Primary Brain Cultures Research Articles

Heat shock, but not the reactive state per se, induces increased expression of the small stress proteins hsp25 and alpha B-crystallin in glial cells in vitro.

The stress proteins hsp25 and alpha B-crystallin are found in increased concentrations in reactive astrocytes of brains undergoing neurodegeneration. In order to characterize this reaction, we investigated the expression of hsp25 and alpha B-crystallin during growth and after stress (heat shock) in glial cells in vitro. In primary rat brain cultures, hsp25 was present in actively dividing astrocytes that were positive for glial fibrillary acidic protein. alpha B-crystallin was found predominantly in oligodendrocytes. Heat shock resulted in increased concentrations of hsp25 and alpha B-crystallin in astrocytes, without any detectable changes in intracellular localization, as detectable with confocal laser microscopy. These results indicate that a neurodegeneration-related increase of the small stress proteins in astrocytes in independent of gliosis per se, and may be a disease-related event.

Prolonged dopamine and serotonin transporter inhibition after exposure to tropanes

Cocaine and tropane analogs are known to interact with biogenic monoamine transporters by inhibiting amine uptake. Previous in vivo studies have demonstrated that some of these tropanes produce a longer lasting behavioral effect compared with cocaine. We have previously examined several tropane analogs and found a difference in their relative affinities for dopamine (DA) and serotonin (5-HT) transporters. The purpose of this study was to determine the recovery time of transporter function in vitro and in vivo comparing cocaine with the tropane analogs WF-11 (PTT, selective for DA transporters), WF-31 (selective for 5-HT transporters) and WF-23 (highly potent at both DA and 5-HT transporters). In vitro, using primary rat brain cultures of either midbrain or raphe regions, the recovery of the ability to transport either [ 3H]dopamine or [ 3H]serotonin, respectively was evaluated at 0, 3, 24, 48, 120 and 240 h after a 1 h exposure to cocaine and tropane analogs. The tropanes exhibited clearance half-lives ranging from 12 to 69 h, while cocaine, on the other hand, exhibited a clearance half-life of ≈6 h. In studies utilizing [ 125I]RTI-55 binding, intraperitoneal injections of cocaine and WF-23 into the rat resulted in striatal clearance half-lives ex vivo that were almost identical to those obtained in vitro. These data suggest that the tropanes bind to and reduce transporter function for prolonged periods of time (up to 10-fold longer than cocaine) and those compounds with the highest affinity may produce a pseudo-irreversible inhibition of transporter function.

Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation.

Two receptor tyrosine kinases, ErB-3 and ErbB-4, mediate signaling by Neu differentiation factors (NDFs, also called neuregulins), while ErbB-1 and ErbB-2 serve as co-receptors. We show that the two NDF/neuregulin receptors differ in spatial and temporal expression patterns: The kinase-defective receptor, ErbB-3, is expressed primarily in epithelial layers of various organs, in the peripheral nervous system, and in adult brain, whereas ErbB-4 is restricted to the developing central nervous system and to the embryonic heart. An example of alternating expression of the two receptors is provided by the developing cerebellum: During postnatal cerebellar development, ErbB-4 expression slightly decreases along with a decline in NDF transcription, whereas ErbB-3 expression commences after the peak of neurogenesis. To study functional differences, we established primary brain cultures and found that ErbB-3 was expressed only in oligodendrocytes, whereas ErbB-4 expression was shared by oligodendrocytes, astrocytes and neurons. Blocking the action of endogenous NDF in vitro, by using a soluble form of ErbB-4, accelerated neurite outgrowth in both primary cultures and in neuronal-type cultures of the P19 teratocarcinoma, suggesting an inhibitory effect of NDF on neural differentiation. Apparently, ErbB-3 is associated with proliferation of P19 cells, whereas ErbB-4 correlates with a differentiated phenotype. We conclude that the two NDF receptors play distinct, rather than redundant, developmental and physiological roles.

IGF-1 is a potent neurotorphic agent in primary brain culture and in neuroblastoma cell-line. Evidence for anti-apoptotic mechanism

IGF-1 is a potent neurotorphic agent in primary brain culture and in neuroblastoma cell-line. Evidence for anti-apoptotic mechanism

Effect of neuroleptics on neuronal cell viability in primary mouse embryo brain cultures and a neuroblastoma cell-line, relevance to tardive dyskinesia

Extrapyramidal side effects (EPS) are a common and serious drawback to the use of conventional antipsychotic drugs. In addition to the discomfort and distress caused to patients, EPS may contribute to poor compliance and ultimately poor treatment outcome. The results of clinical trials with the new generation of atypical antipsychotics suggest that they are associated with a reduction in both early- and late-occurring EPS. All of the new-generation agents produce substantially fewer EPS, and reduce the need for antiparkinsonian medication, in comparison with haloperidol. Data from long-term studies suggest that new-generation antipsychotics are also associated with a reduced risk of tardive dyskinesia, particularly in the elderly.

The mouse calretinin gene promoter region: structural and functional components

The 5′ flanking region of the mouse calretinin gene was cloned and a 1.8 kbp region adjacent to exon 1 was sequenced. Putative upstream promoter and enhancer elements were identified, including appropriately positioned TATA and CAAT boxes (positions −50 and −68, respectively). There was considerable sequence and structural homology between mouse and human upstream elements. Neuron-restrictive activity was demonstrated via transfection of calretinin promoter-reporter constructs into primary embryonic mouse brain cultures expressing calretinin. In promoterless reporter constructs, the proximal upstream 1.5 kbp of the mouse calretinin gene boosted luciferase activity (up to 100-fold) exclusively in the neuronal population. Deletion analysis revealed the minimal promoter to be within the 95-bp proximal to the transcription start site. Transfections with SV40 promoter constructs in these cultures resulted in reporter gene expression predominantly in non-neuronal cells. Inserting the proximal 1.5 kbp of mouse calretinin upstream in SV40 promoter-reporter constructs reduced luciferase activity. Thus, calretinin upstream sequences increased reporter expression in cultured neurons and decreased expression from the SV40 promoter in non-neuronal cultured brain cells. The calretinin promoter contained relevant regulatory element consensus motifs and demonstrated in vitro neuron-restrictive bioactivity.

Astrocyte apoptosis induced by HIV-1 transactivation of the c-kit protooncogene.

HIV-1 infection of the central nervous system (CNS) frequently causes dementia and other neurological disorders. The mechanisms of CNS injury in HIV-1 infection are poorly understood. Apoptosis of neurons and astrocytes is induced by HIV-1 infection in vitro and in brain tissue from AIDS patients, but the apoptotic stimuli have not been identified. We report herein that HIV-1 infection of primary brain cultures induces the receptor tyrosine kinase protooncogene c-kit and that high levels of c-Kit expression are associated with astrocyte apoptosis. Overexpression of c-Kit in an astrocyte-derived cell line in the absence of HIV-1 induces rapid apoptotic death. The apoptotic mechanism requires the c-Kit tyrosine kinase domain. The mechanism of c-kit induction by HIV-1 involves transactivation of the c-kit promoter by the HIV-1 Nef protein. These studies demonstrate that c-Kit can induce astrocyte apoptosis and suggest that this mechanism may play a role in CNS injury caused by HIV-1 infection. We propose that c-Kit can serve dual functions as a growth factor receptor or apoptosis inducer.

Oxidative stress induces dephosphorylation of tau in rat brain primary neuronal cultures.

Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of beta-amyloid is mediated via free radicals, and as the deposition of beta-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein tau that resemble those found in PHF-tau. Contrary to causing an increase in tau phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of tau and so we conclude that oxidative stress is not the direct cause of tau hyperphosphorylation and hence of PHF formation.

The effect of hyperthermia on PGE2 production by primary brain culture after exposure to LPS.

The effect of hyperthermia on PGE2 production by primary brain culture after exposure to LPS.

Different pathways of apoptosis revealed by 2-chloro-adenosine and deoxy-D-ribose in mammalian astroglial cells.

Both the adenosine analogue 2-chloro-adenosine (2-CA) and the reducing sugar deoxy-D-ribose (dRib) induce apoptosis of astroglial cells in rat brain primary cultures (Abbracchio et al.: Biochem Biophys Res Commun 213:908-915, 1995). The present study was undertaken to elucidate by both morphological and cytofluorimetric analyses the intracellular mechanism(s) involved in induction of apoptosis by these two agents. The poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide did not prevent either 2-CA- or dRib-induced cell death, suggesting that activation of PARP is not critically important for induction of apoptosis in astrocytes. The radical scavenger N-acetyl-cysteine (NAC) strongly inhibited dRib- but not 2-CA-induced cell death, suggesting a differential role for radical formation in apoptosis by these two agents. A time-dependent increase of cells with depolarized mitochondria was observed in dRib-, and to a lesser extent, in 2-CA-treated cultures. NAC also prevented dRib- but not 2-CA-induced mitochondrial changes. We conclude that, in mammalian astrocytes, apoptosis can proceed through diverse and multiple pathways, depending upon the apoptotic stimulus. For dRib, apoptosis likely proceeds through generation of radicals and mitochondrial involvement. An adenosine extracellular receptor linked to an as yet unidentified signaling pathway may instead mediate 2-CA-induced cell death, which may have intriguing implications for both nervous system development and brain response to trauma and ischemia.

614 Cloning and characterization of survival-related genes in rat brain primary culture by differential display method

Nerve growth factor (NGF) holds a pivotal role in brain development and maintenance, been also involved in the pathophysiology of neurodegenerative diseases. Here, we provide evidence that a novel C17-spiroepoxy steroid derivative, BNN27, specifically interacts with and activates the TrkA receptor of NGF, inducing phosphorylation of TrkA tyrosine residues and down-stream neuronal survival-related kinase signaling. Additionally, BNN27 potentiates the efficacy of low levels of NGF, by facilitating its binding to the TrkA receptors and differentially inducing fast return of internalized TrkA receptors into neuronal cell membranes. Furthermore, BNN27 synergizes with NGF in promoting axonal outgrowth, effectively rescues from apoptosis NGF-dependent and TrkA positive sympathetic and sensory neurons, in vitro, ex vivo and in vivo in NGF null mice. Interestingly, BNN27 does not possess the hyperalgesic properties of NGF. BNN27 represents a lead molecule for the development of neuroprotective TrkA receptor agonists, with potential therapeutic applications in neurodegenerative diseases and in brain trauma.

Cloning and characterization of survival-related genes in rat brain primary culture by differential display method

Nerve growth factor (NGF) holds a pivotal role in brain development and maintenance, been also involved in the pathophysiology of neurodegenerative diseases. Here, we provide evidence that a novel C17-spiroepoxy steroid derivative, BNN27, specifically interacts with and activates the TrkA receptor of NGF, inducing phosphorylation of TrkA tyrosine residues and down-stream neuronal survival-related kinase signaling. Additionally, BNN27 potentiates the efficacy of low levels of NGF, by facilitating its binding to the TrkA receptors and differentially inducing fast return of internalized TrkA receptors into neuronal cell membranes. Furthermore, BNN27 synergizes with NGF in promoting axonal outgrowth, effectively rescues from apoptosis NGF-dependent and TrkA positive sympathetic and sensory neurons, in vitro, ex vivo and in vivo in NGF null mice. Interestingly, BNN27 does not possess the hyperalgesic properties of NGF. BNN27 represents a lead molecule for the development of neuroprotective TrkA receptor agonists, with potential therapeutic applications in neurodegenerative diseases and in brain trauma.

β-Secretase Processing of the β-Amyloid Precursor Protein in Transgenic Mice Is Efficient in Neurons but Inefficient in Astrocytes

Alzheimer's disease is characterized by the extracellular deposition of beta-amyloid peptide (Abeta) in cerebral plaques and evidence is accumulating that amyloid is neurotoxic. Abeta is derived from the beta-amyloid precursor protein (APP). Proteolytic processing of APP by the enzyme, beta-secretase, produces the N terminus of Abeta, and releases a secreted ectodomain of APP (beta-s-APP). To develop animal models for measuring beta-secretase activity in specific brain cells in vivo, we have targeted the expression of the full-length human APP to either neurons or astrocytes in transgenic mice using the neuron- specific enolase (NSE) promoter or a modified glial fibrillary acidic protein (GFAP) gene, respectively. The APP cDNAs expressed were mutated (KM to NL at 670/671) to encode amino acid substitutions that enhance amyloidogenic processing in vitro. Western analyses revealed abundant production of beta-s-APP in the brains of NSE-APP mice and enzyme-linked immunosorbent assay analyses showed production of Abeta in fetal primary mixed brain cultures and brain homogenates from these transgenic animals. Because the NSE promoter drives expression primarily in neurons, this provides in vivo evidence that the beta-secretase cleavage necessary for generation of beta-s-APP and Abeta is efficiently performed in neurons. In contrast, only little beta-s-APP was detected in brain homogenates of GFAP-APP mice, indicating that astrocytes show very little beta-secretase activity in vivo. This provides strong in vivo evidence that the major source of Abeta in brain is from neurons and not from astrocytes.

Inhibitory effects of HIV-1 gp120 on myelin formation

One of the pathological features of HIV encephalopathy is myelin damage; the cause of demyelination is unknown. To study the role of HIV-1 gp120 in loss of myelin in HIV encephalopathy, the binding of gp120 to various types of neural cells and its effects on myelination were examined in rat primary brain culture. Double staining of cultured cells with gp120 and specific antibodies for different neural cell types showed that gp120 bound to most of the galactocerebroside (GalCer)-positive oligodendrocytes, a small population of type-2-like astrocytes and a few small neurons. gp120 did not bind to type-1-like astrocytes, most neurons, or to macrophage/microglia. To assay myelination, cells were bathed in a myelination medium containing chick embryo extract and a high concentration of glucose, with or without gp120. Seven days after application, myelination in the culture was observed morphologically and by staining with anti-myelin basic protein (MBP) antibody, and was found to be significantly inhibited by the addition of gp120 (50–100 nM). The processes of oligodendrocytes were reduced in length and arborization relative to the control, but MBP production by oligodendrocytes was unaffected. These results show that gp120 can cause a functional disorder of oligodendrocytes and thus could be one of the reasons for the diffuse loss of myelin sheaths in HIV encephalopathy.

Apoptosis induced by HIV-1 infection of the central nervous system.

Apoptosis plays a role in AIDS pathogenesis in the immune system, but its role in HIV-1-induced neurological disease is unknown. In this study, we examine apoptosis induced by HIV-1 infection of the central nervous system (CNS) in an in vitro model and in brain tissue from AIDS patients. HIV-1 infection of primary brain cultures induced apoptosis in neurons and astrocytes in vitro as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidium iodide staining and by electron microscopy. Apoptosis was not significantly induced until 1-2 wk after the time of peak virus production, suggesting induction by soluble factors rather than by direct viral infection. Apoptosis of neurons and astrocytes was also detected in brain tissue from 10/11 AIDS patients, including 5/5 patients with HIV-1 dementia and 4/5 nondemented patients. In addition, endothelial cell apoptosis was frequently detected in the brain of AIDS patients and was confirmed by electron microscopy. Most of the apoptotic cells were not localized adjacent to HIV-1-infected cells, providing further evidence for induction by soluble factors. In six non-AIDS control patients with normal brain, apoptotic cells were absent or limited to rare astrocytes. However, TUNEL-positive neurons and astrocytes were frequently detected in seven patients with Alzheimer's disease or abundant senile plaques. These studies suggest that apoptosis is a mechanism of CNS injury in AIDS which is likely to be induced by soluble factors. The apoptosis of endothelial cells in the CNS raises the possibility that some of these factors may be blood-derived.

Expression of Neuronal Antigens by Astrocytes Derived from EGF-Generated Neuroprogenitor Cells

Previous studies have demonstrated that astrocytes reacting to CNS injury can express antigens normally associated with neurons. The origin of the reactive astrocytes, i.e., whether they are newly differentiated glial cells or preexisting astrocytes somehow triggered to express neuronal markers, remains difficult to determine using anin vivomodel system. Anin vitromodel may prove more manageable. In the present study, primary brain cultures and EGF-generated neuroprogenitor cells were used to study the expression of neuronal antigens by established (primary) and nascent astrocytes, respectively. Astrocytes derived directly from dissociated mouse brains exhibited a flat morphology typical of type 1 astrocytes. These cells were nestin and GFAP positive and, in most cases, the antigens were colocalized. Primary astrocytes did not appear to express the putative neuronal markers GABA,Tau,or MAP2. Nascent astrocytes derived from EGF-generated progenitor cells showed a similar pattern of GFAP and nestin immunoreactivity. Contrary to primary astrocytes, many GFAP-intensive, stellate astrocytes exhibitedTauand MAP2. These cells also exhibited an intense nestin immunoreactivity. These data suggest that the reactive astrocytes expressing neuronal antigens in response to CNS trauma may be derived from neural progenitor cells rather than from previously differentiated astrocytes.

Detyrosination of tubulin is not correlated to cold-adaptation of microtubules in cultured cells from the Atlantic cod (Gadus morhua).

Isolated cod brain microtubules from the cold-adapted Atlantic cod (Gadus morhua) have previously been shown to be highly detyrosinated, a post-translational modification of tubulin usually found in stable subsets of microtubules. In this study we found this was not restricted only to isolated brain microtubules. Microtubules in primary cultures of brain and skin cells were composed of both tyrosinated (Tyr)- and detyrosinated (Glu)-tubulin seen by immunocytochemistry. Immunoelectron microscopy of isolated microtubules showed that individual microtubules were composed of a mixture of Tyr- and Glu-tubulin. Leukocytes with extending lamellopodia contained only microtubules stained with the antibody against Tyr-tubulin, and isolated heart tubulin lacked both Tyr- and Glu-tubulin, suggesting that a relative high level of detyrosination is a characteristic of most, but not all, cod microtubules. Brain cell microtubules were more resistant to mitotic inhibitors than skin cell microtubules, but this was not correlated to a difference in detyrosination. Brain and skin cell microtubules were only partially disassembled when incubated at 0 degrees C. Upon reassembly of microtubules at 12 degrees C, microtubules were still made of mixtures of Tyr- and Glu-tubulin, indicating that detyrosination of assembled microtubules is rapid and/or that in cod cells in contrast to mammalian cells, Glu-tubulin can reassemble to microtubules. Our data show that most cod microtubules are highly detyrosinated, but this is not the cause of their cold adaptation or drug stability.

Selegiline is neuroprotective in primary brain cultures treated with 1-methyl-4-phenylpyridinium

The ability of selegiline to protect against the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been attributed to the inhibition of the conversion of MPTP to 1-methyl-4-phenylpyridinium (MPP +), catalyzed by monoamine oxidase-B. Selegiline, however, has been found to rescue neurons in MPP +-treated mice after they have sustained lethal damage independently of monoamine oxidase-B inhibition. In our present study, we investigate whether selegiline can protect and/or rescue MPP +-injured dopaminergic neurons in co-cultures of mesencephalic and striatal cells of embryonic C57Bl/6 mouse brains. Cells were exposed to selegiline (1, 10, 100 μM) in three different schemes: (i) in control cultures on the 8th day for 48 h; (ii) pretreatment: on the 8th day for 48 h, followed by administration of MPP + (0.5 μM) on the 9th day for 24 h; (iii) delayed treatment: on the 9th day for 48 h, while MPP + was administered on the 8th day and remained in culture during treatment with selegiline. In the delayed scheme, selegiline (1 μM) increased dopamine content, number of tyrosine hydroxylase immunoreactive cells and astrocytes in the cultures. We question whether selegiline protects cells injured by a toxic stressor via an astrocyte-mediated mechanism.

Reversible Inhibitory Effects of Interferon‐γ and Tumour Necrosis Factor‐α on Oligodendroglial Lineage Cell Proliferation and Differentiation In Vitro

We have investigated the effects of the two prominent inflammatory cytokines, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), on oligodendroglial lineage cell development and survival. Purified oligodendrocytes and oligodendrocyte precursors obtained from neonatal rat brain primary cultures were subcultured in a defined, serum-free medium and exposed to IFN-gamma (1-100 U/ml, TNF-alpha (25-100 ng/ml) or both (100 U/ml and 50 ng/ml respectively) from day 1 to day 3 or from day 3 to day 6. While cell survival was not affected in any of the conditions tested, IFN-gamma dose-dependently inhibited [3H]thymidine or bromodeoxyuridine incorporation (by up to 50%) and the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; by up to 33%). TNF-alpha synergized with IFN-gamma, but was ineffective by itself. Moreover, IFN-gamma totally antagonized the induction by basic fibroblast growth factor and platelet-derived growth factor of the proliferation of the oligodendroglial lineage cell population under study. IFN-gamma also blocked the differentiation of oligodendrocyte precursors, as evidenced by cell morphology, immunostaining for early and late differentiation markers (galactocerebroside and myelin basic protein respectively) and activity of ceramide galactosyl transferase. Again, the effect of IFN-gamma was potentiated by TNF-alpha, which was ineffective when tested alone. The inhibitory activity of IFN-gamma was rapidly reversible: 3 days after removal of the cytokine, administered from day 1 to day 3, complete recovery of cll proliferation and differentiation could be documented. The cytokine-induced arrest in the expression of differentiation antigens was accompanied by perturbations in the expression of the corresponding mRNAs, revealed by a semiquantitative reverse transcription-polymerase chain reaction method. In particular, the message for myelin basic protein (and, in the case of treatment from days 3 to 6, also that for myelin associated glycoprotein) was decreased in cultures exposed to IFN-gamma, and further depressed in cultures treated with IFN-gamma and TNF-alpha, while TNF-alpha alone was ineffective. The above observations may help explain the role of IFN-gamma and TNF-alpha in the pathogenesis of inflammatory demyelinating diseases, in which increases in the levels of these substances have been described. In particular, in the case of multiple sclerosis, our results may bear on the problem of defective remyelination and are consistent with the frequent relapsing-remitting course of the disease.

Genotype-dependent sex differentiation of dopaminergic neurons in primary cultures of embryonic mouse brain

In order to investigate genetic factors that interfere with hormone-mediated sex differentiation of dopaminergic neurons, we raised sex-specific primary cultures from embryonic day 13 diencephalon (D) or mesencephalon (M) of three different strains of mice, NMRI, CBA/J, and BALBc/J. Part of the cultures were maintained for 6 or 13 days in vitro (DIV) in medium containing 17β-estradiol or testosterone. The cultures were analyzed for sex differences in numbers of tyrosine hydroxylase-immunoreactive neurons, endogenous dopamine (DA) levels, and specific uptake of [ 3H]DA. Previous results obtained with cultures of embryonic Sprague-Dawley rats had shown that these parameters develop sex-specific characteristics in the absence of sex differences in hormone environment. Similar steroid-independent sex differences as they occur in the rat were found in M cultures of NMRI but not in CBA and BALBc mice. Long-term sex steroid treatment did not affect any of the above parameters in any strain. It is concluded that cell-autonomous realization of the genetic sex of dopaminergic neurons depends on the genetic background.