RESUMEN
African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies. IMPORTANCE: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Sistemas CRISPR-Cas , Edición Génica , Replicación Viral , Animales , Virus de la Fiebre Porcina Africana/genética , Porcinos , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/prevención & control , Edición Génica/métodos , Replicación Viral/genética , Animales Modificados Genéticamente/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Genoma Viral/genéticaRESUMEN
Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain-enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress-specific kinase for eIF2α, leading to stress-induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase-1 cleavage and Il18 expression corroborate microglia-associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.
Asunto(s)
Factor 2 Eucariótico de Iniciación , Inflamasomas , Ratones , Animales , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Inflamasomas/metabolismo , Gránulos de Estrés , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fosforilación , Ratones Noqueados , Gránulos Citoplasmáticos/metabolismoRESUMEN
Synapse loss and memory decline are the primary features of neurodegenerative dementia. However, the molecular underpinnings that drive memory loss remain largely unknown. Here, we report that FAM69C is a kinase critically involved in neurodegenerative dementia. Biochemical analyses uncover that FAM69C is a serine/threonine kinase. We generate the Fam69c knockout mice and show by single-cell RNA sequencing that FAM69C deficiency drives cell-type-specific transcriptional changes relevant to synapse dysfunction. Electrophysiological, morphological, and behavioral experiments demonstrate impairments in synaptic plasticity, dendritic spine density, and memory in Fam69c knockout mice, as well as stress-induced neuronal death. Phosphoproteomic characterizations reveal that FAM69C substrates are involved in synaptic structure and function. Finally, reduced levels of FAM69C are found in postmortem brains of Alzheimer's disease patients. Our study demonstrates that FAM69C is a protective regulator of memory and suggests FAM69C as a potential therapeutic target for memory loss in neurodegenerative dementia.
Asunto(s)
Enfermedad de Alzheimer , Sinapsis , Enfermedad de Alzheimer/genética , Animales , Trastornos de la Memoria/genética , Ratones , Ratones Noqueados , Plasticidad Neuronal/fisiologíaRESUMEN
PTEN (phosphatase and tensin homology deleted on chromosome 10) has multiple functions, and recent studies have shown that the PTEN family has isoforms. The roles of these PTEN family members in biologic activities warrant specific evaluation. Here, we show that PTENα maintains CaMKII in a state that is competent to induce long-term potentiation (LTP) with resultant regulation of contextual fear memory and spatial learning. PTENα binds to CaMKII with its distinctive N terminus and resets CaMKII to an activatable state by dephosphorylating it at sites T305/306. Loss of PTENα impedes the interaction of CaMKII and NR2B, leading to defects in hippocampal LTP, fear-conditioned memory, and spatial learning. Restoration of PTENα in the hippocampus of PTENα-deficient mice rescues learning deficits through regulation of CaMKII. CaMKII mutations in dementia patients inhibit CaMKII activity and result in disruption of PTENα-CaMKII-NR2B signaling. We propose that CaMKII is a target of PTENα phosphatase and that PTENα is an essential element in the molecular regulation of neural activity.