RESUMO
Aging dogs serve as a valuable preclinical model for Alzheimer's disease (AD) due to their natural age-related development of ß-amyloid (Aß) plaques, human-like metabolism, and large brains that are ideal for studying structural brain aging trajectories from serial neuroimaging. Here we examined the effects of chronic treatment with the calcineurin inhibitor (CNI) tacrolimus or the nuclear factor of activated T cells (NFAT)-inhibiting compound Q134R on age-related canine brain atrophy from a longitudinal study in middle-aged beagles (36 females, 7 males) undergoing behavioral enrichment. Annual MRI was analyzed using modern, automated techniques for region-of-interest-based and voxel-based volumetric assessments. We found that the frontal lobe showed accelerated atrophy with age, while the caudate nucleus remained relatively stable. Remarkably, the hippocampus increased in volume in all dogs. None of these changes were influenced by tacrolimus or Q134R treatment. Our results suggest that behavioral enrichment can prevent atrophy and increase the volume of the hippocampus but does not prevent aging-associated prefrontal cortex atrophy.
Assuntos
Envelhecimento , Atrofia , Encéfalo , Tacrolimo , Animais , Cães , Feminino , Atrofia/patologia , Masculino , Envelhecimento/patologia , Encéfalo/patologia , Encéfalo/efeitos dos fármacos , Tacrolimo/farmacologia , Comportamento Animal/efeitos dos fármacos , Imageamento por Ressonância MagnéticaRESUMO
Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+ dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca2+ dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+ transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca2+ signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin-dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus vectors, led to reduced GFAP promoter activity in HHcy diet mice and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e., cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly Alzheimer's disease and other related dementias.SIGNIFICANCE STATEMENT The impact of reactive astrocytes in Alzheimer's disease and related dementias is poorly understood. Here, we evaluated Ca2+ responses and signaling in barrel cortex astrocytes of mice fed with a B-vitamin deficient diet that induces hyperhomocysteinemia (HHcy), cerebral vessel disease, and cognitive decline. Multiphoton imaging in awake mice with HHcy revealed augmented Ca2+ responses in individual astrocytes, but impaired signaling across astrocyte networks. Stimulation-evoked arteriole dilation and elevated red blood cell velocity in capillaries were also impaired in cortex of awake HHcy mice. Astrocyte-specific inhibition of the Ca2+-dependent transcription factor, NFAT, normalized cerebrovascular function in HHcy mice, improved synaptic properties in brain slices, and stabilized cognition. Results suggest that astrocytes are a mechanism and possible therapeutic target for vascular-related dementia.
Assuntos
Doença de Alzheimer , Hiper-Homocisteinemia , Camundongos , Masculino , Feminino , Animais , Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Hiper-Homocisteinemia/metabolismo , Hiper-Homocisteinemia/patologia , Dieta , Fatores de Transcrição/metabolismoRESUMO
The overexpression of calcineurin leads to astrocyte hyperactivation, neuronal death, and inflammation, which are characteristics often associated with pathologic aging and Alzheimer's disease. In this study, we tested the hypothesis that tacrolimus, a calcineurin inhibitor, prevents age-associated microstructural atrophy, which we measured using higher-order diffusion MRI, in the middle-aged beagle brain (n = 30, male and female). We find that tacrolimus reduces hippocampal (p = 0.001) and parahippocampal (p = 0.002) neurite density index, as well as protects against an age-associated increase in the parahippocampal (p = 0.007) orientation dispersion index. Tacrolimus also protects against an age-related decrease in fractional anisotropy in the prefrontal cortex (p < 0.0001). We also show that these microstructural alterations precede cognitive decline and gross atrophy. These results support the idea that calcineurin inhibitors may have the potential to prevent aging-related pathology if administered at middle age.SIGNIFICANCE STATEMENT Hyperactive calcineurin signaling causes neuroinflammation and other neurobiological changes often associated with pathologic aging and Alzheimer's disease (AD). Controlling the expression of calcineurin before gross cognitive deficits are observable might serve as a promising avenue for preventing AD pathology. In this study, we show that the administration of the calcineurin inhibitor, tacrolimus, over 1 year prevents age- and AD-associated microstructural changes in the hippocampus, parahippocampal cortex, and prefrontal cortex of the middle-aged beagle brain, with no noticeable adverse effects. Tacrolimus is already approved by the Food and Drug Administration for use in humans to prevent solid organ transplant rejection, and our results bolster the promise of this drug to prevent AD and aging-related pathology.
Assuntos
Envelhecimento/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Inibidores de Calcineurina/farmacologia , Tacrolimo/farmacologia , Envelhecimento/patologia , Animais , Atrofia/patologia , Cães , Feminino , MasculinoRESUMO
AIMS: The current study utilizes the adeno-associated viral gene transfer system in the CAMKIIα-tTA mouse model to overexpress human wild type TDP-43 (wtTDP-43) and α-synuclein (α-Syn) proteins. The co-existence of these proteins is evident in the pathology of neurodegenerative disorders such as frontotemporal lobar degeneration (FTLD), Parkinson disease (PD), and dementia with Lewy bodies (DLB). METHODS: The novel bicistronic recombinant adeno-associated virus (rAAV) serotype 9 drives wtTDP-43 and α-Syn expression in the hippocampus via "TetO" CMV promoter. Behavior, electrophysiology, and biochemical and histological assays were used to validate neuropathology. RESULTS: We report that overexpression of wtTDP-43 but not α-Syn contributes to hippocampal CA2-specific pyramidal neuronal loss and overall hippocampal atrophy. Further, we report a reduction of hippocampal long-term potentiation and decline in learning and memory performance of wtTDP-43 expressing mice. Elevated wtTDP-43 levels induced selective degeneration of Purkinje cell protein 4 (PCP-4) positive neurons while both wtTDP-43 and α-Syn expression reduced subsets of the glutamate receptor expression in the hippocampus. CONCLUSIONS: Overall, our findings suggest the significant vulnerability of hippocampal neurons toward elevated wtTDP-43 levels possibly via PCP-4 and GluR-dependent calcium signaling pathways. Further, we report that wtTDP-43 expression induced selective CA2 subfield degeneration, contributing to the deterioration of the hippocampal-dependent cognitive phenotype.
Assuntos
Região CA2 Hipocampal/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Potenciação de Longa Duração , Memória , Animais , Região CA2 Hipocampal/fisiologia , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Aprendizagem em Labirinto , Camundongos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , alfa-Sinucleína/metabolismoRESUMO
We present the application of multiphoton in vivo fluorescence correlation spectroscopy (FCS) of fluorescent nanoparticles for the measurement of cerebral blood flow with excellent spatial and temporal resolution. Through the detection of single nanoparticles within the complex vessel architecture of a live mouse, this new approach enables the quantification of nanoparticle dynamics occurring within the vasculature along with simultaneous measurements of blood flow properties in the brain. In addition to providing high resolution blood flow measurements, this approach enables real-time quantification of nanoparticle concentration, degradation, and transport. This method is capable of quantifying flow rates at each pixel with submicron resolution to enable monitoring of dynamic changes in flow rates in response to changes in the animal's physiological condition. Scanning the excitation beam using FCS provides pixel by pixel mapping of flow rates with subvessel resolution across capillaries 300 µm deep in the brains of mice.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica , Nanopartículas , Animais , Circulação Cerebrovascular , Camundongos , Espectrometria de FluorescênciaRESUMO
Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Astrócitos , Calcineurina/genética , Fatores de Transcrição NFATC/genética , Rede Nervosa/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Potenciais Pós-Sinápticos Excitadores , Inativação Gênica , Hipocampo/metabolismo , Aprendizagem em Labirinto , Camundongos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
Ca2+ dysregulation is a hallmark of Alzheimer disease (AD) and affects numerous and diverse signaling cascades linked to neurodegeneration and cognitive decline. Increasing evidence suggests that the protein phosphatase calcineurin (CN) mediates or exacerbates AD pathophysiology through activation of the NFAT family of transcription factors. In this editorial, we discuss work by Hopp et al, , which uncovered a novel role of CN/NFAT signaling in controlling global gene expression in hippocampal neurons of intact mice. Interestingly, the authors showed that elevated CN expression/activity in neurons plays a major role in transcriptional suppression. Many of the genes differentially affected by CN were related to synapse function and NFAT binding, and exhibited similar patterns of downregulation in previous studies on human AD biospecimens. Results are discussed in context with emerging roles for CN/NFATs in astrocyte signaling as they pertain to Ca2+ dysregulation and the progression of neurodegeneration and cognitive loss with AD.
Assuntos
Doença de Alzheimer , Calcineurina , Animais , Encéfalo , Humanos , Camundongos , Fatores de Transcrição NFATC , NeurôniosRESUMO
Vascular contributions to cognitive impairment and dementia (VCID) is understood to be the second most common cause of dementia after Alzheimer's disease, and is also a frequent comorbidity with Alzheimer's disease. While VCID is widely acknowledged as a key contributor to dementia, the mechanistic underpinnings of VCID remain poorly understood. In this review, we address the potential role of astrocytes in the pathophysiology of VCID. The vast majority of the blood vessels in the brain are surrounded by astrocytic end-feet. Given that astrocytes make up a significant proportion of the cells in the brain, and that astrocytes are usually passively connected to one another through gap junctions, we hypothesize that astrocytes are key mediators of cognitive impairment because of cerebrovascular disease. In this review, we discuss the existing body of literature regarding the role of astrocytes at the vasculature in the brain, and the known consequences of their dysfunction, as well as our hypotheses regarding the role astrocytes play in VCID. This article is part of the Special Issue "Vascular Dementia".
Assuntos
Astrócitos/fisiologia , Encéfalo/fisiopatologia , Disfunção Cognitiva/fisiopatologia , Demência Vascular/fisiopatologia , Animais , Astrócitos/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Disfunção Cognitiva/metabolismo , Demência Vascular/metabolismo , HumanosRESUMO
Increasing evidence suggests that the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) mediates deleterious effects of astrocytes in progressive neurodegenerative conditions. However, the impact of astrocytic CN/NFAT signaling on neural function/recovery after acute injury has not been investigated extensively. Using a controlled cortical impact (CCI) procedure in rats, we show that traumatic brain injury is associated with an increase in the activities of NFATs 1 and 4 in the hippocampus at 7 d after injury. NFAT4, but not NFAT1, exhibited extensive labeling in astrocytes and was found throughout the axon/dendrite layers of CA1 and the dentate gyrus. Blockade of the astrocytic CN/NFAT pathway in rats using adeno-associated virus (AAV) vectors expressing the astrocyte-specific promoter Gfa2 and the NFAT-inhibitory peptide VIVIT prevented the injury-related loss of basal CA1 synaptic strength and key synaptic proteins and reduced the susceptibility to induction of long-term depression. In conjunction with these seemingly beneficial effects, VIVIT treatment elicited a marked increase in the expression of the prosynaptogenic factor SPARCL1 (hevin), especially in hippocampal tissue ipsilateral to the CCI injury. However, in contrast to previous work on Alzheimer's mouse models, AAV-Gfa2-VIVIT had no effects on the levels of GFAP and Iba1, suggesting that synaptic benefits of VIVIT were not attributable to a reduction in glial activation per se. Together, the results implicate the astrocytic CN/NFAT4 pathway as a key mechanism for disrupting synaptic remodeling and homeostasis in the hippocampus after acute injury. SIGNIFICANCE STATEMENT: Similar to microglia, astrocytes become strongly "activated" with neural damage and exhibit numerous morphologic/biochemical changes, including an increase in the expression/activity of the protein phosphatase calcineurin. Using adeno-associated virus (AAV) to inhibit the calcineurin-dependent activation of the transcription factor NFAT (Nuclear Factor of Activated T cells) selectively, we have shown that activated astrocytes contribute to neural dysfunction in animal models characterized by progressive/chronic neuropathology. Here, we show that the suppression of astrocytic calcineurin/NFATs helps to protect synaptic function and plasticity in an animal model in which pathology arises from a single traumatic brain injury. The findings suggest that at least some astrocyte functions impair recovery after trauma and may provide druggable targets for treating victims of acute nervous system injury.
Assuntos
Astrócitos/fisiologia , Lesões Encefálicas/terapia , Calcineurina/metabolismo , Hipocampo/fisiologia , Fatores de Transcrição NFATC/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Animais , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Calcineurina/genética , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Masculino , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury.
Assuntos
Astrócitos/metabolismo , Calcineurina/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Especificidade de Anticorpos , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Calcineurina/imunologia , Células Cultivadas , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Humanos , Imuno-Histoquímica , Masculino , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Proteólise , Ratos , Ratos Sprague-DawleyRESUMO
Clinical and animal model studies have implicated inflammation and peripheral immune cell responses in the pathophysiology of Alzheimer's disease (AD). Peripheral immune cells including T cells circulate in the cerebrospinal fluid (CSF) of healthy adults and are found in the brains of AD patients and AD rodent models. Blocking entry of peripheral macrophages into the CNS was reported to increase amyloid burden in an AD mouse model. To assess inflammation in the 5xFAD (Tg) mouse model, we first quantified central and immune cell profiles in the deep cervical lymph nodes and spleen. In the brains of Tg mice, activated (MHCII+, CD45high, and Ly6Chigh) myeloid-derived CD11b+ immune cells are decreased while CD3+ T cells are increased as a function of age relative to non-Tg mice. These immunological changes along with evidence of increased mRNA levels for several cytokines suggest that immune regulation and trafficking patterns are altered in Tg mice. Levels of soluble Tumor Necrosis Factor (sTNF) modulate blood-brain barrier (BBB) permeability and are increased in CSF and brain parenchyma post-mortem in AD subjects and Tg mice. We report here that in vivo peripheral administration of XPro1595, a novel biologic that sequesters sTNF into inactive heterotrimers, reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, XPro1595 treatment in vivo rescued impaired long-term potentiation (LTP) measured in brain slices in association with decreased Aß plaques in the subiculum. Selective targeting of sTNF may modulate brain immune cell infiltration, and prevent or delay neuronal dysfunction in AD. SIGNIFICANCE STATEMENT: Immune cells and cytokines perform specialized functions inside and outside the brain to maintain optimal brain health; but the extent to which their activities change in response to neuronal dysfunction and degeneration is not well understood. Our findings indicate that neutralization of sTNF reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, impaired long-term potentiation (LTP) was rescued by XPro1595 in association with decreased hippocampal Aß plaques. Selective targeting of sTNF holds translational potential to modulate brain immune cell infiltration, dampen neuroinflammation, and prevent or delay neuronal dysfunction in AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Inibidores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/farmacologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Encéfalo/metabolismo , Encéfalo/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Citocinas/metabolismo , Feminino , Potenciação de Longa Duração/fisiologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroimunomodulação/efeitos dos fármacos , Neuroimunomodulação/fisiologia , Placa Amiloide/tratamento farmacológico , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Distribuição Aleatória , Técnicas de Cultura de Tecidos , Fatores de Necrose Tumoral/metabolismoRESUMO
Similar to peripheral immune/inflammatory cells, neuroglial cells appear to rely on calcineurin (CN) signaling pathways to regulate cytokine production and cellular activation. Several studies suggest that harmful immune/inflammatory responses may be the most impactful consequence of aberrant CN activity in glial cells. However, newly identified roles for CN in glutamate uptake, gap junction regulation, Ca2+ dyshomeostasis, and amyloid production suggest that CN's influence in glia may extend well beyond neuroinflammation. The following review will discuss the various actions of CN in glial cells, with particular emphasis on astrocytes, and consider the implications for neurologic dysfunction arising with aging, injury, and/or neurodegenerative disease.
Assuntos
Encéfalo/metabolismo , Calcineurina/metabolismo , Inflamação/metabolismo , Neuroglia/metabolismo , Transdução de Sinais/fisiologia , Animais , HumanosRESUMO
Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.
Assuntos
Astrócitos , Calcineurina , Permeabilidade da Membrana Celular , Conexina 43 , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Conexina 43/metabolismo , Animais , Fosforilação/efeitos dos fármacos , Calcineurina/metabolismo , Ratos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Ratos Sprague-DawleyRESUMO
Research on the cerebrovasculature may provide insights into brain health and disease. Immunohistochemical staining is one way to visualize blood vessels, and digital pathology has the potential to revolutionize the measurement of blood vessel parameters. These tools provide opportunities for translational mouse model research. However, mouse brain tissue presents a formidable set of technical challenges, including potentially high background staining and cross-reactivity of endogenous IgG. Formalin-fixed paraffin-embedded (FFPE) and fixed frozen sections, both of which are widely used, may require different methods. In this study, we optimized blood vessel staining in mouse brain tissue, testing both FFPE and frozen fixed sections. A panel of immunohistochemical blood vessel markers were tested (including CD31, CD34, collagen IV, DP71, and VWF), to evaluate their suitability for digital pathological analysis. Collagen IV provided the best immunostaining results in both FFPE and frozen fixed murine brain sections, with highly-specific staining of large and small blood vessels and low background staining. Subsequent analysis of collagen IV-stained sections showed region and sex-specific differences in vessel density and vessel wall thickness. We conclude that digital pathology provides a useful tool for relatively unbiased analysis of the murine cerebrovasculature, provided proper protein markers are used.
Assuntos
Encéfalo , Colágeno , Masculino , Feminino , Camundongos , Animais , Inclusão em ParafinaRESUMO
BACKGROUND: Calcineurin (CN) is a Ca2+/calmodulin-dependent protein phosphatase. In healthy tissue, CN exists mainly as a full-length (â¼60 kDa) highly-regulated protein phosphatase involved in essential cellular functions. However, in diseased or injured tissue, CN is proteolytically converted to a constitutively active fragment that has been causatively-linked to numerous pathophysiologic processes. These calpain-cleaved CN fragments (∆CN) appear at high levels in human brain at early stages of cognitive decline associated with Alzheimer's disease (AD). NEW METHOD: We developed a monoclonal antibody to ∆CN, using an immunizing peptide corresponding to the C-terminal end of the ∆CN fragment. RESULTS: We obtained a mouse monoclonal antibody, designated 26A6, that selectively detects ∆CN in Western analysis of calpain-cleaved recombinant human CN. Using this antibody, we screened both pathological and normal human brain sections provided by the University of Kentucky's Alzheimer's Disease Research Center. 26A6 showed low reactivity towards normal brain tissue, but detected astrocytes both surrounding AD amyloid plaques and throughout AD brain tissue. In brain tissue with infarcts, there was considerable concentration of 26A6-positive astrocytes within/around infarcts, suggesting a link with anoxic/ischemia pathways. COMPARISON WITH EXISTING METHOD: The results obtained with the new monoclonal are similar to those obtained with a polyclonal we had previously developed. However, the monoclonal is an abundant tool available to the dementia research community. CONCLUSIONS: The new monoclonal 26A6 antibody is highly selective for the ∆CN proteolytic fragment and labels a subset of astrocytes, and could be a useful tool for marking insidious brain pathology and identifying novel astrocyte phenotypes.
Assuntos
Doença de Alzheimer , Calpaína , Camundongos , Animais , Humanos , Calpaína/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Anticorpos Monoclonais/metabolismo , Infarto/metabolismo , Infarto/patologiaRESUMO
Background: Alzheimer's disease (AD) is one of the multifaceted neurodegenerative diseases influenced by many genetic and epigenetic factors. Genetic factors are merely not responsible for developing AD in the whole population. The studies of genetic variants can provide significant insights into the molecular basis of Alzheimer's disease. Our research aimed to show how genetic variants interact with environmental influences in different parts of the world. Methodology: We searched PubMed and Google Scholar for articles exploring the relationship between genetic variations and global regions such as America, Europe, and Asia. We aimed to identify common genetic variations susceptible to AD and have no significant heterogeneity. To achieve this, we analyzed 35 single-nucleotide polymorphisms (SNPs) from 17 genes (ABCA7, APOE, BIN1, CD2AP, CD33, CLU, CR1, EPHA1, TOMM40, MS4A6A, ARID5B, SORL1, APOC1, MTHFD1L, BDNF, TFAM, and PICALM) from different regions based on previous genomic studies of AD. It has been reported that rs3865444, CD33, is the most common polymorphism in the American and European populations. From TOMM40 and APOE rs2075650, rs429358, and rs6656401, CR1 is the common investigational polymorphism in the Asian population. Conclusion: The results of all the research conducted on AD have consistently shown a correlation between genetic variations and the incidence of AD in the populations of each region. This review is expected to be of immense value in future genetic research and precision medicine on AD, as it provides a comprehensive understanding of the genetic factors contributing to the development of this debilitating disease.
Assuntos
Doença de Alzheimer , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Doença de Alzheimer/genética , Doença de Alzheimer/epidemiologia , Humanos , Europa (Continente)/epidemiologia , Ásia/epidemiologia , Estados Unidos/epidemiologia , Variação Genética/genéticaRESUMO
Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimer's disease (AD) and most other neurodegenerative disorders, many astrocytes convert to a chronically "activated" phenotype characterized by morphologic and biochemical changes that appear to compromise protective properties and/or promote harmful neuroinflammatory processes. Activated astrocytes emerge early in the course of AD and become increasingly prominent as clinical and pathological symptoms progress, but few studies have tested the potential of astrocyte-targeted therapeutics in an intact animal model of AD. Here, we used adeno-associated virus (AAV) vectors containing the astrocyte-specific Gfa2 promoter to target hippocampal astrocytes in APP/PS1 mice. AAV-Gfa2 vectors drove the expression of VIVIT, a peptide that interferes with the immune/inflammatory calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, shown by our laboratory and others to orchestrate biochemical cascades leading to astrocyte activation. After several months of treatment with Gfa2-VIVIT, APP/PS1 mice exhibited improved cognitive and synaptic function, reduced glial activation, and lower amyloid levels. The results confirm a deleterious role for activated astrocytes in AD and lay the groundwork for exploration of other novel astrocyte-based therapies.
Assuntos
Doença de Alzheimer/patologia , Astrócitos/fisiologia , Animais , Astrócitos/patologia , Astrócitos/ultraestrutura , Aprendizagem da Esquiva/fisiologia , Western Blotting , Encéfalo/patologia , Inibidores de Calcineurina , Células Cultivadas , Dependovirus/genética , Ensaio de Imunoadsorção Enzimática , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas de Transferência de Genes , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Inflamação/fisiopatologia , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Transgênicos , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/fisiologia , Neurônios/fisiologia , Oligopeptídeos/farmacologia , Transdução de Sinais/fisiologiaRESUMO
Overproduction of proinflammatory cytokines in the CNS has been implicated as a key contributor to pathophysiology progression in Alzheimer's disease (AD), and extensive studies with animal models have shown that selective suppression of excessive glial proinflammatory cytokines can improve neurologic outcomes. The prior art, therefore, raises the logical postulation that intervention with drugs targeting dysregulated glial proinflammatory cytokine production might be effective disease-modifying therapeutics if used in the appropriate biological time window. To test the hypothesis that early stage intervention with such drugs might be therapeutically beneficial, we examined the impact of intervention with MW01-2-151SRM (MW-151), an experimental therapeutic that selectively attenuates proinflammatory cytokine production at low doses. MW-151 was tested in an APP/PS1 knock-in mouse model that exhibits increases in AD-relevant pathology progression with age, including increases in proinflammatory cytokine levels. Drug was administered during two distinct but overlapping therapeutic time windows of early stage pathology development. MW-151 treatment attenuated the increase in microglial and astrocyte activation and proinflammatory cytokine production in the cortex and yielded improvement in neurologic outcomes, such as protection against synaptic protein loss and synaptic plasticity impairment. The results also demonstrate that the therapeutic time window is an important consideration in efficacy studies of drugs that modulate glia biological responses involved in pathology progression and suggest that such paradigms should be considered in the development of new therapeutic regimens that seek to delay the onset or slow the progression of AD.
Assuntos
Envelhecimento/patologia , Doença de Alzheimer/tratamento farmacológico , Citocinas/biossíntese , Progressão da Doença , Piridazinas/farmacologia , Pirimidinas/farmacologia , Sinapses/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Citocinas/antagonistas & inibidores , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Sinapses/metabolismo , Sinapses/patologiaRESUMO
Increased function of neuronal L-type voltage-sensitive Ca(2+) channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal "zipper" slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca(2+) dysregulation can differ substantially between animal models of normal aging and models of pathological aging.
Assuntos
Doença de Alzheimer/fisiopatologia , Canais de Cálcio Tipo L/fisiologia , Modelos Animais de Doenças , Neurônios/fisiologia , Animais , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Numerous preclinical and human tissue studies implicate the protein phosphatase calcineurin (CN) as a pathophysiologic mechanism in Alzheimer's disease (AD) and other neurodegenerative conditions. Using public electronic records of tens of thousands of individuals across the United States, Silva et al. (2023) show that use of the FDA-approved CN inhibitor, tacrolimus (for purposes of immunosuppression) is also associated with reduced prevalence of dementia-related symptoms. Notably, the study controls for age, sex, and race as well as multiple risk factors for AD. The results suggest that tacrolimus, and possibly other immunosuppressants could be repurposed for the treatment of AD-related dementia.