Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

The Pesticide Chlordecone Promotes Parkinsonism-like Neurodegeneration with Tau Lesions in Midbrain Cultures and C. elegans Worms.

Chlordecone (CLD) is an organochlorine pesticide (OCP) that is currently banned but still contaminates ecosystems in the French Caribbean. Because OCPs are known to increase the risk of Parkinson's disease (PD), we tested whether chronic low-level intoxication with CLD could reproduce certain key characteristics of Parkinsonism-like neurodegeneration. For that, we used culture systems of mouse midbrain dopamine (DA) neurons and glial cells, together with the nematode C. elegans as an in vivo model organism. We established that CLD kills cultured DA neurons in a concentration- and time-dependent manner while exerting no direct proinflammatory effects on glial cells. DA cell loss was not impacted by the degree of maturation of the culture. The use of fluorogenic probes revealed that CLD neurotoxicity was the consequence of oxidative stress-mediated insults and mitochondrial disturbances. In C. elegans worms, CLD exposure caused a progressive loss of DA neurons associated with locomotor deficits secondary to alterations in food perception. L-DOPA, a molecule used for PD treatment, corrected these deficits. Cholinergic and serotoninergic neuronal cells were also affected by CLD in C. elegans, although to a lesser extent than DA neurons. Noticeably, CLD also promoted the phosphorylation of the aggregation-prone protein tau (but not of α-synuclein) both in midbrain cell cultures and in a transgenic C. elegans strain expressing a human form of tau in neurons. In summary, our data suggest that CLD is more likely to promote atypical forms of Parkinsonism characterized by tau pathology than classical synucleinopathy-associated PD.

Rescue of Dopamine Neurons from Iron-Dependent Ferroptosis by Doxycycline and Demeclocycline and Their Non-Antibiotic Derivatives.

Several studies have reported that the tetracycline (TC) class antibiotic doxycycline (DOX) is effective against Parkinson's disease (PD) pathomechanisms. The aim of the present work was three-fold: (i) Establish a model system to better characterize neuroprotection by DOX; (ii) Compare the rescue effect of DOX to that of other TC antibiotics; (iii) Discover novel neuroprotective TCs having reduced antibiotic activity. For that, we used cultures of mouse midbrain dopamine (DA) neurons and experimental conditions that model iron-mediated oxidative damage, a key mechanism in PD pathobiology. We found that DOX and the other TC antibiotic, demeclocycline (DMC), provided sustained protection to DA neurons enduring iron-mediated insults, whereas chlortetracycline and non-TC class antibiotics did not. Most interestingly, non-antibiotic derivatives of DOX and DMC, i.e., DDOX and DDMC, respectively, were also robustly protective for DA neurons. Interestingly, DOX, DDOX, DMC, and DDMC remained protective for DA neurons until advanced stages of neurodegeneration, and the rescue effects of TCs were observable regardless of the degree of maturity of midbrain cultures. Live imaging studies with the fluorogenic probes DHR-123 and TMRM revealed that protective TCs operated by preventing intracellular oxidative stress and mitochondrial membrane depolarization, i.e., cellular perturbations occurring in this model system as the ultimate consequence of ferroptosis-mediated lipid peroxidation. If oxidative/mitochondrial insults were generated acutely, DOX, DDOX, DMC, and DDMC were no longer neuroprotective, suggesting that these compounds are mostly effective when neuronal damage is chronic and of low-intensity. Overall, our data suggest that TC derivatives, particularly those lacking antibiotic activity, might be of potential therapeutic utility to combat low-level oxidative insults that develop chronically in the course of PD neurodegeneration.

Neuroprotective Effects of a Novel Demeclocycline Derivative Lacking Antibiotic Activity: From a Hit to a Promising Lead Compound.

The antibiotic tetracycline demeclocycline (DMC) was recently reported to rescue α-synuclein (α-Syn) fibril-induced pathology. However, the antimicrobial activity of DMC precludes its potential use in long-term neuroprotective treatments. Here, we synthesized a doubly reduced DMC (DDMC) derivative with residual antibiotic activity and improved neuroprotective effects. The molecule was obtained by removal the dimethylamino substituent at position 4 and the reduction of the hydroxyl group at position 12a on ring A of DMC. The modifications strongly diminished its antibiotic activity against Gram-positive and Gram-negative bacteria. Moreover, this compound preserved the low toxicity of DMC in dopaminergic cell lines while improving its ability to interfere with α-Syn amyloid-like aggregation, showing the highest effectiveness of all tetracyclines tested. Likewise, DDMC demonstrated the ability to reduce seeding induced by the exogenous addition of α-Syn preformed fibrils (α-SynPFF) in biophysical assays and in a SH-SY5Y-α-Syn-tRFP cell model. In addition, DDMC rendered α-SynPFF less inflammogenic. Our results suggest that DDMC may be a promising drug candidate for hit-to-lead development and preclinical studies in Parkinson's disease and other synucleinopathies.

Modelling α-Synuclein Aggregation and Neurodegeneration with Fibril Seeds in Primary Cultures of Mouse Dopaminergic Neurons.

To model α-Synuclein (αS) aggregation and neurodegeneration in Parkinson's disease (PD), we established cultures of mouse midbrain dopamine (DA) neurons and chronically exposed them to fibrils 91 (F91) generated from recombinant human αS. We found that F91 have an exquisite propensity to seed the aggregation of endogenous αS in DA neurons when compared to other neurons in midbrain cultures. Until two weeks post-exposure, somal aggregation in DA neurons increased with F91 concentrations (0.01-0.75 µM) and the time elapsed since the initiation of seeding, with, however, no evidence of DA cell loss within this time interval. Neither toxin-induced mitochondrial deficits nor genetically induced loss of mitochondrial quality control mechanisms promoted F91-mediated αS aggregation or neurodegeneration under these conditions. Yet, a significant loss of DA neurons (~30%) was detectable three weeks after exposure to F91 (0.5 µM), i.e., at a time point where somal aggregation reached a plateau. This loss was preceded by early deficits in DA uptake. Unlike αS aggregation, the loss of DA neurons was prevented by treatment with GDNF, suggesting that αS aggregation in DA neurons may induce a form of cell death mimicking a state of trophic factor deprivation. Overall, our model system may be useful for exploring PD-related pathomechanisms and for testing molecules of therapeutic interest for this disorder.

Where are the missing gene defects in inherited retinal disorders? Intronic and synonymous variants contribute at least to 4% of CACNA1F-mediated inherited retinal disorders.

Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30-50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene-coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F-related cases have intronic and synonymous disease-causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene-locus sequencing may be a very efficient method in detecting disease-causing variants in clinically well-characterized patients with a diagnosis of IRD, like icCSNB.