ER Stress/UPR Axis in Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis.

Cellular protein homeostasis in the lungs is constantly disrupted by recurrent exposure to various external and internal stressors, which may cause considerable protein secretion pressure on the endoplasmic reticulum (ER), resulting in the survival and differentiation of these cell types to meet the increased functional demands. Cells are able to induce a highly conserved adaptive mechanism, known as the unfolded protein response (UPR), to manage such stresses. UPR dysregulation and ER stress are involved in numerous human illnesses, such as metabolic syndrome, fibrotic diseases, and neurodegeneration, and cancer. Therefore, effective and specific compounds targeting the UPR pathway are being considered as potential therapies. This review focuses on the impact of both external and internal stressors on the ER in idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) and discusses the role of the UPR signaling pathway activation in the control of cellular damage and specifically highlights the potential involvement of non-coding RNAs in COPD. Summaries of pathogenic mechanisms associated with the ER stress/UPR axis contributing to IPF and COPD, and promising pharmacological intervention strategies, are also presented.

Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase ß to Augment Mitochondrial Function and Prevent Neurodegeneration.

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKß to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 µM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 µM) or short hairpin RNA to CaMKKß. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 µM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M1R-modulated, CaMKKß-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Betulin and its derivatives as novel compounds with different pharmacological effects.

Betulin (B) and Betulinic acid (BA) are natural pentacyclic lupane-structure triterpenoids which possess a wide range of pharmacological activities. Recent evidence indicates that B and BA have several properties useful for the treatment of metabolic disorders, infectious diseases, cardiovascular disorders, and neurological disorders. In the current review, we discuss B and BA structures and derivatives and then comprehensively explain their pharmacological effects in relation to various diseases. We also explain antiviral, antibacterial and anti-cancer effects of B and BA. Finally, we discuss the delivery methods, in which these compounds most effectively target different systems.

Poly(ADP-ribose) polymerase-1 inhibits mitochondrial respiration by suppressing PGC-1α activity in neurons.

Poly(ADP-ribose) polymerase-1 (PARP1) is a ubiquitous nuclear enzyme that regulates DNA repair and genomic stability. In oxidative genotoxic conditions, PARP1 activity is enhanced significantly, leading to excessive depletion of nicotinamide adenine dinucleotide (NAD+) and mitochondrial dysfunction. We hypothesized that PARP1-induced NAD+ depletion inhibits NAD+-dependent sirtuin deacetylase activity, thereby interfering with the mitochondrial regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). The DNA alkylator, N'-Nitro-N-nitroso-N-methylguanidine (MNNG), induced NAD+ depletion, inhibited sirtuin deacetylase activity and enhanced acetylation of PGC-1α. This was associated with reduced interaction between PGC-1α and nuclear respiratory factor 1 (NRF-1), which is a nuclear transcription factor that drives mitochondrial replication by regulating mitochondrial transcription factor A (TFAM). MNNG also reduced binding of NRF-1 to the tfam upstream promoter region and reduced TFAM mRNA, mitochondrial DNA copy number and respiratory function. MNNG effects were mitigated by PARP1 inhibition and genetic loss of function, by enhancing intracellular NAD+ levels, and with sirtuin (SIRT1) gain of function, supporting a mechanism dependent on PARP1 activity, NAD+-depletion and SIRT1 inhibition. This and other work from our group supports a destructive sequelae of events related to PARP1-induced sirtuin inhibition and sirtuin-mediated regulation of transcription.

Insulin-like growth factor-1 activates AMPK to augment mitochondrial function and correct neuronal metabolism in sensory neurons in type 1 diabetes.

OBJECTIVE: Diabetic sensorimotor polyneuropathy (DSPN) affects approximately half of diabetic patients leading to significant morbidity. There is impaired neurotrophic growth factor signaling, AMP-activated protein kinase (AMPK) activity and mitochondrial function in dorsal root ganglia (DRG) of animal models of type 1 and type 2 diabetes. We hypothesized that sub-optimal insulin-like growth factor 1 (IGF-1) signaling in diabetes drives loss of AMPK activity and mitochondrial function, both contributing to development of DSPN. METHODS: Age-matched control Sprague-Dawley rats and streptozotocin (STZ)-induced type 1 diabetic rats with/without IGF-1 therapy were used for in vivo studies. For in vitro studies, DRG neurons from control and STZ-diabetic rats were cultured and treated with/without IGF-1 in the presence or absence of inhibitors or siRNAs. RESULTS: Dysregulation of mRNAs for IGF-1, AMPKα2, ATP5a1 (subunit of ATPase), and PGC-1ß occurred in DRG of diabetic vs. control rats. IGF-1 up-regulated mRNA levels of these genes in cultured DRGs from control or diabetic rats. IGF-1 treatment of DRG cultures significantly (P < 0.05) increased phosphorylation of Akt, P70S6K, AMPK and acetyl-CoA carboxylase (ACC). Mitochondrial gene expression and oxygen consumption rate (spare respiratory capacity), ATP production, mtDNA/nDNA ratio and neurite outgrowth were augmented (P < 0.05). AMPK inhibitor, Compound C, or AMPKα1-specific siRNA suppressed IGF-1 elevation of mitochondrial function, mtDNA and neurite outgrowth. Diabetic rats treated with IGF-1 exhibited reversal of thermal hypoalgesia and, in a separate study, reversed the deficit in corneal nerve profiles. In diabetic rats, IGF-1 elevated the levels of AMPK and P70S6K phosphorylation, raised Complex IV-MTCO1 and Complex V-ATP5a protein expression, and restored the enzyme activities of Complex IV and I in the DRG. IGF-1 prevented TCA metabolite build-up in nerve. CONCLUSIONS: In DRG neuron cultures IGF-1 signals via AMPK to elevate mitochondrial function and drive axonal outgrowth. We propose that this signaling axis mediates IGF-1-dependent protection from distal dying-back of fibers in diabetic neuropathy.

Insulin prevents aberrant mitochondrial phenotype in sensory neurons of type 1 diabetic rats.

Diabetic neuropathy affects approximately 50% of diabetic patients. Down-regulation of mitochondrial gene expression and function has been reported in both human tissues and in dorsal root ganglia (DRG) from animal models of type 1 and type 2 diabetes. We hypothesized that loss of direct insulin signaling in diabetes contributes to loss of mitochondrial function in DRG neurons and to development of neuropathy. Sensory neurons obtained from age-matched adult control or streptozotocin (STZ)-induced type 1 diabetic rats were cultured with or without insulin before determining mitochondrial respiration and expression of mitochondrial respiratory chain and insulin signaling-linked proteins. For in vivo studies age-matched control rats and diabetic rats with or without trace insulin supplementation were maintained for 5months before DRG were analyzed for respiratory chain gene expression and cytochrome c oxidase activity. Insulin (10nM) significantly (P<0.05) increased phosphorylation of Akt and P70S6K by 4-fold and neurite outgrowth by 2-fold in DRG cultures derived from adult control rats. Insulin also augmented the levels of selective mitochondrial respiratory chain proteins and mitochondrial bioenergetics parameters in DRG cultures from control and diabetic rats, with spare respiratory capacity increased by up to 3-fold (P<0.05). Insulin-treated diabetic animals exhibited improved thermal sensitivity in the hind paw and had increased dermal nerve density compared to untreated diabetic rats, despite no effect on blood glucose levels. In DRG of diabetic rats there was suppressed expression of mitochondrial respiratory chain proteins and cytochrome c oxidase activity that was corrected by insulin therapy. Insulin elevates mitochondrial respiratory chain protein expression and function in sensory neurons and this is associated with enhanced neurite outgrowth and protection against indices of neuropathy.

MiRNA molecular profiles in human medical conditions: connecting lung cancer and lung development phenomena.

MiRNAs are endogenous, single stranded ~22-nucleotide non-coding RNAs (ncRNAs) which are transcribed by RNA polymerase II and mediate negative post-transcriptional gene regulation through binding to 3'untranslated regions (UTR), possibly open reading frames (ORFs) or 5'UTRs of target mRNAs. MiRNAs are involved in the normal physiology of eukaryotic cells, so dysregulation may be associated with diseases like cancer, and neurodegenerative, heart and other disorders. Among all cancers, lung cancer, with high incidence and mortality worldwide, is classified into two main groups: non-small cell lung cancer and small cell lung cancer. Recent promising studies suggest that gene expression profiles and miRNA signatures could be a useful step in a noninvasive, low-cost and repeatable screening process of lung cancer. Similarly, every stage of lung development during fetal life is associated with specific miRNAs. Since lung development and lung cancer phenomena share the same physiological, biological and molecular processes like cell proliferation, development and shared mRNA or expression regulation pathways, and according to data adopted from various studies, they may have partially shared miRNA signature. Thus, focusing on lung cancer in relation to lung development in miRNA studies might provide clues for lung cancer diagnosis and prognosis.

Sex determination using free fetal DNA at early gestational ages: a comparison between a modified mini-STR genotyping method and real-time PCR.

OBJECTIVE: Recently the use of free fetal deoxyribonucleic acid (DNA) in maternal plasma and serum has been applicable for noninvasive prenatal genetic diagnosis. In this study, we applied a new algorithmic base conventional polymerase chain reaction (PCR) genotyping method and also real-time PCR for detecting fetal X and Y-chromosome sequences in maternal plasma to determine fetal sex in pregnant women in their early gestational ages (5-13 weeks). Finally, we compared the efficiency of each method in sex determination. STUDY DESIGN: DNA was extracted from 106 pregnant women and their husbands' blood samples. Fetus mini-short tandem repeat (STR) genotyping was accomplished through amplification of 19 mini-STRs and 3 non-STR markers using conventional PCR followed by polyacrylamide gel electrophoresis analysis. Simultaneously, TaqMan real-time PCR was done with the use of DYS14-specific primers and probe. RESULTS: In conventional PCR method, 47 cases were diagnosed to be male and 49 to be female. In comparison, real-time PCR amplified DYS14 (Y-marker) sequences in 45 pregnant women plasma samples. Sensitivity and specificity were calculated to be 95.9% and 98% for conventional PCR and 91.8% and 100% for real-time PCR method, respectively. CONCLUSION: According to our study, the conventional PCR method was more sensitive than real-time PCR and it could be employed in future clinical diagnostics singly or in combination with real-time PCR.