La Rocca, Federica et al. (2021) International Worm Meeting "hnRNPQ/hrp-2 role in splicing and neurodegeneration."

Cieri, Federica, Di Schiavi, Elia, La Rocca, Federica, Santonicola, Pamela, Zampi, Giuseppina

[International Worm Meeting, 2021]

A correct splicing of mRNA is globally required in all cells, but neurons seem highly sensitive to perturbations with numerous neurological diseases caused by splicing defects, including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and dementias. Neurons have features that make them vulnerable to mis-splicing events, such as their postmitotic state and longevity. However, the reason of this peculiar sensitivity of neurons to splicing alterations, why only subclasses of neurons are affected and which step of RNA processing is impaired in these diseases is still debated. We are investigating the molecular mechanisms underlying the neurodegeneration caused by splicing defects in SMA, using a neuron specific silencing approach in C.elegans. SMA is a motor neurons (MNs) specific disorder caused by mutations in the Survival Motor Neuron (SMN) gene. RNA-sequencing of iPS-MNs from SMA patients and healthy people allowed us to identify differentially expressed/spliced genes, enriched in RNA motif 7 (CAAAAAG). This motif is specifically bound by hnRNPQ, a protein interacting with SMN in the SMN complex. hrp-2 is the C.elegans homolog of hnRNPQ and we determined that, similarly to smn-1(KO), hrp-2 (ok1278) null mutants arrest as larvae, have a severe decrease in lifespan and locomotion defects. hrp-2 mutants show a reduction of visible D-type MNs and axonal defects, suggesting a specific role in neuron survival. hrp-2 overexpression in D-type MNs rescued the degeneration of MNs and the defects in locomotion observed in smn-1 silenced animals. These data suggest a specific involvement of hrp-2 in MNs survival and in smn-1 pathway, supporting the role of hrp-2 as an smn-1 modifier and as a possible therapeutic target for the disease. Further experiments are ongoing to better understand the genetic interaction between the two genes in MNs and to identify the role of mis-spliced targets in neurodegeneration.

Cieri, Federica et al. (2021) International Worm Meeting "B-Raf contribution to motoneuron degeneration"

Cieri, Federica, Di Schiavi, Elia, Claus, Peter, Santonicola, Pamela, Hensel, Niko, Zampi, Giuseppina

[International Worm Meeting, 2021]

The B-Raf orthologue lin-45 belongs to MAPK/ERK pathway, is involved in the regulation of cellular proliferation and differentiation and shows a Ras GTPase binding activity. Beside its well-known role in vulva formation, lin-45 is also expressed in neurons, including motoneurons, and is critical for chemo- and thermo-sensory behaviors and locomotion. Using a phospho-antibody array combined with a network-biology approach on Spinal Muscular Atrophy (SMA) mice, we identified B-Raf as a signaling hub among different dysregulated pathways involved in motor neuron degeneration. In SMA, motor neurons degenerate because of mutations in the Survival Motor Neuron 1 (SMN1) gene. We investigated in C. elegans the role of B-Raf/lin-45 in degenerating motoneurons (MNs) and observed that lin-45 expression is reduced in pre-symptomatic smn-1(KO) animals. We rescued the neurodegeneration and locomotion defects caused by smn-1 silencing in D-type MNs by re-expressing lin-45 specifically in MNs. We thus demonstrated that lin-45 can play a cell-autonomous neuroprotective role when smn-1 is downregulated. Consistently, a hyperactive isoform of lin-45 (S312A), which is devoid of an inhibitory phospho-site, maximized the rescue effects. The neurodegeneration caused by smn-1 silencing increases with animal ageing and we were able to rescue it by expressing lin-45 from L2 stage, through inducible transgenics. This is important since at L2 stage the degeneration has already started and all 19 D-type motoneurons should have been generated, thus suggesting that lin-45 protects from motoneuron loss rather than interfering with neurogenesis and can block the degeneration when it is already started. Using MAPK pathway drug inhibitors and mek-2 mutants we abrogated the rescue obtained after lin-45 overexpression. Thus, genetic and pharmacological approaches showed that lin-45 rescue is mediated by the MAPK/ERK pathway. The central role of B-Raf was confirmed in a SMA model of SMA and in patient cells and strongly support a role of B-RAF in neurodegeneration and in particular in the Smn1 pathway.

Catalano, Federico et al. (2021) International Worm Meeting "Characterization of a new model of Wilson disease to find innovative targets and pathways to attenuate Cu toxicity"

Santonicola, Pamela, Zampi, Giuseppina, Puchkova, Ludmila, Di Schiavi, Elia, Ilicheva, Ekaterina, Orlov, Iurii, Polishchuk, Roman, Polishchuk, Elena, Catalano, Federico

[International Worm Meeting, 2021]

Copper is an essential nutrient that is required for many vitally important processes, such as respiration, connective tissue biogenesis and iron metabolism. However, excess of copper represents a serious danger, due to its ability to induce free radical-induced oxidative damage, as well as impairments of lipid metabolism and neuronal activity. Wilson disease (WD) represents an excellent system for Cu toxicity studies. WD is caused by mutations in ATP7B pump effluxing excess Cu from hepatocytes into the bile. Loss of ATP7B leads to toxic Cu overload in liver and then in brain, causing fatal hepatic and neurologic abnormalities. Over the last years, C. elegans has emerged as an easy-to-use and highly responsive model of micronutrient metabolism. For this reason, we have generated and characterized a new C. elegans model of WD. CUA-1, the C. elegans ortholog of ATP7B, resides in lysosome-like organelles to sequester excess of copper and, therefore, represents a key component regulating copper supply and detoxification in order to maintain copper homeostasis. Using Crispr-Cas9 technology a cua-1(knu781 [H828Q]) strain was generated, that carries a substitution in conserved histidine corresponding to the most common H1069Q variant of ATP7B causing WD in European and North American population. In order to understand the effect of H828Q mutation on C. elegans phenotype we employed several assays that allow life span, motility, egg laying, larval development and mitochondria damage to be evaluated. Moreover, we are studying the colocalization of the mutant CUA-1 protein with markers of different organelles, such as lysosome, Golgi and ER, as well as with the copper sensor CF4. Our studies established that in absence of copper, cua-1(knu781) does not show any significant phenotypic aberrations. However, mutant worms exhibited very poor resistance to copper compared to the control strain. This manifested in a strong decrease in number of eggs, a delay in the larval development, a shorter lifespan, impaired motility and mitochondrial damage. Taken together our finding suggest that cua-1(knu781) represents an excellent model for Cu toxicity studies in WD. We further plan to use this model for identification and validation of the new therapeutic targets for WD. Moreover, cua-1(knu781) will be used for evaluation of the FDA-approved drugs that emerged from a high throughput screening for compounds reducing Cu toxicity in WD.