A major challenge in neurobiology is identification of the mechanisms by which protein misfolding leads to cellular toxicity. Many neurodegenerative disorders, in which aberrant protein conformers aggregate into pathological inclusions, also present the chronic activation of the PERK branch of the unfolded protein response. The adaptive effects of the PERK pathway include reduction of translation by transient inhibition of eIF2a and antioxidant protein production via induction of the transcription factor Nrf2. In contrast, PERK prolonged activation leads to sustained reduction in protein synthesis and induction of cell death pathways. To further investigate the role of the PERK pathway in neurodegenerative disorders, we focused on Down syndrome (DS), in which aging confers a high risk of Alzheimer-like dementia (AD). Preliminary data investigating human DS frontal cortices and blood derived cells from DS subject suggest an early and sustained activation of PERK associated with the induction of eIF2a and ATF4 downstream signals. We also observed that the Nrf2 response is uncoupled from PERK and its antioxidant effects is repressed in a mechanism implicating the transcription repressor Bach1 (enchoded on chromosome 21).
However, the molecular mechanisms involved are still undetermined. The overall objective of this project is to understand the processes that lead to PERK/NRF2 uncoupling in DS with the idea of identifying compounds able to rescue their physiological interaction and reduce brain damage. The central hypothesis is that trisomy 21 lead to chronic PERK over-activation contributing with BACH1 over-expression, to NRF2 depletion. The reduction of PERK chronic over-activation could allow to the rescue of PERK/NRF2 axis thus re-establishing proper proteostasis and reducing oxidative damage.
During the last decade the scientific community has devoted great attention to age-related cognitive decline and the number of these studies will most likely increase in the near future. Cognitive declines, including AD-like dementia in DS population, have a big impact on both cost of healthcare and the quality of life. Recently, the evidence of a strong contribute of protein dys-homeostasis has emerged in the development of neurodegenerative diseases. The novel idea underlying this project is that early chronic UPR activation represents a key molecular alteration leading to age- related cognitive decline in DS but also in normal population. Although notable strides have been made with regard to discerning the pathophysiological processes associated with dementia, pharmacological treatment trials to date have generally produced minimal or disappointing results. Increased ER stress can be considered a strong risk factor for the cognitive deterioration and, in particular, UPR over-activation may represent a novel target to slow or delay the DS- and age- related cognitive decline. In addition, the uncoupling of PERK/NRF2 axis during AD-like neurodegeneration is a new concept and deepening the knowledge of PERK and NRF2 relationship in DS samples will allow to understand the molecular mechanism that links aberrant proteostasis and increased oxidative stress posing the basis for therapeutic interventions targeting one or the other or both the components. The use of Blood-derived cells from young DS living patients represents a further innovation of this project. Indeed, this is unique model to study early alterations, driven by trisomic condition, in peripheral samples from living subjects, allowing to understands mechanism leading to cell degeneration shared with neuronal cells. Thanks to a collaboration with Dr. Diletta Valentini, pediatrician at Bambino Gesù pediatric hospital (OPBG) in Rome, we have the access to a large number of blood samples from DS children that could allow us to expand the analysis of the molecular mechanisms involved in the alterations observed in DS phenotype. Both primary peripheral blood mononuclear cells (PBMCs) and Epstein Barr virus (EBV) immortalized B-cells cells will be obtained by the same cohort of subjects, which account for about 600 young DS patients and age-matched healthy individuals routinely screened every year. We plan to analyze a total of 100 PBMCs samples (50 DS and 50 HBP) that will be collected from blood of subjects ranging from 1 to 18 years thus allowing to perform experiments across population monitoring the progression of UPR-related alterations. In parallel, blood from selected DS and age-matched healthy individuals (6 each group) will be further processed for the isolation of B-cells, which will be immortalized by Epstein Barr virus (EBV).
These stable cell lines will allow to perform experiments within the same population thus corroborating the consistency and reproducibility of the data. Preliminary data on both primary PBMCs and EBV B-cell confirm shared pathological alterations between blood derived DS cell and frontal cortex from DS human patients and demonstrate, as well, the DS-related hyperactivation of PERK that parallel with NRF2 depletion and increased protein oxidation.
Moreover we will use SUnSET (surface sensing of translation), a non-radioactive technique to measure protein synthesis in cell models. In this novel technique Puromycin, an aminonucleoside antibiotic and structural analog of aminoacyl tRNA, will be added to cell media and it will be incorporated into newly synthesized proteins . Since there are anti-puromycin antibodies, SUnSET will allow the immunostaining of cells highlighting the rate of protein synthesis. This approach is a major advantage over traditional radioactivity-based pulse-chase assays.
In conclusion, early preliminary findings strongly encourage to perform the present project, since the results generated by the investigators, each focusing on targets different but all equally crucial and strictly converging, may offer significant advances in the understanding pathophysiological mechanisms involved in DS- and age- associated cognitive decline. Interestingly the results may suggest novel and promising therapy that hopefully could have the potential not just to alleviate the symptoms but also to modify the course of the declined cognition in DS subjects.