Influence of serum-free medium on the expression of glutamate transporters and the susceptibility to glutamate toxicity in cultured cortical neurons

The presence of glia and glial glutamate transporters seems to modify glutamate-mediated toxicity in neuronal cultures. In this work we cultured cortical cells in serum-containing medium and in a serum-free medium (Neurobasal medium + B27 supplement) and studied the expression of the glutamate transporters GLAST, GLT, and EAAC by immunocytochemistry and RT-PCR. The proportion of glial cells was below 10% in the Neurobasal medium and 46% in the serum-containing medium. Semiquantitative evaluation of the mRNA for the glutamate transporters showed similar amounts in cells grown in serum-free and serum-containing media. We detected immunoreactivity for the three transporters in both media, but EAAC was coexpressed with the neuronal marker MAP2, whereas GLAST and GLT predominated in nonneuronal cells. When the cultures were treated with glutamate for 15 min, the cultures in serum-containing medium showed a clear concentration-dependent neuronal death, whereas cells primed in this medium and switched to Neurobasal medium, as well as cells grown only in the latter, were less sensitive to glutamate concentrations up to 1 mM. A similar difference in the sensitivity to excitotoxicity was observed when the glutamate uptake inhibitor L-trans-2,4-pyrrolidine-dicarboxylate was applied during 6 hr, although the accumulation of extracellular glutamate was similar in the two media. We conclude that glutamate transporters with the culture conditions studied are sensitive to glutamate uptake inhibition and that Neurobasal/B27 medium protects cells against excitotoxicity.     

Differential expression of exons 10 and 11 in normal tau and tau associated with paired helical filaments

Antibodies were raised to two synthetic peptides with amino acid sequences encoded by a variable region of exons 10 and 11 of the tau gene. The affinity-purified antibodies, designated E-10 and E-11, were used to determine whether PHF-tau and normal tau differ in variants containing three or four repeats in the microtubule-binding domain, respectively. Normal adult human brain was shown by gel electrophoresis to contain six isoforms of tau. All of the isoforms reacted with E-11, whereas only four of them with slower electrophoretic mobility were recognized by E-10. Fetal brain tau was readily recognized by E-11 but reacted poorly with E-10. In PHF preparations, E-11 bound to all three polypeptides of PHF-tau of 68 kD, 64 kD, and 60 kD and reacted intensely with a material smearing from the top of the gel to about the 50-kD region. In contrast, E-10 only weakly recognized the two higher molecular weight PHF-tau polypeptides of 68 kD and 64 kD, as well as smeared material, and the binding was not affected by phosphatase treatment. Using recombinant tau with four repeats as a reference, the immunoreactivity of E-10 with PHF-tau was estimated to be approximately 5% of that of E-11. By comparison, the immunoreactivity of E-10 with four isoforms of normal tau was comparable to that of E-11. These results indicate that the ratio of three vs. four repeat variants in PHF-tau is higher than in normal tau and suggest that Alzheimer disease may be associated with the disproportional expression of fetal (or juvenile) forms of tau. Alternatively, the weak reactivity of PHF-tau with E-10 antibody could be due to post-translational modifications other than phosphorylation. © 1995 Wiley-Liss, Inc.     

AMPA-Selective glutamate receptor subunits in astroglial cultures

We analysed AMPA ionotropic receptor subunits at the mRNA level (GluR-1 to -4) and at the protein level (GluR-1 and GluR-2/3/4C) in “primary astroglial cultures” (non-neuronal cell cultures highly enriched in glial fibrillary acidic protein [GFAP] positive cells) prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocamps, and striatum and in “brain non-neuronal cell cultures” (low percentage of GFAP positive cells) prepared from cerebellum, brainstem, mesencephalon, and hypothalamus. For comparison, we also determined ampa subunit mRNA and protein levels in different brain regions. By Northern blot analysis mRNAs for the AMPA receptor subunits (glur-1,-2,-3,-4) were detected in primary rat cerebral hemispheres astroglial cultures. Immunoblotting analysis with anti-GluR-1 and anti GluR-2/3/4C polyclonal antibodies confirmed the presence of low leve of immunoreactive proteins of the same size of those identifice in vivo as GluR subunits. Expression of GluR genes varied depending on the brain area used as starting material for the preparation of the cultures: GluR-1, -2, and -3 were mailly expressed in cortical cultures, while GluR-4 expression predominated in brainstem derived cultures. Interestingly this pattern of expression correlates with that observed in the intact brain, where high levels of GluR-4 mRNA and low levels of the other GluR subunits were found in the brainstem. In conclusion our results confirm the existence of glutmate ionotropic receptors of the AMPA type in primary astroglial cultrues and suggest that GluR-4 is the main AMPA receptor subunit expressed in non neuronal cells of the central nervous system. © 1993 Wiley-Liss, Inc.     

Opioids influence neurotransmitter phenotypic expression in chick embryonic neuronal cultures

There is considerable interest in the role of endogenous opioid peptides in neural growth and differentiation. In this study we used neuron-enriched cultures derived from 3-day-old chick embryos to test the effects of endogenous enkephalins on neurotransmitter phenotypic expression. Cultures were grown in serum-free chemically defined medium and were treated with either Met-enkephalin antiserum (anti-Met) to immunoneutralize enkephalins, or with naloxone, a universal opioid receptor antagonist, to block receptor-mediated actions of released endogenous opioids. The enzyme activities of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) were used as markers for cholinergic and catecholaminergic phenotypic expression, respectively. We found that cultures treated with anti-Met or naloxone exhibited strikingly different neuronal growth patterns as compared to controls. In addition, ChAT activity was enhanced by anti-Met, and TH activity by both anti-Met and naloxone. These findings lend support to the possibility that neuropeptides may be co-localized with neurotransmitters and that peptides released into the microenvironment affect neuronal phenotypic expression by differential receptor subtypes.     

Phospholipid and phospholipid fatty acid composition of mixed murine spinal cord neuronal cultures

The phospholipid and phospholipid fatty acid compositions of mixed murine spinal cord neuronal cultures are reported. The phospholipid composition was primarily comprised of ethanolamine glycerophospholipids (44.8%) and choline glycerophospholipids (43.5%). Plasmalogens made up 29.1% of the ethanolamine glycerophospholipids (13.0% of the total phospholipids) and 4.5% of the choline glycerophospholipids (1.9% of the total phospholipids). Other phospholipids ranged from 2.9% for sphingomyelin to 1.0% for phosphatidylinositol 4-phosphate. The fatty acid compositions of the ethanolamine glycerophospholipids, choline glycerophospholipids, phosphatidylserine, and phosphatidylinositol were also determined. The choline glycerophospholipids were the most saturated and contained the smallest amount of polyunsaturated fatty acids. The ethanolamine glycerophospholipids were the most unsaturated and contained the highest amount of polyunsaturated fatty acids. The phospholipids contained minimal amounts of 20:3 n-9 (Mead acid) and are not considered polyunsaturated fatty acid deficient. Thus, for the mixed neuronal spinal cord cultures, the phospholipid fatty acid compositions were not polyunsaturated fatty acid deficient and contained a large amount of polyenoic fatty acids of both the n-3 and n-6 series. © 1993 Wiley-Liss, Inc.     

P0 mRNA expression in cultures of Schwann cells and neurons that lack basal lamina and myelin.

Schwann cells of the peripheral nervous system depend on the presence of both axons and basal lamina to achieve a myelinating phenotype. Furthermore, removal of axonal influence results in the cessation of myelination and down-regulation of myelin protein expression by Schwann cells. Here we examine whether both axons and basal lamina are required by Schwann cells for the expression of mRNA encoding the major myelin glycoprotein, P0. Cultures of Schwann cells and neurons obtained from dorsal root ganglia of 15 day embryonic rat pups were grown for up to 20 days in vitro under conditions that either allowed or prohibited basal lamina and myelin formation. These cultures were assayed for the expression of P0 mRNA by using an S1 nuclease-protection assay. After 20 days in vitro, the cultures that did not assemble basal lamina and that were incapable of myelin formation expressed P0 mRNA at a level which was comparable to that seen in identically aged, myelinating cultures. Both the myelinating and nonmyelinating cultures demonstrated an appreciable increase in P0 mRNA when compared to the starting embryonic dorsal root ganglia Schwann cells. The latter had a low, but detectable, level of mRNa for this myelin glycoprotein. The cultures that were devoid of basal lamina and myelin showed a clear increase in P0 mRNA by 11-15 days in culture. This increase in expression depended on the presence of neurons/neurites, since Schwann cells which were grown in neuron-depleted cultures expressed little, if any, P0 mRNA. In contrast to the levels of P0 mRNA, the nonmyelinating cultures had a significantly lower amount of P0 glycoprotein than did the cultures which assemble myelin. This suggests that the nonmyelinating Schwann cells regulate the level of this glycoprotein at the translational and/or the posttranslational level. The data presented here suggest that myelin protein mRNA expression and myelin assembly by Schwann cells are separable events, with the former depending on one or more neuronal/axonal factors.     

Angiotensinogen gene expression in neuronal and glial cells in primary cultures of rat brain

Neuronal and glial cells in primary culture prepared from the hypothalamic-brain stem areas of one-day-old rat brains were analyzed for the presence of angiotensinogen messenger RNA (mRNA) to further confirm our previous conclusion that the brain contains an exclusive angiotensin system. Angiotensinogen mRNA was quantitated by Northern analysis using nick-translated angiotensinogen cDNA as the hybridization probe (Kunapuli and Kumar, 1987). Angiotensinogen mRNA sequences were present in the RNA isolated from both neuronal and glial cultures. Quantitative measurements of the mRNA using dot-blot analysis revealed that the level of angiotensinogen mRNA was three times higher in neuronal cultures compared with glial cultures. These observations provide the first evidence for the synthesis of angiotensinogen in neuronal as well as in glial cells form the brain.     

Brain tau expression and correlation with the H1/H1 tau genotype in frontotemporal lobar degeneration patients

Summary Alterations in tau mRNA splicing and association with H1/H1 tau genotype have been described in some sporadic tauopathies. We evaluated the 4R/3R tau mRNA ratio in 18 patients with frontotemporal lobar degeneration (FTLD), and the effect of the H1/H1 genotype on this ratio. The 4R/3R mRNA ratio in frontal cortex was similar in FTLD patients and controls. The H1/H1 genotype carriers showed a significant increase in 4R/3R mRNA ratio, suggesting that this genotype could modulate the tau mRNA splicing. The first two authors contributed equally to this work     

Effects of methadone on ornithine decarboxylase and cyclic nucleotide phosphohydrolase in neuronal and glial cell cultures

Mixed neuronal and nonneuronal cell cultures were obtained from 8-day-old chick embryos cerebral hemispheres and glial-enriched cultures were obtained from fifteen-day-old chick embryo cerebral hemispheres. Cultures were exposed to methadone, a narcotic drug, from days four to six. The activity of ornithine decarboxylase (ODC) was determined at day eight and the activity of cyclic nucleotide phosphohydrolase (CNP) was determined at day fifteen. Both ODC and CNP activity were higher in mixed neuronal-nonneuronal cell cultures treated with methadone as compared to control. No effect was observed in the neuronal-enriched or glial-enriched cultures. These findings are interpreted to reflect that neuronal-glial interaction is important in the response of primary neural cells to methadone.     

Truncated tau expression levels determine life span of a rat model of tauopathy without causing neuronal loss or correlating with terminal neurofibrillary tangle load

We have previously demonstrated in a transgenic rat model of tauopathy that human misfolded truncated tau derived from Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo . We employed this model to investigate the impact of truncated tau expression levels on life span, neuronal loss and the final load of neurofibrillary tangles (NFTs) in transgenic rats. Two independent transgenic lines (SHR72, SHR318), that display different expression levels of truncated tau, were utilized in this study. We found that transgene expression levels in the brain of SHR72 rats were 44% higher than in SHR318 rats and that truncated tau protein levels determined the survival rate of transgenic rats. The line with higher expression levels of truncated tau (SHR72) showed decreased median survival (222.5 days) when compared with the line with lower expression (SHR318; 294.5 days). Interestingly, NFT loads (total NFT/total neurons) were very similar in terminal stages of disease in both transgenic lines (SHR72 – 10.9%; SHR318 – 11.6%), despite significantly different expression levels of truncated tau. Moreover, mean neuron numbers in the hippocampus (CA1–3) and brain stem (gigantocellular reticular nucleus) in the two transgenic rat strains in the terminal stages of disease were similar, and did not differ significantly from those observed in age-matched non-transgenic controls. These findings suggest that the expression levels of misfolded truncated tau determine the life span in a transgenic rat model of tauopathy without causing neuronal loss or correlating with terminal NFT load.     

Familial tauopathy with P364S MAPT mutation: clinical course,neuropathology and ultrastructure of neuronal tau inclusions

Aims

This report presents the clinical course, neuropathology and ultrastructure of neuronal tau inclusions of four Slovene relatives with P364S MAPT mutation.

Methods

The clinical history of three out of four P364S MAPT mutation carriers was taken. After formalin fixation, thorough sampling of the central nervous system was followed by paraffin embedding, H&E, Gallyas, Bielschowsky and immunostaining with AT8, anti‐3R, anti‐4R tau, anti‐amyloid‐β, anti‐TDP43 and anti‐alpha‐synuclein antibodies. The distribution and density of different types of neuronal tau inclusions were semiquantitatively assessed. In addition, the ultrastructure of neuronal tau inclusions was analysed.

Results

Macroscopic examination of the brains was unremarkable. Microscopically, neuronal tau inclusions of almost all known types were widespread and distributed fairly uniformly in all cases. Pick bodies and swollen neurones were found in only one family member. Mutant tau was composed of 3R and 4R isoforms, with a slight predominance of 3R tau. Composite neuronal tau inclusion (CNTI), found in all four relatives, was a hallmark of the P364S MAPT mutation. CNTI showed compartmental differences in H&E and Gallyas staining, tau isoforms immunolabelling and ultrastructure, displaying fuzzy fibrils in the core and paired twisted tubules at the periphery.

Conclusions

P364S MAPT mutation is characterized clinically by a variable combination of frontotemporal dementia, parkinsonism and motor neurone disease of short duration, and neuropathologically by a widespread uniform distribution of all known neuronal tau inclusions in one family member. Two‐compartment CNTI is a unique characteristic of the P364S MAPT mutation.     

Regulation of corticotropin-releasing factor-binding protein expression in amygdalar neuronal cultures

Corticotropin-releasing factor-binding protein (CRF-BP) is known to regulate the bioavailability of CRF and may also play a role in stress behaviours. CRF-BP has been localized in the pituitary as well as central nervous system (CNS) limbic and cortical areas, including the amygdala. The signal transduction pathways which regulate amygdalar CRF-BP are not well understood. In this report, we have examined the effect of protein kinase A and C activators, CRF, dexamethasone and interleukin-6 (IL6) on CRF-BP mRNA and protein expression in dissociated fetal amygdalar cultures. CRF-BP mRNA levels were determined by Northern analysis following 12 h treatment with the following agents: forskolin (1-30 microM), CRF (1-1000 nM), phorbol-12-myristate-13-acetate (TPA; 1-50 nM), dexamethasone (1-100 nM) and IL6 (10-500 pM). Significant increases in CRF-BP mRNA were observed in response to forskolin (30 mM), CRF (100, 1000 nM), IL6 (100, 500 pM), TPA (50 nM) and dexamethasone (100 nM; P<0.05 for all; n=3-6 for all). We extended our observations of CRF-BP expression to the protein level by performing semiquantitative Western analysis of total cellular protein after treatment with the same agents. Twenty-four hour treatment with 30 microM forskolin, 1000 nM CRF, 50 nM TPA, 100 pM IL6 or 100 nM dexamethasone significantly increased CRF-BP expression (P<0.05, n=3 for each treatment). The primary cultures were then transfected with a rat CRF-BP-reporter construct containing 3500 base pairs of CRF-BP 5' flanking DNA. Treatment with all five agents produced statistically significant increases above control (P<0.05; n=3 for each). The results suggest that CRF-BP in the amygdala is stimulated by numerous pathways which may play a significant role in promoting behavioural changes.     

Polymorphisms in the tau gene in sporadic frontotemporal dementia and other neurodegenerative disorders

The tau gene on chromosome 17 is fundamental in the pathogenesis of a number of neurodegenerative disorders. Mutations in tau are found in familial frontotemporal dementia (FTD) and the A0/A0 genotype associated with progressive supranuclear palsy (PSP). This study investigates the hypothesis that polymorphisms in the tau gene are associated with sporadic FTD. Western Australian populations of patients with sporadic frontotemporal dementia, PSP, Alzheimer's disease (AD), Huntington's disease (HD) and normal controls were studied. A new method was developed using fluorescently labelled probes to determine polymorphisms in the GT repeat region of intron 9. The A0/A0 genotype was found in 95% of PSP patients (n=20), 58.3% of FTD patients (n=48), 60.8% of AD patients (n=52), 75% of HD patients (n=40), and 75% of normal controls (n=40). None of these differences in genotype frequency were found to be significant by the Fisher exact test (P > 0.05). There were no significant differences in the frequencies of A0/A3 and A0/A1 haplotypes. We have not observed a significant increase in the A0/A0 genotype frequency in sporadic frontotemporal dementia suggesting that this polymorphism is unlikely to be related to the development of this condition. Furthermore, we have observed an increase in the A0/A0 genotype in PSP which did not reach statistical significance, suggesting that there may be population differences in the role of genetic factors in conferring risks to neurodegenerative disorders. Our work does not exclude that tau may interact with other genetic factors.     

Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex

Excitatory amino acids such as glutamate play important roles in the central nervous system. We previously demonstrated that a neurosteroid, dehydroepiandrosterone (DHEA), has powerful effects on the cell proliferation of human neural progenitor cells (hNPC) derived from the fetal cortex, and this effect is modulated through NMDA receptor signaling. Here, we show that glutamate can significantly increase the proliferation rates of hNPC. The increased proliferation could be blocked by specific NMDA receptor antagonists, but not other glutamate antagonists for kainate-AMPA or metabotropic receptors. The NR1 subunit of the NMDA receptor was detectable in elongated bipolar or unipolar cells with small cell bodies. These NR1-positive cells were colocalized with GFAP immunoreactivity. Detection of the phosphorylation of cAMP response element-binding protein (pCREB) revealed that a subset of NR1-positive hNPC could respond to glutamate. Furthermore, we hypothesized that glutamate treatment may affect mainly the hNPC with a radial morphology and found that glutamate as well as DHEA selectively affected elongated hNPC; these elongated cells may be a type of radial glial cell. Finally we asked whether the glutamate-responsive hNPC had an increased potential for neurogenesis and found that glutamate-treated hNPC produced significantly more neurons following differentiation. Together these data suggest that glutamate stimulates the division of human progenitor cells with neurogenic potential.     

Neuron-selective toxicity of tau peptide in a cell culture model of neurodegenerative tauopathy: essential role for aggregation in neurotoxicity

Intracellular aggregation of tau is a pathological hallmark in Alzheimer's disease and other tauopathies. The mechanisms underlying tau aggregation and the role that these aggregates play in neuronal death have remained controversial. To study these issues, we established a cell culture model of tauopathy using a hexameric peptide with the sequence (306)VQIVYK(311) located within the third microtubule-binding repeat of tau, rendered cell-permeable by a tag of nine arginine residues (R(9)). This peptide (VQIVYK-R(9)), designated as T-peptide, self-assembles in vitro into paired helical filament-like aggregates. Primary neuronal cells treated with T-peptide die within 24 hr. Neurodegeneration correlates with the ability of the peptide to aggregate. Two peptides with mutations in the hexameric core, K-peptide (VQIVKK) and VV-peptide (VQVVVK), that are incapable of aggregating are not toxic, whereas two other mutant peptides, V-peptide (VQVVYK) and F-peptide (VQIVFK), which aggregate, are also neurotoxic. Two other peptides that aggregate in vitro, but are not derived from tau, are not neurotoxic suggesting sequence dependence. Although localizing to the nucleus, T-peptide induces aggregation of cellular proteins in the cytoplasm. These aggregates are not caused by disruption of endogenous tau localization, although endogenous tau is reduced in neurons exposed to T-peptide. Interestingly, nonneuronal cells are less sensitive to T-peptide toxicity, recapitulating in part the selective loss of neurons in tauopathies. Moreover, T-peptide treatment leads to mitochondrial dysfunction, a common feature of neurodegenerative disorders. The model system described here represents a convenient paradigm for studying the mechanisms underlying tau aggregation and neurotoxicity and for identifying compounds that can prevent these effects.     

Glutamatergic regulation of striatal peptide gene expression in rats

Summary The mRNA levels encoding enkephalin and substance P were measured in the rat striatum following cortical ablation, blockade of N-methyl-D-aspartate (NMDA) receptors or inhibition of glutamate release by lamotrigine. Unilateral ablation of the cerebral cortex resulted in a decrease of substance P mRNA levels particularly in the rostral dorsolateral and dorsomedial striatum ipsilateral to the lesion. There was a similar trend for a reduction in levels of enkephalin mRNA. Continuous, intrastriatal infusion of the competitive NMDA receptor antagonist, 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, (CPP, 0.12 and 1.2g/day) decreased both enkephalin mRNA and substance P mRNA in dose-dependent manner evenly throughout the striatum adjacent to the infusion site. Following subchronic administration of the presumed glutamate release inhibitor, lamotrigine (5 and 20 mg/kg IP) there was no significant alterations in either enkephalin mRNA or substance P mRNA levels in the striatum. Both enkephalin mRNA and substance P mRNA expression in the rat striatum appear tonically stimulated through postsynaptic NMDA receptor mediated mechanisms. This contrasts with differential dopaminergic modulation of peptides in striatal output neurons.     

σ receptor-mediated neuroprotection against glutamate toxicity in primary rat neuronal cultures

The role of the putative σ receptor in mediating neuroprotection against glutamate-induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the selective σ₁ ligand (+)-3-PPP, all of the σ ligands tested were neuroprotective, preventing glutamate-induced morphological changes and increases in LDH release. Their rank order of neuroprotective potency (and EC₅₀ values) was as follows: (+)-SKF 10,047 (0.81 μM) > (+)- cyclazocine (2.3 μM) > dextromethorphan (3.1 μM) = haloperidol (3.7 μM) > (+)-pentazocine (8.5 μM) > DTG (42.7 μM) = carbetapentane (46.3 μM). When corrected for relative σ versus PCP binding affinity, it appears that a positive correlation exists between neuroprotective potency and σ₁ site affinity. However, there does not appear to be a significant correlation between neuroprotective potency and the σ₂ site. Critically, none of the σ ligands were neurotoxic when tested alone at concentrations at least 5–30 times their respective neuroprotective EC₅₀ values. Results from preliminary experiments with the selective σ₁ ligand (+)-pentazocine indicated that σ-mediated neuroprotection may involve the buffering of glutamate-induced calcium flux. Collectively, the results of these in vitro experiments demonstrate that σ ligands are neuroprotective and therefore deserve further exploration as potential therapeutic agents in in vivo models of CNS injury and neurodegenerative disorders.