Biology subjects

Ratti, A.

Publications and source records attributed to Ratti, A..

7 recordsLinked to original sources

bioRxiv2025

Pre-clinical evaluation of a gene therapy candidate for SOD1-ALS shows improved survival and signs of inflammation in the CNS of treated mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neurons loss (MN). In 15-20% of familial ALS cases, mutations in the superoxide dismutase 1 (SOD1) gene are the underlying cause. Targeting human SOD1 (hSOD1) toxicity has emerged as a promising approach to treat SOD1-ALS. We previously demonstrated the efficacy of an exon-skipping strategy using a self-complementary AAVrh10-U7-hSOD1 vector in SOD1G93A mice achieving significant hSOD1 silencing. In this study, we optimized the therapeutic protocol by conducting a dose-finding and biodistribution study of scAAVrh10-U7-hSOD1 following a single intracerebroventricular injection in adult SOD1G93A mice. Our findings demonstrate a dose-dependent reduction in mutant hSOD1 levels in the cortex, spinal cord, and peripheral tissues, sustained for up to 60 days post-injection. In vivo, some adverse effects were noted mostly at the highest dose, with inflammation early post-injection and persistent microglial activation in the brain observed around the injection site. Importantly, the medium-dose treatment extended mean survival by up to 27% with a much milder early toxicity, which will provide a great possibility for future applications. Additionally, no major off-target effects were observed in human cell models, highlighting the targeting specificity of this approach and the potential safety for translation. These findings confirm and extend the therapeutic potential of scAAVrh10-U7-hSOD1 gene therapy while emphasizing the need for further technological development to minimize adverse effects and maximize potential clinical benefit.

pharmacology and toxicology↗

bioRxiv2024

PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.

molecular biology↗

bioRxiv2024

NEK1 haploinsufficiency worsens DNA damage but not defective ciliogenesis in C9ORF72 patient-derived iPSC-motoneurons

A hexanucleotide G4C2 repeat expansion (HRE) in C9ORF72 gene is the major cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leading to both loss- and gain-of-function pathomechanisms. The wide clinical heterogeneity among C9ORF72 patients suggests potential modifying genetic factors. Notably, C9ORF72 mutations often co-occur with other variants in ALS/FTD-associated genes, such as NEK1, which encodes for a kinase involved in multiple pathways including DNA damage response and ciliogenesis. In this study, we generated induced pluripotent stem cells (iPSCs) and differentiated motoneurons (iPSC-MNs) derived from an ALS patient carrying both C9ORF72 HRE and a NEK1 loss-of-function mutation to study the effect of the NEK1 haploinsufficiency on C9ORF72 pathology. Double mutant C9ORF72/NEK1 cells showed increased pathological C9ORF72 RNA foci in iPSCs and higher DNA damage levels in iPSC-MNs compared to single mutant C9ORF72 cells. In contrast, ciliogenesis was similarly impaired in both C9ORF72 and C9ORF72/NEK1 iPSC-MNs showing shorter cilia. Altogether, our study supports the use of patient-derived iPSCs to functionally explore the contribution of genetic modifiers in C9ORF72-associated pathology.

neuroscience↗

bioRxiv2024

NEK1 haploinsufficiency impairs ciliogenesis in human iPSC-derived motoneurons and brain organoids

Primary cilia are microtubule-based organelles acting as specialized signalling antennae that respond to specific stimuli to maintain cellular integrity and homeostasis. Recent studies indicate defective primary cilia in post-mortem human brains and animal models of neurodegenerative conditions, including Amyotrophic Lateral Sclerosis (ALS). Heterozygous loss-of-function mutations (LOF) in NEK1 gene are present in about 1% of familial and sporadic ALS cases. The protein kinase NEK1 regulates various cellular processes, including ciliogenesis, but a clear link between NEK1 LOF mutation in ALS and primary cilia is unknown. In this study we generated a human iPSC line carrying a NEK1 LOF mutation by gene editing, leading to NEK1 protein haploinsufficiency. In differentiated iPSC-motoneurons (MNs) we observed that primary cilia were significantly shorter in NEK1-LOF iPSC-MNs compared to wild-type (WT) iPSC-MNs and that also the percentage of ciliated iPSC-MNs was significantly decreased in NEK1-LOF cells. We also investigated ciliogenesis in NEK1-LOF iPSC-brain organoids confirming that primary cilia were thinner with no apparent alteration in the ultrastructure by transmission electron microscopy. Our data suggest that NEK1 protein plays a role in regulating ciliogenesis in both 2D and 3D human iPSC-derived neuronal models and that NEK1 LOF mutations associated to ALS, leading to NEK1 haploinsufficiency and likely to reduced kinase activity, impair primary cilium formation. The involvement of ciliogenesis dysfunction in ALS deserves further investigation providing novel therapeutic targets and strategies to be addressed for this incurable disease.

neuroscience↗

bioRxiv2024

Altered nuclear envelope homeostasis is a key pathogenic event in C9ORF72-linked ALS/FTD

ALS and FTD are complex neurodegenerative disorders that primarily affects motor neurons in the brain and spinal cord, and cortical neurons in the frontal lobe. Although the pathogenesis of ALS/FTD is unclear, recent research spotlights nucleocytoplasmic transport impairment, DNA damage, and nuclear abnormalities as drivers of neuronal death. In this study, we show that loss of nuclear envelope (NE) integrity is a key pathology associated with nuclear pore complex (NPC) injury in C9ORF72 mutant neurons. Importantly, we show that mechanical stresses generated by cytoskeletal forces on the NE can lead to NPC injury, loss of nuclear integrity, and accumulation of DNA damage. Importantly, we demonstrate that restoring NE tensional homeostasis, by disconnecting the nucleus from the cytoskeleton, can rescue NPC injury and reduce DNA damage in C9ORF72 mutant cells. Together, our data suggest that modulation of NE homeostasis and repair may represent a novel and promising therapeutic target for ALS/FTD.

neuroscience↗

bioRxiv2022

Molecular dynamics analysis of Superoxide Dismutase 1 mutations suggests decoupling between mechanisms underlying ALS onset and progression

Mutations in the superoxide dismutase 1 (SOD1) gene are the second most common known cause of ALS. SOD1 variants express high phenotypic variability and over 200 have been reported in people with ALS. Investigating how different SOD1 variants affect the protein dynamics might help in understanding their pathogenic mechanism and explaining their heterogeneous clinical presentation. It was previously proposed that variants can be broadly classified in two groups, wild-type like (WTL) and metal binding region (MBR) variants, based on their structural location and biophysical properties. MBR variants are associated with a loss of SOD1 enzymatic activity. In this study we used molecular dynamics and large clinical datasets to characterise the differences in the structural and dynamic behaviour of WTL and MBR variants with respect to the wild-type SOD1, and how such differences influence the ALS clinical phenotype. Our study identified marked structural differences, some of which are observed in both variant groups, while others are group specific. Moreover, applying graph theory to a network representation of the proteins, we identified differences in the intramolecular contacts of the two classes of variants. Finally, collecting clinical data of approximately 500 SOD1 ALS patients carrying variants from both classes, we showed that the survival time of patients carrying an MBR variant is generally longer (~6 years median difference, p < 0.001) with respect to patients with a WTL variant. In conclusion, our study highlights key differences in the dynamic behaviour of the WTL and MBR SOD1 variants, and wild-type SOD1 at an atomic and molecular level. We identified interesting structural features that could be further investigated to explain the associated phenotypic variability. Our results support the hypothesis of a decoupling between mechanisms of onset and progression of SOD1 ALS, and an involvement of loss-of-function of SOD1 with the disease progression.

neuroscience↗

bioRxiv2020

Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43). The RNA-binding protein TDP-43 is also a component of stress granules (SG), cytoplasmic foci forming to arrest translation under sub-lethal stress conditions. Although commonly considered as distinct structures, a link between SG and pathological TDP-43 inclusions may occur despite evidence that TDP-43 pathology directly arises from SG is still under debate. Primary fibroblasts and iPSC-derived neurons (iPSC-N) from ALS patients carrying mutations in TARDBP (n=3) and C9ORF72 (n=3) genes and from healthy controls (n=3) were exposed to oxidative stress by sodium arsenite. SG formation and cell response to stress was evaluated and quantified by immunofluorescence and electron microscopy analyses. We found that, not only an acute, but also a chronic oxidative insult, better mimicking a persistent condition of stress as in neurodegeneration, is able to induce SG formation in primary fibroblasts and iPSC-N. Importantly, only upon chronic stress, we observed TDP-43 recruitment into SG and the formation of distinct P-TDP-43 aggregates, very similar to the abnormal inclusions observed in ALS/FTD autoptic brains. Moreover, in fibroblasts, cell response to stress was different in control compared with mutant ALS cells, probably due to their different vulnerability. A quantitative analysis revealed also differences in terms of number of SG-forming cells and SG size, suggesting a different composition of foci in acute and chronic stress. In condition of prolonged stress, SG and P-TDP-43 aggregate formation was concomitant with p62 increase and autophagy dysregulation in both ALS fibroblasts and iPSC-N, as confirmed by immunofluorescence and ultrastructural analyses. We found that exposure to a chronic oxidative insult promotes the formation of both SG and P-TDP-43 aggregates in patient-derived cells, reinforcing the idea that SG fail to properly disassemble, interfering with the protein quality control system. Moreover, we obtained a disease cell model recapitulating ALS/FTD P-TDP-43 aggregates, which represents an invaluable bioassay to study TDP-43 pathology and develop therapeutic strategies aimed at disaggregating or preventing the formation of pathological inclusions.

neuroscience↗