Inhibition of lipid synthesis by the HIV integrase strand transfer inhibitor elvitegravir in primary rat oligodendrocyte cultures.

Combined antiretroviral therapy (cART) has greatly decreased mortality and morbidity among persons with HIV; however, neurologic impairments remain prevalent, in particular HIV-associated neurocognitive disorders (HANDs). White matter damage persists in cART-treated persons with HIV and may contribute to neurocognitive dysfunction as the lipid-rich myelin membrane of oligodendrocytes is essential for efficient nerve conduction. Because of the importance of lipids to proper myelination, we examined the regulation of lipid synthesis in oligodendrocyte cultures exposed to the integrase strand transfer inhibitor elvitegravir (EVG), which is administered to persons with HIV as part of their initial regimen. We show that protein levels of genes involved in the fatty acid pathway were reduced, which correlated with greatly diminished de novo levels of fatty acid synthesis. In addition, major regulators of cellular lipid metabolism, the sterol regulatory element-binding proteins (SREBP) 1 and 2, were strikingly altered following exposure to EVG. Impaired oligodendrocyte differentiation manifested as a marked reduction in mature oligodendrocytes. Interestingly, most of these deleterious effects could be prevented by adding serum albumin, a clinically approved neuroprotectant. These new findings, together with our previous study, strengthen the possibility that antiretroviral therapy, at least partially through lipid dysregulation, may contribute to the persistence of white matter changes observed in persons with HIV and that some antiretrovirals may be preferable as life-long therapy.

Differential effects of integrase strand transfer inhibitors, elvitegravir and raltegravir, on oligodendrocyte maturation: A role for the integrated stress response.

Regardless of adherence to combined antiretroviral therapy, white matter and myelin pathologies persist in patients with HIV-associated neurocognitive disorders, a spectrum of cognitive, motor, and behavioral impairments. We hypothesized that antiretroviral therapy alters the maturation of oligodendrocytes which synthesize myelin. We tested whether specific frontline integrase strand transfer inhibitors would alter oligodendrocyte differentiation and myelination. To model the effect of antiretrovirals on oligodendrocytes, we stimulated primary rat oligodendrocyte precursor cells to differentiate into mature oligodendrocytes in vitro in the presence of therapeutically relevant concentrations of elvitegravir or raltegravir and then assessed differentiation with lineage specific markers. To examine the effect of antiretrovirals on myelination, we treated mice with the demyelinating compound cuprizone, for 5 weeks. This was followed by 3 weeks of recovery in absence of cuprizone, during which time some mice received a daily intrajugular injection of elvitegravir. Brains were harvested, sectioned and processed by immunohistochemistry to examine oligodendrocyte maturation and myelination. Elvitegravir inhibited oligodendrocyte differentiation in vitro in a concentration-dependent manner, while raltegravir had no effect. Following cuprizone demyelination, administration of elvitegravir to adult mice reduced remyelination compared with control animals. Elvitegravir treatment activated the integrated stress response in oligodendrocytes in vitro, an effect which was completely blocked by pretreatment with the integrated stress response inhibitor Trans-ISRIB, preventing elvitegravir-mediated inhibition of oligodendrocyte maturation. These studies demonstrate that elvitegravir impairs oligodendrocyte maturation and remyelination and that the integrated stress response mediates this effect and may be a possible therapeutic target.

Reduced sterol regulatory element-binding protein (SREBP) processing through site-1 protease (S1P) inhibition alters oligodendrocyte differentiation in vitro.

The formation of the myelin membrane of the oligodendrocyte in the CNS is a fundamental process requiring the coordinated synthesis of many different components. The myelin membrane is particularly rich in lipids, however, the regulation of this lipid synthesis is not understood. In other cell types, including Schwann cells, the myelin-forming cells of the PNS, lipid synthesis is tightly regulated by the sterol regulatory element-binding protein (SREBP) family of transcription factors, but this has not been previously shown in oligodendrocytes. We investigated SREBPs' role during oligodendrocyte differentiation in vitro. Both SREBP-1 and SREBP-2 were expressed in oligodendrocyte precursor cells and differentiating oligodendrocytes. Using the selective site-1 protease (S1P) inhibitor PF-429242, which inhibits the cleavage of SREBP precursor forms into mature forms, we found that preventing SREBP processing inhibited process growth and reduced the expression level of myelin basic protein, a major component of myelin. Further, process extension deficits could be rescued by the addition of exogenous cholesterol. Blocking SREBP processing reduced mRNA transcription and protein levels of SREBP target genes involved in both the fatty acid and the cholesterol synthetic pathways. Furthermore, de novo levels and total levels of cholesterol synthesis were greatly diminished when SREBP processing was inhibited. Together these results indicate that SREBPs are important regulators of oligodendrocyte maturation and that perturbation of their activity may affect myelin formation and integrity. Cover Image for this issue: doi: 10.1111/jnc.13781.

Altered Oligodendrocyte Maturation and Myelin Maintenance: The Role of Antiretrovirals in HIV-Associated Neurocognitive Disorders.

Despite effective viral suppression through combined antiretroviral therapy (cART), approximately half of HIV-positive individuals have HIV-associated neurocognitive disorders (HAND). Studies of antiretroviral-treated patients have revealed persistent white matter abnormalities including diffuse myelin pallor, diminished white matter tracts, and decreased myelin protein mRNAs. Loss of myelin can contribute to neurocognitive dysfunction because the myelin membrane generated by oligodendrocytes is essential for rapid signal transduction and axonal maintenance. We hypothesized that myelin changes in HAND are partly due to effects of antiretroviral drugs on oligodendrocyte survival and/or maturation. We showed that primary mouse oligodendrocyte precursor cell cultures treated with therapeutic concentrations of HIV protease inhibitors ritonavir or lopinavir displayed dose-dependent decreases in oligodendrocyte maturation; however, this effect was rapidly reversed after drug removal. Conversely, nucleoside reverse transcriptase inhibitor zidovudine had no effect. Furthermore, in vivo ritonavir administration to adult mice reduced frontal cortex myelin protein levels. Finally, prefrontal cortex tissue from HIV-positive individuals with HAND on cART showed a significant decrease in myelin basic protein compared with untreated HIV-positive individuals with HAND or HIV-negative controls. These findings demonstrate that antiretrovirals can impact myelin integrity and have implications for myelination in juvenile HIV patients and myelin maintenance in adults on lifelong therapy.

SCO-spondin derived peptide NX210 induces neuroprotection in vitro and promotes fiber regrowth and functional recovery after spinal cord injury.

In mammals, the limited regenerating potential of the central nervous system (CNS) in adults contrasts with the plasticity of the embryonic and perinatal periods. SCO (subcommissural organ)-spondin is a protein secreted early by the developing central nervous system, potentially involved in the development of commissural fibers. SCO-spondin stimulates neuronal differentiation and neurite growth in vitro. NX210 oligopeptide was designed from SCO-spondin's specific thrombospondin type 1 repeat (TSR) sequences that support the main neurogenic properties of the molecule. The objective of this work was to assess the neuroprotective and neuroregenerative properties of NX210 in vitro and in vivo for the treatment of spinal cord injury (SCI). In vitro studies were carried out on the B104 neuroblastoma cell line demonstrating neuroprotection by the resistance to oxidative damage using hydrogen peroxide and the measure of cell viability by metabolic activity. In vivo studies were performed in two rat models of SCI: (1) a model of aspiration of dorsal funiculi followed by the insertion of a collagen tube in situ to limit collateral sprouting; white matter regeneration was assessed using neurofilament immunostaining; (2) a rat spinal cord contusion model to assess functional recovery using BBB scale and reflex testing. We demonstrate for the first time that NX210 (a) provides neuroprotection to oxidative stress in the B104 neuroblastoma cells, (b) stimulates axonal regrowth in longitudinally oriented neofibers in the aspiration model of SCI and (c) significantly improves functional recovery in the contusive model of SCI.

Dendritic alterations after dynamic axonal stretch injury in vitro.

Traumatic axonal injury (TAI) is the most common and important pathology of traumatic brain injury (TBI). However, little is known about potential indirect effects of TAI on dendrites. In this study, we used a well-established in vitro model of axonal stretch injury to investigate TAI-induced changes in dendrite morphology. Axons bridging two separated rat cortical neuron populations plated on a deformable substrate were used to create a zone of isolated stretch injury to axons. Following injury, we observed the formation of dendritic alterations or beading along the dendrite shaft. Dendritic beading formed within minutes after stretch then subsided over time. Pharmacological experiments revealed a sodium-dependent mechanism, while removing extracellular calcium exacerbated TAI's effect on dendrites. In addition, blocking ionotropic glutamate receptors with the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 prevented dendritic beading. These results demonstrate that axon mechanical injury directly affects dendrite morphology, highlighting an important bystander effect of TAI. The data also imply that TAI may alter dendrite structure and plasticity in vivo. An understanding of TAI's effect on dendrites is important since proper dendrite function is crucial for normal brain function and recovery after injury.

Inflammation-induced preterm birth alters neuronal morphology in the mouse fetal brain.

Adverse neurological outcome is a major cause of long-term morbidity in ex-preterm children. To investigate the effect of parturition and inflammation on the fetal brain, we utilized two in vivo mouse models of preterm birth. To mimic the most common human scenario of preterm birth, we used a mouse model of intrauterine inflammation by intrauterine infusion of lipopolysaccharide (LPS). To investigate the effect of parturition on the immature fetal brain, in the absence of inflammation, we used a non-infectious model of preterm birth by administering RU486. Pro-inflammatory cytokines (IL-10, IL-1beta, IL-6 and TNF-alpha) in amniotic fluid and inflammatory biomarkers in maternal serum and amniotic fluid were compared between the two models using ELISA. Pro-inflammatory cytokine expression was evaluated in the whole fetal brains from the two models. Primary neuronal cultures from the fetal cortex were established from the different models and controls in order to compare the neuronal morphology. Only the intrauterine inflammation model resulted in an elevation of inflammatory biomarkers in the maternal serum and amniotic fluid. Exposure to inflammation-induced preterm birth, but not non-infectious preterm birth, also resulted in an increase in cytokine mRNA in whole fetal brain and in disrupted fetal neuronal morphology. In particular, Microtubule-associated protein 2 (MAP2) staining was decreased and the number of dendrites was reduced (P < 0.001, ANOVA between groups). These results suggest that inflammation-induced preterm birth and not the process of preterm birth may result in neuroinflammation and alter fetal neuronal morphology.

Beyond white matter damage: fetal neuronal injury in a mouse model of preterm birth.

OBJECTIVE: The purpose of this study was to elucidate possible mechanisms of fetal neuronal injury in inflammation-induced preterm birth. STUDY DESIGN: With the use of a mouse model of preterm birth, the following primary cultures were prepared from fetal brains: (1) control neurons (CNs), (2) lipopolysaccharide-exposed neurons (LNs), (3) control coculture (CCC) that consisted of neurons and glia, and (4) lipopolysaccharide-exposed coculture (LCC) that consisted of lipopolysaccharide-exposed neurons and glia. CNs and LNs were treated with culture media from CN, LN, CCC, and LCC after 24 hours in vitro. Immunocytochemistry was performed for culture characterization and neuronal morphologic evidence. Quantitative polymerase chain reaction was performed for neuronal differentiation marker, microtubule-associated protein 2, and for cell death mediators, caspases 1, 3, and 9. RESULTS: Lipopolysaccharide exposure in vivo did not influence neuronal or glial content in cocultures but decreased the expression of microtubule-associated protein 2 in LNs. Media from LNs and LCCs induced morphologic changes in control neurons that were comparable with LNs. The neuronal damage caused by in vivo exposure (LNs) could not be reversed by media from control groups. CONCLUSION: Lipopolysaccharide-induced preterm birth may be responsible for irreversible neuronal injury.

Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases.

Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.

Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro.

The vulnerability of brain cells to neurologic insults varies greatly, depending on their neuronal subpopulation. However, cells surviving pathological insults such as ischemia or brain trauma may undergo structural changes, e.g., altered process growth, that could compromise brain function. In this study, we examined the effect of glutamate excitotoxicity on dendrite growth from surviving cortical GABAergic neurons in vitro. Glutamate exposure did not affect GABAergic neuron viability, however, it significantly reduced dendrite growth from GABAergic neurons. This effect was blocked by the AMPA receptor antagonists NBQX and CFM-2, and mimicked by AMPA, but not NMDA. Glutamate excitotoxicity also caused an NMDA receptor-mediated decrease in the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) immunoreactivity from GABAergic neurons, measured using immunocytochemical and Western blot techniques. GAD is necessary for GABA synthesis; however, reduction of GABA by 3-mercaptopropionic acid (3-MPA), which inhibits GABA synthesis, did not alter dendrite growth. These results suggest that GABAergic cortical neurons are relatively resistant to excitotoxic-induced cell death, but they can display morphological and biochemical alterations which may impair their function.

Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro.

Glutamate is an important regulator of dendrite development that may inhibit, (during ischemic injury), or facilitate (during early development) dendrite growth. Previous studies have reported mainly on the N-methyl-D-aspartate (NMDA) receptor-mediated dendrite growth-promoting effect of glutamate. In this study, we examined how the non-NMDA receptor agonist kainate influenced dendrite growth. E18 mouse cortical neurons were grown for 3 days in vitro and immunolabeled with anti-microtubule-associated protein 2 (MAP2) and anti-neurofilament (NF-H), to identify dendrites and axons, respectively. Exposure of cortical neurons to kainate increased dendrite growth without affecting neuron survival. This effect was dose-dependent, reversible and blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor antagonist NBQX and the low-affinity kainate receptor antagonist NS-102, but not by the AMPA receptor antagonist CFM-2. In addition, the NMDA receptor antagonist MK-801 had no effect on kainate-induced dendrite growth. Immunolabeling and Western blot analysis of kainate receptors using antibodies against the GluR6 and KA2 subunits, demonstrated that the immature cortical neurons used in this study express kainate receptor proteins. These results suggest that kainate-induced non-NMDA receptor activation promotes dendrite growth, and in particular primary dendrite number and length, from immature cortical neurons in vitro, and that kainate receptors may be directly involved in this process. Furthermore, these data support the possibility that like NMDA receptors, kainate receptor activation may also contribute to early neurite growth from cortical neurons in vitro.

Growth of rat cortical neurons on DuraGen, a collagen-based dural graft matrix.

OBJECTIVES: DuraGen, a collagen-based dural graft matrix, is frequently used in clinical neurosurgery. In the present study we examined whether DuraGen influenced neuron survival of or process growth from cerebral cortex neurons in culture. METHODS: Dissociated E19 rat cerebral cortical neurons were cultured at low density on poly-L-lysine or on cryostat-sectioned DuraGen. Neuron survival was assessed using morphological criteria, fluorescein diacetate (FDA) and propidium iodide (PI), nuclear staining and TUNEL labeling. Process growth was analysed using specific antibodies against MAP2 and the 200 kDa neurofilament subunit (NF-H) to identify dendrites and axons, respectively. RESULTS: In immature cultures (3 days in vitro, DIV), nearly 70% of the neurons remained viable in control and DuraGen-exposed cells. In mature cultures (10 DIV), approximately 45% of the neurons were viable. Survival was similar in DuraGen cultures and controls. Cell viability also was similar when DuraGen conditioned the medium, but was not in contact with the neurons. When 10-day-old cultures were treated with glutamate (100 mumol/l for 24 hours) to elicit excitotoxic injury, a 40% decrease in neuron survival was observed. DuraGen's presence neither exacerbated nor attenuated glutamate-induced excitotoxic neuron death. The amount of necrotic or apoptotic cells also was similar in control and DuraGen cultures. Finally, DuraGen had an equal ability to support both axon and dendrite growth as poly-L-lysine. CONCLUSION: Our findings demonstrate that DuraGen has no adverse effect on survival of or process growth from cerebral cortical neurons in vitro. These data support DuraGen's biosafety as a dural substitute in clinical neurosurgery.

Beta-amyloid-induced reactive astrocytes display altered ability to support dendrite and axon growth from mouse cerebral cortical neurons in vitro.

OBJECTIVES: The presence of beta-amyloid (betaA) deposition, induction of reactive gliosis and dystrophic neurites, is a characteristic feature of neuritic plaques in Alzheimer's disease. In vitro, betaA-exposed astrocytes become reactive, similar to astrocytes in contact with betaA plaques in vivo. How betaA-exposed reactive astrocytes support neuron process growth, however, is not well defined. Therefore, we used neuron/astrocyte co-cultures in which astrocytes had been grown on betaA, to assess whether process growth was altered. METHODS: Purified rat cortical astrocytes were plated on the betaA peptide's neurotoxic fragment (25-35), the scrambled (35-25) peptide, or poly-D-lysine alone and grown to confluency before mouse cortical neurons were seeded at low density onto the astrocyte monolayer. Cell survival was assessed using trypan blue, lactate dehydrogenase release and propidium iodide. Process growth was analyzed using specific antibodies against MAP2 and the 200 kDa neurofilament subunit (NF-H) to identify dendrites and axons, respectively. RESULTS: betaA-exposed astrocytes changed dramatically from their flat polygonal shape into stellate process-bearing morphology. Viability however, was not affected. Immunocytochemical analysis of neuronal processes using anti-MAP2 and anti-NF-H, demonstrated that betaA (25-35)induced reactive astrocytes had an altered ability to support dendrite and axon growth after 3 days in vitro. Indeed, primary dendrite number and axon length were decreased by 30 and 26%, respectively, compared with control astrocytes, whereas individual primary dendrite length increased by 20%. Astrocyte support of dendritic branching, however, was not affected by betaA. DISCUSSION: We conclude that an astrocyte reaction to betaA may contribute, in part, to neuronal dystrophy associated with betaA plaques.

Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors.

Glutamate is an important regulator of dendrite development; however, during cerebral ischemia, massive glutamate release can lead to neurodegeneration and death. An early consequence of glutamate excitotoxicity is dendrite injury, which often precedes cell death. We examined the effect of glutamate on dendrite growth from embryonic day 18 (E18) mouse cortical neurons grown for 3 days in vitro (DIV) and immunolabeled with anti-microtubule-associated protein (MAP)2 and anti-neurofilament (NF)-H, to identify dendrites and axons, respectively. Cortical neurons exposed to excess extracellular glutamate (100 microM) displayed reduced dendrite growth, which occurred in the absence of cell death. This effect was mimicked by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and blocked by the ionotropic glutamate receptor antagonist kynurenic acid and the NMDA receptor-specific antagonist MK-801. The non-NMDA receptor agonist AMPA, however, did not affect process growth. Neither NMDA nor AMPA influenced neuron survival. Immunolabeling and Western blot analysis of NMDA receptors using antibodies against the NR1 subunit, demonstrated that immature cortical neurons used in this study, express NMDA receptors. These results suggest that excess glutamate decreases dendrite growth through a mechanism resulting from NMDA receptor subclass activation. Furthermore, these data support the possibility that excess glutamate activation of NMDA receptors mediate both cell death in mature neurons and the inhibitory effect of excess glutamate on dendrite growth in immature neurons or in the absence of cell death.

Decreased dendrite growth from cultured mouse cortical neurons surviving excitotoxic activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptors.

During cerebral ischemia, massive glutamate release leads to cell death through ionotropic glutamate receptor activation. An early consequence of this excitotoxicity is dendrite injury, which can precede cell death. We therefore tested whether cells that survived an excitotoxic insult triggered by overactivation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate (KA) subtype of ionotropic glutamate receptors displayed altered dendrite growth. We demonstrate that 24 h exposure of cultured cortical neurons to AMPA or KA dramatically reduced dendrite growth from surviving neurons. AMPA or KA exposure decreased primary dendrite number and length, and also reduced dendritic branching. The AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the deleterious effect of AMPA and KA on dendrite growth. These results suggest that AMPA/KA receptor overactivation may contribute to dendritic injury from neurons that survive an ischemic insult.

BMP-7 and excess glutamate: opposing effects on dendrite growth from cerebral cortical neurons in vitro.

Glutamate is an important regulator of dendrite development. During cerebral ischemia, however, there is massive release of glutamate reaching millimolar concentrations in the extracellular space. An early consequence of this excess glutamate is reduced dendrite growth. Bone morphogenetic protein-7 (BMP-7) a member of the transforming growth factor-beta (TGF-beta) superfamily has been demonstrated to enhance dendrite output from cerebral cortical and hippocampal neurons in vitro. However, it is not known whether BMP-7can prevent the reduced dendrite growth associated with excess glutamate or enhance dendrite growth after glutamate exposure. Therefore we quantified axon and primary, secondary, and total dendrite growth from embryonic mouse cortical neurons (E18) grown at low density in vitro in a chemically defined medium and exposed to glutamate (1 or 2 mM) for 48 h. Morphology and double immunolabeling (MAP2, NF-H) were used to identify cortical dendrites and axons after 3 DIV. In these short-term cultures, glutamate did not influence neuron survival. The addition of glutamate to cortical neurons, however, significantly attenuated dendrite output. This effect was mimicked by the addition of NMDA but not AMPA agonists and inhibited by the specific NMDA receptor antagonist MK-801. The reduction in dendrite growth mediated by excess glutamate was ameliorated by the administration of 30 or 100 ng/ml of BMP-7. In addition, when administered in a delayed fashion between 1 and 24 h after the initial glutamate exposure, BMP-7 was able to enhance dendrite growth, including primary dendrite number, primary dendrite length, and secondary dendritic branching. These findings demonstrate that BMP-7 can ameliorate reduced dendrite growth from cerebral cortical neurons associated with excess glutamate in vitro and are important because they may help explain why BMP-7 administration is associated with enhanced functional recovery in models of cerebral ischemia.