A nonlinear meccano for Alzheimer's emergence by amyloid ß-mediated glutamatergic hyperactivity.

The pathophysiological process of Alzheimer's disease (AD) is believed to begin many years before the formal diagnosis of AD dementia. This protracted preclinical phase offers a crucial window for potential therapeutic interventions, yet its comprehensive characterization remains elusive. Accumulating evidence suggests that amyloid-ß (Aß) may mediate neuronal hyperactivity in circuit dysfunction in the early stages of AD. At the same time, neural activity can also facilitate Aß accumulation through intricate feed-forward interactions, complicating elucidating the conditions governing Aß-dependent hyperactivity and its diagnostic utility. In this study, we use biophysical modeling to shed light on such conditions. Our analysis reveals that the inherently nonlinear nature of the underlying molecular interactions can give rise to the emergence of various modes of hyperactivity. This diversity in the mechanisms of hyperactivity may ultimately account for a spectrum of AD manifestations.

27-hydroxycholesterol promotes oligodendrocyte maturation: Implications for hypercholesterolemia-associated brain white matter changes.

Oxidized cholesterol metabolite 27-hydroxycholesterol (27-OH) is a potential link between hypercholesterolemia and neurodegenerative diseases since unlike peripheral cholesterol, 27-OH is transported across the blood-brain barrier. However, the effects of high 27-OH levels on oligodendrocyte function remain unexplored. We hypothesize that during hypercholesterolemia 27-OH may impact oligodendrocytes and myelin and thus contribute to the disconnection of neural networks in neurodegenerative diseases. To test this idea, we first investigated the effects of 27-OH in cultured oligodendrocytes and found that it induces cell death of immature O4+ /GalC+ oligodendrocytes along with stimulating differentiation of PDGFR+ oligodendrocyte progenitors (OPCs). Next, transgenic mice with increased systemic 27-OH levels (Cyp27Tg) underwent behavioral testing and their brains were immunohistochemically stained and lysed for immunoblotting. Chronic exposure to 27-OH in mice resulted in increased myelin basic protein (MBP) but not 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) or myelin oligodendrocyte glycoprotein (MOG) levels in the corpus callosum and cerebral cortex. Intriguingly, we also found impairment of spatial learning suggesting that subtle changes in myelinated axons of vulnerable areas like the hippocampus caused by 27-OH may contribute to impaired cognition. Finally, we found that 27-OH levels in cerebrospinal fluid from memory clinic patients were associated with levels of the myelination regulating CNPase, independently of Alzheimer's disease markers. Thus, 27-OH promotes OPC differentiation and is toxic to immature oligodendrocytes as well as it subtly alters myelin by targeting oligodendroglia. Taken together, these data indicate that hypercholesterolemia-derived higher 27-OH levels change the oligodendrocytic capacity for appropriate myelin remodeling which is a crucial factor in neurodegeneration and aging.

Functional characterization of cannabidiol effect on the serotonergic neurons of the dorsal raphe nucleus in rat brain slices.

Cannabidiol (CBD), the main non-psychoactive cannabinoid found in the cannabis plant, elicits several pharmacological effects via the 5-HT1A receptor. The dorsal raphe nucleus (DRN) is the main serotonergic cluster in the brain that expresses the 5-HT1A receptor. To date, the effect of CBD on the neuronal activity of DRN 5-HT cells and its interaction with somatodendritic 5-HT1A autoreceptors have not been characterized. Our aim was to study the effect of CBD on the firing activity of DRN 5-HT cells and the 5-HT1A autoreceptor activation by electrophysiological and calcium imaging techniques in male Sprague-Dawley rat brain slices. Perfusion with CBD (30 µM, 10 min) did not significantly change the firing rate of DRN 5-HT cells or the inhibitory effect of 5-HT (50-100 µM, 1 min). However, in the presence of CBD (30 µM, 10 min), the inhibitory effects of 8-OH-DPAT (10 nM) and ipsapirone (100 nM) were reduced by 66% and 53%, respectively. CBD failed to reverse ipsapirone-induced inhibition, whereas perfusion with the 5-HT1A receptor antagonist WAY100635 (30 nM) completely restored by 97.05 ± 14.63% the firing activity of 5-HT cells. Administration of AM251 (1 µM), MDL100907 (30 nM), or picrotoxin (20 µM) did not change the blockade produced by CBD (30 µM) on ipsapirone-induced inhibition. Our study also shows that CBD failed to modify the KCl (15 mM, 4 min)-evoked increase in [Ca2+]i or the inhibitory effect of ipsapirone (1 µM, 4 min) on KCl-evoked [Ca2+]i. In conclusion, CBD does not activate 5-HT1A autoreceptors, but it hindered the inhibitory effect produced by selective 5-HT1A receptor agonists on the firing activity of DRN 5-HT cells through a mechanism that does not involve CB1, 5-HT2A, or GABAA receptors. Our data support a negative allosteric modulation of DRN somatodendritic 5-HT1A receptor by CBD.

Recombinant Integrin ß1 Signal Peptide Blocks Gliosis Induced by Aß Oligomers.

Glial cells participate actively in the early cognitive decline in Alzheimer's disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aß) oligomers and integrin ß1. Here, we have generated four recombinant peptides including the extracellular domain of integrin ß1, and evaluated their capacity both to bind in vitro to Aß oligomers and to prevent in vivo Aß oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin ß1 that binds to Aß oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin ß1 N-terminal domain. This recombinant integrin ß1 signal peptide prevented Aß oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin ß1 signal peptide combined with or without Aß oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin ß1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aß oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aß oligomer/integrin ß1 signaling.

A Neuron, Microglia, and Astrocyte Triple Co-culture Model to Study Alzheimer's Disease.

Glial cells are essential to understand Alzheimer's disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated and optimized a 2D triple co-culture model with murine astrocytes, neurons and microglia, based on sequential seeding of each cell type. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aß (oAß) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory marker Arginase I, and reduced pro-inflammatory iNOS and IL-1ß, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Anti-inflammatory marker TGF-ß1 was also increased in the triple co-culture. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAß in the triple co-culture reduced synaptic markers and increased CD11b in microglia, which are hallmarks of AD. Consequently, we developed a straightforward and reproducible triple co-cultured model, where cells resemble physiological conditions better than in individual primary cultures: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology. Moreover, we are able to recapitulate Aß-induced synaptic loss and CD11b increase. This model emerges as a powerful tool to study neurodegeneration and neuroinflammation in the context of AD and other neurodegenerative diseases.

Amyloid ß / PKC-dependent alterations in NMDA receptor composition are detected in early stages of Alzheimer´s disease.

Amyloid beta (Aß)-mediated synapse dysfunction is an early event in Alzheimer's disease (AD) pathogenesis and previous studies suggest that NMDA receptor (NMDAR) dysregulation may contribute to these pathological effects. Although Aß peptides impair NMDAR expression and activity, the mechanisms mediating these alterations in the early stages of AD are unclear. Here, we observed that NMDAR subunit NR2B and PSD-95 levels were aberrantly upregulated and correlated with Aß42 load in human postsynaptic fractions of the prefrontal cortex in early stages of AD patients, as well as in the hippocampus of 3xTg-AD mice. Importantly, NR2B and PSD95 dysregulation was revealed by an increased expression of both proteins in Aß-injected mouse hippocampi. In cultured neurons, Aß oligomers increased the NR2B-containing NMDAR density in neuronal membranes and the NMDA-induced intracellular Ca2+ increase, in addition to colocalization in dendrites of NR2B subunit and PSD95. Mechanistically, Aß oligomers required integrin ß1 to promote synaptic location and function of NR2B-containing NMDARs and PSD95 by phosphorylation through classic PKCs. These results provide evidence that Aß oligomers modify the contribution of NR2B to NMDAR composition and function in the early stages of AD through an integrin ß1 and PKC-dependent pathway. These data reveal a novel role of Aß oligomers in synaptic dysfunction that may be relevant to early-stage AD pathogenesis.

RNA Localization and Local Translation in Glia in Neurological and Neurodegenerative Diseases: Lessons from Neurons.

Cell polarity is crucial for almost every cell in our body to establish distinct structural and functional domains. Polarized cells have an asymmetrical morphology and therefore their proteins need to be asymmetrically distributed to support their function. Subcellular protein distribution is typically achieved by localization peptides within the protein sequence. However, protein delivery to distinct cellular compartments can rely, not only on the transport of the protein itself but also on the transport of the mRNA that is then translated at target sites. This phenomenon is known as local protein synthesis. Local protein synthesis relies on the transport of mRNAs to subcellular domains and their translation to proteins at target sites by the also localized translation machinery. Neurons and glia specially depend upon the accurate subcellular distribution of their proteome to fulfil their polarized functions. In this sense, local protein synthesis has revealed itself as a crucial mechanism that regulates proper protein homeostasis in subcellular compartments. Thus, deregulation of mRNA transport and/or of localized translation can lead to neurological and neurodegenerative diseases. Local translation has been more extensively studied in neurons than in glia. In this review article, we will summarize the state-of-the art research on local protein synthesis in neuronal function and dysfunction, and we will discuss the possibility that local translation in glia and deregulation thereof contributes to neurological and neurodegenerative diseases.

Astrocytes in Alzheimer's Disease: Pathological Significance and Molecular Pathways.

Astrocytes perform a wide variety of essential functions defining normal operation of the nervous system and are active contributors to the pathogenesis of neurodegenerative disorders such as Alzheimer's among others. Recent data provide compelling evidence that distinct astrocyte states are associated with specific stages of Alzheimer´s disease. The advent of transcriptomics technologies enables rapid progress in the characterisation of such pathological astrocyte states. In this review, we provide an overview of the origin, main functions, molecular and morphological features of astrocytes in physiological as well as pathological conditions related to Alzheimer´s disease. We will also explore the main roles of astrocytes in the pathogenesis of Alzheimer´s disease and summarize main transcriptional changes and altered molecular pathways observed in astrocytes during the course of the disease.

Sephin1 Protects Neurons against Excitotoxicity Independently of the Integrated Stress Response.

Sephin1 is a derivative of guanabenz that inhibits the dephosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) and therefore may enhance the integrated stress response (ISR), an adaptive mechanism against different cellular stresses, such as accumulation of misfolded proteins. Unlike guanabenz, Sephin1 provides neuroprotection without adverse effects on the α2-adrenergic system and therefore it is considered a promising pharmacological therapeutic tool. Here, we have studied the effects of Sephin1 on N-methyl-D-aspartic acid (NMDA) receptor signaling which may modulate the ISR and contribute to excitotoxic neuronal loss in several neurodegenerative conditions. Time-course analysis of peIF2α levels after NMDA receptor overactivation showed a delayed dephosphorylation that occurred in the absence of activating transcription factor 4 (ATF4) and therefore independently of the ISR, in contrast to that observed during endoplasmic reticulum (ER) stress induced by tunicamycin and thapsigargin. Similar to guanabenz, Sephin1 completely blocked NMDA-induced neuronal death and was ineffective against AMPA-induced excitotoxicity, whereas it did not protect from experimental ER stress. Interestingly, both guanabenz and Sephin1 partially but significantly reduced NMDA-induced cytosolic Ca2+ increase, leading to a complete inhibition of subsequent calpain activation. We conclude that Sephin1 and guanabenz share common strong anti-excitotoxic properties with therapeutic potential unrelated to the ISR.

Early Effects of Aß Oligomers on Dendritic Spine Dynamics and Arborization in Hippocampal Neurons.

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to impaired memory and cognitive deficits. Spine loss as well as changes in spine morphology correlates with cognitive impairment in this neurological disorder. Many studies in animal models and ex vivo cultures indicate that amyloid ß-peptide (Aß) oligomers induce synaptic damage early during the progression of the disease. Here, in order to determine the events that initiate synaptic alterations, we acutely applied oligomeric Aß to primary hippocampal neurons and an ex vivo model of organotypic hippocampal cultures from a mouse after targeted expression of EGFP to allow high-resolution imaging and algorithm-based evaluation of spine changes. Dendritic spines were classified as thin, stubby or mushroom, based on morphology. In vivo, time-lapse imaging showed that the three spine types were relatively stable, although their stability significantly decreased after treatment with Aß oligomers. Unexpectedly, we observed that the density of total dendritic spines increased in organotypic hippocampal slices treated with Aß compared to control cultures. Specifically, the fraction of stubby spines significantly increased, while mushroom and thin spines remained unaltered. Pharmacological tools revealed that acute Aß oligomers induced spine changes through mechanisms involving CaMKII and integrin ß1 activities. Additionally, analysis of dendritic complexity based on a 3D reconstruction of the whole neuron morphology showed an increase in the apical dendrite length and branching points in CA1 organotypic hippocampal slices treated with Aß. In contrast to spines, the morphological changes were affected by integrin ß1 but not by CaMKII inhibition. Altogether, these data indicate that the Aß oligomers exhibit early dual effects by acutely enhancing dendritic complexity and spine density.

Mitochondrial division inhibitor 1 disrupts oligodendrocyte Ca2+ homeostasis and mitochondrial function.

Mitochondrial fission mediated by cytosolic dynamin related protein 1 (Drp1) is essential for mitochondrial quality control but may contribute to apoptosis as well. Blockade of Drp1 with mitochondrial division inhibitor 1 (mdivi-1) provides neuroprotection in several models of neurodegeneration and cerebral ischemia and has emerged as a promising therapeutic drug. In oligodendrocytes, overactivation of AMPA-type ionotropic glutamate receptors (AMPARs) induces intracellular Ca2+ overload and excitotoxic death that contributes to demyelinating diseases. Mitochondria are key to Ca2+ homeostasis, however it is unclear how it is disrupted during oligodendroglial excitotoxicity. In the current study, we have analyzed mitochondrial dynamics during AMPAR activation and the effects of mdivi-1 on excitotoxicity in optic nerve-derived oligodendrocytes. Sublethal AMPAR activation triggered Drp1-dependent mitochondrial fission, whereas toxic AMPAR activation produced Drp1-independent mitochondrial swelling. Accordingly, mdivi-1 efficiently inhibited Drp1-mediated mitochondrial fission and did not prevent oligodendrocyte excitotoxicity. Unexpectedly, mdivi-1 also induced mitochondrial depolarization, ER Ca2+ depletion and modulation of AMPA-induced Ca2+ signaling. These off-target effects of mdivi-1 sensitized oligodendrocytes to excitotoxicity and ER stress and eventually produced oxidative stress and apoptosis. Interestingly, in cultured astrocytes mdivi-1 induced nondetrimental mitochondrial depolarization and oxidative stress that did not cause toxicity or sensitization to apoptotic stimuli. In summary, our results provide evidence of Drp1-mediated mitochondrial fission during activation of ionotropic glutamate receptors in oligodendrocytes, and uncover a deleterious and Drp1-independent effect of mdivi-1 on mitochondrial and ER function in these cells. These off-target effects of mdivi-1 limit its therapeutic potential and should be taken into account in clinical studies.

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome.

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENT Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.

Oligodendrocyte Differentiation and Myelination Is Potentiated via GABAB Receptor Activation.

Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the central nervous system (CNS). Several growth factors and neurotransmitters like GABA are postulated as important regulators of that process, and different protein kinases may also participate in OL differentiation and myelination. However, the molecular mechanisms underlying the regulation of myelination by neurotransmitters are only partially known. In the present study, we provide evidence showing that GABA receptors (GABARs) play an important role in OL differentiation. First, we observed that OPCs and OLs synthesize GABA and expressed GABAR and transporters, both in vitro and in vivo and, in contrast to GABAARs, the subunits GABAB1R and GABAB2R are expressed in OLs over time. Then, we found that exogenous GABA increases the number of myelin segments and MBP expression in DRG-OPC cocultures, indicating that GABA regulates myelination when OLs are in contact with axons. Notably, in purified rat OPC cultures, chronic treatment with GABA and baclofen, specific GABABR agonist, accelerates OPC differentiation by enhancing the processes branching and myelin protein expression, effects that are reverted in presence of GABABR specific antagonist CGP55845. Exposure of OPCs to baclofen promotes the Src-phosphorylation, and the baclofen-induced maturation is attenuated in presence of the Src-family kinases inhibitor PP2. None of these effects are mediated by the GABAAR agonist muscimol. Together, these results highlight the relevance of the GABAergic system in OL differentiation, and indicate that this functional role is mediated through GABABR involving the participation of Src-family kinases. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Aß oligomers promote oligodendrocyte differentiation and maturation via integrin ß1 and Fyn kinase signaling.

Alzheimer´s disease (AD) is characterized by a progressive cognitive decline that correlates with the levels of amyloid ß-peptide (Aß) oligomers. Strong evidences connect changes of oligodendrocyte function with the onset of neurodegeneration in AD. However, the mechanisms controlling oligodendrocyte responses to Aß are still elusive. Here, we tested the role of Aß in oligodendrocyte differentiation, maturation, and survival in isolated oligodendrocytes and in organotypic cerebellar slices. We found that Aß peptides specifically induced local translation of 18.5-kDa myelin basic protein (MBP) isoform in distal cell processes concomitant with an increase of process complexity of MBP-expressing oligodendrocytes. Aß oligomers required integrin ß1 receptor, Src-family kinase Fyn and Ca2+/CaMKII as effectors to modulate MBP protein expression. The pharmacological inhibition of Fyn kinase also attenuated oligodendrocyte differentiation and survival induced by Aß oligomers. Similarly, using ex vivo organotypic cerebellar slices Aß promoted MBP upregulation through Fyn kinase, and modulated oligodendrocyte population dynamics by inducing cell proliferation and differentiation. Importantly, application of Aß to cerebellar organotypic slices enhanced remyelination and oligodendrocyte lineage recovery in lysolecithin (LPC)-induced demyelination. These data reveal an important role of Aß in oligodendrocyte lineage function and maturation, which may be relevant to AD pathogenesis.

Corrigendum to "Contribution of Neurons and Glial Cells to Complement-Mediated Synapse Removal during Development, Aging and in Alzheimer's Disease".

[This corrects the article DOI: 10.1155/2018/2530414.].

Contribution of Neurons and Glial Cells to Complement-Mediated Synapse Removal during Development, Aging and in Alzheimer's Disease.

Synapse loss is an early manifestation of pathology in Alzheimer's disease (AD) and is currently the best correlate to cognitive decline. Microglial cells are involved in synapse pruning during development via the complement pathway. Moreover, recent evidence points towards a key role played by glial cells in synapse loss during AD. However, further contribution of glial cells and the role of neurons to synapse pathology in AD remain not well understood. This review is aimed at comprehensively reporting the source and/or cellular localization in the CNS-in microglia, astrocytes, or neurons-of the triggering components (C1q, C3) of the classical complement pathway involved in synapse pruning in development, adulthood, and AD.

Mangiferin and Morin Attenuate Oxidative Stress, Mitochondrial Dysfunction, and Neurocytotoxicity, Induced by Amyloid Beta Oligomers.

Amyloid beta- (Aß-) mediated ROS overproduction disrupts intraneuronal redox balance and exacerbates mitochondrial dysfunction which leads to neuronal injury. Polyphenols have been investigated as therapeutic agents that promote neuroprotective effects in experimental models of brain injury and neurodegenerative diseases. The aim of this study was to identify the neuroprotective effects of morin and mangiferin against Aß oligomers in cultured cortical neurons and organotypic slices as well as their mechanisms of action. Cell death caused by Aß oligomers in neuronal cultures was decreased in the presence of micromolar concentrations of mangiferin or morin, which in turn attenuated oxidative stress. The neuroprotective effects of antioxidants against Aß were associated with the reduction of Aß-induced calcium load to mitochondria; mitochondrial membrane depolarization; and release of cytochrome c from mitochondria, a key trigger of apoptosis. Additionally, we observed that both polyphenols activated the endogenous enzymatic antioxidant system and restored oxidized protein levels. Finally, Aß induced an impairment of energy homeostasis due to a decreased respiratory capacity that was mitigated by morin and mangiferin. Overall, the beneficial effects of polyphenols in preventing mitochondrial dysfunction and neuronal injury in AD cell models suggest that morin and mangiferin hold promise for the treatment of this neurological disorder.

Isolation, Expansion, and Maturation of Oligodendrocyte Lineage Cells Obtained from Rat Neonatal Brain and Optic Nerve.

Oligodendrocytes are the myelin-forming cells in the central nervous system (CNS) and their loss or dysfunction is a hallmark of CNS demyelinating diseases, such as multiple sclerosis (MS), hypoxic-ischemic demyelination, or spinal cord injury. In the rodent CNS, oligodendrocyte progenitor cells (OPCs) arise in multiple ventral and dorsal locations of the forebrain during late embryogenesis and early postnatal periods. OPCs migrate out from these germinal zones and disperse throughout the CNS, to populate the developing white and gray matter. There, OPCs can begin to mature through a series of intermediate states characterized by the expression of stage-specific proteins until completely differentiated into postmitotic myelinating oligodendrocytes. Elucidating the cellular and molecular mechanisms that control oligodendrocyte maturation requires isolating OPCs and premyelinating oligodendrocytes by rapid and reliable methods that provide high yield, pure and viable culture, being a powerful tool to characterize their differentiation and potential capacity for myelin repair after injury. This chapter describes in detail two simple and efficient protocols for the preparation of highly enriched rat OPC populations and immature oligodendrocytes derived from mixed glial cultures and optic nerves, respectively. Functional oligodendrocytes obtained with these protocols can be cocultured with primary neurons to study myelination.

Aß1-42 triggers the generation of a retrograde signaling complex from sentinel mRNAs in axons.

Neurons frequently encounter neurodegenerative signals first in their periphery. For example, exposure of axons to oligomeric Aß1-42 is sufficient to induce changes in the neuronal cell body that ultimately lead to degeneration. Currently, it is unclear how the information about the neurodegenerative insult is transmitted to the soma. Here, we find that the translation of pre-localized but normally silenced sentinel mRNAs in axons is induced within minutes of Aß1-42 addition in a Ca2+-dependent manner. This immediate protein synthesis following Aß1-42 exposure generates a retrograde signaling complex including vimentin. Inhibition of the immediate protein synthesis, knock-down of axonal vimentin synthesis, or inhibition of dynein-dependent transport to the soma prevented the normal cell body response to Aß1-42 These results establish that CNS axons react to neurodegenerative insults via the local translation of sentinel mRNAs encoding components of a retrograde signaling complex that transmit the information about the event to the neuronal soma.

Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling.

Excessive dynamin related protein 1 (Drp1)-triggered mitochondrial fission contributes to apoptosis under pathological conditions and therefore it has emerged as a promising therapeutic target. Mitochondrial division inhibitor 1 (mdivi-1) inhibits Drp1-dependent mitochondrial fission and is neuroprotective in several models of brain ischemia and neurodegeneration. However, mdivi-1 also modulates mitochondrial function and oxidative stress independently of Drp1, and consequently the mechanisms through which it protects against neuronal injury are more complex than previously foreseen. In this study, we have analyzed the effects of mdivi-1 on mitochondrial dynamics, Ca2+ signaling, mitochondrial bioenergetics and cell viability during neuronal excitotoxicity in vitro. Time-lapse fluorescence microscopy revealed that mdivi-1 blocked NMDA-induced mitochondrial fission but not that triggered by sustained AMPA receptor activation, showing that mdivi-1 inhibits excitotoxic mitochondrial fragmentation in a source specific manner. Similarly, mdivi-1 strongly reduced NMDA-triggered necrotic-like neuronal death and, to a lesser extent, AMPA-induced toxicity. Interestingly, neuroprotection provided by mdivi-1 against NMDA, but not AMPA, correlated with a reduction in cytosolic Ca2+ ([Ca2+]cyt) overload and calpain activation indicating additional cytoprotective mechanisms. Indeed, mdivi-1 depolarized mitochondrial membrane and depleted ER Ca2+ content, leading to attenuation of mitochondrial [Ca2+] increase and enhancement of the integrated stress response (ISR) during NMDA receptor activation. Finally, lentiviral knockdown of Drp1 did not rescue NMDA-induced mitochondrial fission and toxicity, indicating that neuroprotective activity of mdivi-1 is Drp1-independent. Together, these results suggest that mdivi-1 induces a Drp1-independent protective phenotype that prevents predominantly NMDA receptor-mediated excitotoxicity through the modulation of mitochondrial function and intracellular Ca2+ signaling.