Mitochondrial and protease dysfunction in progeroid Cockayne syndrome cells and during age-associated processes – CS_AGE

To assess mitochondrial components that are affected in CS cells, we used subcellular
fractionation, gradient fractionation, lentivirus-mediated silencing, and overexpression of
genes of interest, co-immunoprecipitation, super-resolution microscopy, and other
molecular and cellular biology approaches in patient-derived and immortalized fibroblasts.
In patient-derived cells we performed genome-wide DNA methylation, RNAseq,
bioinformatic analysis of genes and pathways.

To address the question of the large clinical variability of CS in patients carrying very
close mutations, we generated isogenic cellular models (mutations and wild type in
otherwise identical genomes), with CSB mutations found in patients with either a very
severe or a mild form of the disease. A strength of the CS paradigm is the occurrence of a
disease (UVSS) due to the same mutations as CS, and characterize by photosensitivity
(CSB is involved in the repair of UV-induced DNA damage) but no progeroid and
neurodegenerative phenotype. We focus on mechanistic defects present in CS and absent
in UVSS, to identify the factors leading to progeria and neurodegeneration.

Due to the limitation of cell types from patients and their restricted amplification potential,
we generated iPSCs and cerebral organoids (COs) from patient fibroblasts.
COs are 3D neural structures that contain multiple cell types and a cytoarchitecture
that resembles the early developing human brain and has been used as advanced models
for neurodegenerative conditions. We selected COs because of the severe
neurodegeneration in CS. Isogenic iPSCs and COs have been also generated to
disentangle functional defects in these structures due to the CSB mutation alone from
its expression in a given genetic context.

1) We have identified a mechanism that depletes mitochondrial POLG1, a key defect in CS cells and which is directly linked to the progeroid phenotype. This mechanism is of relevance also for normal cells, since it was not known how the homeostasis of the essential POLG1 protein is regulated. We discovered that in normal cells this process is activated during replicative senescence, providing a further mechanistic link between a progeroid defect and a process linked to ageing.
Manuscript in preparation (Ms1_Fernandez Molina et al). The HTRA3 protease/chaperone stabilizes cathepsin B for degradation of mitochondrial POLG1 in progeroid Cockayne syndrome and senescent cells.

2) DNAmethylation analysis has shown epigenetic changes in three main categories of genes: developmental transcription factors, transmembrane transporters, and cell adhesion factors, that appear as major accelerated aging triggers.
We have also identified DNAm that are specific to accelerated ageing (CS alone or in common with other progeroid diseases) and genes that are shared with regular ageing. We have this established a DNAm signature for accelerated ageing (CS) and in common with regular ageing. A few genes of either category are being functionally analysed.
BioRxiv_2021_445308, and manuscript under revision (Ms2_Crochemore et al). Epigenomic signature of the progeroid Cockayne syndrome exposes distinct features of accelerated ageing.

3) We have generated more isogenic cellular models than initially anticipated to overcome an unexpected problem (see above), finally resulting in a more robust and more diverse paradigm for the ongoing studies than originally anticipated.

4) We have generated cerebral organoids (COs) derived from patient cells and the respective healthy controls. For this, there has been intense technology transfer from P3-Yates (Sup’Biotech) to Institut Pasteur (P1-Ricchetti). Both labs are now autonomous and we are improving the technology aiming to increase the homogeneity of COs samples. Molecular, cellular, and structural analysis of COs has been started, with approaches that we have optimized for this goal. Phenotypically CS-COs appear smaller than WT-COs, a defect that we are now analysing under multiples aspects and that may have mechanistical links with microcephaly observed in CS patients. The state of the art and the potential of these models seems to exceed the predictions indicated in the initial project.
5) We have also elaborated on the role on multiple reactive species in Cockayne syndrome and more in general in progeroid diseases and ageing. This review has been accepted in the influential journal Antioxidant Redox Signalling.
Antioxidant Redox Signalling, (Ms_3, Crochemore et al), Reactive species in progeroid syndromes and ageing-related processes. Epub ahead of print, PMID: 34428933, DOI: 10.1089/ars.2020.8242

We have developed our project with a remarkable fit to the plans, despite the partial closure and reduced activity due to the Covid-19 situation. We will continue following the roadmap indicated in the project, as it showed to be well-planned.

We will take advantage of the development of cerebral organoids (COs) that we developed at better level than originally anticipated, and will analyse defective mechanisms in these structures, using biochemical, molecular biology, and cellular biology approaches. We will also perform immunofluorescence analysis to assess possible alterations of the cell content, cell organization, and cytoarchitecture in CS- as well as UVSS-derived COs. We will perform these analyses also in isogenic models to identify the role of the different types of mutations in CO cytoarchitecture, and evaluate whether the genetic backgrounds does play a role in these alterations. We may reprogram more iPSCs from patient fibroblasts derived from patients with neurodegenerative processes of interest, and will then derive the corresponding COs. We will also perform experiments on neurons differentiated from iPSCs. The role of the rescue molecule MnTBAP will be tested at different stages of CO formation (or 2D neuron differentiation) to evaluate whether potential defects are recovered. (Task 3).

We will perform whole cell (fibroblasts) transcriptome analysis to identify genes and pathway that are altered in CS in general or in some forms of the disease (severe, common, mild forms), and again those that are in common with other ageing conditions (other progeroid diseases, physiological ageing). We will also analyse the correlation between DNA methylation and gene expression changes, to understand which epigenetic changes modulate gene expression in CS. These studies will be extended to selected samples of neurons and iPSCs. (Tasks 2 and 4).

We will analyse mechanistic changes leading to mitochondrial dysfunction in CS fibroblasts by assessing replication and transcription of mitochondrial DNA, as well as the mitochondrial DNA replication complex, as indicated in the project. Part of these analyses will be also conducted in iPSC-derived neurons form CS with various severities, UVSS, and healthy controls to assess the persistence and extent of CS defects also in neural cells. (Task 1). This part has of the project was partially delayed because of the lab restrictions due to Covid-19, as we had to maintain long-term cell cultures (fibroblasts, iPSCs and cerebral organoids) active in priority (Tasks 2-4). This part will be therefore particularly developed in the second half of the project.

Clément Crochemore, Claudia Chica, Paolo Garagnani, Giovanna Lattanzi, Steve Horvath, Alain Sarasin, Claudio Franceschi, Maria Giulia Bacalini, and Miria Ricchetti. Epigenomic signature of the progeroid Cockayne syndrome exposes distinct features of accelerated ageing. BioRxiv_2021_445308.

Clément Crochemore, Chiara Cimmaruta, Cristina Fernandez Molina, Miria Ricchetti. Reactive species in progeroid syndromes and ageing-related processes. Antioxidant Redox Signalling. Epub ahead of print, PMID: 34428933, DOI: 10.1089/ars.2020.8242

The mechanisms governing ageing, which is a multifactorial process, have not been resolved and constitute a fundamental open question in cell and organismal biology research worldwide. Exceptionally, in rare genetic diseases like the Cockayne syndrome (CS), the ageing phenotype is greatly accelerated1. Understanding the molecular defects responsible for these premature ageing diseases is critical to develop treatments, which are dramatically missing to date, and also elucidate physiological ageing in general.
We recently identified a completely hitherto undiscovered pathway that is altered in cells from CS patients2 (Partner1-Ricchetti). This pathway is uncoupled from the well-documented DNA repair defect and rather involves mitochondrial and metabolic impairments. Although mitochondrial defects have been described in CS cells (mutated in the DNA repair factor CSA or CSB)1, they have been essentially considered consequences of impaired DNA repair in the nucleus and in the organelle. Conversely, our findings show that in CS cells oxidative and nitrosative stress (ROS/RNS) promotes overexpression of the HTRA3 protease, which results in the degradation of the key mitochondrial DNA polymerase POLG1 responsible for replication of the organelle genome, and triggers mitochondrial dysfunction. Importantly, we rescued defects in CS patient cells by scavenging oxidative/nitrosative molecules (with MnTBAP), paving the path to therapeutic approaches for CS that are missing to date. These findings led to an international patent application (WO2012123588), and the designation by the EMA (European Medicines Agency) of MnTBAP as Orphan Drug for CS (P1-Ricchetti and Partner2-Laugel).
Our findings represent a paradigm shift to understand and possibly challenge the causes of precocious ageing in CS, and perhaps in normal ageing. It urges now to identify the underlying mechanisms and evaluate the impact of these cellular alterations on progeroid dysfunction. Importantly, our (unpublished) results reveal genome-wide epigenetic changes that are correlated with the severity of disease, making possible the identification of novel key effectors in the establishment of CS. Based on a consortium that has the largest collection of research and clinical paradigms to study CS, and a robust set of preliminary data, we launch a research program to elucidate how CSA/B deficiency alters the HTRA3/POLG1/mitochondrial pathway and the epigenetic landscape in the presence of nitroso-redox imbalance. This study has also implications for the clinical treatment of CS and possible interventions for physiological ageing.
The present study has four aims: 1) assess the mechanism by which HTRA3 overexpression results in POLG1 depletion and in turn mitochondrial dysfunction; 2) link epigenetic modifications specific to CS fibroblasts (manuscript, ms1, in preparation) to transcriptome changes, and to iPSC-derived neuronal cells/structures, for the identification of novel effectors of CS defects, and assess whether they are reverted upon MnTBAP treatment; 3) correlate CS cell alterations with clinical phenotypes for diagnostic and therapeutic purposes, taking also advantage from iPSC-derived neuronal organoids (to date produced only by our consortium) for personalized rescue strategies with MnTBAP (bench-to-bedside developments); and 4) investigate CS-specific alterations during regular ageing, by assessing the mechanism responsible for their occurrence in the senescence of normal cells (a process linked to ageing), that we have identified (ms2 to submit), and validating common epigenetic changes in CS and during normal ageing.