Astrocytes accumulate A beta 42 and give rise to astrocytic amyloid plaques in Alzheimer disease brains

beta-Amyloid(1-42) (A beta 42), a major component of amyloid plaques, accumulates within pyramidal neurons in the brains of individuals with Alzheimer's disease (AD) and Down syndrome. In brain areas exhibiting AD pathology, A beta 42-immunopositive material is observed in astrocytes. In the present study, single- and double-label immunohistochemistry were used to reveal the origin and fate of this material in astrocytes. Our findings suggest that astrocytes throughout the entorhinal cortex of AD patients gradually accumulate A beta 42-positive material and that the amount of this material correlates positively with the extent of local AD pathology. A beta 42-positive material within astrocytes appears to be of neuronal origin, most likely accumulated via phagocytosis of local degenerated dendrites and synapses, especially in the cortical molecular layer. The co-localization of neuron-specific proteins, alpha 7 nicotinic acetylcholine receptor and choline acetyltransferase, in A beta 42-burdened, activated astrocytes supports this possibility. Our results also suggest that some astrocytes containing A beta 42-positive deposits undergo lysis, resulting in the formation of astrocyte-derived amyloid plaques in the cortical molecular layer in brain regions showing moderate to advanced AD pathology. These astrocytic plaques can be distinguished from those arising from neuronal lysis by virtue of their smaller size, their nearly exclusive localization in the subpial portion of the molecular layer of the cerebrocortex, and by their intense glial fibrillary acidic protein immunoreactivity. Overall, A beta 42 accumulation and the selective lysis of A beta 42-burdened neurons and astrocytes appear to make a major contribution to the observed amyloid plaques in AD brains.     

Production of nerve growth factor by beta-amyloid-stimulated astrocytes induces p75NTR-dependent tau hyperphosphorylation in cultured hippocampal neurons

Reactive astrocytes surround amyloid depositions and degenerating neurons in Alzheimer's disease (AD). It has been previously shown that beta-amyloid peptide induces inflammatory-like responses in astrocytes, leading to neuronal pathology. Reactive astrocytes up-regulate nerve growth factor (NGF), which can modulate neuronal survival by signaling through TrkA or p75 neurotrophin receptor (p75NTR). Here, we analyzed whether soluble Abeta peptide 25-35 (Abeta) stimulated astrocytic NGF expression, modulating the survival of cultured embryonic hippocampal neurons. Hippocampal astrocytes incubated with Abeta up-regulated NGF expression and release to the culture medium. Abeta-stimulated astrocytes increased tau phosphorylation and reduced the survival of cocultured hippocampal neurons. Neuronal death and tau phosphorylation were reproduced by conditioned media from Abeta-stimulated astrocytes and prevented by caspase inhibitors or blocking antibodies to NGF or p75NTR. Moreover, exogenous NGF was sufficient to induce tau hyperphosphorylation and death of hippocampal neurons, a phenomenon that was potentiated by a low steady-state concentration of nitric oxide. Our findings show that Abeta-activated astrocytes potently stimulate NGF secretion, which in turn causes the death of p75-expressing hippocampal neurons, through a mechanism regulated by nitric oxide. These results suggest a potential role for astrocyte-derived NGF in the progression of AD.     

Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival.

Astrocytes are intimately involved in both glutamate and gamma-aminobutyric acid (GABA) synthesis, and ischemia-induced disruption of normal neuroastrocytic interactions may have important implications for neuronal survival. The effects of middle cerebral artery occlusion (MCAO) on neuronal and astrocytic intermediary metabolism were studied in rats 30, 60, 120, and 240 minutes after MCAO using in vivo injection of [1-13C]glucose and [1,2- 13C]acetate combined with ex vivo 13C magnetic resonance spectroscopy and high-performance liquid chromatography analysis of the ischemic core (lateral caudoputamen and lower parietal cortex) and penumbra (upper frontoparietal cortex). In the ischemic core, both neuronal and astrocytic metabolism were impaired from 30 minutes MCAO. There was a continuous loss of glutamate from glutamatergic neurons that was not replaced as neuronal glucose metabolism and use of astrocytic precursors gradually declined. In GABAergic neurons astrocytic precursors were not used in GABA synthesis at any time after MCAO, and neuronal glucose metabolism and GABA-shunt activity declined with time. No flux through the tricarboxylic acid cycle was found in GABAergic neurons at 240 minutes MCAO, indicating neuronal death. In the penumbra, the neurotransmitter pool of glutamate coming from astrocytic glutamine was preserved while neuronal metabolism progressively declined, implying that glutamine contributed significantly to glutamate excitotoxicity. In GABAergic neurons, astrocytic precursors were used to a limited extent during the initial 120 minutes, and tricarboxylic acid cycle activity was continued for 240 minutes. The present study showed the paradoxical role that astrocytes play in neuronal survival in ischemia, and changes in the use of astrocytic precursors appeared to contribute significantly to neuronal death, albeit through different mechanisms in glutamatergic and GABAergic neurons.     

PPARβ/δ-agonist GW0742 ameliorates dysfunction in fatty acid oxidation in PSEN1ΔE9 astrocytes

Astrocytes are the gatekeepers of neuronal energy supply. In neurodegenerative diseases, bioenergetics demand increases and becomes reliant upon fatty acid oxidation as a source of energy. Defective fatty acid oxidation and mitochondrial dysfunctions correlate with hippocampal neurodegeneration and memory deficits in Alzheimer's disease (AD), but it is unclear whether energy metabolism can be targeted to prevent or treat the disease. Here we show for the first time an impairment in fatty acid oxidation in human astrocytes derived from induced pluripotent stem cells of AD patients. The impairment was corrected by treatment with a synthetic peroxisome proliferator activated receptor delta (PPARβ/δ) agonist GW0742 which acts to regulate an array of genes governing cellular metabolism. GW0742 enhanced the expression of CPT1a, the gene encoding for a rate-limiting enzyme of fatty acid oxidation. Similarly, treatment of a mouse model of AD, the APP/PS1-mice, with GW0742 increased the expression of Cpt1a and concomitantly reversed memory deficits in a fear conditioning test. Although the GW0742-treated mice did not show altered astrocytic glial fibrillary acidic protein-immunoreactivity or reduction in amyloid beta (Aβ) load, GW0742 treatment increased hippocampal neurogenesis and enhanced neuronal differentiation of neuronal progenitor cells. Furthermore, GW0742 prevented Aβ-induced impairment of long-term potentiation in hippocampal slices. Collectively, these data suggest that PPARβ/δ-agonism alleviates AD related deficits through increasing fatty acid oxidation in astrocytes and improves cognition in a transgenic mouse model of AD.     

The effect of amyloid associated proteins on the expression of genes involved in amyloid-β clearance by adult human astrocytes

Astrocytes appear to be important mediators in the clearance of amyloid beta1-42 (Aβ), the key component of senile plaques characteristic of Alzheimer's disease (AD). Recently, we found the amyloid associated proteins (AAPs) α1-antichymotrypsin (ACT), apolipoprotein J and E (ApoJ and ApoE) and a mixture of serum amyloid P (SAP) and C1q (SAP-C1q) to modify Aβ-uptake by human astrocytes. Here we investigated the effect of oligomeric (Aβoligo) and fibrillar Aβ (Aβfib), alone and in combination with a panel of AAPs on the astrocytic expression of genes proposed to be involved in Aβ-uptake and degradation. Primary human astrocytes (isolated from non-demented control (n=4) and AD patient (n=4) brain specimens) were exposed to either Aβoligo or Aβfib preparations with or without the above mentioned AAPs. Quantitative gene expression analysis of Aβ-receptors Scavenger receptor B1 (SCARB1), macrophage receptor with collagenous structure (MARCO) and low density lipoprotein receptor related protein-2 (LRP2 or megalin) as well as of Aβ-degrading enzymes neprilysin (NEP), insulin-degrading enzyme (IDE) and metalloproteinase-9 (MMP-9) was performed by real-time PCR. Basal expression of NEP, IDE and SCARB1 was easily detected whereas expression of MARCO, LRP2 and MMP-9 could only be detected upon pre-amplification. Basal expression of NEP, IDE and SCARB1 did not change upon exposure to Aβoligo or Aβfib alone in any of the investigated astrocyte cultures. Interestingly NEP expression was increased upon exposure to ApoE in combination with both Aβ-preparations, and also SCARB1 expression was induced upon treatment with ApoE in combination with Aβfib in astrocytes from non-demented controls. Further, SAP-C1q increased SCARB1 expression in control astrocytes when combined with Aβoligo. These alterations were not found in astrocytes from AD patients. Thus, we conclude that Aβ alone apparently does not affect the astrocytic expression of IDE, NEP or SCARB1. However, NEP and SCARB1 expression is increased in astrocytes from non-demented subjects when exposed to Aβ combined with AAPs like ApoE. These astrocytic gene expression-regulatory mechanisms appear to be defective in AD and thus might contribute to the development and progression of AD pathology.     

Peroxiredoxin 6 in human brain: molecular forms,cellular distribution and association with Alzheimer’s disease pathology

Peroxiredoxin 6 is an antioxidant enzyme and is the 1-cys member of the peroxiredoxin family. Using two-dimensional electrophoresis and Western blotting, we have shown for the first time that, in human control and brain tissue of patient's with Alzheimer's disease (AD), this enzyme exists as three major and five minor forms with pIs from 5.3 to 6.1. Using specific cellular markers, we have shown that peroxiredoxin 6 is present in astrocytes with very low levels in neurons, but not detectable in microglia or oligodendrocytes. In control brains, there was a very low level of peroxiredoxin 6 staining in astrocytes that was confined to a "halo" around the nucleus. In AD, there were marked increases in the number and staining intensity of peroxiredoxin 6 positive astrocytes in both gray and white matter in the midfrontal cortex, cingulate, hippocampus and amygdala. Confocal microscopy using antibodies to A beta peptide, tau and peroxiredoxin 6 showed that peroxiredoxin 6 positive astrocytes are closely involved with diffuse plaques and to a lesser extent with neuritic plaques, suggesting that plaques are producing reactive oxygen species. There appeared to be little astrocytic response to tau containing neurons. Although peroxiredoxin 6 positive astrocytes were seen to make multiple contacts with tau positive neurons, there was no intraneuronal colocalization. In brain tissue of patients with AD, many blood vessels exhibited peroxiredoxin 6 staining that appeared to be due to the astrocytic foot processes. These results suggest that oxidative stress conditions exist in AD and that peroxiredoxin 6 is an important antioxidant enzyme in human brain defenses.     

Endothelial nitric oxide synthetase (eNOS) in astrocytes: another source of nitric oxide in neocortex.

The distribution of the endothelial form of nitric oxide synthetase (eNOS) was examined in the visual cortex of three species of primate and in the rat using immunocytochemistry. Labeled cells were found in both the gray and white matter. These cells were stellate in appearance and labeled cell processes were seen contacting blood vessels or the pia, suggesting that, by morphological criteria, the cells were astrocytes. All eNOS positive cells were double labeled with an antibody against S100beta. Although all cells were double labeled in the white matter, in the gray matter, some S100beta positive cells did not contain detectable levels of eNOS. eNOS positive astrocytic processes appeared to form prominent and distinctive structures next to neurons, especially in cortical layer IIIC. We postulate that these eNOS-positive structures form astrocytic perisynaptic sheaths on neuronal somas in the cortex. If this is true, then nitric oxide can influence neuronal transmission directly at axosomatic synapses in the cortex. In addition, the presence of eNOS in astrocytes and in their processes that contact blood vessels suggests that the link between local cortical activity and changes in cerebral blood flow could be mediated by astrocytic release of nitric oxide.     

Changes in the cellular distribution of glutamine synthetase in Alzheimer's disease.

The intracellular localization of glutamine synthetase (GS) in the inferior temporal cortices of non-demented elderly individuals was compared with that in brains affected by Alzheimer's disease (AD). The present study confirmed previous reports of a general decrease in GS expression in astrocytes and the expression of GS in some neurons. Several new observations were made: the morphology of astrocytes is generally unaffected by the presence of plaques, GS labeling is present in some diffuse plaques and occasional neuritic plaques, whereas the overall density of astrocytes increases 1.4-fold in AD. In addition, the present study found that the reduction in GS expression is almost entirely due to a loss of GS from perisynaptic regions of the neuropil and from the astrocytic endfeet that normally abut cortical blood vessels. These changes implicate astrocytes in glutamate excitotoxicity and ammonia neurotoxicity. It is suggested that it may be more fruitful to regard AD not as a neuronal disease, but as a disorder of astrocyte-neuron interactions.     

Correlation between astrocyte apoptosis and Alzheimer changes in gray matter lesions in Alzheimer's disease

The relationships between astrocytic apoptosis and both senile plaques and neurofibrillary tangles (NFT) in gray matter lesions were examined quantitatively in Alzheimer's disease (AD) brains. Seven cortical regions were examined in seven AD brains by terminal dUTP nick end-labeling and immunolabeling with antibodies to glial fibrillary acidic protein, phosphorylated tau protein (AT180), apoptosis-related proteins (caspase-3, bcl-2, and CD95), and beta amyloid protein. Senile plaques showed the lowest density in the cornu ammonis. The density of apoptotic astrocytes was significantly correlated with the density of uncored and cored senile plaques. Neuronal caspase-3 and CD95 expression levels were too low to allow statistical assessment, but Bcl-2 was expressed strongly in the astrocytes and neurons with and without NFT. The correlation of the density of apoptotic astrocytes with apoptotic neurons and NFT was not statistically significant. The density of Bcl2-positive neurons correlated significantly with those of NFT and cored senile plaques, but Bcl2-positive astrocyte density showed no correlation with density of senile plaques or apoptotic astrocytes. These observations suggest that senile plaques may be a cause of astrocytic apoptosis in the gray matter, and that Bcl-2 protein is associated with NFT formation.     

Astrocytes down-regulate neuronal β-amyloid precursor protein expression and modify its processing in an apolipoprotein E isoform-specific manner

Alzheimer's disease is the most frequent neurodegenerative disorder in the aged population and is characterized by the deposition of the 40/42-residue amyloid beta protein (A beta), a proteolytic fragment of the beta-amyloid precursor protein (APP). A common apolipoprotein E (apoE) polymorphism is associated with an increased risk of developing the disease. In order to assess the putative relationship between apoE and amyloidogenesis in the CNS, we prepared primary cortical neurons overexpressing humanized APP695 bearing the Swedish mutation (hAPP(695sw)) and we analysed APP expression and processing after: (i) coculture with primary astrocytes from wild-type, apoE-deficient (E0) mice, or mice overexpressing human apoE2, E3, or E4; (ii) treatment with conditioned media from apoE0, E2, E3 or E4 astrocytes; and (iii) treatment with human recombinant ApoE or human apoE purified from conditioned media of stably transfected RAW264 cells (E2, E3 and E4). Interestingly, a strong decrease in APP expression was observed only when neurons were cocultured with astrocytes (and independently of the apoE genotype considered), suggesting that cell-cell contact is required. Moreover, apoE4-secreting astrocytes, but not recombinant or purified apoE4, significantly increased A beta production and decrease sAPP alpha secretion only when cultured in direct contact with neurons, whereas apoE2 astrocytes had a protective effect. We conclude that astrocytes: (i) strongly regulate neuronal APP expression in primary neurons, and (ii) promote the amyloidogenic pathway in an apoE4-dependent manner. Thus, apoE and astrocytic factor(s) may modulate the pathogenesis of Alzheimer's disease.     

Glutamatergic neurotransmission and protein kinase C play a role in neuron-glia communication during the development of methamphetamine-induced psychological dependence

Methamphetamine (METH) is a strongly addictive psychostimulant that dramatically affects the central nervous system (CNS). On the other hand, protein kinase C (PKC) plays a major role in cellular regulatory and signalling processes that involve protein phosphorylation. The purpose of this study was to investigate the role of neuronal and astrocytic PKC in changes in the central glutamatergic system induced by METH. We show here that in vitro treatment with METH caused the phosphorylation of both neuronal and astrocytic PKC and the activation of astrocytes in cortical neuron/glia co-cultures. Treatment of cortical neuron/glia co-cultures with either the PKC activator phorbol 12,13-dibutyrate (PDBu) or glutamate also caused the PKC-dependent activation of astrocytes. The PKC inhibitor chelerythrine suppressed the Ca2+ responses to glutamate in both cortical neurons and astrocytes. Moreover, a low concentration of PDBu significantly enhanced the Ca2+ responses to glutamate, but not to dopamine, in both cortical neurons and astrocytes. Notably, treatment with METH also enhanced the Ca2+ responses to glutamate in cortical neurons. The activation of astrocytes induced by METH was also reversed by co-treatment with glutamate receptor antagonists (ifenprodil, DNQX or MPEP) in cortical neuron/glia co-cultures. In the conditioned place preference paradigm, intracerebroventricular administration of glutamate receptor antagonists (ifenprodil, DNQX or MPEP) attenuated the METH-induced rewarding effect. These findings provide evidence that the changes in PKC-dependent neuronal and astrocytic glutamatergic transmission induced by METH may, at least in part, contribute to the development of psychological dependence on METH.     

LRP and senile plaques in Alzheimer's disease: colocalization with apolipoprotein E and with activated astrocytes

The low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor which is present on senile plaques in Alzheimer's disease (AD). It is suggested to play an important role in the balance between amyloid beta (Abeta) synthesis and clearance mechanisms. One of its ligands, apolipoprotein E (apoE), is also present on senile plaques and has been implicated as a risk factor for AD, potentially affecting the deposition, fibrillogenesis and clearance of Abeta. Using immunohistochemistry we show that LRP was present only on cored, apoE-containing senile plaques, in both PDAPP transgenic mice and human AD brains. We detected strong LRP staining in neurons and in reactive astrocytes, and immunostaining of membrane-bound LRP showed colocalization with fine astrocytic processes surrounding senile plaques. LRP was not present in plaques in young transgenic mice or in plaques of APOE-knockout mice. As LRP ligands associated with Abeta deposits in AD brain may play an important role in inducing levels of LRP in both neurons and astrocytes, our findings support the idea that apoE might be involved in upregulation of LRP (present in fine astrocytic processes) and act as a local scaffolding protein for LRP and Abeta. The upregulation of LRP would allow increased clearance of LRP ligands as well as clearance of Abeta/ApoE complexes.     

beta-Amyloid immunoreactivity in astrocytes in Alzheimer's disease brain biopsies: an electron microscope study.

The deposition of amyloid beta (A beta) protein plays a central role in the neuropathology of Alzheimer's disease (AD) and it constitutes the core of classical senile plaques. However, little is known about its intracellular distribution. An immunogold electron microscope study was therefore carried out on biopsies of brain tissue from patients with AD using a monoclonal antibody raised against residues 8 to 17 of the A beta protein. Specific A beta immunogold labeling was observed over extracellular amyloid fibrils associated with senile plaques. In addition, widespread intracellular A beta immunolabeling was observed adjacent to granular structures (30-40 nm in diameter) within membrane-bound processes. Pretreatment of some sections with amylase or omission of lead citrate staining from others strongly suggests that the electron-dense granular structures associated with A beta immunoreactivity are glycogen. Some of the A beta-immunolabeled processes contained gliofilaments and immunolabeling of alternate sections for glial fibrillary acidic protein confirmed that the A beta-immunolabeled processes were astrocytic. A beta immunolabeling was not observed over neuronal or microglial processes. Whether the presence of A beta protein in astrocytes is the result of synthetic or degradation processes requires further investigation.     

Neuronal and nonneuronal quantitative BACE immunocytochemical expression in the entorhinohippocampal and frontal regions in Alzheimer's disease

In this study, we quantitatively investigated the expression of beta-site amyloid precursor protein cleaving enzyme (BACE) in the entorhinohippocampal and frontal cortex of Alzheimer's disease (AD) and old control subjects. The semiquantitative estimation indicated that the intensity of BACE overall immunoreactivity did not differ significantly between AD and controls, but that a significantly stronger staining was observed in the hippocampal regions CA3-4 compared to other regions in both AD patients and controls. The quantitative estimation confirmed that the number of BACE-positive neuronal profiles was not significantly decreased in AD. However, some degeneration of BACE-positive profiles was attested by the colocalization of neurons expressing BACE and exhibiting neurofibrillary tangles (NFT), as well as by a decrease in the surface area of BACE-positive profiles. In addition, BACE immunocytochemical expression was observed in and around senile plaques (SP), as well as in reactive astrocytes. BACE-immunoreactive astrocytes were localized in the vicinity or close to the plaques and their number was significantly increased in AD entorhinal cortex. The higher amount of beta-amyloid SP and NFT in AD was not correlated with an increase in BACE immunoreactivity. Taken together, these data accent that AD progression does not require an increased neuronal BACE protein level, but suggest an active role of BACE in immunoreactive astrocytes. Moreover, the strong expression in controls and regions less vulnerable to AD puts forward the probable existence of alternate BACE functions.     

Rapid up-regulation of the neuronal serotoninergic phenotype by brain-derived neurotrophic factor and cyclic adenosine monophosphate: relations with raphe astrocytes

Up-regulation of the neuronal serotoninergic phenotype in relation to astrocytic population was studied in primary cultures of rat embryonic rostral raphe. Short treatments (18 hr at day in vitro 4) with brain-derived neurotrophic factor (BDNF) or dibutyryl-cAMP (dBcAMP) increased the number of serotoninergic neurons by approximately 80% and approximately 40%, respectively, and markedly enhanced the branching (by 11-fold and 5-fold, respectively) and total length (by 4-fold and 2.5-fold, respectively) of their neurites. Concomitantly, under BDNF treatment, the astrocyte population was decreased by half and became mostly protoplasmic-like. In contrast, dBcAMP treatment also reduced the astrocytic cell density (by one-third) but induced a stellate morphology. Similar short treatment with the astrocyte-derived S100beta factor induced no modification of the serotonin (5-HT) neuronal phenotype nor of astrocytes morphology. Both BDNF- and cAMP-induced effects were abolished by simultaneous treatment with the specific tyrosine kinase inhibitor genistein, suggesting a role for the high-affinity BDNF receptor tyrosine kinase (TrkB). These data suggest that BDNF and cAMP, but not S100beta, rapidly induce both an up-regulation of the 5-HT neuronal phenotype and modifications of the neighboring astrocytes in a TrkB-dependent manner.     

A central role for astrocytes in the inflammatory response to beta-amyloid; chemokines, cytokines and reactive oxygen species are produced.

Alzheimer's disease (AD) is the commonest form of adult onset dementia and is characterised neuropathologically by the accumulation of plaques containing beta-amyloid (A beta) fibrils, reactive astrocytes, activated microglia, and leukocytes. A beta plays a role in the pathology of AD by directly causing neuronal cytotoxicity and stimulating microglia to secrete cytokines and reactive oxygen species (ROS) which also damage neurons. Here, we demonstrate that A beta activates astrocytes and oligodendrocytes (the most common cell types in the brain) to produce chemokines, in particular MCP-1 and RANTES, which serve as potent in vitro microglial and macrophage chemoattractants. Furthermore, we have shown that A beta activates astrocytes to upregulate pro-inflammatory cytokine expression and enhances the production of ROS. We propose therefore that A beta-mediated astrocyte activation initiates an inflammatory cascade which could be targeted for therapeutic intervention in AD.     

Minocycline inhibits neuronal death and glial activation induced by beta-amyloid peptide in rat hippocampus

Minocycline, a second-generation tetracycline compound, has been examined as a neuroprotectant in beta-amyloid (A beta)-injected rat hippocampus. At 7 days post-injection, A beta(1-42) caused a significant loss of granule cell layer neurons (28% reduction) compared to control uninjected hippocampus. Hippocampal injection of A beta peptide also led to marked gliosis with numbers of microglia (increased by 26-fold) and immunoreactivity of astrocytes (increased by 11-fold) relative to control, as determined from immunohistochemical analysis. Intraperitoneal administration of minocycline significantly reduced neuronal loss induced by A beta(1-42) (by 80%) and also diminished numbers of microglia (by 69%) and astrocytes (by 36%) relative to peptide alone. Peptide injection increased expression of cyclooxygenase-2 (COX-2) in most (about 70%) of granule cells, a subset (about 20%) of microglia, but not in astrocytes; in the presence of minocycline, COX-2 immunostaining was abolished in microglia. The results from this study suggest that minocycline may have efficacy in the treatment of AD.     

Guidance of dopaminergic neuritic growth by immature astrocytes in organotypic cultures of rat fetal ventral mesencephalon.

Astrocytes, with their many functions in producing and controlling the environment in the brain, are of great interest when it comes to studying regeneration after injury and neurodegenerative diseases such as in grafting in Parkinson's disease. This study was performed to investigate astrocytic guidance of growth derived from dopaminergic neurons using organotypic cultures of rat fetal ventral mesencephalon. Primary cultures were studied at different time points starting from 3 days up to 28 days. Cultures were treated with either interleukin-1 beta (IL-1 beta), which has stimulating effects on astrocytic proliferation, or the astrocytic inhibitor cytosine arabinoside (Ara-C). Tyrosine hydroxylase (TH)-immunohistochemistry was used to visualize dopaminergic neurons, and antibodies against glial fibrillary acidic protein (GFAP) and S100 beta were used to label astrocytes. The results revealed that a robust TH-positive nerve fiber production was seen already at 3 days in vitro. These neurites had disappeared by 5 days. This early nerve fiber outgrowth was not guided by direct interactions with glial cells. Later, at 7 days in vitro, a second wave of TH-positive neuritic outgrowth was clearly observed. GFAP-positive astrocytic processes guided these neurites. TH-positive neurites arborized overlying S100 beta-positive astrocytes in an area distal to the GFAP-positive astrocytic processes. Treatment with IL-1 beta resulted in an increased area of TH-positive nerve fiber network. In cultures treated with Ara-C, neither astrocytes nor outgrowth of dopaminergic neurites were observed. In conclusion, this study shows that astrocytes play a major role in long-term dopaminergic outgrowth, both in axonal elongation and branching of neurites. The long-term nerve fiber growth is preceded by an early transient outgrowth of dopamine neurites.     

Neurotrophic effects of basic and acidic fibroblast growth factors are not mediated through glial cells

Basic and acidic fibroblast growth factors (bFGF, aFGF) increase the survival of fetal hippocampal pyramidal neurons in serum-free cultures. bFGF is also a mitogen for astrocytes either in highly purified glial cultures or as a contaminant in neuronal cultures. The possibility that bFGF enhances neuronal survival indirectly through stimulating glial proliferation is unlikely. In the presence of 1 ng/ml bFGF, the total number of contaminating astrocytes (as defined by immunohistochemical staining for glial fibrillary acidic protein (GFAP] was increased to 4.3% vs 0.9% in control hippocampal cultures. aFGF did not significantly increase astrocyte number while supporting neuronal survival. Two other agents which stimulated equal or greater astrocytic proliferation, epidermal growth factor (EGF) and 10% serum, did not support neurons, and bFGF still significantly increased neuronal survival in their presence. When glial proliferation was inhibited by aphidicolin, contamination decreased to 0.1% in controls and 1.0% with 1 ng/ml bFGF, yet the neurons remained responsive to FGF. Cultures lacking any detectable GFAP-positive cells were identified, and even in the absence of glial cells, aFGF and bFGF increased neuronal survival. Because there is no significant correlation between the neuronal response and astrocyte number, it appears that bFGF and aFGF can directly support neuronal survival.