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.
In one study we took advantage of advances in solid-state nuclear magnetic resonance (ssNMR) spectroscopy and cryogenic electron microscopy (cryoEM) that have revealed the structures of many amyloid fibrils revealing the polymorphic nature of the amyloid state of proteins. To address this issue, we combined computational tools to predict the specific regions of the sequence forming the β-spine of amyloid fibrils with the availability of 30, 83 and 24 amyloid structures deposited in the Protein Data Bank (PDB) and Amyloid Atlas (AAt) for the amyloid β (Aβ) peptide, α-synuclein (αS), and the 4R isoforms of tau, associated with Alzheimer's disease, Parkinson's disease, and various tauopathies, respectively. We determnined for any sequence residue n the fraction of PDB/AAt structures in which that residue adopts a β-sheet conformation (Fβ(n)) to generate an experimental, structure-based profile of Fβ(n) vs n, which represents the β-conformational preference of that given residue n in the amyloid state. The peaks in the respective Fβ(n) profiles of the three proteins correspond very well to the peaks identified with five predictive algorithms, indicating that, despite amyloid polymorphism, sequence regions most often forming the structural core of amyloid have high hydrophobicity, high intrinsic β-sheet propensity and low electrostatic charge across the sequence. This study is published in Biochemical J (see reference below).
In another study, in which two members of the PI group were involved as co-authors, solid-state nuclear magnetic resonance (ssNMR), cryogenic electron microscopy (cryo-EM), and biophysical methods, were used to characterize intermediate amyloid fibrils of αS forming during the aggregation from liquid-like spherical condensates to mature amyloids. These transient amyloid intermediates were found to be toxic and stabilized by a small core in an antiparallel β-sheet conformation, with a disordered N-terminal region of the protein remaining available to mediate membrane binding. Instead, mature amyloid fibrils exhibited different structural and biological properties, including low toxicity, a rearranged structured core embedding also the N-terminal region, and a reduced propensity to interact with the membrane. This study is published in J. Ac. Chem. Soc. (see reference below).
We have also published a review in which the PI of this Taks was a coauthor (published in iScience, as seen in the reference below).