Leader: Fabrizio Chiti (UNIFI); Other collaborator(s):
Proteins associated with neurodegeneration and their mutants will be purified and converted into misfolded oligomers thought to be relevant in neurodegeneration. The structure of such relevant protein complexes will be studied by biophysical techniques, such as circular dichroism, fluorescence, FTIR, dynamic light scattering, sold-state NMR, atomic force and electron microscopy. Their toxicity will be evaluated on cell cultures (including primary neurons and iPSC-derived neurons), organotypic hippocampal cultures and animal models and mitochondrial status, ROS, calcium influx, caspase-3 activation, will be used as readouts of cell dysfunction. This will allow us to define a structure-toxicity relationship to identify proteotoxicity biomarkers
Brief description of the activities and of the intermediate results
Since Alzheimer disease (AD) is characterized by a generic failure of the proteostasis network, we compared cerebrospinal fluid (CSF) samples extracted from AD and non-AD cases to detect aggregated/proteotoxic proteins. The two CSF groups are distinguishable for the population of large protein species detected with dynamic light scattering, which indicates aggregation, and oligomeric species evoking an influx of calcium ions across the cell membrane of cultured cells, which indicates proteotoxicity. Scatter plots with pairs of these parameters and optimized Youden’s indexes provide well defined separations between non-AD and AD cases, indicating that these two novel AD biomarkers go beyond the concept of protein-specific biomarkers and rather implies a widespread proteome-based misfolding/aggregation/toxicity in the CSF, following a proteostasis failure.
Main policy, industrial and scientific implications
Aging represents the primary risk factor for major human pathologies, including Alzheimer disease (AD). Assessing aging and AD biomarkers in cerebral spinal fluid (CSF) holds of paramount importance.
The identification of two novel biomarkers for AD can implement the current availability of biomarkers, paves the way to a longitudinal study to assess whether they are early or late AD biomarkers sand and opens the possibility that they are also valid for other protein deposition diseases.
Scatter plots with pairs of these parameters and optimized Youden’s indexes provide well defined separations between non-AD and AD cases. Moreover, scatter plots with one of these parameters and a classical biomarkers such as CSF Ab42/Ab40 ratio, CSF tau or phosphorylated tau and again optimized Youden’s indexes provide better separations between non-AD and AD cases, which are similar in terms of sensitivity and specificity to scatter plots obtained with pairs of classical biomarkers. This indicates that these two novel AD biomarkers go beyond the concept of protein-specific biomarkers and rather implies a widespread proteome-based misfolding/aggregation/toxicity in the CSF, following a PN failure. This study is published in Transl Neurodegener (see reference below).
In another study we set up a cell culture model based on neuroblastoma and primary neurons to study the hyperexcitability observed in an aging phase that even precedes the preclinical stage of AD detectable only with CSF and PET amyloid biomarkers and in which cognitive and memory impairments are not yet apparent under clinical investigation. To this aim, we used ADDLs as misfolded Ab42 oligomers and Glutamate as a neurotransmitter at concentrations that do not cause neuronal toxicity, but still stimulate an entry of calcium ions through AMPA and NMDA receptors. Indeed, increases of these two species is known to occur in such an hyperactivity phase preceding preclinical AD in both humans and mouse models. We identified sub-lethal concentrations of the two species and revealed the key biochemical changes occurring in this phase. This study is summarised in a manuscript in preparation.