RESUMO
Alzheimer's disease (AD) and osteoporosis are two diseases that mainly affect elderly people, with increases in the occurrence of cases due to a longer life expectancy. Several epidemiological studies have shown a reciprocal association between both diseases, finding an increase in incidence of osteoporosis in patients with AD, and a higher burden of AD in osteoporotic patients. This epidemiological relationship has motivated the search for molecules, genes, signaling pathways and mechanisms that are related to both pathologies. The mechanisms found in these studies can serve to improve treatments and establish better patient care protocols.
Assuntos
Doença de Alzheimer , Osteoporose , Humanos , Doença de Alzheimer/epidemiologia , Osteoporose/epidemiologia , IncidênciaRESUMO
In humans and rats, aging is associated with a progressive deterioration of spatial learning and memory. These functional alterations are correlated with morphological and molecular changes in the hippocampus. Here, we assessed age-related changes in DNA methylation (DNAm) landscape in the rat hippocampus and the correlation of spatial memory with hippocampal DNAm age in 2.6- and 26.6-month-old rats. Spatial memory performance was assessed with the Barnes maze test. To evaluate learning ability and spatial memory retention, we assessed the time spent by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. The rat pan-tissue clock was applied to DNAm data from hippocampal tissue. An enrichment pathway analysis revealed that neuron fate commitment, brain development, and central nervous system development were processes whose underlying genes were enriched in hypermethylated CpGs in the old rats. In the old rat hippocampi, the methylation levels of CpG proximal to transcription factors associated with genes Pax5, Lbx1, Nr2f2, Hnf1b, Zic1, Zic4, Hoxd9; Hoxd10, Gli3, Gsx1 and Lmx1b, and Nipbl showed a significant regression with spatial memory performance. Regression analysis of different memory performance indices with hippocampal DNAm age was significant. These results suggest that age-related hypermethylation of transcription factors related to certain gene families, such as Zic and Gli, may play a causal role in the decline in spatial memory in old rats. Hippocampal DNAm age seems to be a reliable index of spatial memory performance in young and old rats.
Assuntos
Metilação de DNA , Memória Espacial , Animais , Ratos , Envelhecimento/genética , Proteínas de Ciclo Celular/genética , Epigênese Genética , Hipocampo , Aprendizagem em Labirinto/fisiologia , Memória Espacial/fisiologia , Fatores de Transcrição/genéticaRESUMO
Hippocampus serves as a pivotal role in cognitive and emotional processes, as well as in the regulation of the hypothalamus-pituitary axis. It is known to undergo mild neurodegenerative changes during normal aging and severe atrophy in Alzheimer's disease. Furthermore, dysregulation in the hippocampal function leads to epilepsy and mood disorders. In the first section, we summarized the most salient knowledge on the role of glial cell-line-derived neurotrophic factor and its receptors focused on aging, cognition and neurodegenerative and hippocampal-related neurological diseases mentioned above. In the second section, we reviewed the therapeutic approaches, particularly gene therapy, using glial cell-line-derived neurotrophic factor or its gene, as a key molecule in the development of neurological disorders. In the third section, we pointed at the potential of regenerative medicine, as an emerging and less explored strategy for the treatment of hippocampal disorders. We briefly reviewed the use of partial reprogramming to restore brain functions, non-neuronal cell reprogramming to generate neural stem cells, and neural progenitor cells as source-specific neuronal types to be implanted in animal models of specific neurodegenerative disorders.
RESUMO
Spatial memory performance declines in both normal aging and Alzheimer's disease. This cognitive deficit is related to hippocampus dysfunction. Gene therapy using neurotrophic factors like Glial cell line-derived neurotrophic factor (GDNF) emerges as a promising approach to ameliorate age-related cognitive deficits. We constructed a two vector regulatable system (2VRS) which consists of a recombinant adenoviral vector (RAd) harboring a Tet-Off bidirectional promoter flanked by GDNF and Green Fluorescent Protein (GFP) genes. A second adenovector, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are cotransduced by the 2VRS, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is silenced. We tested the 2VRS in CHO-K1 cells where we observed a dose-dependent GFP expression that was completely inhibited by DOX (1 mg/ml). The 2VRS injected in the hippocampal CA1 region transduced both neurons and astrocytes and was efficiently inhibited by DOX added to the drinking water. In order to assess GDNF biological activity we injected 2VRS and its Control (CTRL) vector in the hypothalamus and monitored body weight for one month. The results showed that GDNF retards weight recovery 6 days more than CTRL. In conclusion, our 2VRS demonstrated optimal GFP expression and showed a bioactive effect of transgenic GDNF in the brain.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Adenoviridae , Animais , Células CHO , Cricetinae , Cricetulus , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , RatosRESUMO
Several countries have established self-help cryonics groups whose mission is to cryopreserve human bodies or brains after legal death and ship them to cryonics organizations. The objective of this study was to report the first case of human brain cryopreservation in Argentina and complementary experiments in rats. After legal death, the body of a 78-year-old Caucasian woman was transported to a funeral home where her head was submitted to intracarotid perfusion with 5 L cold physiologic saline followed by the same volume of cold saline containing 13% dimethyl sulfoxide and 13% glycerol. The brain was removed, temporarily frozen at -80°C, and shipped to a U.S. cryostasis facility. Three groups of rats were intracardially perfused with fixative but not frozen (Reference group), vitrification solution VM1 (Control group), or the cryoprotection solution used in the patient (Experimental group). Control and Experimental brains were stored at -80°C and subsequently assessed by immunohistochemistry for the adult neuron marker (NeuN), the immature neuron marker doublecortin (DCX), the dopaminergic neuron marker tyrosine hydroxylase, and the presynaptic marker synaptophysin (SYN). The number of NeuN-positive neurons remained unchanged in the experimental brain cortex, whereas the number of immature DCX neurons in the hippocampus fell markedly in the cryoprotected brains. The results were highly variable for hypothalamic dopaminergic neurons. Confocal microscopy for SYN revealed that cryopreservation did not affect the synaptic network in the hippocampus. To our knowledge, this is the first report correlating a human cryoprotection procedure with results in complementary experiments in laboratory animals.
Assuntos
Encéfalo , Criopreservação , Modelos Animais , Idoso , Animais , Encéfalo/anatomia & histologia , Cadáver , Criopreservação/métodos , Crioprotetores , Proteína Duplacortina , Feminino , Congelamento , Hipocampo , Humanos , Ratos , Fixação de Tecidos , VitrificaçãoRESUMO
Sporadic Alzheimer's disease (sAD) is the most prevalent neurodegenerative pathology with no effective therapy until date. This disease promotes hippocampal degeneration, which in turn affects multiple cognitive domains and daily life activities. In this study, we hypothesized that long-lasting therapy with mesenchymal stem cells (MSC) would have a restorative effect on the behavioral alterations and cognitive decline typical of sAD, as they have shown neurogenic and immunomodulatory activities. To test this, we chronically injected intravenous human MSC in a sAD rat model induced by the intracerebroventricular injection of streptozotocin (STZ). During the last 2 weeks, we performed open field, Barnes maze, and marble burying tests. STZ-treated rats displayed a poor performance in all behavioral tests. Cell therapy increased exploratory behavior, decreased anxiety, and improved spatial memory and marble burying behavior, the latter being representative of daily life activities. On the hippocampus, we found that STZ promotes neuronal loss in the Cornus Ammoni (CA1) field and decreased neurogenesis in the dentate gyrus. Also, STZ induced a reduction in hippocampal volume and presynaptic protein levels and an exacerbated microgliosis, relevant AD features. The therapy rescued CA1 neurodegeneration but did not reverse the decrease of immature neurons, suggesting that the therapy effect varied among hippocampal neuronal populations. Importantly, cell therapy ameliorated microgliosis and restored hippocampal atrophy and some presynaptic protein levels in the sAD model. These findings, by showing that intravenous injection of human MSC restores behavioral and hippocampal alterations in experimental sAD, support the potential use of MSC therapy for the treatment of neurodegenerative diseases.
Assuntos
Comportamento Animal , Hipocampo/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Ansiedade/complicações , Ansiedade/patologia , Ansiedade/fisiopatologia , Comportamento Exploratório , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/complicações , Gliose/patologia , Masculino , Aprendizagem em Labirinto , Memória , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Neurônios/patologia , Tamanho do Órgão , Ratos Sprague-Dawley , Aprendizagem Espacial , Estreptozocina , Sinapses/metabolismoRESUMO
There is a growing interest in the potential of adult stem cells for implementing regenerative medicine in the brain. We assessed the effect of intracerebroventricular (icv) administration of human umbilical cord perivascular cells (HUCPVCs) on spatial memory of senile (27 mo) female rats, using intact senile counterparts as controls. Approximately one third of the animals were injected in the lateral ventricles with a suspension containing 4.8 X 105 HUCPVC in 8 µl per side. The other third received 4.8 X 105 transgenic HUCPVC overexpressing Insulin-like growth factor-1 (IGF-1) and the last third of the rats received no treatment. Spatial memory performance was evaluated using a modified version of the Barnes maze test. In order to evaluate learning ability as well as spatial memory retention, we assessed the time spent (permanence) by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. Fluorescence microscopy revealed the prescence of Dil-labeled HUCPVC in coronal sections of treated brains. The HUCPVC were located in close contact with the ependymal cells with only a few labeled cells migrating into the brain parenchyma. After treatment with naïve or IGF-1 transgenic HUCPVC, permanence in GS1 and GS3 increased significantly whereas there were no changes in the intact animals. We conclude that HUCPVC injected icv are effective to improve some components of spatial memory in senile rats. The ready accessibility of HUCPVC constitutes a significant incentive to continue the exploration of their therapeutic potential on neurodegenerative diseases.
Assuntos
Envelhecimento , Encéfalo/fisiopatologia , Transplante de Células , Transtornos da Memória/terapia , Memória Espacial , Cordão Umbilical , Animais , Feminino , Humanos , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Ratos , Ratos Sprague-DawleyRESUMO
There is a growing interest in the potential of mesenchymal stem cells (MSCs) for implementing regenerative medicine in the brain as they have shown neurogenic and immunomodulatory activities. We assessed the effect of intracerebroventricular (icv) administration of human bone marrow-derived MSCs (hBM-MSCs) on spatial memory and hippocampal morphology of senile (27 months) female rats, using 3-months-old counterparts as young controls. Half of the animals were injected in the lateral ventricles (LV) with a suspension containing 5 × 105hBM-MSCs in 8 µl per side. The other half received no treatment (senile controls). Spatial memory performance was assessed with a modified version of the Barnes maze test. We employed one probe trial, one day after training in order to evaluate learning ability as well as spatial memory retention. Neuroblast (DCX) and microglial (Iba-1 immunoreactive) markers were also immunohistochemically quantitated in the animals by means of an unbiased stereological approach. In addition, hippocampal presynaptic protein expression was assessed by immunoblotting analysis. After treatment, the senile MSC-treated group showed a significant improvement in spatial memory accuracy and extended permanence in a one- and 3-hole goal sectors as compared with senile controls. The MSC treatment increased the number of neuroblasts in the hippocampal dentate gyrus, reduced the number of reactive microglial cells, and restored presynaptic protein levels as compared to senile controls. We conclude that icv injected hBM-MSCs are effective in improving spatial memory in senile rats and that the strategy improves some functional and morphologic brain features typically altered in aging rats.
Assuntos
Envelhecimento/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais/métodos , Memória Espacial/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Proteína Duplacortina , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Microglia/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Memória Espacial/fisiologia , Lobo Temporal/efeitos dos fármacos , Lobo Temporal/metabolismoRESUMO
Biological rejuvenation by partial cell reprogramming is an emerging avenue of research. In this context, regulatable pluripotency gene expression systems are the most widely used at present. We have constructed a regulatable bidirectional adenovector expressing the humanized green fluorescent protein (GFP) and oct4, sox2, klf4, and c-myc genes (known as the Yamanaka genes or OSKM). The OSKM genes are arranged as a bicistronic tandem (hSTEMCCA tandem), which is under the control of a Tet-Off bidirectional promoter that also controls the expression of the gFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. Vector DNA was transfected in HEK293 Cre cells, which were additionally infected with the helper adenovector H14, unable to package its DNA due to the Cre recombinase produced by the HEK293 Cre cells. The newly generated vector was expanded by six iterated coinfections of the above cells which were lysed at the end of the process and the adenovector purified by ultracentrifugation in a CsCl gradient. The titer of the initial preparation was 1.2 × 1012 physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the four Yamanaka genes. Additionally, 3 days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced the GFP expression. This adenovector constitutes a promising tool for implementing nonintegrative partial cell reprogramming.
Assuntos
Encéfalo/fisiologia , Terapia Genética/métodos , Fatores de Transcrição Kruppel-Like/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Regeneração , Fatores de Transcrição SOXB1/genética , Adenoviridae/genética , Animais , Células Cultivadas , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOXB1/metabolismoRESUMO
Sporadic Alzheimer's disease (SAD) is the most common form of dementia; therefore, there is an urgent need for a model that recapitulates the main pathologic hallmarks of this disease. The intracerebroventricular (icv) injection of streptozotocin (icv-STZ) in rats constitutes a promising model, and thus, icv-STZ rats develop insulin-resistant brain state and cognitive impairments. Even though a great piece of studies has hitherto described this system as a model for SAD, further behavioral and morphometric studies are still needed to fully characterize it. In this study, using Sprague Dawley rats, we evaluated short-term effects on behavior and hippocampus morphometry of the icv-STZ injection at two doses: 1 (STZ1) and 3 mg/kg (STZ3). We found that, following icv-STZ injection, STZ3 animals, but not STZ1, exhibited impairments in spatial reference learning and memory (Barnes maze test) and in recognition memory (object recognition test). Furthermore, the results from behavioral and morpho-histochemical data are compatible. STZ3 rats displayed Stratum Radiatum volume reduction and a decreased NeuN immunoreactivity (neuron loss) in hippocampal CA1 region, together with an increased immunoreactivity for microglial (Iba1) and astroglial (GFAP) markers (neuroinflammation). Sholl analysis revealed the vulnerability of hippocampal astrocytes to STZ in CA1 and CA3. Thus, both doses induced a reduction in process length and in the number of main processes, accompanied by a frank decrease in branching complexity. The present study provides important knowledge of this AD rat model. Overall, we found that the only high STZ dose induced severe and acute neurodegenerative lesions, associated with an inflammation process.