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1.
Nat Nanotechnol ; 18(10): 1241-1251, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37430038

RESUMEN

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.


Asunto(s)
Encéfalo , Callithrix , Humanos , Animales , Recién Nacido , Chlorocebus aethiops , Macaca mulatta/genética , Callithrix/genética , Encéfalo/fisiología , Técnicas de Transferencia de Gen , Neuronas , Vectores Genéticos/genética
2.
Res Sq ; 2023 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-36789432

RESUMEN

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.

3.
Nat Metab ; 3(5): 618-635, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-34031590

RESUMEN

Macrophages generate mitochondrial reactive oxygen species and mitochondrial reactive electrophilic species as antimicrobials during Toll-like receptor (TLR)-dependent inflammatory responses. Whether mitochondrial stress caused by these molecules impacts macrophage function is unknown. Here, we demonstrate that both pharmacologically driven and lipopolysaccharide (LPS)-driven mitochondrial stress in macrophages triggers a stress response called mitohormesis. LPS-driven mitohormetic stress adaptations occur as macrophages transition from an LPS-responsive to LPS-tolerant state wherein stimulus-induced pro-inflammatory gene transcription is impaired, suggesting tolerance is a product of mitohormesis. Indeed, like LPS, hydroxyoestrogen-triggered mitohormesis suppresses mitochondrial oxidative metabolism and acetyl-CoA production needed for histone acetylation and pro-inflammatory gene transcription, and is sufficient to enforce an LPS-tolerant state. Thus, mitochondrial reactive oxygen species and mitochondrial reactive electrophilic species are TLR-dependent signalling molecules that trigger mitohormesis as a negative feedback mechanism to restrain inflammation via tolerance. Moreover, bypassing TLR signalling and pharmacologically triggering mitohormesis represents a new anti-inflammatory strategy that co-opts this stress response to impair epigenetic support of pro-inflammatory gene transcription by mitochondria.


Asunto(s)
Reprogramación Celular , Metabolismo Energético , Tolerancia Inmunológica , Macrófagos/inmunología , Macrófagos/metabolismo , Mitocondrias/metabolismo , Acetilcoenzima A/metabolismo , Antiinflamatorios/farmacología , Estrógenos/metabolismo , Regulación de la Expresión Génica , Lipopolisacáridos/inmunología , Activación de Macrófagos , Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico
4.
Mol Cell ; 81(10): 2094-2111.e9, 2021 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-33878293

RESUMEN

Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.


Asunto(s)
Autoinmunidad , Neoplasias/enzimología , Neoplasias/prevención & control , Quinasa Syk/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Animales , Antígenos CD19/metabolismo , Linfocitos B , Calcio/metabolismo , Diferenciación Celular , Transformación Celular Neoplásica , Activación Enzimática , Humanos , Tolerancia Inmunológica , Linfoma de Células B/enzimología , Linfoma de Células B/patología , Ratones , Modelos Genéticos , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal
6.
Nature ; 588(7838): 491-497, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33149299

RESUMEN

Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection1-3. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3-/- naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3-/- B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3-/- B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.


Asunto(s)
Linfocitos B/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Animales , Antígenos CD19/metabolismo , Linfocitos B/enzimología , Linfocitos B/inmunología , Linfocitos B/patología , Transformación Celular Neoplásica , Femenino , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/patología , Humanos , Integrinas/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Modelos Moleculares , Fosforilación , Receptores de Antígenos de Linfocitos B/metabolismo
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