Development and application of N-tail terminomics workflows for the characterization of proteolytic events of pathological relevance in ocular diseases

Reference Year 5X1000: 2022

Project Start: 01/01/2024

Project End: 31/12/2025

Project Summary – Abstract: Diseases of ophthalmological relevance, such as glaucoma, diabetic retinopathy, age-related macular generation (AMD), retinal detachment, as well as corneal diseases (e.g. Fuchs syndrome), have a significant impact on the patient’s quality of life, as well as on the social and economic costs borne by national health systems. Although the pathogenesis follows different trajectories, as well as the affected ocular tissues, the onset, but also the progression, of these diseases is shared by alterations in the remodeling of ocular extracellular tissues. However, as with many human pathologies affecting other anatomical districts, the molecular mechanisms underlying these processes are poorly understood. By virtue of these considerations, a fine characterization of endogenous proteolytic fragments, i.e. peptides released by enzymatic digestion of proteins, within ocular fluids, such as aqueous and vitreous humor, represents a promising approach to identify enzymatic degradation phenomena specific to the pathological phenomenon in question. In fact, the occurrence of proteolytic events of pathological relevance to ocular tissues, such as trabeculate, retinal layers, corneal tissues, can be reconstructed, with a reasonable margin of reliability, by examining the peptide fragments of the two fluids, aqueous and vitreous humour in the course of comparative analyses between samples isolated from subjects suffering from a specific disease and control patients (defined on the basis of the pathology under examination). In addition, exploring the repertoire of protein fragments in samples of a cellular nature, such as those identifiable in corneal endothelial sampling, can provide important parameters for correlating the origin of the peptide fragment and the tissue to which it belongs. By virtue of the reduced concentration of proteins, and peptides, within the fluids in question, experimental approaches with high sensitivity represent the only technological solution to provide an answer to the scientific problem posed. In this sense, mass spectrometry is undoubtedly the most suitable approach to obtain the widest possible coverage of biological data. Classic strategies (“shot-gun”) of proteomics for the characterization of the proteome of a biological matrix involve the fragmentation of the proteins contained in it by digestion with trypsin before injecting the sample into the mass spectrometer. This instrument then performs the characterization of the mass (and charge) of the peptides present in solution. Identification is followed by bioinformatics approaches necessary to trace the identification of proteins present in solution. However, this approach does not optimally distinguish peptide fragments derived from endogenous proteolysis from artificial tryptic proteolysis. In order to characterize the endogenous proteolytic fragments, specific proteomics workflows were then elaborated, such as the N-tail terminomics to which this project refers. Conceptually, the approach is based on the chemical labelling, in vitro, of all N-termini of proteins and peptides present in solution prior to trypsin digestion. In this way, endogenous peptide fragments can be chemically protected from further trypsin digestion events in vitro and characterized by conventional mass spectrometer approaches.

Date of receipt of 5×1000 funds: 28/09/2023

Total project cost: 29.703,73

Total 5×1000 shares: 29.703,73

Statement of allocation of resources 5×1000 2022:

• Initial economic sheet of funds expenditure
• Summary sheet of resource assignment
• Illustrative report of the project

From clinical practice to the laboratory: an inverse path for the research of biomarkers in retinal detachment and development of cell cultures with the use of eye fluids

Reference Year 5X1000: 2021

Project Start: 01/03/2023

Project End: 31/01/2024

Project Summary – Abstract: Translational medicine is an interdisciplinary branch of the biomedical field based on three main pillars: “benchside, bedside and community”.

In this area, personalised or precision medicine aims to provide the right treatment to the right patient, but above all at the right time. In recent years, this goal has been enhanced by advancing technologies in genomics, disease model systems, and many other paradigms followed by data science. It was fundamental to obtain a systemic perspective on science, complemented by multiple and

multidisciplinary
skills. The aim of this project is, first and foremost, to collect data through cross-talking between the operating units directly involved in patient care in the operating room for retinal diseases and the research laboratory. Therefore, starting directly from the patient, his disease and the clinical profile, the classic “bench-to-bedside” path is traced back in reverse.

Specifically, a part of the project (Part A) will be dedicated to proteome analysis in patients with primary retinal detachment. This aims to compare the repertoire of proteins present in the vitreous body, with particular attention to the identification of mediators of inflammation and to identify possible biomarkers of pathology that can be correlated with clinical parameters of severity and extension. In the second part, on the other hand, the study (Part B) involves the characterization and validation of an in vitro model consisting of epithelial cells of the anterior/posterior capsule exposed to a pathological microenvironment (diabetes, hypertension and other systemic pathologies) in order to deepen our knowledge of the biology of native epithelial cells and the mechanistic role they play in retinal pathologies.
.

Date of receipt of 5×1000 funds: 23/09/2022

Total cost of the project: € 31.892,40

Total 5 x 1000 shares: € 31.892,40
Resource allocation
statement 5×1000 2021:

• Initial economic sheet of funds expenditure
• Summary sheet of resource assignment
• Illustrative report of the project

Molecular and functional characterization of the ubiquitin-proteasome system during hypoxic damage in an experimental model of Muller Glia

Reference Year 5X1000: 2020

Project Start: 01/02/2022

Project End: 31/01/2024

Project Summar – Abstract: Diabetic retinopathy (DR) and glaucoma, two of the main causes of irreversible blindness worldwide, represent two ocular degenerative diseases with different etiology, whose clinical progression is characterized by ischemic-hypoxic insults, on a vascular, neuro-inflammatory and mechanical basis. Therefore, hypoxia is a stress with a central role in triggering the morphological and functional alterations to which the retina and the optic nerve are subjected. However, the molecular mechanisms, as well as the contribution of each retinal cell histotype to the perception and adaptation to this metabolic stress are poorly characterized. Hypoxia is known to activate multiple intracellular pathways, including the Hypoxia-Inducible Factor (HIF), which modulates the pro-angiogenic response through the synthesis and release of VEGF, and NF-kB, the main pro-inflammatory pathway that regulates the synthesis and release of pro-inflammatory cytokines. At the same time, it is known as hypoxic preconditioning, understood as transient exposure to a modest hypoxic event, constitutes a protective mechanism through which the cell acquires a greater capacity to tolerate a subsequent more severe hypoxic event. The molecular factors involved in this mechanism are not known, but they represent a promising research objective for the definition of therapeutic and preventive treatments of ischemic events. Adaptation to metabolic insults, including hypoxia, as well as the acquisition of specific phenotypic properties, such as resistance to certain environmental factors, are primarily regulated through an intracellular proteolytic pathway known as the ubiquitin-proteasome system (UPS), already the subject of in-depth studies aimed at understanding the molecular basis of neurodegeneration and identifying unconventional therapeutic strategies. In particular, the conjugation of intracellular proteins with ubiquitin, a small protein with key functions in the regulation of the cellular proteome and capable of directing the localization as well as the half-life of each cellular protein, represents an interesting and largely unexplored area of study to understand how the cell is able to adapt its metabolism in response to the environmental stimuli to which it is subjected. Therefore, the purpose of the present project is to set up an experimental platform, based on a newly developed specific proteomics technique, known as diGLY proteomics, to identify molecular determinants involved in hypoxic response and pre-hypoxic conditioning by investigating the ubiquitin repertoire in an experimental model of Muller Glia, rMC1 cells, subjected to controlled hypoxia and hypoxic pre-conditioning in vitro. The high-throughput strategy that characterizes the proposed approach suggests that it will be possible to identify the presence of numerous functional alterations of the UPS system in the experimental conditions under investigation, which may therefore allow the identification of new biological targets. In parallel, a proteomic investigation is proposed to characterize the ubiquitin repertoire also in the aqueous humor of patients with glaucoma and diabetic retinopathy (patients with cataracts will be used as healthy controls) in order to evaluate the possible presence of molecular determinants related to the heterogeneity of ubiquitin also in ocular biological fluids.

Date of receipt of 5×1000 funds: 19/10/2021

Total cost of the project: € 35.735,24

Total shares 5×1000: € 35.735,24
Resource allocation
statement 5×1000 2020:

• Initial economic sheet of funds expenditure
• Summary sheet of resource assignment
• Illustrative report of the Hypoxia project

Modulation of neuronal connectivity along optical pathways in glaucomatous patients: multimodal morpho-functional study

Reference Year 5X1000: 2019

Project Start: 01/04/2021

Project End: 30/03/2023

Project Summary – Abstract: Open-angle glaucoma is considered as a neurodegenerative process of the optic nerve that determines visual field deficits. In the open-angle glaucoma there are modifications of retinal ganglion cells (RGCs) and optic nerve fibers detectable by Optical Coherence Tomography (OCT). Functional deficits of RGCs and optic nerve can be assessed respectively by recording by Pattern ERGs (PERG) and Visual Evoked Potentials (VEP). Post-retinal functional deficit can be estimated using an electrophysiological index: Retinocortical time (RCT). Neuroimaging studies have shown in glaucomatous patients a neuronal loss at the level of the structures of the post-retinal optical pathways (lateral geniculate nucleus (LGN) and visual cortex). In glaucomatous patients, Citicoline has been observed to reduce the progression of glaucomatous perimeter deficit through mechanisms of action of: 1) neuroenhancement, improving the functionality of RGCs; 2) neuroprotection, reducing the loss of RGCs; 3) neuromodulation, improving synaptic connectivity between RGCs and NGLs. The objectives of this research project will be: 1) primary objective: to evaluate whether treatment with Citicoline in oral solution can produce an improvement in post-retinal nerve conduction, which is basically altered in patients with open-angle glaucoma. 2) Secondary objective: to evaluate in patients with open-angle glaucoma whether the possible changes in post-retinal nerve conduction induced by treatment with Citicoline in oral solution (information obtained through electrophysiological recordings) are associated or not with morphological and functional changes in the nerve structures that form the optical pathways (LGN, optic tract, visual cortex, information obtained through the acquisition of structural and functional magnetic resonance imaging) and whether both conditions can be related to morpho-functional changes in RGCs and CV. The study will be conducted double-blind, on 60 patients with glaucoma, randomized into two groups: one group will be treated with Citicoline oral solution, while the other group will be treated with placebo for a total duration of 12 months. At baseline, after 6 and 12 months in all enrolled patients, the following changes will be evaluated: post-retinal nerve conduction (through the simultaneous recording of PERG and PEV), morphological condition of the nerve structures that form the optical pathways (through neuro-imaging-MRI assessments) and RGCs (through OCT), perimeter deficit (CV assessment). The expected results are as follows: 1) as far as the primary objective is concerned, the expected result is that Citicoline in oral solution can improve post-retinal nerve conduction. 2) Regarding the secondary objectives, the expected result is the possibility of detecting that any improvements in post-retinal nerve conduction after treatment with Citicoline in oral solution may or may not be associated with changes in the structure and function of the nerve stations that form the optical pathways (evaluated by MRI) depending on or independent of the morpho-functional condition of the RGCs and the CV. The clinical relapses can be summarized as follows: 1) at present, the main therapeutic purpose in glaucoma is to reduce ocular pressure which is considered the main risk factor for the genesis of perimeter deficits. 2) Being able to highlight that treatment with Citicoline, based on its various actions (neuroenhancement, neuroprotection and neuromodulation) may be able to modulate neuronal connectivity, lays the foundations for a greater understanding of deficits in neuronal connectivity and its possible improvement with a consequent reduction in glaucomatous perimeter deficit.

Date of receipt of 5×1000 funds: 30/09/2020

Total cost of the project: € 153.588,12

Total shares 5 x 1000: € 53.588,12
Statement of allocation of resources 5×1000 2019:

• • Initial economic sheet of funds expenditure 2019
  • Summary sheet of resource assignment
  • Illustrative report of the project

Study of the role of intracellular proteolytic pathways, UPS and autophagy, in the pathogenesis of diabetic retinopathy and glaucoma

Reference Year 5X1000: 2018

Project Start: 03 Gennaio 2021

Project End: 30 Gennaio 2023

Project Synthesis– Abstract: A previous in vitro molecular study on a cellular model of rat Muller Glia (rMC1 cells) allowed to document a rapid (within 30 minutes) and unconventional (independent of the phosphorylation of IkBα, the natural inhibitor of the pathway) activation of the main proinflammatory pathway, NF-kB, and specifically of the heterodimer p65-p50, following the administration of 25 mmol/L glucose (experimental condition of hyperglycemia). The purpose of this research is to demonstrate that the described pathway capable of determining the activation of Muller Glia in vitro is reproducible and also operates in a cell system with increased complexity, such as primary retinal culture. The objective of the following research proposal is to analyze, through a multidisciplinary approach of biochemistry, molecular biology and immunohistochemistry, the role of the Ca2+, calmodulin-dependent (CamKII) proteasome axis in determining the functional activation, in a proinflammatory sense, of Muller Glia, in the context of a primary retinal culture, in the presence of a hyperglycemic stimulus (25 mmol/L D+ glucose).

Date of receipt of 5X1000 funds: June 12, 2020

Total cost of the project: € 56,506.35

Total shares 5×1000: € 56,506.35
Resource allocation
statement 5×1000 2018:

• • Initial economic sheet of funds expenditure 2018
  • Summary sheet of resource assignment
  • Illustrative report of the project