RESUMO
The current focus of researchers is to create certain types of cells in vitroas transplantation materials. The problem of this approach is that terminally differentiated cells may not integrate into the host. To overcome this problem, we may want to transplant premature cells, which can migrate and differentiate due to environmental cues received from the host, allowing for intrinsic proper functioning of the cells. Thus, we have to consider the effects that the pathological environment might have on the transplanted cells. Here, we show the effects of amyloid precursor protein and reelin on neural stem cell (NSC) differentiation, and demonstrate how we have regulated this effect to produce desirable cells under pathological conditions. We found that amyloid precursor protein increases glial differentiation via the notch and cytokine-signaling pathway, while reelin induces radial glial differentiation followed by neuronal differentiation via increasing phosphorylation of adapter protein disabled-1. Since amyloid and reelin are found in plaques within Alzheimer's disease, these findings may closely associate with NSC biology in the context of its pathology. By regulating these factors in Alzheimer's disease, we may be able to not only guide differentiation of transplanted NSCs, but also to modify progression of disease by guiding differentiation of endogenous NSCs.
Assuntos
Doença de Alzheimer/patologia , Diferenciação Celular/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/farmacologia , Animais , Moléculas de Adesão Celular Neuronais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Humanos , Modelos Biológicos , Proteínas do Tecido Nervoso/farmacologia , Neuroglia/efeitos dos fármacos , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína Reelina , Serina Endopeptidases/farmacologia , Células-Tronco/efeitos dos fármacosRESUMO
We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 kDa form of the glutamic acid decarboxylase (GAD(67)) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in rats with spinal cord injury (SCI). One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. Rats with SCI without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40-45% and 38-40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD(67) mRNA and protein levels were significantly increased in the L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group, while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD(67)-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO after SCI predominantly through the activation of spinal GABA(A) receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for treatment of neurogenic DO.
Assuntos
Terapia Genética/métodos , Glutamato Descarboxilase/genética , Simplexvirus/genética , Traumatismos da Medula Espinal/complicações , Bexiga Urinária Hiperativa/terapia , Animais , Estudos de Viabilidade , Feminino , Expressão Gênica/genética , Vetores Genéticos , Glutamato Descarboxilase/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Transgenes , Bexiga Urinária/fisiopatologia , Bexiga Urinária Hiperativa/etiologia , Bexiga Urinária Hiperativa/fisiopatologiaRESUMO
We have demonstrated that aged animals show significant improvements in cognitive function and neurogenesis after brain transplantation of human neural stem cells or of human adult mesenchymal stem cells that have been dedifferentiated by transfection of the embryonic stem cell gene. We have also demonstrated that peripheral administration of a pyrimidine derivative increased cognition, endogenous brain stem cell proliferation and neurogenesis. These results indicate a bright future for stem cell therapies in Alzheimer's disease (AD). Before this is realized, however, we need to consider the affect of AD pathology on stem cell biology to establish an effective stem cell therapy for this disease. Although amyloid-beta (Abeta) deposition is a hallmark of AD, an absence of a phenotype in the beta-amyloid precursor protein (APP) knockout mouse, might lead one to underestimate the potential physiological functions of APP and suggest that it is unessential or can be compensated for. We have found, however, that APP is needed for differentiation of neural stem cells (NSCs) in vitro, and that NSCs transplanted into a APP-knockout mouse did not migrate or differentiate -- indicating that APP plays an important role in differentiation or migration process of NSCs in the brain. Then again, treatment with high a concentration of APP or its over-expression increased glial differentiation of NSCs. Human NSCs transplanted into APP-transgenic mouse brain exhibited less neurogenesis and active gliosis around the plaque like formations. Treatment of such animals with the compound, (+)-phenserine, that is known to reduce APP protein levels, increased neurogenesis and suppressed gliosis. These results suggest APP levels can regulate NSC biology in the adult brain, that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases, and that a combination of stem cell therapy and regulation of APP levels could provide a treatment strategy for these disorders.
Assuntos
Doença de Alzheimer/cirurgia , Transplante de Células-Tronco/métodos , Células-Tronco/fisiologia , Animais , Terapia Genética , HumanosRESUMO
The human genome is composed of long-range G+C% (GC%) mosaic structures thought to be related to chromosome bands. We previously reported a boundary of megabase-sized GC% mosaic domains at the junction area between major histocompatibility complex (MHC) classes II and III, proposing it as a possible chromosome band boundary. DNA replication timing during the S phase is known to be correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. In this study, to identify to the nucleotide sequence level the replication switch point during the S phase, we determined the precise DNA replication timing for MHC classes II and III, focusing on the junction area. To do this, we used PCR-based quantitation of nascent DNA obtained from synchronized human myeloid leukemia HL60 cells. The replication timing changed precisely in the boundary region with a 2-h difference between the two sides, supporting the prediction that this region may be a chromosome band boundary. We supposed that replication fork movement terminates (pauses) or significantly slows in the switch region, which contains dense Alu clusters; polypurine/polypyrimidine tracts; di-, tri-, or tetranucleotide repeats; and medium-reiteration-frequency sequences. Because the nascent DNA in the switch region was recovered at low efficiency, we investigated whether this region is associated with the nuclear scaffold and found three scaffold-associated regions in and around the switch region.
Assuntos
Replicação do DNA , Complexo Principal de Histocompatibilidade , Sequência de Bases , Evolução Biológica , Primers do DNA , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Replicon , Fatores de TempoRESUMO
We compared the effects of melatonin, an antioxidant and sleep inducer in humans, and rilmazafone hydrochloride, a hypnotic, in elderly patients with nocturia. Patients received either melatonin (2 mg/day; n = 20) or rilmazafone (2 mg/day; n = 22) for 4 weeks. There were no significant differences in the mean age, the quality of life (QoL) score and the serum melatonin levels between the two groups at baseline. After 4 weeks' treatment, the number of nocturnal urinations was significantly decreased and the QoL score was significantly improved in both groups. There was no significant difference between the patient-reported effectiveness ratings between the two groups. The serum melatonin level was significantly increased in the melatonin-treated group, but it remained unchanged in the rilmazafone-treated group. Melatonin and rilmazafone were equally effective for nocturia in the elderly. We recommend that the problems of sleep disturbance should be considered when choosing a therapy for nocturia.
Assuntos
Hipnóticos e Sedativos/uso terapêutico , Melatonina/uso terapêutico , Noctúria/tratamento farmacológico , Triazóis/uso terapêutico , Idoso , Feminino , Humanos , Masculino , Melatonina/sangue , Noctúria/fisiopatologia , Qualidade de VidaRESUMO
Although amyloid beta (Abeta) deposition has been a hallmark of Alzheimer's disease (AD), the absence of a phenotype in the beta amyloid precursor protein (APP) knockout mouse, tends to detract our attention away from the physiological functions of APP. Although much attention has been focused on the neurotoxicity of Abeta, many studies suggest the involvement of APP in neuroplasticity. We found that secreted amyloid precursor protein (sAPP) increased the differentiation of human neural stem cells (hNSCs) in vitro, while an antibody-recognizing APP dose-dependently inhibited these activities. With a high dose of sAPP treatment or wild-type APP gene transfection, hNSCs were differentiated into astrocytes rather than neurons. In vivo, hNSCs transplanted into APP-transgenic mouse brain exhibited glial differentiation rather than neural differentiation. Our results suggest that APP regulates neural stem cell biology in the adult brain, and that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/farmacologia , Diferenciação Celular/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Animais , Apoptose , Movimento Celular/efeitos dos fármacos , Transplante de Células , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We have found much evidence that the brain is capable of regenerating neurons after maturation. In our previous study, human neural stem cells (HNSCs) transplanted into aged rat brains differentiated into neural cells and significantly improved the cognitive functions of the animals, indicating that HNSCs may be a promising candidate for cell-replacement therapies for neurodegenerative diseases including Alzheimer's disease (AD). However, ethical and practical issues associated with HNSCs compel us to explore alternative strategies. Here, we report novel technologies to differentiate adult human mesenchymal stem cells, a subset of stromal cells in the bone marrow, into neural cells by modifying DNA methylation or over expression of nanog, a homeobox gene expressed in embryonic stem cells. We also report peripheral administrations of a pyrimidine derivative that increases endogenous stem cell proliferation improves cognitive function of the aged animal. Although these results may promise a bright future for clinical applications used towards stem cell strategies in AD therapy, we must acknowledge the complexity of AD. We found that glial differentiation takes place in stem cells transplanted into amyloid-( precursor protein (APP) transgenic mice. We also found that over expression of APP gene or recombinant APP treatment causes glial differentiation of stem cells. Although further detailed mechanistic studies may be required, RNA interference of APP or reduction of APP levels in the brain can significantly reduced glial differentiation of stem cells and may be useful in promoting neurogenesis after stem cell transplantation.
Assuntos
Doença de Alzheimer/terapia , Neurônios/citologia , Transplante de Células-Tronco , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/fisiologia , Diferenciação Celular , Proteína Glial Fibrilar Ácida/análise , Humanos , Presenilina-1/genética , RNA Interferente Pequeno/farmacologia , Células-Tronco/citologiaRESUMO
Messenger RNA (mRNA) molecules encoding proteins related to the presynaptic cholinergic and neurotrophin systems were quantitated in the hippocampus and basal forebrain of Long-Evans rats with spatial learning ability assessed in the Morris water maze. The reverse transcriptase-polymerase chain reaction showed that the mRNAs for the low-affinity neurotrophin receptor (p75-NTR) and the growth-associated protein GAP-43 were decreased in level in the basal forebrain of aged-impaired rats. In the hippocampus of these aged-impaired rats, the mRNA for VGF, another neurotrophin-inducible gene, also was decreased. In situ hybridization histochemistry revealed that mRNAs for nerve growth factor (NGF) and brain-derived neurotrophic factor increased in level in the aged rat hippocampus; when age effects were removed, NGF mRNA level remained significantly correlated with maze performance. Enzyme-linked immunosorbent assay indicated that NGF protein was expressed at normal levels in the aged rat hippocampus. These mRNA and protein alterations may signify that a defect in neurotrophin signaling exists in the brains of aged Long-Evans rats, underlying reduced plasticity responses in the basal forebrain cholinergic system.
Assuntos
Envelhecimento/fisiologia , Envelhecimento/psicologia , Cognição/fisiologia , Hipocampo/fisiologia , Fatores de Crescimento Neural/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Biomarcadores , Colina O-Acetiltransferase/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteína GAP-43/metabolismo , Hipocampo/metabolismo , Hibridização In Situ , Aprendizagem em Labirinto , Fatores de Crescimento Neural/metabolismo , Sistema Nervoso Parassimpático/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Transcriptional activity of RNA polymerase II is modulated during the cell cycle. We previously identified a temperature-sensitive mutation in the largest (catalytic) subunit of RNA polymerase II (RPB1) that causes cell cycle arrest and genome instability. We now characterize a different cell line that has a temperature-sensitive defect in cell cycle progression, and find that it also has a mutation in RPB1. The temperature-sensitive mutant, tsAF8, of the Syrian hamster cell line, BHK21, arrests at the non-permissive temperature in the mid-G(1) phase. We show that RPB1 in tsAF8--which is found exclusively in the nucleus at the permissive temperature--is also found in the cytoplasm at the non-permissive temperature. Comparison of the DNA sequences of the RPB1 gene in the wild-type and mutant shows the mutant phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one RPB1 allele; this gives rise to an ala-to-asp substitution at residue 315 in the protein. Aligning the amino acid sequences from various species reveals that ala(315) is highly conserved in eukaryotes.
Assuntos
Ciclo Celular/fisiologia , Fase G1/fisiologia , RNA Polimerase II/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico/genética , Ciclo Celular/genética , Linhagem Celular , DNA Complementar/química , DNA Complementar/genética , Fase G1/genética , Genótipo , Dados de Sequência Molecular , Mutação , Filogenia , RNA Polimerase II/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
In the course of the Schizosaccharomyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1alpha, EF-1beta, EF-1gamma, EF-2 and EF-3. With the exception of the EF-1gamma gene, the nucleotide (nt) sequence of S. pombe elongation factors has not been previously reported. For EF-1alpha, we found three genes whose amino acid (aa) sequences are quite homologous each other (99.5%), but whose 3' untranslated regions (UTRs) are completely different. Southern blot indicated that those three EF-1alpha genes are located at different loci. Northern analysis indicated that one of three EF-1alpha genes was inducible with UV-irradiation, while the level of expression for another of three EF-1alpha genes was repressed by UV and heat-shock (HS) treatments. The aa sequence predicted from the nt sequence of the S. pombe EF-1beta cDNA clone covered almost all the coding sequence (CDS) of EF-1beta except the first methionine which has 55.4% identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their aa sequences deduced from nt sequences are identical (100%), but they have different 3' UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced aa sequence has a 76% identity with those of other yeasts and fungi.
Assuntos
Proteínas Fúngicas , Fatores de Alongamento de Peptídeos/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Fúngico , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Fator 2 de Elongação de Peptídeos , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de AminoácidosRESUMO
The cDNA and genomic clones for the human counterpart of the mouse mammary tumor gene Int3 were isolated and sequenced. We designated this human major histocompatibility complex (MHC) class III gene as NOTCH4, since very recently, by sequencing cDNA clones, the complete form of the mouse proto-oncogene Int3 has been clarified and named Notch4. The present human NOTCH4 sequence is the first example of the genomic sequence for the extracellular portion of the mammalian Notch4, and by comparing it with the mouse Notch4 cDNA sequence, the exon/intron organization was clarified. The comparison of the predicted amino acid sequence of human NOTCH4 with those of other Notch homologues of a wide range of species revealed four subfamilies for mammalian Notch. In the protein coding region of human NOTCH4, we found (CTG)n repeats showing a variable number tandem repeat (VNTR) polymorphism for different human leukocyte antigen (HLA) haplotypes. Ten genes mapped on 6p21.3, including NOTCH4, were found to have counterparts structurally and functionally similar to those mostly mapped on 9q33-q34, indicating segmental chromosome duplication during the course of evolution. Similarity of genes on chromosomes 1, 6, 9 and 19 was also discussed.
Assuntos
Polimorfismo Genético , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores de Superfície Celular , Repetições de Trinucleotídeos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Humanos , Complexo Principal de Histocompatibilidade/genética , Camundongos , Dados de Sequência Molecular , Filogenia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/química , Receptor Notch4 , Receptores Notch , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
In order to analyze the mutation sites related to abnormal induction of sister chromatid exchanges (SCEs) in the RNA polymerase II largest subunit (RpII LS) gene of the Chinese hamster CHO-KI cell mutant, we have completely sequenced the whole region of the RpII LS cDNAs obtained from normal and mutant cells. By comparing both sequences, a mutation that results in an amino acid (aa) change in the RpII LS gene was found. This aa change was Pro (CCC) to Ser (TCC) at position 1006. Multiple alignment for aa sequences of RpII LS from various species revealed that this Pro residue was highly conserved throughout the eukaryotes. Considering the differences in physico-chemical properties between Pro and Ser residues, the Pro-->Ser substitution may alter the RpII LS structure.
Assuntos
Mutação Puntual , RNA Polimerase II/genética , Troca de Cromátide Irmã/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , DNA Complementar , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes is reviewed.
Assuntos
Núcleo Celular/metabolismo , Transcrição Gênica/fisiologia , Nucléolo Celular/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Células HeLa , Humanos , Fatores de Transcrição/metabolismoRESUMO
Neurons and glia within the hippocampus of aged, spatial learning-impaired Long-Evans rats exhibit uniquely altered gene expression profiles, and we have postulated oxidative stress as the basis for this. To test this hypothesis we quantitated the extent of protein and nucleic acid oxidative damage, evaluated the status of mitochondrial DNA integrity, and examined several signaling entities and molecular indicators frequently associated with oxidative stress and gliosis. Immunoblotting demonstrated elevated heme oxygenase-1 in the aged-impaired hippocampus and immunocytochemistry suggested that heme oxygenase-1 is largely cytosolic and at least partly neuronal in nature. In the aged-impaired group, immunoreactivity to 8-hydroxy-2'-deoxyguanosine, an oxidative nucleic acid adduct, was found to be elevated in the dentate gyrus and in area CA1 of the hippocampal formation. Isolated mitochondrial DNA was found to be significantly damaged in the aged-impaired group. In the aged learning-impaired rats only, proteins in a 65-kDa band were found to contain excessive levels of carbonyl residues. Glial activation was examined by in situ hybridization histochemistry to tumor necrosis factor alpha and by immunocytochemistry with OX-6, which detects activated microglia. White matter in aged brains exhibited a modest up-regulation of tumor necrosis factor alpha mRNA and OX-6 immunoreactivity, but the hippocampal formation expressed tumor necrosis factor alpha mRNA equivalent to young animals and few OX-6-positive microglia. The mRNA for manganese-dependent superoxide dismutase, which is elevated in the aged hippocampus, was found preferentially expressed in neurons. We conclude that aged hippocampal neurons appear to be under oxidative stress and this is more severe in the learning-impaired subjects, suggesting a possible basis for age-induced cognitive decline.
Assuntos
Envelhecimento/fisiologia , Hipocampo/metabolismo , Deficiências da Aprendizagem/metabolismo , Estresse Oxidativo , Percepção Espacial/fisiologia , Tirosina/análogos & derivados , Animais , Comportamento Animal , DNA Mitocondrial/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Deficiências da Aprendizagem/psicologia , Masculino , Neurônios/metabolismo , Ácidos Nucleicos/metabolismo , Oxirredução , RNA Mensageiro/metabolismo , Ratos , Superóxido Dismutase/genética , Fator de Necrose Tumoral alfa/genética , Tirosina/metabolismoRESUMO
Superoxide dismutase (SOD) protects cells exposed to an excess of the free radical nitric oxide, by preventing the formation of peroxynitrite. Certain central cholinergic neurons express constitutive nitric oxide synthase (nNOS), and presumably they are at risk from peroxynitrite intoxication. Immunocytochemistry for choline acetyltransferase (ChAT) was combined with in situ hybridization histochemistry (ISHH) to examine whether brain cholinergic populations differ with respect to their expression of the messenger RNA molecules (mRNAs) for the manganese-dependent (Mn-SOD) and copper/zinc-dependent superoxide dismutases (Cu /Zn-SOD). The cholinergic neurons located in the reticular formation of the upper brainstem (the laterodorsal tegmental nucleus [LDTN] and the pedunculopontine nucleus [PPN]) were found to express relatively high levels of Mn-SOD mRNA, whereas cholinergic neurons located in the basal forebrain (substantia innominata [SI], diagonal band [DB], medial septum [MS], and the nucleus basalis magnocellularis [nBM]), and the striatal cholinergic interneurons expressed low to intermediate levels of Mn-SOD mRNA. The rank order of median Mn-SOD mRNA density per cholinergic cell was LDTN > PPN > SI > striatum = nBM = DB > MS. This is similar to the rank order of nNOS mRNA densities in the cholinergic cells in these regions (R = 0.9, p < 0.02). The rank order of Cu/Zn-SOD mRNA levels in cholinergic populations (DB > LDTN = PPN =MS > SI = nBM = striatum) was not correlated with nNOS mRNA (R = 0.29, P > 0.05). Thus, for cholinergic neurons, Mn-SOD may be important for protection from NO-related oxidative stress.
Assuntos
Encéfalo/enzimologia , Neurônios/enzimologia , Superóxido Dismutase/genética , Transcrição Gênica , Animais , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
The expression of mRNA for the calmodulin-dependent form of brain nitric oxide synthase (NOS) was examined in cholinergic cells of the rat brain using a method combining in situ hybridization histochemistry with immunocytochemistry for choline acetyltransferase (ChAT) in the same brain sections. We constructed a riboprobe specific for brain NOS by subcloning a 493 bp fragment of the coding region which displayed low homology to other forms of NOS. The general distribution of NOS mRNA was in excellent agreement with previous studies using the full-length probe or NADPH diaphorase histochemistry. NOS mRNA was observed in many brain structures and relative levels were quantitated using grain counting procedures in a number of cholinergic and non-cholinergic neuronal groups throughout the brain. In the forebrain, ChAT-immunoreactive cells or cell groups were observed in medial septum (MS), vertical limbs of diagonal band (DBV) and horizontal limbs of diagonal band (DBH), nucleus basalis magnocellularis (NBM), substantia innominata (SI), and striatum (ST). In the brainstem, the cholinergic groups studied included those located in the pedunculopontine tegmental nucleus (PPTN), the laterodorsal tegmental nucleus (LDTN), the nucleus parabigeminalis and several motor nuclei. For NOS mRNA quantitation, silver grains overlying ChAT-stained neuronal profiles in sections on emulsion-dipped slides were counted digitally. In the LDTN and PPTN, virtually all the ChAT-positive cells expressed NOS mRNA at high levels. In MS, DBV and SI, about 30-50% of the ChAT-positive cells expressed NOS mRNA at low-to-moderate levels. Less than 20% of ChAT-positive neurons in the other cholinergic populations studied expressed NOS mRNA; the NBM was one of these low-expressing populations. Many scattered non-cholinergic cells expressing NOS mRNA were found in the striatum and cerebral cortex. In other non-cholinergic regions, high NOS mRNA expression was observed in the islands of Calleja, thalamic and hypothalamic nuclei, several amygdaloid nuclei, regions related to the optic tract, the interpeduncular nucleus, and the supramammillary nucleus. The heterogeneous distribution of NOS mRNA implies complex roles for nitric oxide neurotransmission in brain function, including for the cholinergic phenotype. Additionally, given the postulated involvement of nitric oxide in neurodegeneration, the widely varying levels of expression of NOS within identified central cholinergic neurons may relate to differential vulnerability of this phenotype in disease or aging.
Assuntos
Aminoácido Oxirredutases/biossíntese , Encéfalo/enzimologia , Fibras Colinérgicas/enzimologia , Proteínas do Tecido Nervoso/biossíntese , Aminoácido Oxirredutases/classificação , Aminoácido Oxirredutases/genética , Animais , Autorradiografia , Biomarcadores/análise , Encéfalo/citologia , Calmodulina , Colina O-Acetiltransferase/análise , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Hibridização In Situ , Masculino , NADP , Proteínas do Tecido Nervoso/genética , Óxido Nítrico Sintase , Ratos , Ratos Sprague-DawleyRESUMO
A number of pharmacological, anatomical, and immunological studies have previously addressed the subtype identity of the hippocampal muscarinic pre-synaptic autoreceptor. A preponderance of findings indicate that it is of the M2 pharmacological type. Both the m2 and m4 molecular subtypes exhibit M2 pharmacology and there are few drugs that differentiate between these receptors. Pharmacological attempts at defining the hippocampal autoreceptor have yielded conflicting results. The basal forebrain is relatively enriched in m2 muscarinic receptor mRNA and protein, and lesions that denervate the hippocampus of its basal forebrain cholinergic input have shown a decrement in m2, but not m4, receptor protein in the hippocampus. Thus, the anatomical data obtained to date tend to support the view that the m2 subtype is expressed as the hippocampal autoreceptor. We have combined in situ hybridization histochemistry (ISHH) with immunocytochemistry to choline acetyltransferase to examine whether mRNA for the m4 subtype of muscarinic receptor is expressed in central cholinergic neurons. The m4 muscarinic mRNA was found at moderate levels in all subdivisions of the cholinergic basal forebrain, including the medial septum/diagonal band complex (MS/DB). The m4 mRNA was also found in striatal cholinergic interneurons, in the cholinergic reticular core of the upper brainstem, and in brainstem cholinergic motor neurons. Muscarinic m4 receptor mRNA was also found in many non-cholinergic cells in the brain. For example, the hippocampal pyramidal neurons, dentate gyrus granule cells, and entorhinal cortical pyramidal neurons express relatively high levels of m4 mRNA, while in the brainstem the dorsal raphe and pontine reticular nuclei express relatively high levels of this mRNA. The finding of m4 mRNA in the MS/DB cholinergic neurons suggests that this receptor protein might be expressed as an autoreceptor in hippocampal cholinergic terminals.
Assuntos
Encéfalo/metabolismo , Colina O-Acetiltransferase/análise , Neurônios/metabolismo , RNA Mensageiro/biossíntese , Receptores Muscarínicos/genética , Animais , Encéfalo/citologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Glial activation and oxidative stress are both consequences of brain aging. To investigate whether glial activation causes oxidative stress or not, the immune activator, lipopolysaccharide (LPS), was intraventricularly injected into the rat brain. The expression of candidate genes were examined by in situ hybridization histochemistry (ISHH) combined with immunohistochemistry for glial markers over a period of time up to 24 h after the LPS injection. The mRNA for glial fibrillary acidic protein (GFAP) was elevated around the injection site by 2 h, and the volume of elevated expression spread to the entire brain after 6 h, with higher levels present in the injected hemisphere. The level of inducible isoform of nitric oxide synthase (i-NOS) mRNA increased in a punctate-like pattern in the region of the injection by 6 h and this response spread to the entire brain after 12 h. These results indicate that the glia are activated for at least 24 h after a single LPS injection. The mRNAs for a heat-shock protein (HSP70) and for the manganese-dependent superoxide dismutase (Mn-SOD) were elevated in the ipsilateral hemisphere as early as 2 h post-injection, but these responses subsided nearly to basal levels by 4 h. These levels of mRNAs for these genes increased again after 6 h of the LPS injection; thus, the earlier increases of the messages appeared to be associated with the survival surgery procedure. With microautoradiographic analysis, scattered OX-42 positive cells expressed i-NOS mRNA after 6 h post-injection, but elevation of Mn-SOD mRNA was not detected in either microglia or astrocytes at any time point examined. The level for Cu/Zn-SOD mRNA did not alter at any time point. The beta-amyloid precursor protein (betaAPP) mRNAs were elevated beginning at 6 h. These results indicate that chronic glial activation leads to a condition of oxidative stress in the brain. The data also suggest that LPS injection could be used to study the effects of chronic glial activation on the survival of neuronal populations that could be at risk from oxidative stress.
Assuntos
Encéfalo/efeitos dos fármacos , Ventrículos Cerebrais/fisiologia , Endotoxinas/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Neuroglia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Ventrículos Cerebrais/efeitos dos fármacos , Endotoxinas/administração & dosagem , Indução Enzimática , Lateralidade Funcional , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/biossíntese , Imuno-Histoquímica , Hibridização In Situ , Infusões Parenterais , Cinética , Lipopolissacarídeos/administração & dosagem , Masculino , Neuroglia/metabolismo , Neuroglia/patologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo I , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/análise , Superóxido Dismutase/biossíntese , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
To identify whether the process of apoptosis bears a topographic relationship to selected aspects of Alzheimer's disease (AD) pathology, we used an in situ nick translation method (TUNEL) to map DNA fragmentation in hippocampal sections immunostained for abnormally phosphorylated tau, which exists in the neurofibrillary tangles (NFTs) and in the dystrophic neurites associated with senile plaques. To ascertain associations of DNA fragmentation with glia, TUNEL was combined with immunohistochemistry for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial antigen OX-42. Consistent with previous reports, the incidence of putative DNA fragmentation detected by TUNEL was much higher in the AD brain, compared to non-demented subjects. While most TUNEL-positive cells did not exhibit any systematic topographic relationship to senile plaques, which were visualized by immunostain of abnormally phosphorylated tau for dystrophic neurites, DNA fragmentation was found frequently within cells containing NFTs. In hippocampal sections prepared to visualize glia, DNA fragmentation was not observed in GFAP-positive astrocytes, but some OX-42-positive microglia exhibited TUNEL signals. Other TUNEL-positive cells were found frequently in proximity to glia. The data suggest that cells compromised by the deposition of NFTs are prone to initiate the process of apoptosis. Furthermore, some glial populations appear to be apoptotic in the AD brain.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Fragmentação do DNA , Hipocampo/patologia , Hipocampo/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/patologia , Neuroglia/fisiologia , Valores de ReferênciaRESUMO
A unilateral ibotenic acid lesion of the nucleus basalis magnocellularis in the rat, which is known to produce a reduction in cortical choline acetyltransferase activity and acetylcholine release, produces a decrease of 125I-alpha-bungarotoxin and 125I-kappa-bungarotoxin binding sites in the frontoparietal cortex of the lesioned hemisphere. This decrease can be observed at two weeks following the lesion and persists for up to twelve weeks. The results suggest that a population of bungarotoxin binding sites may have a presynaptic localization.