Adipose-Derived Stem Cells Spontaneously Express Neural Markers When Grown in a PEG-Based 3D Matrix.

Neurological diseases are among the leading causes of disability and death worldwide and remain difficult to treat. Tissue engineering offers avenues to test potential treatments; however, the development of biologically accurate models of brain tissues remains challenging. Given their neurogenic potential and availability, adipose-derived stem cells (ADSCs) are of interest for creating neural models. While progress has been made in differentiating ADSCs into neural cells, their differentiation in 3D environments, which are more representative of the in vivo physiological conditions of the nervous system, is crucial. This can be achieved by modulating the 3D matrix composition and stiffness. Human ADSCs were cultured for 14 days in a 1.1 kPa polyethylene glycol-based 3D hydrogel matrix to assess effects on cell morphology, cell viability, proteome changes and spontaneous neural differentiation. Results showed that cells continued to proliferate over the 14-day period and presented a different morphology to 2D cultures, with the cells elongating and aligning with one another. The proteome analysis revealed 439 proteins changed in abundance by >1.5 fold. Cyclic nucleotide 3'-phosphodiesterase (CNPase) markers were identified using immunocytochemistry and confirmed with proteomics. Findings indicate that ADSCs spontaneously increase neural marker expression when grown in an environment with similar mechanical properties to the central nervous system.

Identification of some calcium binding proteins and neural cell markers in rat testis and epididymis during postnatal development.

Calcium-binding proteins (CaBPs) have an essential role in male reproduction by modulating calcium ion metabolism. Although the brain and testis are structurally and functionally different, they show a high degree of transcriptomic and proteomic similarities. The purpose of the present study was to explore some CaBPs (Iba-1, Calbindin, Calretinin and Parvalbumin) and neural cell markers (CNPase, NG2 and Drebrin) expression in rat testis and epididymis during postnatal periods by using immunohistochemistry. Iba-1, calbindin, calretinin, parvalbumin, CNPase, NG2 and drebrin were expressed in the epididymal epithelium in each postnatal period. Iba-1 and calbindin expression showed stage-dependent expression in spermatids. CaBPs and neural cell markers showed a positive reaction in Leydig cells in the postpubertal and mature periods. Sertoli cells, gonocytes, spermatogonium, and peritubular myoid cells showed heterogeneity in the expression of CaBPs and nerve markers throughout postnatal development. Interestingly, CNPase, NG2 and drebrin were positive in spermatocytes, spermatids, and sperm. Expression dynamics of calcium-binding proteins and nerve markers showed differences in germ cells and somatic cells during postnatal development. The expression of these proteins in the testis and epididymis supports that they may have important roles in reproductive physiology.

Sirt3 increases CNPase enzymatic activity through deacetylation and facilitating substrate accessibility.

Myocardial 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) metabolizes a nucleoside 2',3'-cyclic phosphate to a nucleoside 2'-phosphate. Recently, the roles of CNPase in the pathophysiological processes of heart failure have emerged. The mitochondrial acylome subjected to SIRT3 regulation give us comprehensive understanding of acylation modifications to a vast array of protein targets, and the list of acetylated mitochondrial proteins is still growing. However, it remains elusive whether CNPase is subjected to the regulation of acetylation and deacetylation, and the effects of which on CNPase enzymatic activity are still unknown. In this study, the mitochondrial distribution of CNPase was identified by immunofluorescence and cytosol/mitochondria fractioning. The immunofluorescence staining pattern of CNPase and Sirt3 overlapped on the same focal plane. Moreover, Sirt3 associates directly with CNPase, and the CNPase enzymatic activity was subjected to Sirt3 activity. Then biochemical methods using acetic anhydride was employed to acetylate the CNPase proteins, the enzymatic activity of CNPase decreased. Furthermore, co-immunoprecipitation coupled mass spectrometry identifies K196, K379, K128 as the main acetylation sites. Molecular dynamic simulation shows that acetylation modification suppressed the CNPase enzymatic activity through decreasing the opening probability of the binding pocket and restricting substrate accessibility. Together with these findings, this study reveals a molecular mechanism underlying Sirt3 regulating CNPase enzymatic activity, and suggests that targeting CNPase's post-translational modifications represents a promising therapeutic strategy.

CNPase, a 2',3'-Cyclic-nucleotide 3'-phosphodiesterase, as a Therapeutic Target to Attenuate Cardiac Hypertrophy by Enhancing Mitochondrial Energy Production.

Heart failure is the end-stage of all cardiovascular diseases with a ~25% 5-year survival rate, and insufficient mitochondrial energy production to meet myocardial demand is the hallmark of heart failure. Mitochondrial components involved in the regulation of ATP production remain to be fully elucidated. Recently, roles of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) in the pathophysiological processes of heart diseases have emerged, implicated by evidence that mitochondrial CNPase proteins are associated with mitochondrial integrity under metabolic stress. In this study, a zebrafish heart failure model was established, by employing antisense morpholino oligonucleotides and the CRISPR-Cas9 gene-editing system, which recapitulates heart failure phenotypes including heart dysfunction, pericardial edema, ventricular enlargement, bradycardia, and premature death. The translational implications of CNPase in the pathophysiological process of heart failure were tested in a pressure overload-induced heart hypertrophy model, which was carried out in rats through transverse abdominal aorta constriction (TAAC). AAV9-mediated myocardial delivery of CNPase mitigated the hypertrophic response through the specific hydrolysis of 2'-3'-cyclic nucleotides, supported by the decrease of cardiac hypertrophy and fibrosis, the integrity of mitochondrial ultrastructure, and indicators of heart contractility in the AAV9-TAAC group. Finally, the biometrics of a mitochondrial respiration assay carried out on a Seahorse cellular energy analyzer demonstrated that CNPase protects mitochondrial respiration and ATP production from AngII-induced metabolic stress. In summary, this study provides mechanistic insights into CNPase-2',3'-cyclic nucleotide metabolism that protects the heart from energy starvation and suggests novel therapeutic approaches to treat heart failure by targeting CNPase activity.

Effect of Melatonin on Rat Heart Mitochondria in Acute Heart Failure in Aged Rats.

Excessive generation of reactive oxygen species (ROS) in mitochondria and the opening of the nonselective mitochondrial permeability transition pore are important factors that promote cardiac pathologies and dysfunction. The hormone melatonin (MEL) is known to improve the functional state of mitochondria via an antioxidant effect. Here, the effect of MEL administration on heart mitochondria from aged rats with acute cardiac failure caused by isoprenaline hydrochloride (ISO) was studied. A histological analysis revealed that chronic intake of MEL diminished the age-dependent changes in the structure of muscle fibers of the left ventricle, muscle fiber swelling, and injury zones characteristic of acute cardiac failure caused by ISO. In acute heart failure, the respiratory control index (RCI) and the Ca2+ retention capacity in isolated rat heart mitochondria (RHM) were reduced by 30% and 40%, respectively, and mitochondrial swelling increased by 34%. MEL administration abolished the effect of ISO. MEL partially prevented ISO-induced changes at the subunit level of respiratory complexes III and V and drastically decreased the expression of complex I subunit NDUFB8 both in control RHM and in RHM treated with ISO, which led to the inhibition of ROS production. MEL prevents the mitochondrial dysfunction associated with heart failure caused by ISO. It was shown that the level of 2',3'-cyclicnucleotide-3'-phosphodiasterase (CNPase), which is capable of protecting cells in aging, increased in acute heart failure. MEL also retained the CNPase content in RHM both in control experiments and after ISO-induced heart damage. We concluded that an increase in the CNPase level promotes cardioprotection.

Effect of skilled reaching training and enriched environment on generation of oligodendrocytes in the adult sensorimotor cortex and corpus callosum.

BACKGROUND: Increased motor activity or social interactions through enriched environment are strong stimulators of grey and white matter plasticity in the adult rodent brain. In the present study we evaluated whether specific reaching training of the dominant forelimb (RT) and stimulation of unspecific motor activity through enriched environment (EE) influence the generation of distinct oligodendrocyte subpopulations in the sensorimotor cortex and corpus callosum of the adult rat brain. Animals were placed in three different housing conditions: one group was transferred to an EE, a second group received daily RT, whereas a third group remained in the standard cage. Bromodeoxyuridine (BrdU) was applied at days 2-6 after start of experiments and animals were allowed to survive for 10 and 42 days. RESULTS: Enriched environment and daily reaching training of the dominant forelimb significantly increased the number of newly differentiated GSTπ+ oligodendrocytes at day 10 and newly differentiated CNPase+ oligodendrocytes in the sensorimotor cortex at day 42. The myelin level as measured by CNPase expression was increased in the frontal cortex at day 42. Distribution of newly differentiated NG2+ subpopulations changed between 10 and 42 days with an increase of GSTπ+ subtypes and a decrease of NG2+ cells in the sensorimotor cortex and corpus callosum. Analysis of neuronal marker doublecortin (DCX) showed that more than half of NG2+ cells express DCX in the cortex. The number of new DCX+NG2+ cells was reduced by EE at day 10. CONCLUSIONS: Our results indicate for the first time that specific and unspecific motor training conditions differentially alter the process of differentiation from oligodendrocyte subpopulations, in particular NG2+DCX+ cells, in the sensorimotor cortex and corpus callosum.

Discovery and Roles of 2',3'-cAMP in Biological Systems.

In 2009, investigators using ultra-performance liquid chromatography-tandem mass spectrometry to measure, by selected reaction monitoring, 3',5'-cAMP in the renal venous perfusate from isolated, perfused kidneys detected a large signal at the same m/z transition (330 → 136) as 3',5'-cAMP but at a different retention time. Follow-up experiments demonstrated that this signal was due to a positional isomer of 3',5'-cAMP, namely, 2',3'-cAMP. Soon thereafter, investigative teams reported the detection of 2',3'-cAMP and other 2',3'-cNMPs (2',3'-cGMP, 2',3'-cCMP, and 2',3'-cUMP) in biological systems ranging from bacteria to plants to animals to humans. Injury appears to be the major stimulus for the release of these unique noncanonical cNMPs, which likely are formed by the breakdown of RNA. In mammalian cells in culture, in intact rat and mouse kidneys, and in mouse brains in vivo, 2',3'-cAMP is metabolized to 2'-AMP and 3'-AMP; and these AMPs are subsequently converted to adenosine. In rat and mouse kidneys and mouse brains, injury releases 2',3'-cAMP, 2'-AMP, and 3'-AMP into the extracellular compartment; and in humans, traumatic brain injury is associated with large increases in 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine in the cerebrospinal fluid. These findings motivate the extracellular 2',3'-cAMP-adenosine pathway hypothesis: intracellular production of 2',3'-cAMP → export of 2',3'-cAMP → extracellular metabolism of 2',3'-cAMP to 2'-AMP and 3'-AMP → extracellular metabolism of 2'-AMP and 3'-AMP to adenosine. Since 2',3'-cAMP has been shown to activate mitochondrial permeability transition pores (mPTPs) leading to apoptosis and necrosis and since adenosine is generally tissue protective, the extracellular 2',3'-cAMP-adenosine pathway may be a protective mechanism [i.e., removes 2',3'-cAMP (an intracellular toxin) and forms adenosine (a tissue protectant)]. This appears to be the case in the brain where deficiency in CNPase (the enzyme that metabolizes 2',3'-cAMP to 2-AMP) leads to increased susceptibility to brain injury and neurological diseases. Surprisingly, CNPase deficiency in the kidney actually protects against acute kidney injury, perhaps by preventing the formation of 2'-AMP (which turns out to be a renal vasoconstrictor) and by augmenting the mitophagy of damaged mitochondria. With regard to 2',3'-cNMPs and their downstream metabolites, there is no doubt much more to be discovered.

Role of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the renal 2',3'-cAMP-adenosine pathway.

Energy depletion increases the renal production of 2',3'-cAMP (a positional isomer of 3',5'-cAMP that opens mitochondrial permeability transition pores) and 2',3'-cAMP is converted to 2'-AMP and 3'-AMP, which in turn are metabolized to adenosine. Because the enzymes involved in this "2',3'-cAMP-adenosine pathway" are unknown, we examined whether 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) participates in the renal metabolism of 2',3'-cAMP. Western blotting and real-time PCR demonstrated expression of CNPase in rat glomerular mesangial, preglomerular vascular smooth muscle and endothelial, proximal tubular, thick ascending limb and collecting duct cells. Real-time PCR established the expression of CNPase in human glomerular mesangial, proximal tubular and vascular smooth muscle cells; and the level of expression of CNPase was greater than that for phosphodiesterase 4 (major enzyme for the metabolism of 3',5'-cAMP). Overexpression of CNPase in rat preglomerular vascular smooth muscle cells increased the metabolism of exogenous 2',3'-cAMP to 2'-AMP. Infusions of 2',3'-cAMP into isolated CNPase wild-type (+/+) kidneys increased renal venous 2'-AMP, and this response was diminished by 63% in CNPase knockout (-/-) kidneys, whereas the conversion of 3',5'-cAMP to 5'-AMP was similar in CNPase +/+ vs. -/- kidneys. In CNPase +/+ kidneys, energy depletion (metabolic poisons) increased kidney tissue levels of adenosine and its metabolites (inosine, hypoxanthine, xanthine, and uric acid) without accumulation of 2',3'-cAMP. In contrast, in CNPase -/- kidneys, energy depletion increased kidney tissue levels of 2',3'-cAMP and abolished the increase in adenosine and its metabolites. In conclusion, kidneys express CNPase, and renal CNPase mediates in part the renal 2',3'-cAMP-adenosine pathway.

Neurogenic marker expression in differentiating human adipose derived adult mesenchymal stem cells.

Background: Adipose-derived stem cells (ADSCs) are increasingly utilised in the field of neural regeneration due to their high accessibility and capacity for differentiation into neural like cells. Culturing ADSCs in the presence of various growth factors, small molecules and combinations thereof have shown promise in this regard; however, these protocols are generally complex, time-consuming and costly. The need for commercially available and chemically defined growth media/supplements is required to facilitate further developments in this area. Methods: In this study, we have examined the neural differentiation and proliferation potential of the commercially available supplements B27, CultureOne (C1) and N2 on human ADSCs (hADSCs). Through a combination of immunocytochemistry, cytokine analysis, and CNPase enzymatic assays, we provide novel insight into the neural differentiation effects of B27, C1 and N2 on hADSCs. Results: The study found that C1 and N2 supplements initiated neural differentiation of the cells, with C1 pushing differentiation towards an oligodendrocytic lineage and N2 initiating neuronal differentiation. This suggests that C1 and N2 supplements can be used to drive neural differentiation in hADSCs. However, B27 did not show significant differentiation in the time frame in which the experiments took place and therefore is unsuitable for this purpose. Conclusions: These findings highlight the utility of commercially available supplements in the neural differentiation of ADSCs and may assist in establishing simpler, more affordable differentiation protocols.

Impact of chronic sleep deprivation and sleep recovery on hippocampal oligodendrocytes, anxiety-like behavior, spatial learning and memory of rats.

Sleep and its quality play an important role in memory, cognition, and quality of life. Sleep deprivation-induced changes in hippocampal neurons and behavior have been studied widely, in contrast, the extent of damage to oligodendrocytes have not been fully understood. The present study aims to investigate chronic sleep deprivation (CSD) and sleep recovery-induced changes in oligodendrocytes of the hippocampus, cognition, and behavior of rats. Male Sprague-Dawley rats (n = 48) were grouped as control, sham control (SC), CSD, and CSD+sleep recovery (CSD+SR) (n = 12/group). CSD and CSD+SR group rats were sleep deprived for 21-days. After CSD, the CSD+SR group rats sleep recovered for 21-days. Oxidative markers, CNPase+ve oligodendrocytes, CNPase intensity, and CNPase gene expression were measured in the hippocampus, and the anxiety-like behavior, spatial learning, and memory were assessed. The 21-days of CSD significantly (p < 0.001) increased oxidative stress and significantly (p < 0.001) reduced the number of CNPase+ve oligodendrocytes, CNPase intensity, and CNPase gene expression when compared to controls. The increased oxidative stress was correlated with reduced CNPase+ve oligodendrocytes, CNPase intensity, and CNPase gene expression (r = -0.9). In-line with cellular changes, an increased (p < 0.01) anxiety-like behavior and impaired spatial memory were observed in the CSD group compared to controls. The 21-days of sleep recovery significantly (p < 0.01) reduced oxidative stress and anxiety-like behavior, improved spatial memory, increased CNPase intensity and CNPase gene expression, and non-significant (p > 0.05) increase in CNPase+ve oligodendrocytes compared to CSD. Overall, the 21-days of CSD reduced the number of CNPase+ve oligodendrocytes in the hippocampus, increased anxiety, and impaired spatial memory in rats. Though the 21-day sleep recovery showed an improvement in all parameters, it was not sufficient to completely reverse the CSD-induced changes to the control level.

Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

EGF-Coupled Gold Nanoparticles Increase the Expression of CNPase and the Myelin-Associated Proteins MAG, MOG, and MBP in the Septal Nucleus Demyelinated by Cuprizone.

Current pharmacological therapies against demyelinating diseases are not quite satisfactory to promote remyelination. Epidermal growth factor (EGF) can expand the population of oligodendrocyte precursor cells (OPCs) that may help with the remyelination process, but its delivery into the injured tissue is still a biomedical challenge. Gold nanoparticles (GNPs) may be a useful tool for drug delivery into the brain. To evaluate remyelination in the septal nucleus, we administered intracerebral GNPs coupled with EGF (EGF-GNPs). C57BL6/J mice were demyelinated with 0.4% cuprizone (CPZ) and divided into several groups: Sham, Ctrl, GNPs, EGF, and EGF-GNPs. We evaluated the remyelination process at two time-points: 2 weeks and 3 weeks post-injection (WPI) of each treatment. We used the rotarod for evaluating motor coordination. Then, we did a Western blot analysis myelin-associated proteins: CNPase, MAG, MOG, and MBP. EGF-GNPs increase the expression of CNPase, MAG, and MOG at 2 WPI. At 3 WPI, we found that the EGF-GNPs treatment improves motor coordination and increases MAG, MOG, and MBP. EGF-GNPs enhance the expression of myelin-associated proteins and improve the motor coordination in mice. Thus, EGF-associated GNPs may be a promising pharmacological vehicle for delivering long-lasting drugs into the brain.

Diffuse Traumatic Injury in the Mouse Disrupts Axon-Myelin Integrity in the Cerebellum.

Cerebellar dysfunction after traumatic brain injury (TBI) is commonly suspected based on clinical symptoms, although cerebellar pathology has rarely been investigated. To address the hypothesis that the cerebellar axon-myelin unit is altered by diffuse TBI, we used the central fluid percussion injury (cFPI) model in adult mice to create widespread axonal injury by delivering the impact to the forebrain. We specifically focused on changes in myelin components (myelin basic protein [MBP], 2',3'-cyclic nucleotide 3'-phosphodiesterase [CNPase], nodal/paranodal domains [neurofascin (Nfasc), ankyrin-G], and phosphorylated neurofilaments [SMI-31, SMI-312]) in the cerebellum, remote from the impact, at two, seven, and 30 days post-injury (dpi). When compared with sham-injured controls, cerebellar MBP and CNPase protein levels were decreased at 2 dpi that remained reduced up to 30 dpi. Diffuse TBI induced different effects on neuronal (Nfasc 186, Nfasc 140) and glial (Nfasc 155) neurofascin isoforms that play a key role in the assembly of the nodes of Ranvier. Expression of Nfasc 140 in the cerebellum increased at 7 dpi, in contrast to Nfasc 155 levels, which were decreased. Although neurofascin binding partner ankyrin-G protein levels decreased acutely after cFPI, its expression levels increased at 7 dpi and remained unchanged up to 30 dpi. The TBI-induced reduction in neurofilament phosphorylation (SMI-31) observed in the cerebellum was closely associated with decreased levels of the myelin proteins MBP and CNPase. This is the first evidence of temporal and spatial structural changes in the axon-myelin unit in the cerebellum, remote from the location of the impact site, in a diffuse TBI model in mice.

Histopathological and Immunohistochemical Evaluation of Canine Nerve Sheath Tumors and Proposal for an Updated Classification.

Nerve sheath tumors are a group of tumors originating from Schwann cells, fibroblasts, and perineurial cells. In veterinary pathology, the terminology for nerve sheath tumors remains inconsistent, and many pathologists follow the human classification of such tumors in practice. Immunohistochemistry plays an important role in the diagnosis of nerve sheath tumors, but specific immunohistochemical and molecular biomarkers are lacking. In our study, we histopathologically reevaluated 79 canine nerve sheath tumors and assessed their reactivity for the immunohistochemical markers Sox10, claudin-1, GFAP, CNPase, and Ki-67. Based on the results, we classified the tumors according to the most recent human classification. Twelve cases were diagnosed as benign nerve sheath tumors, including six neurofibromas, three nerve sheath myxomas, two hybrid nerve sheath tumors (perineurioma/neurofibroma and perineurioma/schwannoma), and one schwannoma. Sixty-seven tumors were malignant nerve sheath tumors, including fifty-six conventional, four perineural, one epithelioid malignant nerve sheath tumor, and six malignant nerve sheath tumors with divergent differentiation. We believe that with the application of the proposed panel, an updated classification of canine nerve sheath tumors could largely follow the recent human WHO classification of tumors of the cranial and paraspinal nerves, but prospective studies would be needed to assess its prognostic value.

Co-Ultramicronized Palmitoylethanolamide/Luteolin-Induced Oligodendrocyte Precursor Cell Differentiation is Associated With Tyro3 Receptor Upregulation.

Remyelination in patients with multiple sclerosis frequently fails, especially in the chronic phase of the disease promoting axonal and neuronal degeneration and progressive disease disability. Drug-based therapies able to promote endogenous remyelination capability of oligodendrocytes are thus emerging as primary approaches to multiple sclerosis. We have recently reported that the co-ultramicronized composite of palmitoylethanolamide and the flavonoid luteolin (PEALut) promotes oligodendrocyte precursor cell (OPC) maturation without affecting proliferation. Since TAM receptor signaling has been reported to be important modulator of oligodendrocyte survival, we here evaluated the eventual involvement of TAM receptors in PEALut-induced OPC maturation. The mRNAs related to TAM receptors -Tyro3, Axl, and Mertk- were all present at day 2 in vitro. However, while Tyro3 gene expression significantly increased upon cell differentiation, Axl and Mertk did not change during the first week in vitro. Tyro3 gene expression developmental pattern resembled that of MBP myelin protein. In OPCs treated with PEALut the developmental increase of Tyro3 mRNA was significantly higher as compared to vehicle while was reduced gene expression related to Axl and Mertk. Rapamycin, an inhibitor of mTOR, prevented oligodendrocyte growth differentiation and myelination. PEALut, administered to the cultures 30 min after rapamycin, prevented the alteration of mRNA basal expression of the TAM receptors as well as the expression of myelin proteins MBP and CNPase. Altogether, data obtained confirm that PEALut promotes oligodendrocyte differentiation as shown by the increase of MBP and CNPase and Tyro3 mRNAs as well as CNPase and Tyro3 immunostainings. The finding that these effects are reduced when OPCs are exposed to rapamycin suggests an involvement of mTOR signaling in PEALut effects.

Peripheral Nerve Sheath Tumors Resembling Human Atypical Neurofibroma in Goldfish (Carassius auratus, Linnaeus, 1758).

Skin spindle cell tumors (SSTs) frequently occur in fishes, with peripheral nerve sheath tumors (PNSTs) being the most commonly reported neoplasms in goldfish. However, distinguishing PNSTs from other SCTs is not always possible when relying exclusively on routine cytological and histopathological findings. Therefore, the aim of this study is to characterize six skin nodules, resembling atypical neurofibromas in humans, found in six cohabiting goldfish (Carassius auratus), and to determine a minimal subset of special stains required to correctly identify PNSTs in this species. Routine cytology and histopathology were indicative of an SCT with nuclear atypia in all cases, with randomly distributed areas of hypercellularity and loss of neurofibroma architecture. Muscular and fibroblastic tumors were excluded using Azan trichrome staining. Alcian blue and Gomori's reticulin stains revealed the presence of intratumoral areas of glycosaminoglycans or mucins and basement membrane fragments, respectively. PAS and PAS-diastase stains confirmed the latter finding and revealed intra- and extracellular glycogen granules. Immunohistochemistry displayed multifocal, randomly distributed aggregates of neoplastic cells positive for S100 protein and CNPase, intermingled with phosphorylated and non-phosphorylated neurofilament-positive axons. Collectively, these findings are consistent with a PNST resembling atypical neurofibroma in humans, an entity not previously reported in goldfish, and suggest that Azan trichrome staining, reticulin staining, and immunohistochemistry for S100 protein and CNPase represent a useful set of special stains to identify and characterize PNSTs in this species.

Quantitative morphometric and cell-type-specific population analysis of microglia-enriched cultures subcloned to high purity from newborn rat brains.

Morphological and functional characterizations of cultured microglia are essential for the improved understanding of their roles in neuronal health and disease. Although some studies (phenotype analysis, phagocytosis) can be carried out in mixed or microglia-enriched cultures, in others (gene expression) pure microglia must be used. If the use of genetically modified microglial cells is not feasible, isolation of resident microglia from nervous tissue must be carried out. In this study, mixed primary cultures were established from the forebrains of newborn rats. Secondary microglia-enriched cultures were then prepared by shaking off these cells from the primary cultures, which were subsequently used to establish tertiary cultures by further shaking off the easily detachable microglia. The composition of these cultures was quantitatively analyzed by immunocytochemistry of microglia-, astrocyte-, oligodendrocyte- and neuron-specific markers to determine yield and purity. Microglia were quantitatively characterized regarding morphological and proliferation aspects. Secondary and tertiary cultures typically exhibited 73.3% ± 17.8% and 93.1% ± 6.0% purity for microglia, respectively, although the total number of microglia in the latter was much smaller. One in seven attempts of culturing the tertiary cultures had ~99% purity for microglia. The overall yield from the number of cells plated at DIV0 to the Iba1-positive microglia in tertiary cultures was ~1%. Astrocytic and neuronal contamination progressively decreased during subcloning, while oligodendrocytes were found sporadically throughout culturing. Although the tertiary microglia cultures had a low yield, they produced consistently high purity for microglia; after validation, such cultures are suitable for purity-sensitive functional screenings (gene/protein expression).

The Effects of PK11195 and Protoporphyrin IX Can Modulate Chronic Alcohol Intoxication in Rat Liver Mitochondria under the Opening of the Mitochondrial Permeability Transition Pore.

Decades of active research have shown that mitochondrial dysfunction, the associated oxidative stress, impaired anti-stress defense mechanisms, and the activation of the proapoptotic signaling pathways underlie pathological changes in organs and tissues. Pathologies caused by alcohol primarily affect the liver. Alcohol abuse is the cause of many liver diseases, such as steatosis, alcoholic steatohepatitis, fibrosis, cirrhosis, and, potentially, hepatocellular cancer. In this study, the effect of chronic alcohol exposure on rat liver mitochondria was investigated. We observed an ethanol-induced increase in sensitivity to calcium, changes in the level of protein kinase Akt and GSK-3ß phosphorylation, an induction of the mitochondrial permeability transition pore (mPTP), and strong alterations in the expression of mPTP regulators. Moreover, we also showed an enhanced effect of PK11195 and PPIX, on the parameters of the mPTP opening in rat liver mitochondria (RLM) isolated from ethanol-treated rats compared to the RLM from control rats. We suggest that the results of this study could help elucidate the mechanisms of chronic ethanol action on the mitochondria and contribute to the development of new therapeutic strategies for treating the effects of ethanol-related diseases.

Blockade of receptor for advanced glycation end products promotes oligodendrocyte autophagy in spinal cord injury.

Receptor for advanced glycation end product (RAGE) is involved in neuronal inflammation, cell cycle and differentiation. However, the role of RAGE in autophagy in the process of spinal cord injury (SCI) is yet unknown. The present study investigated the effect of RAGE blockade on autophagy in SCI. A rat Allen SCI model was established and the animals were micro-injected with rabbit RAGE neutralizing antibody or rabbit polyclonal Ig G immediately after the injury. The oligodendrocytes(OLs) marker, 2', 3'-cyclic nucleotide 3'-phosphodiesterase(CNPase) and autophagy-related marker microtubule associated protein light chain 3B(LC3B) were evaluated by Western blot. Furthermore, myelin basic protein (MBP) and LC3B double staining were observed in the SCI via immunofluorescence. The results showed that RAGE blockade reduced the expression of CNPase, promoted LC3B-II/I and p62 expression after SCI. In addition, the MBP/LC3B double positive oligodendrocytes-expressing LC3B was up-regulated by RAGE blockade. Moreover, RAGE blockade attenuated the neuronal survival at ventral horn after SCI. The present study revealed the role of RAGE in maintaining oligodendrocyte autophagy to promote neuronal regeneration post-SCI.

Oxidative stress induces release of 2'-AMP from microglia.

BACKGROUND: Microglia metabolize exogenous 2'-AMP and 3'-AMP (non-canonical nucleotides) to adenosine and exogenous 2'-AMP and 3'-AMP (via conversion to adenosine) inhibit the production of inflammatory cytokines by microglia. This suggests that if microglia release endogenous 2'-AMP and/or 3'-AMP in response to injurious stimuli, this would complete an autocrine/paracrine mechanism that attenuates the over-activation of microglia during brain injury. Here we investigated in microglia (and for comparison astrocytes and neurons) the effects of injurious stimuli on extracellular and intracellular levels of 2',3'-cAMP (2'-AMP and 3'-AMP precursor), 2'-AMP, and 3'-AMP. METHODS: Experiments were conducted in primary cultures of rat microglia, astrocytes, and neurons. Cells were exposed to oxygen/glucose deprivation, iodoacetate plus 2,4-dinitrophenol (metabolic inhibitors), glutamate, or H2O2 for one hour, and extracellular and intracellular 2',3'-cAMP, 2'-AMP, and 3'-AMP were measured by UPLC-MS/MS. KEY RESULTS: In microglia, H2O2 increased extracellular levels of 2'-AMP, but not 3'-AMP, by ∼16-fold (from 0.17 ±â€¯0.11 to 2.78 ±â€¯0.27 ng/106 cells; n = 13; mean ±â€¯SEM; P < 0.000005). H2O2 also induced oxidative changes in cellular proteins as detected by an increased number of carbonyl groups in protein side chains. In contrast, oxygen/glucose deprivation, metabolic inhibitors, or glutamate had no effect on either extracellular 2'-AMP or 3'-AMP levels. In astrocytes and neurons, none of the injurious stimuli increased extracellular 2'-AMP or 3'-AMP. CONCLUSIONS: Oxidative stress (but not oxygen/glucose deprivation, energy deprivation, or excitotoxicity) induces microglia (but not astrocytes or neurons) to release 2'-AMP, but not 3'-AMP. The 2',3'-cAMP/2'-AMP/adenosine pathway mechanism may serve to prevent over-activation of microglia in response to oxidative stress.