Neuropeptide Y immunoreactive neurons in murine trisomy 16 cortical cultures

Neuropeptide Y (NPY)-containing neurons are depleted in the cortices of individuals with Alzheimer disease (AD), yet spared in the striatum of patients with Huntington chorea. It is unknown whether this neuronal phenotype is inherently susceptible to the neurodegenerative processes that are a hallmark of AD. To study this question, the murine trisomy 16 model of Down syndrome and Alzheimer disease was investigated. Since trisomic fetuses diein utero, studies were carried out on primary cultures of dissociated cortical neurons. These were prepared from 15-d gestational trisomy 16 fetuses and their littermate euploid controls, and examined by immunocytochemical staining for neuropeptide Y at 7 and 12 d in vitro. Trisomy 16 neurons were also grown on euploid glial carpets, whereas euploid neurons were grown on trisomic glia. The results demonstrate a significant increase in the number of NPY neurons and a stunting in the dendritic arbor of these neurons in trisomic vs euploid cortex. Both of these parameters could be normalized by direct contact with euploid glia. When euploid cortex was plated on trisomic glia, the number of NPY neurons and their morphology were altered so that they began to resemble trisomic NPY cortical neurons. These results indicate a dysregulation of NPY neuronal expression and differentiation in trisomy 16 cortex that are modifiable by interaction with euploid glia and imply an abnormal trophic (glial) environment in trisomic cortex.     

Expression of glial fibrillary acidic protein and glutamine synthetase genes in the natural scrapie of sheep

Gene expression of two astroglial markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), was investigated in cerebellum and brainstem from scrapie-affected sheep. The GFAP and GFAP-mRNA concentrations were increased in the two cerebral regions studied in the scrapie-affected animals as compared to the controls. The good correlation between the increase in GFAP and GFAP-mRNA concentrations found in scrapie-affected sheep indicates a significantde novo synthesis of GFAP in this pathology. In contrast to these results, in scrapie no significant differences in GS-mRNA content appeared in either brain area from scrapie-affected sheep as compared to the controls. This fact could suggest some specificity of GFAP expression changes in this pathology. The over-expression of GFAP gene could be related to a possible interaction between GFAP and scrapie infectious agent in astrocytes. The relative increase in the GFAP and its encoding message in affected animals was higher in the cerebellum than in the brainstem, which would suggest regional comparative differences in the effect here described.     

Elevated levels of dolichol in the brains of mucopolysaccharidosis and related disorders

The contents of total dolichol were measured in the cerebral cortex of various patients with lysosomal storage disorders, including mucopolysaccharidosis. Strikingly high levels of dolichol were demonstrated in G_M1-gangliosides, Sanfilippo B syndrome, and a severe type of Hunter syndrome as well as neuronal ceroid-lipofuscinosis. An increased level of dolichol in cerebral cortex in neuronal ceroid-lipofuscinosis (NCL) was once regarded as pathognomonic for NCL. Our data, however, suggest that an increased level of dolichol in cerebral cortex is a nonspecific phenomenon related to some lysosomal dysfunction secondary to various neurodegenerative disorders.     

Immunoreactivity patterns in neurofibrillary tangles of the inferior temporal cortex in Alzheimer disease

The distributions of various immunohistochemical markers of neurofibrillary tangles (NFT) were compared to that of a normal nerve cell cytoskeletal marker, SMI32, in the inferior temporal cortex of Alzheimer brains and normal aged controls. NFT markers included antibodies to the microtubule-associated proteins tau, ubiquitin, or amyloid P component (AP). The results showed that, in our group of patients, the decrease of SMI32 immunoreactivity in the Alzheimer temporal cortex is paralleled by an increase in AP immunoreactivity in neurons and neurofibrillary tangles. This suggests that AP may play an important role in NFT formation or evolution in Alzheimer disease.     

mRNA levels of Ca2+-independent forms of protein kinase C in postischemic gerbil brain by Northern blot analysis

To investigate the role of Ca²⁺-independent forms of protein kinase C (PKC) in ischemic neuronal injury, mRNA expression of PKC was studied by Northern blot analysis. Ischemia was produced in gerbils by 10-min bilateral carotid artery occlusion and was followed by recirculation for 15 min, 6 h, and 24 h. Brains of postischemic and sham-operated animals were removed, forebrains fresh frozen, and processed for Northern blot analysis. Three synthetic oligonucleotide probes based on published cDNA sequences of rat brain PKC for the isozymes δ, ε, and ζ were utilized for hybridization. Northern blot analysis showed increased hybridization signal for all three PKC isozymes examined in the 6- and 24-h postischemic groups. Of these, the twofold increases in the expression of PKC δ and ζ were statistically significant in comparison to the control. These results suggest that the mRNA levels of Ca²⁺-independent forms of PKC, in particular, δ and ζ, are temporally stimulated by ischemic injury in the brain and may imply an important role of the enzyme in postischemic neuronal damage. However, since the protein itself was not examined in this study, the significance of the increased expression cannot be ascertained. However, it may reflect a compensatory response to the loss of PKC reported to occur in the reperfusion phase.     

Anomalous binding of radiolabeled oligonucleotide probes to plaques and tangles in Alzheimer disease hippocampus

Several reports indicate that Alzheimer disease (AD) brain contains elevated levels of heat shock 70 proteins. To determine the cellular localization of the heat shock 70mRNAs, specific oligonucleotide probes werein situ hybridized to AD and control brains. When oligonucleotides werein situ hybridized to brain sections with no AD neuropathology, hybridization was cell-specific and prior ribonuclease (RNase) treatment of adjacent sections resulted in no hybridization signal. However,in situ hybridization to AD hippocampus resulted in heavy grain deposition over senile plaques and neurofibrillary tangles. Despite altering a number of experimental variables, we observed a similar pattern of grain deposition with most of the oligonucleotides tested, including one oligonucleotide specific for glutamic acid decarboxylase mRNA.In situ hybridization with either an RNA probe for glutamic acid decarboxylase or an oligonucleotide probe specific for 18S rRNA did not show this pattern of grain deposition. In control studies a sense hsc70 oligonucleotide showed no grain deposition in either cerebellum or hippocampus. Sections from AD hippocampus pretreated with RNase prior toin situ hybridization demonstrated enhanced grain deposition with the majority of probes tested. Anomalousin situ hybridization to AD hippocampus was usually eliminated by removing formamide from the posthybridization weshes, although post-RNase sticking often remained intense. These findings indicate that artifactual probe binding to senile plaques and neurofibrillary tangles may complicate the analysis ofin situ hybridization studies using oligonucleotide probes to determine mRNA distribution in AD brain.     

Redistribution of glutamate and GABA in the cerebral neocortex and hippocampus of the mongolian gerbil after transient ischemia

The redistribution of glutamate and GABA in postischemic brains was examined immunocytochemically using the gerbil model of unilateral 1 h cerebral ischemia. In the cerebral neocortex, the majority of neurons underwent recovery processes after 5 h of recirculation, while neurons in the hippocampus were irreversibly damaged. Glutamate-like immunoreactivity (LI) was highly increased in the degenerating hippocampal CA3 pyramidal cells after recirculation, while in the neocortex and the hippocampal CA1 sector, the pyramidal cells showed only slightly increased glutamate-LI. GABA-LI-positive punctae in the neuropil, corresponding to neuronal processes of GABAergic neurons, were accentuated after recirculation both in the cerebral neocortex and the hippocampus. Although the astrocytes on the non-ischemic side showed neither glutamate-LI nor GABA-LI, the swollen astrocytes and their foot processes, which were observed after recirculation, often showed strong glutamate-LI and GABA-LI. These data suggest (1) the accumulation of glutamate or glutamate-like substances, especially in the CA3 pyramidal cells, (2) the excitation of the GABAergic neurons and their subsequent uptake of GABA, and (3) the sequestration of the extracellular neurotransmitters by astrocytes in the postischemic period.     

Induction of gene expression of amyloid precursor protein (APP) in activated human lymphoblastoid cells and lymphocytes

To understand the possible role of amyloid precursor protein (APP) in human lymphocytes, and the regulation of APP gene expression in this cell type, we determined levels of cellular APP protein and of mRNA in human T-cell-derived Jurkat cells that were treated with lectin, phorbol ester, and calcium ionophore. We also related these levels to cell aggregation and adhesion. Cell-cell aggregation and cell-plastic adhesion were observed over a 24-h period after incubating cells for 2 h with phytohemagglutinin or phorbol myristate acetate. Cells treated with a calcium ionophore showed no aggregation or adhesion. Western blots indicated no obvious alteration in the level of cellular APP with different treatments. Northern blots showed a significant transient increase of APP mRNA after incubation with the calcium ionophore, whereas phorbol ester treatment showed a slight increase of APP mRNA. We analyzed the level of APP mRNA in human peripheral T cells which had been separated from peripheral lymphocytes. The level increased transiently by up to threefold after treatment with calcium ionophore plus phorbol esters. These data suggest that cell-cell aggregation and cell-matrix adhesion by human lymphocytes are not associated with an increased level of cellular APP protein or of mRNA.     

Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA

Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol- and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar granule cells acutely treated with TPA. TPA had no effect on basal, GTPγS?, NaF?, and calcium-stimulated phospholipase C when compared with membranes prepared from vehicle-treated cells. The effects of pulsing with TPA (30-min pulse, 10 nM) on agonis-stimulated PI hydrolysis were studied 1, 3, and 5 or 6 d after TPA treatment. TPA treatment results in a statistically significant decrease in glutamate-stimulated PI hydrolysis, and a slight reduction of carbachol-stimulated PI hydrolysis when compared to temporally matched controls. Measurements in membranes prepared from TPA-treated vs control cells 1, 3, and 5 d after treatment showed that calcium- and NaF-stimulated phospholipase C activity was significantly decreased at all days tested, whereas GTPγS-stimulated phospholipase C activity was significantly decreased only at d 3. These data demonstrate differences in the acute vs long-term effects of TPA treatment on agonist-stimulated PH hydrolysis, and suggest that the acute effects may be mediated at the level of the receptor, whereas long-term effects of TPA on PI hydrolysis may be mediated by deficits in effector function.     

Transforming growth factor β1 inhibits cytokine-induced CNS endothelial cell activation

Postcapillary endothelium at the sites of inflammation undergoes a series of changes collectively termed endothelial cell activation. Activated endothelium expresses immunologically relevant surface proteins that include MHC class II antigens (Ags) and adhesion proteins, as well as exhibits a number of functional changes. Endothelial activation has not been thoroughly studied in CNS endothelium. We have examined cytokine-mediated endothelial activation in isolated rat CNS microvessels. Freshly isolated rat CNS microvessels are viable in culture for at least 72 h. Untreated microvessels express no endothelial activation antigens, but do exhibit constitutive expression of the transferrin receptor (tfR). INFγ induces a dose-dependent increase in both MHC class II antigens and tfR measured by immunofluorescent staining and quantitated by laser cytometry. IFNγ-mediated endothelial cell activation could be inhibited with as little as 2 ng/mL TGF-β1, although 100% inhibition was seen with 10 ng/mL TGF-β1. Cytokinepreactivated endothelial expression of class II Ag and tfR could also be inhibited by TGF-β1. TGF-β1-treated microvessels become anergic to IFNγ stimulation. Results suggest that TGF-β1 may have a regulatory role in endothelial activation.     

Neuroprotective activity of HU-211, a novel NMDA antagonist,in global ischemia in gerbils

HU-211, a nonpsychotropic cannabinoid and a noncompetitive NMDA antagonist, was tested in a global ischemia model in the Mongolian gerbil. Male Mongolian gerbils underwent a 10-min bilateral common carotid artery occlusion. HU-211, administered iv at 4 mg/kg, 30 min postischemia, induced statistically significant neuroprotection of the CA1 subfield of the hippocampus. A dose-response study demonstrated an inverted U curve in which the 4mg/kg dose induced the best neuroprotection in the CA1 subfield of the hippocampus (p<0.05 ANOVA followed by Duncan's post-hoc test). The therapeutic window was then investigated, and in another study, HU-211 4 mg/kg were administered iv at 30, 60, 120, and 180 min postinsult. A statistically significant neuroprotection was detected at 30 and 60 min administration postinsult.     

The glutamate uptake inhibitorl-trans-2,4-pyrrolidine dicarboxylate is neurotoxic in neonatal rat brain

High-affinity glutamate uptake (HAGU) transporters rapidly remove released glutamate from the synaptic cleft. If HAGU is suppressed, neurotoxic concentrations of excitatory amino acids may accumulate. To seek further evidence in support of the neurotoxicity of endogenous glutamate in the developing brain, we assessed the neurotoxicity of the selective HAGU inhibitorl-trans-2,4-pyrrolidine dicarboxylate (L-PDC) in postnatal day 7 (PND 7) rats. The hippocampus of PND 7 rats is susceptible to EAA agonist-mediated injury; features of injury include atrophy and neuronal loss. Since HAGU is energy-dependent, we hypothesized that moderate hypoxia would increasel-PDC-mediated injury by further suppressing HAGU.l-PDC was stereotaxically injected into dorsolateral hippocampus of PND 7 rats (568 nmol,n=20). Prior to return to the dam, rats were divided into two groups, one of which was subjected to moderate hypoxia (3 h, F_iO₂=0.08) (n=11; 2 died acutely). On PND 12, hippocampal neuropathology was assessed by a blinded observer using a five-point scale and also by measuring hippocampal cross-sectional areas with computerized image analysis. Three brains were excluded from analysis, since markedly asymmetric tissue sectioning precluded valid side-to-side comparison of hippocampal areas. Injection ofl-PDC alone elicited focal pyramidal cell loss (6/7); in the (l-PDC + hypoxia) group, injury was significantly increased (median scores:l-PDC=2; [l=PDC + hypoxia]=3.5;p<0.005). Hippocampal atrophy was noted only afterl-PDC + hypoxia (4/8) (percent right-left difference in mean hippocampal area [±SE]:l-PDC=2.5% [±2.6]; [l-PDC + hypoxia]=8.9% [±3.2];p<0.02). In tissue from PND 7 rats,l-PDC (10 μM) inhibited hippocampal synaptosomal HAGU by >85%; at the same concentration,l-PDC did not displace [³H]glutamate from NMDA- or AMPA-sensitive hippocampal binding sites. These results support the hypothesis that increased synaptic accumulation of endogenous excitatory amino acid neurotransmitters may produce hippocampal injury in perinatal rodents.     

Freed-amino acids in human cerebrospinal fluid of alzheimer disease,multiple sclerosis,and healthy control subjects

This is the first report of the presence of freeD-amino acids in lumbar and ventricular human cerebrospinal fluid (CSF) of individuals with Alzheimer disease (AD) compared with CSF of normal control subjects and with individuals affected by multiple sclerosis, as an unrelated neurologic disorder. Freed-amino acids are present at significantly higher levels in AD CSF than normal CSF, whereas in the CSF of patients affected by multiple sclerosis,d-amino acids occurs at the same level as in the normal controls. The totald-amino acid content in ventricular CSF was 1.48 times higher in the AD than controls (26.4 vs 17.9 nmol/mL,p=0.025). The totald-amino acid content was 1.43 times higher in AD lumbar CSF than controls (1.89 vs. 1.32 nmol/mL,p=0.001).d-Aspartate in particular was 2.74 times higher in AD ventricular CSF compared to normal ventricular CSF (3.34 vs 1.22 nmol/mL,p=0.029). In lumbar CSF,d-aspartate was 1.5 times higher in AD than controls (0.054 vs 0.036 nmol/mL,p=0.041). Previously we reported thatd-amino acids are elevated in AD brain proteins associated with neurofibrillary tangles compared to normal brain proteins (D'Aniello et al., 1992c; Fisher et al., 1992a,b). Thus, thed-amino acids present in CSF may originate from degradation of brain proteins.     

Preferential binding of the epidermal growth factor receptor to ganglioside GM3 coated plates

Ganglioside GM3 has been shown to modulate epidermal growth factor receptor function. These observations have lead to the hypothesis that GM3 may bind to the epidermal growth factor receptor. An enzyme-linked immunosorbant assay was designed to test this hypothesis. In these experiments, receptor-rich vesicle preparations were incubated with ganglioside GM1 or GM3 coated 96-well microtiter plates and the amount of bound receptor was compared. Plates coated with GM3 consistently bound more epidermal growth factor receptor than did GM1 coated wells appeared to be specific and saturable. These results suggest that GM3 may modulate epidermal growth factor receptor function owing to a specific association of the two molecules.     

The identification of glioblastoma-associatied,fucose-containing glycoproteins induced by retinoic acid

We have used a tumorigenic glioblastoma cell line, SNB-19, as a model system to identify fucose-containing glycoprotein candidates for tumor suppressor function. Glycoproteins were analyzed after treatment with a variety of chemical differentiating agents by two-dimensional SDS-PAGE, followed by electroblotting and visualization using the fucose-specific lectin,Ulex europeaus I. Approximately 25 fucose-containing glycoproteins (FUCGLAPs) were routinely visualized in control extracts using 60–70 μg of protein per gel and staining with Vectastain ABC kits. Retinoic acid induced the most marked change in FUCGLAP expression, causing a fivelold increase in one FUCGLAP (M _r =125 kDa,pI=6.6) Neither butyric acid, dibutyryl cAMP, nor combinations of these compounds gave a similar result. Using this model system and analytical approach, it should be possible to identify, isolate, and evaluate glycoprotein oligosaccharides for their tumor modulating capability.     

In vivo and in vitro models of medulloblastomas and other primitive neuroectodermal brain tumors of childhood

Recent advances in understanding the basic biology of the neoplastic cells that populate childhood primitive neuroectodermal tumors (PNET) of the central nervous system (CNS) underline several unique properties of these common pediatric brain neoplasms. For example, studies of posterior fossa cerebellar medulloblastomas (MB), a prototypical group of brain tumors that comprise the largest class of PNET, suggest that the molecular phenotype of subpopulations of neoplastic cells in MB partially recapitulates stages in the acquisition of the neuronal phenotype by normal developing human CNS progenitor cells. However, as reviewed here, it appears that the neoplastic cells in MB exhibit one or more molecular defects in the sequence of normal maturational events that enable CNS progenitor cells to exit the cell cycle, become committed to the neuronal lineage, and undergo terminal differentiation into fully mature, permanently postmitotic CNS neurons. Indeed, since PNET emerge almost exclusively in early childhood, the induction of PNET may result from genetic lesions that arise in developing CNS progenitor cells thereby preventing these neural precursors from executing normal programs of lineage commitment and differentiation in the CNS. Clarification of how lineage commitment and maturation in PNET comprised of neuron-like tumor cells deviate from normal CNS development may clarify how oncogenes and tumor suppressor genes exert their effects in a cell type specific manner at different stages in the normal maturation of CNS cells. Recently, a number of potentially effective in vitro and in vivo model systems of PNET have been developed. Since these model systems could facilitate efforts to elucidate mechanisms of neoplastic transformation and tumor progression in the CNS, we review, the potential utility of several recently described in vitro (e.g., MB cell lines) and in vivo (e.g., transgenic mice) experimental systems as models of authentic childhood CNS neoplasms.     

Altered RNA turnover in carcinogenesis

Excretion of urinary modified nucleosides is frequently elevated in patients with oncogenic disease. Increases of urinary pseudouridine excretion are now demonstrated in patients with a variety of brain tumors. The potential use of urinary modified base excretion as a cancer marker is discussed and possible sources of the elevated nucleosides are detailed. The specific steps in RNA metabolism that result in increased levels of RNA nucleoside excretion are poorly understood. This knowledge will be necessary to understand the molecular mechanism and the clinical significance of urinary nucleoside excretion in treatment and diagnosis of oncogenic disease.     

Loop diuretic derivative L-644, 711 inhibits K+-stimulated cellular injury in neonatal guinea pig cortical astrocytes

An early pathological rise in extracellular K⁺ following acute hypoxia results in Cl^? uptake into astrocytes through the Cl^?/HCO ₃ ^? exchanger with an osmotic equivalent of water. This study addressed effects of the anion transport inhibitor, L-644,711, (5,6,-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl-1H-fluroen-7-yl)oxyacetic acid. Confluent primary cultures from neonatal guinea pigs, characterized as >95% astrocytes with antiserum to glial fibrillary acidic protein, were manipulated by incubation in either basal buffer (BB) with the ionic composition of Dulbecco’s minimum essential media (DMEM) or one with high extracellular K⁺ (HiK). Incubation in 27 or 60 mM Hik significantly reduced cell viability and precipitated a time-dose dependent increase in lactate dehydrogenase (LDH) efflux (30 min to 4h). L-644,711 was not cytotoxic, and significantly inhibited HiK-stimulated LDH efflux. The optimal effective dose of L-644,711 for preventing injury in guinea pig astrocytes was 10^?11 M when administered simultaneously with the HiK paradigm or in reversing injury when administered 30 min after exposing cells to HiK. These findings indicate the potential usefulness of agents which modify ion transport processes in hypoxic-ischemic cerebral injury.