Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

As neurons age, they show a decrease in their ability to degrade proteins and membranes. Because undegraded material is a source of toxic products, defects in degradation are associated with reduced cell function and survival. However, there are very few dead neurons in the aging brain, suggesting the action of compensatory mechanisms. We show in this work that ageing neurons in culture show large multivesicular bodies (MVBs) filled with intralumenal vesicles (ILVs) and secrete more small extracellular vesicles than younger neurons. We also show that the high number of ILVs is the consequence of the accumulation of cholesterol in MVBs, which in turn is due to decreased levels of the cholesterol extruding protein NPC1. NPC1 down-regulation is the consequence of a combination of upregulation of the NPC1 repressor microRNA 33, and increased degradation, due to AktmTOR targeting of NPC1 to the phagosome. Although releasing more exosomes can be beneficial to old neurons, other cells, neighbouring and distant, can be negatively affected by the waste material they contain.Fil: Guix, Francesc X.. Universidad Autónoma de Madrid; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Marrero Capitán, Ana. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Casadomé Perales, Álvaro. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Palomares Pérez, Irene. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Lopez del Castillo, Irene. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Miguel, Verónica. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Goedeke, Leigh. University of Yale; Estados UnidosFil: Martín, Mauricio Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Lamas, Santiago. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Peinado, Héctor. Centro Nacional de Investigaciones Oncológicas; EspañaFil: Fernández Hernando, Carlos. University of Yale; Estados UnidosFil: Dotti, Carlos. Universidad Autónoma de Madrid; España. Consejo Superior de Investigaciones Científicas; Españ

Aging increases hippocampal DUSP2 by a membrane cholesterol loss-mediated RTK/p38MAPK activation mechanism

Numerous studies suggest that the increased activity of p38MAPK plays an important role in the abnormal immune and inflammatory response observed in the course of neurodegenerative diseases such as Alzheimer's disease. On the other hand, High levels of p38MAPK are present in the brain during normal aging, suggesting the existence of mechanisms that keep the p38MAPK-regulated pro-inflammatory activity within physiological limits. In this study, we show that high p38MAPK activity in the hippocampus of old mice is in part due to the reduction in membrane cholesterol that constitutively occurs in the aging brain. Mechanistically, membrane cholesterol reduction increases p38MAPK activity through the stimulation of a subset of tyrosine kinase receptors (RTKs). In turn, activated p38MAPK increases the expression and activity of the phosphatase DUSP2, which is known to reduce the activity of different MAPKs, including p38MAPK. These results suggest that the loss of membrane cholesterol that constitutively occurs with age takes part in a negative-feedback loop that keeps p38MAPK activity levels within physiological range. Thus, conditions that increase p38MAPK activity such as cellular stressors or that inhibit DUSP2 will amplify inflammatory activity with its consequent deleterious functional changes.Ministry of Economy and Competitiveness grants Consolider CSD2010-00064 and SAF2013-45392 and SAF201676722 to CD. AN was supported by the Consolider grant CSD2010-0045. PF is the recipient of a Ramon y Cajal Award RYC-2014-16410 from the Spanish Ministry of Economy and Competitiveness

Extracellular Vesicles Derived from Young Neural Cultures Attenuate Astrocytic Reactivity In Vitro

Extracellular vesicles (EVs) play an important role in intercellular communication and are involved in both physiological and pathological processes. In the central nervous system (CNS), EVs secreted from different brain cell types exert a sundry of functions, from modulation of astrocytic proliferation and microglial activation to neuronal protection and regeneration. However, the effect of aging on the biological functions of neural EVs is poorly understood. In this work, we studied the biological effects of small EVs (sEVs) isolated from neural cells maintained for 14 or 21 days in vitro (DIV). We found that EVs isolated from 14 DIV cultures reduced the extracellular levels of lactate dehydrogenase (LDH), the expression levels of the astrocytic protein GFAP, and the complexity of astrocyte architecture suggesting a role in lowering the reactivity of astrocytes, while EVs produced by 21 DIV cells did not show any of the above effects. These results in an in vitro model pave the way to evaluate whether similar results occur in vivo and through what mechanisms

Age-associated cholesterol reduction triggers brain insulin resistance by facilitating ligand-independent receptor activation and pathway desensitization

In the brain, insulin plays an important role in cognitive processes. During aging, these faculties decline, as does insulin signaling. The mechanism behind this last phenomenon is unclear. In recent studies, we reported that the mild and gradual loss of cholesterol in the synaptic fraction of hippocampal neurons during aging leads to a decrease in synaptic plasticity evoked by glutamate receptor activation and also by receptor tyrosine kinase (RTK) signaling. As insulin and insulin growth factor activity are dependent on tyrosine kinase receptors, we investigated whether the constitutive loss of brain cholesterol is also involved in the decay of insulin function with age. Using long-term depression (LTD) induced by application of insulin to hippocampal slices as a read-out, we found that the decline in insulin function during aging could be monitored as a progressive impairment of insulin-LTD. The application of a cholesterol inclusion complex, which donates cholesterol to the membrane and increases membrane cholesterol levels, rescued the insulin signaling deficit and insulin-LTD. In contrast, extraction of cholesterol from hippocampal neurons of adult mice produced the opposite effect. Furthermore, in vivo inhibition of Cyp46A1, an enzyme involved in brain cholesterol loss with age, improved insulin signaling. Fluorescence resonance energy transfer (FRET) experiments pointed to a change in receptor conformation by reduced membrane cholesterol, favoring ligand-independent autophosphorylation. Together, these results indicate that changes in membrane fluidity of brain cells during aging play a key role in the decay of synaptic plasticity and cognition that occurs at this late stage of life.Ministry of Science and Spanish Ministry of Economy and Competitivenes

Inhibition of p38 MAPK in the brain through nasal administration of p38 inhibitor loaded in chitosan nanocapsules

[Aim]: To determine whether a p38 MAPK inhibitor incorporated into nanoemulsion-based chitosan nanocapsules can reduce the activity of this kinase in the brain through their nasal administration in mice.[Materials & methods]: We selected the p38 MAPK inhibitor PH797804, an ATP-competitive inhibitor of p38α encapsulated in nanoemulsion-based chitosan nanocapsules. Biological effect was evaluated in microglial and neuronal cells in vitro and in ex vivo and in vivo systems, in a mouse model of Alzheimer’s disease.[Results]: Encapsulated inhibitor retains enzymatic inhibitory activity and tissue penetration capacity in vitro, ex vivo and in vivo.[Conclusion]: Nasal administration of chitosan nanocapsules can be an effective approach for brain-restricted reduction of p38 MAPK activity, thus reducing the side effects of systemic administration.Peer reviewe