Laura Ciapponi

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and intellectual disability. Mutations in over 30 genes, nearly half linked to centrosome biogenesis or microtubule (MT) dynamics, highlight spindle defects in disease aetiology, yet these alone do not fully explain MCPH. Here, we show that the Drosophila orthologs of ASPM/MCPH5 (asp) and CENPJ/MCPH6 (Sas4) contribute to safeguard nuclear architecture and chromatin organization during brain development. Loss of either gene perturbs MT organization and centromere clustering, leading to reduced Lamin and HP1α levels, and to deformed nuclear lamina. Mutants also display a global reduction in heterochromatin-associated histone marks, H3K9me2/3 and H3K27me3, along with an increase in the euchromatin-associated mark H3K4me3 and elevated DNA damage with delayed repair. Notably, inhibiting demethylases with methylstat restores H3K9me3 and nuclear morphology. These findings suggest a previously unreported role for centrosome proteins in regulating chromatin organization, providing new insights into the mechanisms underlying MCPH pathogenesis.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant, multisystemic disorder caused by the expansion of CCTG repeats in the first intron of the CNBP gene. Repeat-associated non-AUG (RAN) translation of the expanded CCTG RNA generates two tetrapeptide repeat proteins, polyQAGR and polyLPAC, but their roles in DM2 pathogenesis remain unclear. To define their individual contributions, we expressed codon-optimized polyQAGR and polyLPAC peptides with an ATG start codon in Drosophila melanogaster. Expression of both tetrapeptide repeat proteins reduced viability and lifespan and induced eye degeneration and locomotor defects. We found that polyQAGR accumulated in the nucleolus, disrupted nucleolar integrity and impaired rRNA processing. It also interfered with autophagy, promoting Atg5 and Atg7 transcription and accumulation of Atg8a- and Ref(2)P-positive aggregates. Overexpression of Atg8a or Ref(2)P mitigated polyQAGR-induced eye toxicity, whereas knockdown of autophagy genes worsened it. Conversely, PolyLPAC expression increased the cytoplasmic pool of TIAR in human cells and in DM2 patient-derived myoblasts. Together, these findings show that polyQAGR and polyLPAC exert distinct toxic effects that likely converge to drive DM2 pathogenesis and may represent promising therapeutic targets.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant, multisystemic disorder caused by the expansion of CCTG repeats in the first intron of the CNBP gene. Repeat-associated non-AUG (RAN) translation of the expanded CCTG RNA may produce two tetrapeptide repeat proteins (TPRs), poly-QAGR and poly-PACL, whose roles in DM2 pathogenesis remain poorly understood. To investigate their individual contributions, we expressed codon-optimized QAGR and PACL peptides with ATG start codon in Drosophila melanogaster . Expression of QAGR and PACL TPRs significantly compromised fly viability and lifespan, induces eye degeneration and locomotor defects. We found that QAGR accumulated in the nucleolus, disrupted nucleolar integrity, and compromised rRNA processing. Moreover, QAGR expression interfered with autophagy, leading to the accumulation of Atg8a– and Ref(2)P-positive aggregates. Genetic interaction studies showed that overexpression of Atg8a or Ref(2)P mitigated QAGR-induced eye-toxicity, while knockdown of autophagy genes exacerbated it. Conversely, PACL repeats promoted stress granule formation, as indicated by their colocalization with TIAR in human cells and their epistatic interaction with the Drosophila orthologue Rox8. Notably, cytoplasmic PACL aggregates were observed in myoblasts of DM2 patient. Together these findings demonstrate that QAGR and PACL peptides exert distinct toxic effects, impairing nucleolar function and autophagy, or altering stress granule dynamics, respectively. Both mechanisms likely converge to drive DM2 pathogenesis and represent promising therapeutic targets.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and non-progressive intellectual disability. Mutations in over 30 genes have been linked to MCPH. Nearly a half of the genes identified by these mutations encode proteins involved in centrosome biogenesis or microtubule (MT) dynamics, suggesting a central role for mitotic spindle organization and division plane orientation in disease aetiology. However, it has been suggested that disruptions in spindle positioning alone are not sufficient to lead to microcephaly. Here, we investigate the contribution of the Drosophila orthologs of ASPM / MCPH5 ( asp ) and CENPJ/MCPH6 ( Sas4 ) to nuclear architecture, chromatin organization, and genome stability. We show that loss of either Sas4 or Asp leads to aberrant microtubule architecture, mislocalization of the LINC complex, and deformation of the nuclear lamina. These defects are accompanied by reduced levels of both lamin and HP1α and impaired centromere clustering in interphase cells. Sas4 and asp mutants also exhibit a global reduction in heterochromatin-associated histone marks (H3K9me2/3 and H3K27me3) and increased levels of the euchromatin-associated mark H3K4me3. Remarkably, treatment with Methylstat, a demethylase inhibitor, reduced nuclear invaginations by partially restoring H3K9me3 levels. Additionally, Sas4 or Asp depletion leads to DNA damage, increased sensitivity to genotoxic stress, and delayed DNA repair. Together, these findings reveal a previously underappreciated role for Asp and Sas4 in preserving nuclear architecture and chromatin integrity, offering new insight into the pathogenesis of MCPH. GRAPHICAL

Caspase-8 is a cysteine protease historically regarded as anti-neoplastic protein, thanks to its role in apoptosis. However, Caspase-8 expression is retained or even enhanced in several tumors, including glioblastoma (GBM), where it plays pro-tumor functions. We previously reported that it is a negative prognostic factor and contributes to resistance against DNA damaging agents, such as ionizing radiations (IR) and Temozolomide, commonly used in standard GBM treatment. We therefore investigated whether Caspase-8 may sustain DNA repair pathways proficiency in GBM. Here we uncover a novel role of Caspase-8 as promoter of the Homologous Recombination Repair (HRR). Importantly, IR promote Caspase-8 transient nuclear translocation and its recruitment to the chromatin. Moreover, Caspase-8 sustains the expression and the recruitment to the chromatin upon IR of RAD51 and CtIP, two key players of the HRR. Consistently, we identify a synthetically lethal interaction between Caspase-8 and PARP inhibition, that may ameliorate GBM response to IR. Remarkably, by using Caspase-8-/- murine embryo fibroblasts and a Drosophila melanogaster Caspase-8 mutant, we demonstrate that Caspase-8 plays an evolutionary conserved role in DNA repair. GRAPHICAL

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by the progressive degeneration of motor neurons. One of the key pathogenic factors implicated in ALS is TDP-43 (TAR DNA-binding protein 43), an RNA-binding protein encoded by the TARDBP gene. Under normal physiological conditions, TDP-43 predominantly resides in the nucleus, where it plays a critical role in regulating gene expression, alternative splicing, RNA transport, and stability. In ALS, TDP-43 undergoes pathological mislocalization from the nucleus to the cytoplasm, disrupting its normal function and contributing to disease progression. The nuclear loss of TDP-43 leads to widespread dysregulation of RNA metabolism. Moreover, mislocalized TDP-43 aggregates in the cytoplasm, acquires toxic properties that sequester essential RNA molecules and proteins. Importantly, deviations in TDP-43 levels, whether excessive or reduced, can lead to cellular dysfunction, and contribute to disease progression, highlighting the delicate balance required for neuronal health. Emerging evidence suggests that epigenetic mechanisms may play a crucial role in regulating TARDBP expression and, consequently, TDP-43 cellular levels. Epigenetic modifications such as DNA methylation, histone modifications, and non-coding RNAs are increasingly recognized as modulators of gene expression and cellular function in neurodegenerative diseases, including ALS. Dysregulation of these processes could contribute to aberrant TARDBP expression, amplifying TDP-43-associated pathologies. This review explores and summarizes the recent findings on how specific epigenetic modifications influence TDP-43 expression and discusses their possible implications for disease progression.

Aging progressively modifies the physiological balance of the organism increasing susceptibility to both genetic and sporadic neurodegenerative diseases. These changes include epigenetic chromatin remodeling events that may modify the transcription levels of disease-causing genes affecting neuronal survival. However, how these events interconnect is not well understood. Here, we found that Su(var)3-9 causes increased methylation of histone H3K9 in the promoter region of TDP-43, the most frequently altered factor in amyotrophic lateral sclerosis (ALS), affecting the mRNA and protein expression levels of this gene through epigenetic modifications that appear to be conserved in agedDrosophilabrains, mouse, and human cells. Remarkably, augmented Su(var)3-9 activity causes a decrease in TDP-43 expression followed by early defects in locomotor activities. In contrast, decreasing Su(var)3-9 action promotes higher levels of TDP-43 expression, improving motility parameters in old flies. The data uncover a novel role of this enzyme in regulating TDP-43 expression and locomotor senescence and indicate conserved epigenetic mechanisms that may play a role in the pathogenesis of ALS.

Myotonic dystrophy 2 (DM2) is a genetic multi-systemic disease primarily affecting skeletal muscle. It is caused by CCTGn expansion in intron 1 of the CNBP gene, which encodes a zinc finger protein. DM2 disease has been successfully modeled in Drosophila melanogaster, allowing the identification and validation of new pathogenic mechanisms and potential therapeutic strategies. Here, we describe the principal tools used in Drosophila to study and dissect molecular pathways related to muscular dystrophies and summarize the main findings in DM2 pathogenesis based on DM2 Drosophila models. We also illustrate how Drosophila may be successfully used to generate a tractable animal model to identify novel genes able to affect and/or modify the pathogenic pathway and to discover new potential drugs.

Aging progressively modifies the physiological balance of the organism increasing susceptibility to both genetic and sporadic neurodegenerative diseases. These changes include epigenetic chromatin remodeling events that may modify gene transcription. However, how aging interconnects with disease-causing genes is not well known. Here, we found that Su(var)3-9 causes increased methylation of histone H3K9 in the promoter region of TDP-43, the most frequently altered factor in amyotrophic lateral sclerosis (ALS), affecting the mRNA and protein expression levels of this gene through epigenetic modifications in chromatin organization that appear to be conserved in aged Drosophila brains, mouse and human cells. Remarkably, augmented Su(var)3-9 activity causes a decrease in TDP-43 expression followed by early defects in locomotor activities. In contrast, decreasing Su(var)3-9 action promotes higher levels of TDP-43 expression and reinvigorates motility parameters in old flies, uncovering a novel role of this enzyme in regulating TDP-43 expression and locomotor senescence. The data indicate how conserved epigenetic mechanisms may link aging with neuronal diseases and suggest that Su(var)3-9 may play a role in the pathogenesis of ALS.