Within the microenvironment of multiple sclerosis lesions, oligodendrocytes are subject to metabolic stress reflecting effects of focal ischemia and inflammation. Previous studies have shown that ...under optimal conditions in vitro, the respiratory activity of human adult brain-derived oligodendrocytes is lower and more predominantly glycolytic compared to oligodendrocytes differentiated in vitro from post natal rat brain oligodendrocyte progenitor cells. In response to sub-lethal metabolic stress, adult human oligodendrocytes reduce overall energy production rate impacting the capacity to maintain myelination. Here, we directly compare the metabolic profiles of oligodendrocytes derived from adult rat brain with oligodendrocytes newly differentiated in vitro from oligodendrocyte progenitor cells obtained from the post natal rat brain, under both optimal culture and metabolic stress (low/no glucose) conditions. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. Our findings indicate that under optimal conditions, adult rat oligodendrocytes preferentially use glycolysis whereas newly differentiated post natal rat oligodendrocytes, and the oligodendrocyte progenitor cells from which they are derived, mainly utilize oxidative phosphorylation to produce ATP. Metabolic stress increases the rate of ATP production via oxidative phosphorylation and significantly reduces glycolysis in adult oligodendrocytes. The rate of ATP production was relatively unchanged in newly differentiated post natal oligodendrocytes under these stress conditions, while it was significantly reduced in oligodendrocyte progenitor cells. Our study indicates that both age and maturation influence the metabolic profile under optimal and stressed conditions, emphasizing the need to consider these variables for in vitro studies that aim to model adult human disease.
Dendritic polyglycerols (dPG), particularly dendritic polyglycerol sulfates (dPGS), have been intensively studied due to their intrinsic anti-inflammatory activity. As related to brain pathologies ...involving neuroinflammation, the current study examined if dPG and dPGS can (i) regulate neuroglial activation, and (ii) normalize the morphology and function of excitatory postsynaptic dendritic spines adversely affected by the neurotoxic 42 amino acid amyloid-β (Aβ
) peptide of Alzheimer disease (AD). The exact role of neuroglia, such as microglia and astrocytes, remains controversial especially their positive and negative impact on inflammatory processes in AD. To test dPGS effectiveness in AD models we used primary neuroglia and organotypic hippocampal slice cultures exposed to Aβ
peptide. Overall, our data indicate that dPGS is taken up by both microglia and astrocytes in a concentration- and time-dependent manner. The mechanism of action of dPGS involves binding to Aβ
, i.e., a direct interaction between dPGS and Aβ
species interfered with Aβ fibril formation and reduced the production of the neuroinflammagen lipocalin-2 (LCN2) mainly in astrocytes. Moreover, dPGS normalized the impairment of neuroglia and prevented the loss of dendritic spines at excitatory synapses in the hippocampus. In summary, dPGS has desirable therapeutic properties that may help reduce amyloid-induced neuroinflammation and neurotoxicity in AD.
The amyloid-β42 (Aβ42) peptide is believed to be the main culprit in the pathogenesis of Alzheimer disease (AD), impairing synaptic function and initiating neuronal degeneration. Soluble Aβ42 ...oligomers are highly toxic and contribute to progressive neuronal dysfunction, loss of synaptic spine density, and affect long-term potentiation (LTP). We have characterized a short, L-amino acid Aβ-oligomer Interacting Peptide (AIP) that targets a relatively well-defined population of low-n Aβ42 oligomers, rather than simply inhibiting the aggregation of Aβ monomers into oligomers. Our data show that AIP diminishes the loss of Aβ42-induced synaptic spine density and rescues LTP in organotypic hippocampal slice cultures. Notably, the AIP enantiomer (comprised of D-amino acids) attenuated the rough-eye phenotype in a transgenic Aβ42 fly model and significantly improved the function of photoreceptors of these flies in electroretinography tests. Overall, our results indicate that specifically "trapping" low-n oligomers provides a novel strategy for toxic Aβ42-oligomer recognition and removal.
Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. Here we report that a novel signal for delaying ...neutrophil apoptosis is the classic acute phase reactant, C-reactive protein (CRP). However, this anti-apoptotic activity is expressed only when the cyclic pentameric structure of CRP is lost, resulting in formation of modified or monomeric CRP (mCRP), which may be formed in inflamed tissues. By contrast, native pentameric CRP and CRP peptides 77-82, 174-185, and 201-206 failed to affect neutrophil apoptosis. The apoptosis delaying action of mCRP was markedly attenuated by an antibody against the low affinity IgG immune complex receptor (CD16) but not by an anti-CD32 antibody. mCRP evoked a transient concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to inhibition of caspase-3 and consequently to delaying apoptosis. Consistently, pharmacological inhibition of either ERK or Akt reversed the anti-apoptotic action of mCRP; however, they did not produce additive inhibition. Thus, mCRP, but not pentameric CRP or peptides derived from CRP, promotes neutrophil survival and may therefore contribute to amplification of the inflammatory response.
Alzheimer's disease is characterized by deposition of the amyloid β-peptide (Aβ) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can ...potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aβ peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation. In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aβ solution. We find that sulindac sulfide induced Aβ aggregates are structurally homogeneous. The C-terminal part of the peptide adopts a β-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in wild type fibril structures. (13)C-(19)F transferred echo double resonance experiments suggest that sulindac sulfide colocalizes with the Aβ peptide in the aggregate.
Oligodendrocytes (OLGs) produce and maintain myelin in the central nervous system (CNS). In the demyelinating autoimmune disease multiple sclerosis, OLGs are damaged and those remaining fail to fully ...remyelinate CNS lesions. Therefore, current therapies directed to restrain the inflammation process with approaches that protect and reconstitute oligodendrocyte density would be essential to pave the way of myelin repair. A critical signal for oligodendrocytes is insulin‐like growth factor‐1 (IGF‐1), which promotes their development and ultimately myelin formation. PTEN inhibits the phosphoinositide 3‐kinase (PI3K)/Akt signaling, a convergence downstream pathway for growth factors such as IGF‐1. In this report, we temporarily inhibited PTEN activity by treating rat and human oligodendrocyte progenitors (OLPs) cultured alone or with dorsal root ganglion neurons (DRGNs) with bisperoxovanadium (phen). Our findings show that phen potentiates IGF‐1 actions by increasing proliferation of OLPs in a concentration‐dependent manner, and caused a sustained and time‐dependent activation of the main pathways: PI3K/Akt/mammalian target of rapamycin (mTOR) and MEK/ERK. At low concentrations, IGF‐1 and phen stimulated the differentiation of rat and human OLPs. Concordantly, the PTEN inhibitor together with IGF‐1 robustly augmented myelin basic protein accumulation in rat newborn and human fetal OLGs co‐cultured with DRGNs in a longer timeframe by promoting the elaboration of organized myelinated fibers as evidenced by confocal microscopy. Thus, our results suggest that a transient suppression of a potential barrier for myelination in combination with other therapeutic approaches including growth factors may be promising to improve the functional recovery of CNS injuries. GLIA 2013;62:64–77
Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by ...α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Since prolonged agonist stimulation can regulate the expression of various families of receptors, we examined this possibility. Pretreatment of progenitor cultures with 100 μM kainic acid (KA) for 1–24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced 45Ca2+ uptake. The maximum effect (70–80% decrease), observed in the 24 h-pretreated cells, was accompanied by a significant reduction in AMPA receptor subunits, as determined by Western blotting. GluR2/3 and GluR4 subunits were the most affected. Receptor down-regulation and 45Ca2+ uptake were only partially reversible upon KA removal. Furthermore, 24 h co-treatment of cultures with CNQX blocked the KA-induced decreases in calcium uptake. To address whether calpain, a calcium-activated protease, was implicated in the regulation of the AMPA receptor subunits, cultures were treated with the specific inhibitor PD150606 alone or in combination with KA for 24 h. Calpain inhibition significantly increased GluR1 in both conditions and partly reversed downregulation of GluR4 by KA. Collectively, these results indicate that calpain is not involved in the agonist-induced down-regulation of AMPA receptors subunits 2/3 in oligodendrocyte progenitors, while it downregulates GluR1 and GluR4.
•We have studied AMPA receptor subunit regulation in oligodendrocyte progenitors.•AMPA receptors are downregulated in differentiated oligodendrocytes.•Chronic agonist exposure downregulates AMPA receptor expression.•AMPA receptors downregulation is partially recovered 24 h after.•Calpain is involved in agonist-induced GluR1 and GluR4 AMPA receptor downregulation.
Fyn, a nonreceptor Src-like tyrosine kinase (SLK), plays an important role in oligodendrocyte differentiation and myelination in the brain. However, its role in myelination of peripheral nerves ...remains undefined. Here we report that selective inhibitors of SLKs (PP2 and SU6656) caused a dose-dependent decrease in the accumulation of several myelin proteins, including myelin basic protein (MBP), protein zero (P0) and myelin-associated glycoprotein (MAG) in rat Schwann cell-dorsal root ganglion neuron (SC-DRGN) co-cultures. Interestingly, SLK inhibition was insufficient to completely abrogate myelin synthesis, as removal of PP2 after several days of treatment permitted a partial recovery of myelin proteins expression. Furthermore, fewer and shorter myelinated segments formed in the continuous presence of PP2, although the myelin formed was normally compacted. PP2 also decreased the number of SCs expressing Krox-20, a master-regulatory transcription factor expressed by myelinating SCs, by 50%. These results were corroborated by selective knockdown of Fyn and Lyn kinases using siRNA. Extracellular matrix is important to SC differentiation and peripheral myelination. Using phospho-specific antibodies, we showed that addition of extracellular matrix extracts to SC-DRGN co-cultures resulted in the activation of ERK, Akt and p38 MAPK, three protein kinases involved in SC proliferation, differentiation and peripheral myelination. PP2 blocked the phosphorylation of all three kinases. Our results support a role for SLKs in the initiation of peripheral myelination via the activation of p38, Akt and ERK, which regulate Krox-20 expression and peripheral myelination.
► Inhibition of Src-like kinases using PP2 inhibits peripheral myelination ► PP2 treatment decreases the number and length of myelinated segments ► Krox-20 expression is reduced by PP2 and selective knockdown of Fyn and Lyn ► PP2 blocked ECM-induced ERK, Akt and p38 MAPK activation
Cadmium toxicity has been associated with learning disabilities and Parkinsonian symptoms in humans. We have previously shown that cultured oligodendrocytes are directly damaged by cadmium exposure. ...Here, we characterized the molecular mechanisms underlying cadmium-induced cell death in oligodendrocyte progenitors (OLP). Cadmium caused a concentration-dependent decrease in cell viability as assessed by mitochondrial dehydrogenase activity and by the cellular release of lactate dehydrogenase (LDH). A short exposure (1
h) to cadmium (25–100
μM), followed by several hours of recovery, produced a predominant apoptotic mechanism of cell death, involving the mitochondrial intrinsic pathway, as evidenced by nuclear condensation, DNA fragmentation, bax integration into the outer mitochondrial membrane, cytochrome
c release, and activation of caspases-9 and -3. Pretreatment of OLPs with the pan-caspase inhibitor, zVAD-fmk, prevented caspase-3 activation but only slightly reduced cell death 11
h after cadmium exposure and failed to prevent cadmium-induced bax insertion into the mitochondrial membrane. In contrast, the anti-oxidant N-acetyl cysteine blocked caspase-3 activation and significantly protected OLPs from cadmium-induced cell death. Continuous exposure (18–48
h) of OLPs to low micromolar concentrations (0.001–25
μM) of cadmium significantly decreased mitochondrial metabolic activity, increased LDH leakage starting at 5
μM and maximally activated caspase-3. These results suggest that cadmium induces OLP cell death mainly by apoptosis, and at higher concentrations or with prolonged exposure to the heavy metal there is an increase in cytoplasmic membrane damage, an index of necrosis. More importantly, transient exposure to cadmium is sufficient to damage OLPs and could in principle impair myelination in the neonate.
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