cAMP and chaperones: Potential therapeutic strategies to prevent inflammation-linked Tau pathology in Alzheimer's disease.

摘要

Senile plaques and neurofibrillary tangles are hallmarks of Alzheimer's disease (AD). The main component of neurofibrillary tangles (NFTs) is Tau, a highly soluble microtubule-associated protein whose major function is to stabilize microtubules, specifically in axons, in a phosphorylation-dependent manner. Neurodegenerative diseases collectively designated "Tauopathies" are linked to Tau mutations and/or Tau post-translational modifications. Accordingly, Tau hyperphosphorylation and cleavage are important events leading to Tau intracellular accumulation, aggregation and neuronal cell death. Caspase-cleaved Tau is detected in NFTs supporting the view that the apoptosis cascade is involved in the formation of NFTs. It is thought that Tau cleavage at its C-terminus by caspases renders Tau prone to aggregation and formation of NFTs. At the sites of damage, AD brains also exhibit signs of chronic inflammation manifested by reactive astrocytes and microglia, which produce cytotoxic agents among them prostaglandins. There is a profound gap in our understanding of how cyclooxygenases and their prostaglandin products redirect cellular events to promote neurodegeneration.;In our studies we treated E18 cortical neurons with prostaglandin J2 (PGJ2), because it is potently neurotoxic. We show that PGJ2, a neurotoxic product of inflammation, induces the formation of the aggregate-prone form of Tau (Tau cleaved at Asp421, Delta-Tau) in a time- and dose-dependent manner. Furthermore, PGJ2 activates caspase 8 (extrinsic apoptotic pathway) and the effector caspase 3, thus inducing apoptosis in the cortical neuronal cultures.;In addition, we addressed the potential of increasing cAMP levels to prevent the toxic effects of neuroinflammation. Our studies focused on increasing cAMP because PGJ2 signals through a Gi protein-coupled receptor that reduces cAMP levels, promoting neuronal loss. Notably, increasing intracellular cAMP levels with dibutyryl-cAMP (db-cAMP) or PACAP27 prior to PGJ2 treatment decreased the levels of Delta-Tau and caspase activation, mitigating the loss of cell viability. These protective results of cAMP were only observed at early time points (4h and 8h) upon treatment with PGJ2, indicating that they are only effective when applied before the neurons reach a point of no return.;We confirmed that PGJ2 treatment for 24h inhibits the proteasome and induces the accumulation and aggregation of ubiquitinated proteins. Elevating cAMP moderately increased the activity of the 26S proteasome. Surprisingly, db-cAMP or PACAP27 pre-treatment failed to prevent the accumulation/aggregation of ubiquitinated proteins induced by PGJ2.;We also addressed the potential of targeting molecular chaperones, such as Hsp90 and Hsp105 to prevent the toxic effects of neuroinflammation. Our studies focused on Hsp105 because it is a newly characterized chaperone that is highly abundant in the brain, and it is active under low ATP conditions. Nevertheless its role in neurodegeneration has yet to be determined. Promoting Hsp105 overexpression by its transient transfection in human neuroblastoma SK-N-SH cells improved cell survival upon treatment with PGJ2 for 24h. Furthermore, we investigated the protective effect of EC102, a small molecule Hsp90 inhibitor, in PGJ2-induced cell toxicity. EC102 blocks the Hsp90 ATPase activity, inhibiting its foldase capacity, instead targeting substrates for degradation by the proteasome. Rat E18 primary cortical neurons showed an increase in cell viability when cells were pre-treated with EC102 prior to PGJ2 treatment. These beneficial effects of molecular chaperones support targeting them as a potential therapeutic target for AD.;Based on our studies, we propose a model in which any stimulus (physical, chemical or infectious) capable of inducing inflammation in a particular brain area activates microglia and astrocytes. The toxic products released by glia, including PGJ2, act on the neighboring neurons causing among other effects, intracellular protein misfolding. If these proteins fail to be cleared by the ubiquitin/proteasome pathway (UPP) or fail to be refolded by chaperones, apoptosis is triggered launching caspase-mediated proteolysis. Caspase activation is responsible for generating protein fragments, including truncated Tau, which serve as seeds for cytotoxic protein aggregation. This sequence of events could explain many pathological features of the AD neurodegenerative process.;In conclusion, these studies showed that products of inflammation affect proteasome activity and alter protein turnover, which leads to protein aggregation, and neuronal injury. All of these processes are relevant to the pathology in AD. Moreover, our data indicate that the accumulation/aggregation of ubiquitinated proteins is a very stable phenomenon, and that once formed, the cell has difficulty in removing these aggregates. Overall, our studies suggest a new potential therapeutic approach for AD that involves maintenance of intracellular levels of cAMP and, in a separate approach, enhancing the activity of heat shock proteins.;Elucidation of neurotoxic mechanisms linked to products of inflammation is highly significant, as it will offer new targets for anti-inflammatory drugs that more effectively prevent AD neurodegeneration linked to chronic inflammation and protein aggregation.