RESUMO
Alzheimer's disease (AD) is an age-dependent incurable neurodegenerative disorder accompanied by neuroinflammation, amyloid accumulation, and memory impairment. It begins decades before the first clinical symptoms appear, and identifying early biomarkers is key for developing disease-modifying therapies. We show now in a mouse model of AD that before any amyloid deposition the brains of 1.5-month-old mice contain increased levels of pro-inflammatory cytokines IL-1ß and IL-6, decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain and brain mitochondria and increased amounts of α7 nAChR-bound Aß1-42, along with impaired episodic memory and increased risk of apoptosis. Both acute (1-week-long) and chronic (4-month-long) treatments with α7-selective agonist PNU282987, starting at 1.5 months of age, were well tolerated. The acute treatment did not affect the levels of soluble Aß1-42 but consistently upregulated the α7 nAChR expression, decreased the level of α7-Aß1-42 complexes, and improved episodic memory of 1.5-month-old mice. The chronic treatment, covering the disease development phase, strongly upregulated the expression of all abundant brain nAChRs, reduced both free and α7-coupled Aß1-42 within the brain, had anti-inflammatory and antiapoptotic effects, and potently upregulated cognition, thus identifying α7 nAChRs as both early biomarker and potent therapeutic target for fighting this devastating disease.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Encéfalo , Modelos Animais de Doenças , Receptor Nicotínico de Acetilcolina alfa7 , Animais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Camundongos , Fragmentos de Peptídeos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compostos Bicíclicos com Pontes/farmacologia , Benzamidas/farmacologia , Apoptose/efeitos dos fármacosRESUMO
Introduction: Invasion of the central nervous system (CNS) is the most serious consequence of Trypanosoma brucei infection, which causes sleeping sickness. Recent experimental data have revealed some more insights into the disease during the meningoencephalitic stage. However, detailed cellular processes befalling the CNS during the disease are poorly understood. Methods: To further address this issue, we implanted a cranial window on the cortex of B6.129P2(Cg)-Cx3cr1tm1Litt/J mice, infected them with Trypanosoma brucei expressing RFP via intraperitoneal injection, and monitored microglial cells and parasites longitudinally over 30 days using in vivo 2-photon imaging. We correlated the observed changes with histological analyses to evaluate the recruitment of peripheral immune cells. Results and discussion: We uncovered an early involvement of microglia that precedes invasion of the CNS by the parasite. We accomplished a detailed characterization of the progressive sequence of events that correlates with microglial morphological changes and microgliosis. Our findings unveiled a heterogeneous microglial response in places of initial homeostatic disruption near brain barriers and pointed out an exceptional capability of microglia to hamper parasite proliferation inside the brain. We also found early signs of inflammation in the meninges, which synchronize with the microglial response. Moreover, we observed a massive infiltration of peripheral immune cells into the parenchyma as a signature in the final disease stage. Overall, our study provides new insights into the host-pathogen immune interactions in the meningeal and parenchymal compartments of the neocortex.
Assuntos
Trypanosoma brucei brucei , Tripanossomíase Africana , Camundongos , Animais , Microglia/patologia , Encéfalo , Sistema Nervoso Central/patologiaRESUMO
The rodent olfactory bulb (OB) is continuously supplied with adult-born cells maturing into GABAergic neurons. Using in vivo ratiometric Ca2+ imaging to readout ongoing and sensory-driven activity, we asked whether mature adult-born cells (mABCs) in the glomerular layer of the bulb become functionally identical to resident GABAergic (ResGABA) neurons. In awake head-restrained mice the two cell populations differed significantly in terms of ongoing spontaneous activity, with 24% of mABCs contributing to a strongly active cell cluster, absent among ResGABA cells. Odor-evoked responses of mABCs were sparse, less reliable, and had smaller amplitudes compared with ResGABA cells. The opposite was seen under anesthesia, with response reliability increasing and response size of mABCs becoming larger than that of ResGABA cells. Furthermore, ongoing activity of mABCs showed increased sensitivity to ketamine/xylazine and was selectively blocked by the antagonist of serotonin receptors methysergide. These functional features of mABCs clearly distinguish them from other OB interneurons.
Assuntos
Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Bulbo Olfatório/metabolismo , Animais , Camundongos , OdorantesRESUMO
Peripheral inflammation is known to trigger a mirror inflammatory response in the brain, involving brain's innate immune cells - microglia. However, the functional phenotypes, which these cells adopt in the course of peripheral inflammation, remain obscure. In vivo two-photon imaging of microglial Ca2+ signaling as well as process motility reveals two distinct functional states of cortical microglia during a lipopolysaccharide-induced peripheral inflammation: an early "sensor state" characterized by dramatically increased intracellular Ca2+ signaling but ramified morphology and a later "effector state" characterized by slow normalization of intracellular Ca2+ signaling but hypertrophic morphology, substantial IL-1ß production in a subset of cells as well as increased velocity of directed process extension and loss of coordination between individual processes. Thus, lipopolysaccharide-induced microglial Ca2+ signaling might represent the central element connecting receptive and executive functions of microglia.
Assuntos
Inflamação , Microglia , Encéfalo , Humanos , Lipopolissacarídeos , Transdução de SinaisRESUMO
Microglia, resident immune cells of the brain, react to the presence of pathogens/danger signals with a large repertoire of functional responses including morphological changes, proliferation, chemotaxis, production/release of cytokines, and phagocytosis. In vitro studies suggest that many of these effector functions are Ca2+-dependent, but our knowledge about in vivo Ca2+ signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca2+ indicators. Here, we introduce a novel approach, utilizing a microglia-specific microRNA-9-regulated viral vector, enabling the expression of a genetically-encoded ratiometric Ca2+ sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca2+ signals and allows for the first time to monitor the steady state intracellular Ca2+ levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca2+ levels. However, the levels increase significantly after acute slice preparation and cell culturing along with an increase in the expression of activation markers CD68 and IL-1ß. These data identify the steady state intracellular Ca2+ level as a versatile microglial activation marker, which is highly sensitive to the cell's environment.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Microglia/metabolismo , Imagem Molecular , Animais , Linhagem Celular , Células Cultivadas , Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos , Humanos , Camundongos , Imagem Molecular/métodosRESUMO
The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Doenças Priônicas/prevenção & controle , Príons/imunologia , Príons/isolamento & purificação , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Período de Incubação de Doenças Infecciosas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Priônicas , Ligação Proteica , Fatores de TempoRESUMO
Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrP(C), efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrP(C) selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrP(Sc) and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.