RESUMO
Immunotherapies designed to treat neurodegenerative tauopathies that primarily engage extracellular tau may have limited efficacy as tau is primarily intracellular. We generated tau-targeting single-chain variable fragments (scFvs) and intrabodies (iBs) from the phosphorylated tau-specific antibodies CP13 and PHF1 and the pan-tau antibody Tau5. Recombinant adeno-associated virus (rAAV) was utilized to express these antibody fragments in homozygous JNPL3 P301L tau mice. Two iBs (CP13i, PHF1i) and one scFv (PHF1s) abrogated tau pathology and delayed time to severe hindlimb paralysis. In a second tauopathy model (rTg4510), CP13i and PHF1i reduced tau pathology, but cognate scFvs did not. These data demonstrate that (1) disease-modifying efficacy does not require antibody effector functions, (2) the intracellular targeting of tau with phosphorylated tau-specific iBs is more effective than extracellular targeting with the scFvs, and (3) robust effects on tau pathology before neurodegeneration only resulted in modest disease modification as assessed by delay of severe motor phenotype.
Assuntos
Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapia , Via Secretória/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Proteínas tau/antagonistas & inibidores , Animais , Terapia Combinada , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Camundongos , Camundongos Transgênicos , Doenças Neurodegenerativas/etiologia , Resultado do Tratamento , Proteínas tau/metabolismoRESUMO
The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-ß (Aß) production. In cells, CRF treatment increases Aß production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by ß-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aß. These data collectively link CRF to increased Aß through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aß and in some cases preferentially increase Aß42 via complex effects on γ-secretase.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Hormônio Liberador da Corticotropina/metabolismo , Modelos Biológicos , Estresse Fisiológico/fisiologia , Doença de Alzheimer/etiologia , Análise de Variância , Animais , Western Blotting , AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Sistema Hipotálamo-Hipofisário/fisiologia , Imunoprecipitação , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Sistema Hipófise-Suprarrenal/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Hormônio Liberador da Corticotropina/metabolismoRESUMO
Brain expression of AAV-Ifn-γ leads to reactive gliosis, nigrostriatal degeneration and midbrain calcification in wild type mice. This mouse model phenocopies idiopathic basal ganglia calcification which is associated with Parkinsonian symptoms. To understand how the nigro-striatal pathway is selectively vulnerable to Ifn-γ, we determined if the phenotype is driven by canonical signaling intermediates, Ifngr1 and Stat1. Using focused bioinformatic analysis and rotarod testing, we show that neuroinflammation and motor abnormalities precede the appearance of midbrain neuropathologies in the brains of Ifn-γ mouse model. To test whether canonical Ifn-γ signaling is a key driver of progressive nigrostriatal degeneration, we overexpressed Ifn-γ in the brains of Ifngr1-/- and Stat1-/- mice. Expression of Ifn-γ in Ifngr1-/- mice did not result in any neuroinflammation, midbrain calcinosis or nigrostriatal degenerative pathology. Interestingly, in Stat1-/- mice, Ifn-γ expression resulted in gliosis without recapitulating the neurodegenerative phenotype. Overall, our data shows that canonical Ifn-γ signaling triggers midbrain calcinosis and nigrostriatal neurodegeneration, providing mechanistic insights into cytokine-driven selective neuronal vulnerability. Our study establishes the broader relevance of inflammatory signaling in neurodegenerative diseases and can potentially identify novel immunological targets for Parkinsonian syndromes.
Assuntos
Encéfalo/patologia , Interferon gama/metabolismo , Degeneração Neural/metabolismo , Receptores de Interferon/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Doenças dos Gânglios da Base/metabolismo , Doenças dos Gânglios da Base/patologia , Encéfalo/metabolismo , Calcinose/metabolismo , Calcinose/patologia , Camundongos , Camundongos Knockout , Degeneração Neural/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Transdução de Sinais/fisiologia , Receptor de Interferon gamaRESUMO
Mechanisms underlying α-synuclein (αSyn) mediated neurodegeneration are poorly understood. Intramuscular (IM) injection of αSyn fibrils in human A53T transgenic M83+/- mice produce a rapid model of α-synucleinopathy with highly predictable onset of motor impairment. Using varying doses of αSyn seeds, we show that αSyn-induced phenotype is largely dose-independent. We utilized the synchrony of this IM model to explore the temporal sequence of αSyn pathology, neurodegeneration and neuroinflammation. Longitudinal tracking showed that while motor neuron death and αSyn pathology occur within 2â¯months post IM, astrogliosis appears at a later timepoint, implying neuroinflammation is a consequence, rather than a trigger, in this prionoid model of synucleinopathy. Initiating at 3â¯months post IM, immune activation dominates the pathologic landscape in terminal IM-seeded M83+/- mice, as revealed by unbiased transcriptomic analyses. Our findings provide insights into the role of neuroinflammation in αSyn mediated proteostasis and neurodegeneration, which will be key in designing potential therapies.
Assuntos
Neurônios Motores/metabolismo , Degeneração Neural/metabolismo , alfa-Sinucleína/biossíntese , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/imunologia , Neurônios Motores/patologia , Degeneração Neural/imunologia , Degeneração Neural/patologia , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , alfa-Sinucleína/imunologiaRESUMO
Inflammatory activation precedes and correlates with accumulating τ lesions in Alzheimer's disease and tauopathies. However, the relationship between neuroinflammation and etiology of pathologic τ remains elusive. To evaluate whether inflammatory signaling may promote or accelerate neurofibrillary tangle pathology, we explored the effect of recombinant adeno-associated virus (rAAV)-mediated overexpression of a master inflammatory cytokine, IFN-γ, on τ phosphorylation. In initial studies in primary neuroglial cultures, rAAV-mediated expression of IFN-γ did not alter endogenous τ production or paired helical filament τ phosphorylation. Next, we tested the effect of rAAV-mediated expression of IFN-γ in the brains of 2 mouse models of tauopathy: JNPL3 and rTg4510. In both models, IFN-γ increased 1) signal transducer and activator of transcription 1 levels and gliosis, and 2) hyperphosphorylation and conformational alterations of soluble τ compared with control cohorts. However, sarkosyl-insoluble phosphorylated τ levels and ubiquitin staining were unaltered in the IFN-γ cohorts. Notably, IFN-γ-induced τ hyperphosphorylation was associated with release of the inhibitory effect of glycogen synthase kinase 3ß function by decreasing Ser9 phosphorylation. Our data suggest that type II IFN signaling can promote τ phosphorylation by modulating cellular kinase activity, though this is insufficient in accelerating neuritic tangle pathology.
Assuntos
Interferon gama/metabolismo , Emaranhados Neurofibrilares/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Dependovirus/genética , Ativação Enzimática , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Immunoblotting , Imuno-Histoquímica , Interferon gama/genética , Masculino , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Emaranhados Neurofibrilares/genética , Neuroglia/metabolismo , Fosforilação , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genética , Tauopatias/genéticaRESUMO
The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Encéfalo/metabolismo , Dependovirus/genética , Terapia Genética/métodos , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Capsídeo/metabolismo , Cisterna Magna/metabolismo , Cisterna Magna/patologia , Dependovirus/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Injeções Espinhais , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transdução GenéticaRESUMO
In order to further evaluate the parameters whereby intracerebral administration of recombinant α-synuclein (αS) induces pathological phenotypes in mice, we conducted a series of studies where αS fibrils were injected into the brains of M83 (A53T) and M47 (E46K) αS transgenic (Tg) mice, and non-transgenic (nTg) mice. Using multiple markers to assess αS inclusion formation, we find that injected fibrillar human αS induced widespread cerebral αS inclusion formation in the M83 Tg mice, but in both nTg and M47 Tg mice, induced αS inclusion pathology is largely restricted to the site of injection. Furthermore, mouse αS fibrils injected into nTg mice brains also resulted in inclusion pathology restricted to the site of injection with no evidence for spread. We find no compelling evidence for extensive spread of αS pathology within white matter tracts, and we attribute previous reports of white matter tract spreading to cross-reactivity of the αS pSer129/81A antibody with phosphorylated neurofilament subunit L. These studies suggest that, with the exception of the M83 Tg mice which appear to be uniquely susceptible to induction of inclusion pathology by exogenous forms of αS, there are significant barriers in mice to widespread induction of αS pathology following intracerebral administration of amyloidogenic αS.
Assuntos
Amiloidose/metabolismo , Amiloidose/patologia , Encéfalo/metabolismo , Encéfalo/patologia , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Escherichia coli , Técnicas de Transferência de Genes , Humanos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Vias Neurais/metabolismo , Vias Neurais/patologia , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , alfa-Sinucleína/genéticaRESUMO
The neonatal intraventricular injection of adeno-associated virus has been shown to transduce neurons widely throughout the brain, but its full potential for experimental neuroscience has not been adequately explored. We report a detailed analysis of the method's versatility with an emphasis on experimental applications where tools for genetic manipulation are currently lacking. Viral injection into the neonatal mouse brain is fast, easy, and accesses regions of the brain including the cerebellum and brainstem that have been difficult to target with other techniques such as electroporation. We show that viral transduction produces an inherently mosaic expression pattern that can be exploited by varying the titer to transduce isolated neurons or densely-packed populations. We demonstrate that the expression of virally-encoded proteins is active much sooner than previously believed, allowing genetic perturbation during critical periods of neuronal plasticity, but is also long-lasting and stable, allowing chronic studies of aging. We harness these features to visualise and manipulate neurons in the hindbrain that have been recalcitrant to approaches commonly applied in the cortex. We show that viral labeling aids the analysis of postnatal dendritic maturation in cerebellar Purkinje neurons by allowing individual cells to be readily distinguished, and then demonstrate that the same sparse labeling allows live in vivo imaging of mature Purkinje neurons at a resolution sufficient for complete analytical reconstruction. Given the rising availability of viral constructs, packaging services, and genetically modified animals, these techniques should facilitate a wide range of experiments into brain development, function, and degeneration.
Assuntos
Encéfalo , Dependovirus/genética , Mosaicismo , Transdução Genética/métodos , Animais , Animais Recém-Nascidos , Vetores Genéticos/genética , CamundongosRESUMO
OBJECTIVE: The 5-lipoxygenase (5LO) enzyme is upregulated in Alzheimer disease (AD), and its genetic absence reduces Aß levels in APP mice. However, its functional role in modulating tau neuropathology remains to be elucidated. METHODS: To this end, we generated triple transgenic mice (3xTg-AD) overexpressing neuronal 5LO and investigated their phenotype. RESULTS: Compared with controls, 3xTg-AD mice overexpressing 5LO manifested an exacerbation of memory deficits, plaques, and tangle pathologies. The elevation in Aß was secondary to an upregulation of γ-secretase pathway, whereas tau hyperphosphorylation resulted from an activation of the Cdk5 kinase. In vitro study confirmed the involvement of this kinase in the 5LO-dependent tau phosphorylation, which was independent of the effect on Aß. INTERPRETATION: Our findings highlight the novel functional role that neuronal 5LO plays in exacerbating AD-related tau pathologies. They provide critical preclinical evidence to justify testing selective 5LO inhibitors for AD treatment.
Assuntos
Doença de Alzheimer , Araquidonato 5-Lipoxigenase/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Transtornos da Memória , Proteínas tau/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM10 , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Quinase 5 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/genética , Humanos , Aprendizagem em Labirinto , Proteínas de Membrana/metabolismo , Transtornos da Memória/etiologia , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos , Camundongos Transgênicos , Fatores de Transcrição NFI/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilação/genética , Presenilina-1/genética , Radioimunoensaio , Distribuição Aleatória , Transdução de Sinais/genética , TransfecçãoRESUMO
Reactive gliosis surrounding amyloid beta (Abeta) plaques is an early feature of Alzheimer's disease pathogenesis and has been postulated to represent activation of the innate immune system in an apparently ineffective attempt to clear or neutralize Abeta aggregates. To evaluate the role of IFN-gamma-mediated neuroinflammation on the evolution of Abeta pathology in transgenic (Tg) mice, we have expressed murine IFN-gamma (mIFN-gamma) in the brains of Abeta precursor protein (APP) Tg mice using recombinant adeno-associated virus serotype 1. Expression of mIFN-gamma in brains of APP TgCRND8 mice results in robust noncell autonomous activation of microglia and astrocytes, and a concomitant significant suppression of Abeta deposition. In these mice, mIFN-gamma expression upregulated multiple glial activation markers, early components of the complement cascade as well as led to infiltration of Ly-6c positive peripheral monocytes but no significant effects on APP levels, APP processing or steady-state Abeta levels were noticed in vivo. Taken together, these results suggest that mIFN-gamma expression in the brain suppresses Abeta accumulation through synergistic effects of activated glia and components of the innate immune system that enhance Abeta aggregate phagocytosis.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas do Sistema Complemento/biossíntese , Interferon gama/fisiologia , Placa Amiloide/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Linhagem Celular , Galinhas , Proteínas do Sistema Complemento/genética , Dependovirus/genética , Dependovirus/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Feminino , Humanos , Imunidade Inata/genética , Interferon gama/biossíntese , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Fagocitose/genética , Fagocitose/imunologia , Placa Amiloide/genética , Placa Amiloide/patologia , Processamento de Proteína Pós-Traducional/genética , Regulação para Cima/imunologiaRESUMO
Proinflammatory stimuli, after amyloid beta (Abeta) deposition, have been hypothesized to create a self-reinforcing positive feedback loop that increases amyloidogenic processing of the Abeta precursor protein (APP), promoting further Abeta accumulation and neuroinflammation in Alzheimer's disease (AD). Interleukin-6 (IL-6), a proinflammatory cytokine, has been shown to be increased in AD patients implying a pathological interaction. To assess the effects of IL-6 on Abeta deposition and APP processing in vivo, we overexpressed murine IL-6 (mIL-6) in the brains of APP transgenic TgCRND8 and TG2576 mice. mIL-6 expression resulted in extensive gliosis and concurrently attenuated Abeta deposition in TgCRND8 mouse brains. This was accompanied by up-regulation of glial phagocytic markers in vivo and resulted in enhanced microglia-mediated phagocytosis of Abeta aggregates in vitro. Further, mIL-6-induced neuroinflammation had no effect on APP processing in TgCRND8 and had no effect on APP processing or steady-state levels of Abeta in young Tg2576 mice. These results indicate that mIL-6-mediated reactive gliosis may be beneficial early in the disease process by potentially enhancing Abeta plaque clearance rather than mediating a neurotoxic feedback loop that exacerbates amyloid pathology. This is the first study that methodically dissects the contribution of mIL-6 with regard to its potential role in modulating Abeta deposition in vivo.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Gliose/patologia , Interleucina-6/fisiologia , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Gliose/induzido quimicamente , Humanos , Inflamação/fisiopatologia , Camundongos , Camundongos Transgênicos , FagocitoseRESUMO
Parkinson's disease (PD) and related synucleinopathies are characterized by chronic neuroinflammation leading to the premise that anti-inflammatory therapies could ameliorate synucleinopathy and associated sequelae. To test this idea, we used recombinant adeno-associated viruses (AAV) to express the anti-inflammatory cytokine, Interleukin (Il)-10, in Line M83 transgenic mice that expresses the PD-associated A53T mutant human α-synuclein (αSyn). Contrary to our expectations, we observed that intraspinal Il-10 expression initiated at birth upregulated microgliosis and led to early death in homozygous M83+/+ mice. We further observed that Il-10 preconditioning led to reduced lifespan in the hemizygous M83+/- mice injected with preformed αSyn aggregates in hindlimb muscles. To determine the mechanistic basis for these adverse effects, we took advantage of the I87A variant Il-10 (vIl-10) that has predominantly immunosuppressive properties. Sustained intraspinal expression of vIl-10 in preformed αSyn-aggregate seeded M83+/- mice resulted in earlier death, accelerated αSyn pathology, pronounced microgliosis, and increased apoptosis compared to control mice. AAV-vIl-10 expression robustly induced p62 and neuronal LC3B accumulation in these mice, indicating that Il-10 signaling mediated preconditioning of the neuraxis can potentially exacerbate αSyn accumulation through autophagy dysfunction in the neurons. Together, our data demonstrate unexpected adverse effects of both Il-10 and its immunosuppressive variant, vIl-10, in a mouse model of PD, highlighting the pleiotropic functions of immune mediators and their complex role in non-cell autonomous signaling in neurodegenerative proteinopathies.
RESUMO
Imaging biomarkers for immune activation may be valuable for early-stage detection, therapeutic testing, and research on neurodegenerative conditions. In the present study, we determined whether diffusion magnetic resonance imaging-derived free water signal is a sensitive marker for neuroinflammatory effects of interferon-gamma (Ifn-γ). Neonatal wild-type mice were injected in the cerebral ventricles with recombinant adeno-associated viruses expressing the inflammatory cytokine Ifn-γ. Groups of mice expressing Ifn-γ and age-matched controls were imaged at 1, 5 and 8 months. Mice deficient in Ifngr1-/- and Stat1-/- were scanned at 5 months as controls for the signaling cascades activated by Ifn-γ. The results indicate that Ifn-γ affected fractional anisotropy (FA), mean diffusivity (MD), and free water (FW) in white matter structures, midline cortical areas, and medial thalamic areas. In these structures, FA and MD decreased progressively from 1 to 8 months of age, while FW increased significantly. The observed reductions in FA and MD and increased FW with elevated brain Ifn-γ was not observed in Ifngr1-/- or Stat1-/- mice. These results suggest that the observed microstructure changes involve the Ifn-gr1 and Stat1 signaling. Interestingly, increases in FW were observed in midbrain of Ifngr1-/- mice, which suggests alternative Ifn-γ signaling in midbrain. Although initial evidence is offered in relation to the sensitivity of the FW signal to neurodegenerative and/or inflammatory patterns specific to Ifn-γ, further research is needed to determine applicability and specificity across animal models of neuroinflammatory and degenerative disorders.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Encefalite/diagnóstico por imagem , Encefalite/patologia , Interferon gama/metabolismo , Substância Branca/diagnóstico por imagem , Substância Branca/patologia , Animais , Anisotropia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encefalite/induzido quimicamente , Feminino , Interferon gama/administração & dosagem , Interferon gama/genética , Masculino , Camundongos Knockout , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Água/análise , Substância Branca/efeitos dos fármacos , Substância Branca/metabolismoRESUMO
BACKGROUND: Self-assembly of the amyloid-ß (Aß) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer's disease (AD). However, it is clear that not all forms of Aß are equally harmful and that linking a specific aggregate to toxicity also depends on the assays and model systems used (Haass et al., J Biol. Chem 269:17741-17748, 1994; Borchelt et al., Neuron 17:1005-1013, 1996). Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aß deposition and neurodegeneration. METHODS: In this study, we examined familial mutations of Aß that increase aggregation and oligomerization, E22G and ΔE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aß fusion technology and rAAV2/1-based somatic brain transgenesis in mice to selectively express individual mutant Aß species in vivo. In parallel, we generated PhiC31-based transgenic Drosophila melanogaster expressing wild-type (WT) and Aß40 and Aß42 mutants, fused to the Argos signal peptide to assess the extent of Aß42-induced toxicity as well as to interrogate the combined effect of different Aß40 and Aß42 species. RESULTS: When expressed in the mouse brain for 6 months, Aß42 E22G, Aß42 E22Q/D23N, and Aß42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aß40WT showed no distinctive phenotype, Aß40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aß40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aß42 E22G, E22Q, and S26C, but not Aß40, were toxic to the eye of Drosophila. In contrast, flies expressing a copy of Aß40 (WT or mutants), in addition to Aß42WT, showed improved phenotypes, suggesting possible protective qualities for Aß40. CONCLUSIONS: These studies suggest that while some Aß40 mutants form unique amyloid aggregates in mouse brains, they do not exacerbate Aß42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.
Assuntos
Doença de Alzheimer , Drosophila , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Encéfalo/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Camundongos , Fragmentos de Peptídeos/toxicidadeRESUMO
BACKGROUND: Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. METHODS: We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. RESULTS: There was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. CONCLUSIONS: Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.
Assuntos
Dependovirus , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Transdução Genética/métodos , Animais , Encéfalo , Terapia Genética/métodos , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Camundongos , Neuroglia , NeurôniosRESUMO
It has been challenging to produce ex vivo models of the inclusion pathologies that are hallmark pathologies of many neurodegenerative diseases. Using three-dimensional mouse brain slice cultures (BSCs), we have developed a paradigm that rapidly and robustly recapitulates mature neurofibrillary inclusion and Lewy body formation found in Alzheimer's and Parkinson's disease, respectively. This was achieved by transducing the BSCs with recombinant adeno-associated viruses (rAAVs) that express α-synuclein or variants of tau. Notably, the tauopathy BSC model enables screening of small molecule therapeutics and tracking of neurodegeneration. More generally, the rAAV BSC "toolkit" enables efficient transduction and transgene expression from neurons, microglia, astrocytes, and oligodendrocytes, alone or in combination, with transgene expression lasting for many months. These rAAV-based BSC models provide a cost-effective and facile alternative to in vivo studies, and in the future can become a widely adopted methodology to explore physiological and pathological mechanisms related to brain function and dysfunction.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Dependovirus/genética , Doença de Parkinson/patologia , Doença de Alzheimer/virologia , Animais , Encéfalo/metabolismo , Encéfalo/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Humanos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Microrganismos Geneticamente Modificados , Mutação , Neurônios/patologia , Técnicas de Cultura de Órgãos , Doença de Parkinson/virologia , Transdução Genética , Transgenes , alfa-Sinucleína/genética , Proteínas tau/genéticaRESUMO
Processing of amyloid-ß (Aß) precursor protein (APP) by γ-secretase produces multiple species of Aß: Aß40, short Aß peptides (Aß37-39), and longer Aß peptides (Aß42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aß42 but increase the relative abundance of short Aß peptides. To evaluate the pathological relevance of these peptides, we expressed Aß36-40 and Aß42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aß42 toxicity. In contrast to Aß42, the short Aß peptides were not toxic and, when coexpressed with Aß42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aß38 and Aß40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aß42 deposition, Aß38 and Aß40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aß42 by raising the levels of short Aß peptides could attenuate the toxic effects of Aß42.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Amiloide/genética , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Drosophila melanogaster , Olho/metabolismo , Olho/patologia , Olho/ultraestrutura , Feminino , Locomoção , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fenótipo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismoRESUMO
There is considerable interest in harnessing innate immunity to treat Alzheimer's disease (AD). Here, we explore whether a decoy receptor strategy using the ectodomain of select TLRs has therapeutic potential in AD. AAV-mediated expression of human TLR5 ectodomain (sTLR5) alone or fused to human IgG4 Fc (sTLR5Fc) results in robust attenuation of amyloid ß (Aß) accumulation in a mouse model of Alzheimer-type Aß pathology. sTLR5Fc binds to oligomeric and fibrillar Aß with high affinity, forms complexes with Aß, and blocks Aß toxicity. Oligomeric and fibrillar Aß modulates flagellin-mediated activation of human TLR5 but does not, by itself, activate TLR5 signaling. Genetic analysis shows that rare protein coding variants in human TLR5 may be associated with a reduced risk of AD. Further, transcriptome analysis shows altered TLR gene expression in human AD. Collectively, our data suggest that TLR5 decoy receptor-based biologics represent a novel and safe Aß-selective class of biotherapy in AD.
Assuntos
Doença de Alzheimer , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Receptor 5 Toll-Like/uso terapêutico , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/imunologia , Animais , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/imunologiaRESUMO
Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1-9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter-driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies.
RESUMO
The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid ß 1-25 (Aß1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.