RESUMO
BACKGROUND: Cerebral amyloid angiopathy (CAA) is typified by the cerebrovascular deposition of amyloid. The mechanisms underlying the contribution of CAA to neurodegeneration are not currently understood. Although CAA is highly associated with the accumulation of amyloid beta (Aß), other amyloids are known to associate with the vasculature. Alzheimer's disease (AD) is characterized by parenchymal Aß deposition, intracellular accumulation of tau, and significant neuroinflammation. CAA increases with age and is present in 85-95% of individuals with AD. A substantial amount of research has focused on understanding the connection between parenchymal amyloid and glial activation and neuroinflammation, while associations between vascular amyloid pathology and glial reactivity remain understudied. METHODS: Here, we dissect the glial and immune responses associated with early-stage CAA with histological, biochemical, and gene expression analyses in a mouse model of familial Danish dementia (FDD), a neurodegenerative disease characterized by the vascular accumulation of Danish amyloid (ADan). Findings observed in this CAA mouse model were complemented with primary culture assays. RESULTS: We demonstrate that early-stage CAA is associated with dysregulation in immune response networks and lipid processing, severe astrogliosis with an A1 astrocytic phenotype, and decreased levels of TREM2 with no reactive microgliosis. Our results also indicate how cholesterol accumulation and ApoE are associated with vascular amyloid deposits at the early stages of pathology. We also demonstrate A1 astrocytic mediation of TREM2 and microglia homeostasis. CONCLUSION: The initial glial response associated with early-stage CAA is characterized by the upregulation of A1 astrocytes without significant microglial reactivity. Gene expression analysis revealed that several AD risk factors involved in immune response and lipid processing may also play a preponderant role in CAA. This study contributes to the increasing evidence that brain cholesterol metabolism, ApoE, and TREM2 signaling are major players in the pathogenesis of AD-related dementias, including CAA. Understanding the basis for possible differential effects of glial response, ApoE, and TREM2 signaling on parenchymal plaques versus vascular amyloid deposits provides important insight for developing future therapeutic interventions.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Angiopatia Amiloide Cerebral/metabolismo , Angiopatia Amiloide Cerebral/patologia , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Gliose/metabolismo , Gliose/patologia , Humanos , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Cerebral amyloid angiopathy (CAA) is typified by the cerebrovascular deposition of amyloid. Currently, there is no clear understanding of the mechanisms underlying the contribution of CAA to neurodegeneration. Despite the fact that CAA is highly associated with the accumulation of Aß, other types of amyloids have been shown to associate with the vasculature. Interestingly, in many cases, vascular amyloidosis has been associated with an active immune response and perivascular deposition of hyperphosphorylated tau. Despite the fact that in Alzheimer's disease (AD) a major focus of research has been the understanding of the connection between parenchymal amyloid plaques, tau aggregates in the form of neurofibrillary tangles (NFTs), and immune activation, the contribution of tau and neuroinflammation to neurodegeneration associated with CAA remains understudied. In this review, we discussed the existing evidence regarding the amyloid diversity in CAA and its relation to tau pathology and immune response, as well as the possible contribution of molecular and cellular mechanisms, previously associated with parenchymal amyloid in AD and AD-related dementias, to the pathogenesis of CAA. The detailed understanding of the "amyloid-tau-neuroinflammation" axis in the context of CAA could open the opportunity to develop therapeutic interventions for dementias associated with CAA that are currently being proposed for AD and AD-related dementias.
Assuntos
Amiloide/metabolismo , Encéfalo/metabolismo , Angiopatia Amiloide Cerebral/metabolismo , Inflamação/metabolismo , Proteínas tau/metabolismo , Animais , HumanosRESUMO
Alzheimer's disease is a neurological disorder characterized by the overproduction and aggregation of amyloid-beta and the phosphorylation and intraneuronal accumulation of tau. These events promote synaptic dysfunction and loss, leading to neurodegeneration and cognitive deficits. Astrocytes are intimately associated with synapses and become activated under pathological conditions, becoming neurotoxic and detrimentally affecting synapses. Although it has been established that reducing neuronal tau expression prevents amyloid-beta-induced toxicity, the role of astrocytic tau in this setting remains understudied. Herein, we performed a series of astrocytic and neuronal primary cultures to evaluate the effects of decreasing astrocytic tau levels on astrocyte-mediated amyloid-beta-induced synaptic degeneration. Our results suggest that the downregulation of tau in astrocytes mitigates the loss of synapses triggered by their exposure to amyloid-beta. Additionally, the absence of tau from astrocytes promotes the upregulation of several synaptoprotective genes, followed by increased production of the neuroprotective factor Pentraxin 3. These results expand our understanding of the contribution of astrocytic tau to the neurodegenerative process induced by amyloid-beta-stimulation and how reducing astrocytic tau could improve astrocyte function by stimulating the expression of synaptoprotective factors. Reducing endogenous astrocytic tau expression could be a potential strategy to prevent synaptic damage in Alzheimer's disease and other neurological conditions.
RESUMO
Reactive astrogliosis is a universal response of astrocytes to abnormal events and injuries. Studies have shown that proinflammatory microglia can polarize astrocytes (designated A1 astrocytes) toward a neurotoxic phenotype characterized by increased Complement Component 3 (C3) expression. It is still unclear if inflammatory stimuli from other cell types may also be capable of inducing a subset of C3+ neurotoxic astrocytes. Here, we show that a subtype of C3+ neurotoxic astrocytes is induced by activated endothelial cells that is distinct from astrocytes activated by microglia. Furthermore, we show that endothelial-induced astrocytes have upregulated expression of A1 astrocytic genes and exhibit a distinctive extracellular matrix remodeling profile. Finally, we demonstrate that endothelial-induced astrocytes are Decorin-positive and are associated with vascular amyloid deposits but not parenchymal amyloid plaques in mouse models and AD/CAA patients. These findings demonstrate the existence of potentially extensive and subtle functional diversity of C3+-reactive astrocytes.
Assuntos
Astrócitos , Células Endoteliais , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Microglia/metabolismo , Placa AmiloideRESUMO
Cerebral amyloid angiopathy (CAA) is typified by the cerebrovascular deposition of amyloid. The mechanisms underlying the contribution of CAA to neurodegeneration are not currently understood. Although CAA is highly associated with the accumulation of ß-amyloid (Aß), other amyloids are known to associate with the vasculature. Alzheimer's disease (AD) is characterized by parenchymal Aß deposition and intracellular accumulation of tau as neurofibrillary tangles (NFTs), affecting synapses directly, leading to behavioral and physical impairment. CAA increases with age and is present in 70%-97% of individuals with AD. Studies have overwhelmingly focused on the connection between parenchymal amyloid accumulation and synaptotoxicity; thus, the contribution of vascular amyloid is mostly understudied. Here, synaptic alterations induced by vascular amyloid accumulation and their behavioral consequences were characterized using a mouse model of Familial Danish dementia (FDD), a neurodegenerative disease characterized by the accumulation of Danish amyloid (ADan) in the vasculature. The mouse model (Tg-FDD) displays a hyperactive phenotype that potentially arises from impairment in the GABAergic synapses, as determined by electrophysiological analysis. We demonstrated that the disruption of GABAergic synapse organization causes this impairment and provided evidence that GABAergic synapses are impaired in patients with CAA pathology. Understanding the mechanism that CAA contributes to synaptic dysfunction in AD-related dementias is of critical importance for developing future therapeutic interventions.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Angiopatia Amiloide Cerebral/genética , Doenças Neurodegenerativas/genética , Animais , Angiopatia Amiloide Cerebral/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Doenças Neurodegenerativas/patologiaRESUMO
The clinical benefit of therapies using Mesenchymal Stem Cells (MSCs) is attributable to their pleiotropic effect over cells and tissues, mainly through their secretome. This paracrine effect is mediated by secreted growth factors and extracellular vesicles (EV) including small EV (sEV). sEV are extra-cellular, membrane encompassed vesicles of 40 to 200 nm diameter that can trigger and signal many cellular responses depending on their cargo protein and nucleic acid repertoire. sEV are purified from cell culture conditioned media using several kits and protocols available that can be tedious and time-consuming, involving sequences of ultracentrifugations and density gradient separations, making their production a major challenge under Good Manufacturing Practices (GMP) conditions. We have developed a method to efficiently enrich cell culture media with high concentrations of sEV by encapsulating cells in semipermeable cellulose beads that allows selectively the release of small particles while offering a 3D culture condition. This method is based on the pore size of the capsules, allowing the release of particles of ≤ 200 nm including sEV. As a proof-of-principle, MSCs were encapsulated and their sEV release rate (sEV-Cap) was monitored throughout the culture and compared to sEV isolated from 2D seeded cells (sEV-2D) by repetitive ultracentrifugation cycles or a commercial kit. The isolated sEV expressed CD63, CD9, and CD81 as confirmed by flow cytometry analysis. Under transmission electron microscopy (TEM), they displayed the similar rounded morphology as sEV-2D. Their corresponding diameter size was validated by nanoparticle tracking analysis (NTA). Interestingly, sEV-Cap retained the expected biological activities of MSCs, including a pro-angiogenic effect over endothelial cells, neuritic outgrowth stimulation in hippocampal neurons and immunosuppression of T cells in vitro. Here, we successfully present a novel, cost, and time-saving method to generate sEV from encapsulated MSCs. Future applications include using encapsulated cells as a retrievable delivery device that can interact with the host niche by releasing active agents in vivo, including sEV, growth factors, hormones, and small molecules, while avoiding cell clearance, and the negative side-effect of releasing undesired components including apoptotic bodies. Finally, particles produced following the encapsulation protocol display beneficial features for their use as drug-loaded delivery vehicles.
RESUMO
In the last few decades, it has been established that astrocytes play key roles in the regulation of neuronal morphology. However, the contribution of astrocyte-derived small extracellular vesicles (sEVs) to morphological differentiation of neurons has only recently been addressed. Here, we showed that cultured astrocytes expressing a GFP-tagged version of the stress-regulated astrocytic enzyme Aldolase C (Aldo C-GFP) release small extracellular vesicles (sEVs) that are transferred into cultured hippocampal neurons. Surprisingly, Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) displayed an exacerbated capacity to reduce the dendritic complexity in developing hippocampal neurons compared to sEVs derived from control (i.e., GFP-expressing) astrocytes. Using bioinformatics and biochemical tools, we found that the total content of overexpressed Aldo C-GFP correlates with an increased content of endogenous miRNA-26a-5p in both total astrocyte homogenates and sEVs. Notably, neurons magnetofected with a nucleotide sequence that mimics endogenous miRNA-26a-5p (mimic 26a-5p) not only decreased the levels of neuronal proteins associated to morphogenesis regulation, but also reproduced morphological changes induced by Aldo-C-GFP sEVs. Furthermore, neurons magnetofected with a sequence targeting miRNA-26a-5p (antago 26a-5p) were largely resistant to Aldo C-GFP sEVs. Our results support a novel and complex level of astrocyte-to-neuron communication mediated by astrocyte-derived sEVs and the activity of their miRNA content.
Assuntos
Astrócitos/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Animais , Astrócitos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Dendritos/metabolismo , Feminino , Frutose-Bifosfato Aldolase/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Cerebral amyloid angiopathy (CAA) is typified by the cerebrovascular deposition of amyloid. Currently, there is no clear understanding of the mechanisms underlying the contribution of CAA to neurodegeneration. Despite the fact that CAA is highly associated with accumulation of Aß, other types of amyloids have been shown to associate with the vasculature. Interestingly, in many cases, vascular amyloidosis is accompanied by significant tau pathology. However, the contribution of tau to neurodegeneration associated to CAA remains to be determined. We used a mouse model of Familial Danish Dementia (FDD), a neurodegenerative disease characterized by the accumulation of Danish amyloid (ADan) in the vasculature, to characterize the contribution of tau to neurodegeneration associated to CAA. We performed histological and biochemical assays to establish tau modifications associated with CAA in conjunction with cell-based and electrophysiological assays to determine the role of tau in the synaptic dysfunction associated with ADan. We demonstrated that ADan aggregates induced hyperphosphorylation and misfolding of tau. Moreover, in a mouse model for CAA, we observed tau oligomers closely associated to astrocytes in the vicinity of vascular amyloid deposits. We finally determined that the absence of tau prevents synaptic dysfunction induced by ADan oligomers. In addition to demonstrating the effect of ADan amyloid on tau misfolding, our results provide compelling evidence of the role of tau in neurodegeneration associated with ADan-CAA and suggest that decreasing tau levels could be a feasible approach for the treatment of CAA.
Assuntos
Angiopatia Amiloide Cerebral/genética , Angiopatia Amiloide Cerebral/metabolismo , Proteínas tau/deficiência , Proteínas tau/genética , Sequência de Aminoácidos , Amiloidose/genética , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Ataxia Cerebelar/genética , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Angiopatia Amiloide Cerebral/patologia , Surdez/genética , Surdez/metabolismo , Surdez/patologia , Demência/genética , Demência/metabolismo , Demência/patologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
Caspases are a family of cysteine-proteases, activated upon several different stimuli, which execute apoptosis in many cell death models. Previous work of our group has shown rats have the highest rate of apoptosis during the first wave of spermatogenesis (between 20 and 25 days after birth), as evaluated by TUNEL and caspase activity. However, the hierarchical order of caspase activation and the relevance of each caspase during germ cell apoptosis are not clear. Thus, the goal of this work is to take a pharmacological approach to dissect the apoptosis pathway of caspase activation. Results showed that intratesticular injection of a caspase-8 inhibitor (z-IETD-fmk), or a pan-caspase inhibitor (z-VAD- fmk), significantly decreased the cleavage of p115 and PARP, two endogenous substrates of caspases, in 22-day-old rats. Additionally, these inhibitors promoted a significant reduction in the number of apoptotic germ cells. On the other hand, intratesticular injection of two different inhibitors of the intrinsic pathway (z-LEHD-fmk and minocycline) did not have any effect upon caspase substrates cleavage (p115 and PARP) or the number of apoptotic germ cells. Therefore, we conclude that the extrinsic pathway of apoptosis plays an important role in physiological germ cell apoptosis during the first round of spermatogenesis in the rat.
Assuntos
Apoptose/fisiologia , Caspase 8/metabolismo , Maturidade Sexual/fisiologia , Espermatogênese/fisiologia , Espermatozoides/enzimologia , Animais , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Humanos , Masculino , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Maturidade Sexual/efeitos dos fármacos , Espermatogênese/efeitos dos fármacosRESUMO
Hypothyroxinemia (Hpx) is a highly frequent condition characterized by low thyroxine (T4) and normal 3,3',5'-triiodothyronine (T3) and thyroid stimulating hormone (TSH) levels in the blood. Gestational Hpx is closely related to cognitive impairment in the human offspring. In animal models gestational Hpx causes impairment at glutamatergic synapsis, spatial learning, and the susceptibility to suffer strong autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying these phenotypes are unknown. On the other hand, it has been shown that astrocytes and microglia affect the outcome of EAE. In fact, the activation of astrocytes and microglia in the central nervous system (CNS) contributes to EAE progression. Thus, in this work, the reactivity of astrocytes and microglia from rats gestated in Hpx was evaluated aiming to understand whether these cells are targets of gestational Hpx. Interestingly, microglia derived from the offspring gestated in Hpx were less reactive compared to microglia derived from offspring gestated in euthyroidism. Instead, astrocytes derived from the offspring gestated in Hpx were significantly more reactive than the astrocytes from the offspring gestated in euthyroidism. This work contributes with novel information regarding the effects of gestational Hpx over astrocytes and microglia in the offspring. It suggests that astrocyte could react strongly to an inflammatory insult inducing neuronal death in the CNS.
Assuntos
Astrócitos/patologia , Inflamação/sangue , Inflamação/patologia , Microglia/patologia , Tiroxina/sangue , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Quimiocina CXCL2/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Interleucina-1beta/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Gravidez , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Repetitive stress negatively affects several brain functions and neuronal networks. Moreover, adult neurogenesis is consistently impaired in chronic stress models and in associated human diseases such as unipolar depression and bipolar disorder, while it is restored by effective antidepressant treatments. The adult neurogenic niche contains neural progenitor cells in addition to amplifying progenitors, neuroblasts, immature and mature neurons, pericytes, astrocytes, and microglial cells. Because of their particular and crucial position, with their end feet enwrapping endothelial cells and their close communication with the cells of the niche, astrocytes might constitute a nodal point to bridge or transduce systemic stress signals from peripheral blood, such as glucocorticoids, to the cells involved in the neurogenic process. It has been proposed that communication between astrocytes and niche cells depends on direct cell-cell contacts and soluble mediators. In addition, new evidence suggests that this communication might be mediated by extracellular vesicles such as exosomes, and in particular, by their miRNA cargo. Here, we address some of the latest findings regarding the impact of stress in the biology of the neurogenic niche, and postulate how astrocytic exosomes (and miRNAs) may play a fundamental role in such phenomenon.
RESUMO
Gestational hypothyroxinemia, characterized by low levels of maternal thyroxine (T4) during gestation, is closely associated with cognitive impairment in offspring. Studies in animal models have shown that this condition alters neuronal glutamatergic synapses in the hippocampus. Given that astrocytes critically contribute to the establishment and functioning of synapses, the aim of this study was to determine the effects of gestational hypothyroxinemia on the capacity of astrocytes to regulate glutamatergic synapses. In an in vitro co-culture model of astrocytes and hippocampal neurons, gestational hypothyroxinemia profoundly affected the synaptic patterns of GluN1 and CD3ζ in an astrocyte-dependent manner. These effects were associated with impaired plasticity that was dependent on both neuronal and astrocyte contributions. These results highlight the importance of neuron-astrocyte interplay in the deleterious effects of gestational hypothyroxinemia and the timely diagnosis and treatment of this condition during gestation to ensure proper central nervous system development in offspring.
Assuntos
Astrócitos/metabolismo , Comunicação Celular , Glutamatos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Sinapses/metabolismo , Tiroxina/sangue , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Comunicação Celular/efeitos dos fármacos , Contagem de Células , Técnicas de Cocultura , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Feminino , Glicina/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Gravidez , Transporte Proteico/efeitos dos fármacos , Ratos Sprague-Dawley , Sinapses/efeitos dos fármacosRESUMO
BACKGROUND: Maternal thyroid hormones play a fundamental role in appropriate fetal development during gestation. Offspring that have been gestated under maternal hypothyroidism suffer cognitive impairment. Thyroid hormone deficiency during gestation can significantly impact the central nervous system by altering the migration, differentiation, and function of neurons, oligodendrocytes, and astrocytes. Given that gestational hypothyroidism alters the immune cell ratio in offspring, it is possible that this condition could result in higher sensitivity for the development of autoimmune diseases. METHODS: Adult mice gestated under hypothyroidism were induced with experimental autoimmune encephalomyelitis (EAE). Twenty-one days after EAE induction, the disease score, myelin content, immune cell infiltration, and oligodendrocyte death were evaluated. RESULTS: We observed that mice gestated under hypothyroidism showed higher EAE scores after disease induction during adulthood compared to mice gestated in euthyroidism. In addition, spinal cord sections of mice gestated under hypothyroidism that suffered EAE in adulthood showed higher demyelination, CD4(+) and CD8(+) infiltration, and increased oligodendrocyte death. CONCLUSIONS: These results show for the first time that a deficiency in maternal thyroid hormones during gestation can influence the outcome of a central nervous system inflammatory disease, such as EAE, in their offspring. These data strongly support evaluating thyroid hormones in pregnant women and treating hypothyroidism during pregnancy to prevent increased susceptibility to inflammatory diseases in the central nervous system of offspring.
Assuntos
Encefalomielite Autoimune Experimental/fisiopatologia , Hipotireoidismo/fisiopatologia , Linfócitos/imunologia , Medula Espinal/fisiopatologia , Animais , Proliferação de Células , Encefalomielite Autoimune Experimental/imunologia , Feminino , Hipotireoidismo/imunologia , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Índice de Gravidade de Doença , Medula Espinal/imunologiaRESUMO
Apoptosis is a type of cell death characterized by the activation of a family of cysteine-proteases called caspases. We made a comparative study to determine the presence of several caspases and other regulators of apoptosis in rat, mouse, and hamster spermatozoa. Our results showed that the three species have both active and inactive caspases-8 and -3, the proapoptotic protein BID, p53, and the endogenous caspase inhibitor cIAP-1. However, we did not find evidence for the presence of active caspase-9. The acrosome reaction (i.e., the exocytic process of sperm acrosome) and sperm viability were not affected by the presence of a general caspase inhibitor. On the other hand, valinomycin, which promotes caspase-dependent cell death in somatic cells, induced caspase-independent cell death in spermatozoa. TRAIL, a ligand whose receptor induces apoptosis in malignant cells, did not have any effect in the viability of mouse spermatozoa, despise the presence of its receptor in rat and mouse, but not in hamster spermatozoa. Therefore, our results strongly suggest that rodent spermatozoa have some components of the apoptotic pathway. However, the role of caspases in mammalian spermatozoa appears to be unrelated to sperm survival or to the acrosome reaction under physiological conditions.