RESUMEN
Excessive production and accumulation of amyloid-beta (Aß) in the brain are one of the hallmarks of Alzheimer's disease (AD). Although oxidative stress is known to trigger and promote the progression of AD, the molecular relationship between oxidative stress and Aß production is not yet fully understood. In this study, we demonstrate that microtubule acetylation induced by oxidative stress plays a critical role in Aß production and secretion by altering the subcellular distribution of Aß precursor protein (APP)-containing lysosomal vesicles. Under oxidative stress, both H4-APPSwe/Ind and HEK293T-APPSwe/Ind cell lines showed increased microtubule acetylation and Aß secretion. Knockdown (KD) of alpha-tubulin N-acetyltransferase 1 (ATAT1) by using a lentiviral shRNA not only inhibited the generation of intermediate APP fragments, such as ß-CTF and AICD, but also suppressed Aß secretion. Oxidative stress promoted the dispersion of LAMP1-positive vesicles to the periphery of the cell through microtubule acetylation, leading to the formation of neutralized lysosomal vesicles (NLVs), which was inhibited by ATAT1 KD. Treatment of the cells with the dynein ATPase inhibitor EHNA or downregulation of LIS1, a regulator of dynein-mediated intracellular transport, increased the peripheral localization of NLVs and promoted Aß secretion, whereas KD of ADP ribosylation factor like GTPase 8B showed the opposite result. ATAT1 KD in the hippocampal region of the 5×FAD AD mouse model also showed significant reductions in Aß plaque accumulation and memory loss. Taken together, these findings suggest that oxidative stress-induced microtubule acetylation promotes the peripheral localization of lysosomal vesicles to form NLVs, thereby enhancing Aß secretion.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Animales , Humanos , Ratones , Acetilación , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lisosomas/metabolismo , Microtúbulos/metabolismo , Estrés Oxidativo , Línea CelularRESUMEN
Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1ß (IL-1ß) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1ß secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X7 receptor (P2X7R), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2X7R, and blocked by cell pre-treatment with the P2X7R antagonist A438079. Interestingly, ATP-induced PNP release from glial cells partly occurred through the secretion of lysosomal vesicles in the extracellular medium. Thus, during inflammatory cerebral events PNP secretion promoted by extracellular ATP accumulation might concur to control extracellular purine signals. Further studies could elucidate whether, in these conditions, a consensual activity of enzymes downstream of PNP in the purine metabolic cascade avoids accumulation of extracellular purine bases that might concur to brain injury by unusual formation of reactive oxygen species.