Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Understanding the contribution of factors-mediated pathogenetic mechanisms in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder due to motor neuron degeneration and characterized by muscle atrophy, weakness and, ultimately, muscle paralysis. It is associated in 20% of the familial cases with mutations in the gene coding for the superoxide dismutase-1 (SOD1), which is a scavenger enzyme involved in the antioxidant cellular response. ALS is considered as a multi-systemic and multifactorial disease that involves different molecular mechanisms, cell types and tissues (Musarò, 2013). In particular, skeletal muscle plays an important role in ALS pathogenesis; indeed, according to the “Dying back” hypothesis, retrograde signals from muscle to nerve can contribute to axon and neuromuscular synapses damage (Dadon-Nachum, 2011). Our recent works have revealed that skeletal muscle is a primary target of SOD1G93A mediated toxicity (Dobrowolny et al., 2008) and demonstrated that Protein kinase C theta (PKCθ), physiologically involved in synapses pruning (Nelson, 2004), is chronically activated in a transgenic mouse model overexpressing the mutated SOD1G93A gene under the control of a muscle specific promoter (MLC/SOD1G93A mice) (Dobrowolny et al., 2018). PKCθ pharmacological inhibition in the MLC/SOD1G93A mice was able to rescue mitochondrial functionality, neuromuscular junction (NMJ) architecture and its turnover (Dobrowolny et al., 2018). Therefore, based on these previous results, we elucidated whether PKCθ inhibition can counteract muscle functional decline associated with disease progression in ALS mouse model. To unravel this point, we used two alternative approaches, namely genetic ablation, and pharmacological inhibition of PKCθ in ALS SOD1G93A mice. While complete genetic ablation of PKCθ confirmed the physiologic role of the kinase during the maturation stage of NMJ, the pharmacologic inhibition at the stage of the diseases associated with NMJ alteration (symptomatic phase) can ameliorate clinical outcomes, NMJ morphology and muscle and NMJ functionality in ALS SOD1G93A mice. PKCθ plays also an important role in skeletal muscle metabolic homeostasis: it can induce a metabolic shift towards an oxidative metabolism by the regulation of factors that govern the fast-to-slow shift in skeletal muscle fibers (Camerino et al., 2014). Moreover, it has been reported that both ALS patients (O’Reilly et al., 2013) and ALS mouse model (Dupuis et al., 2017) show a hypermetabolic condition and a reduction in body mass index (BMI) at the presymptomatic phase, correlating with higher risk of ALS pathogenesis and poor prognosis. In this context PKCθ can be considered a molecular link between muscle metabolic adaptations and NMJ alteration. Our recent work has disclosed that muscle expression of SOD1G93A is associated with metabolic changes and in particular we have demonstrated that metabolic alterations in muscle fibers occur independently of motor neuron degeneration, underlining skeletal muscle as an important therapeutic target in ALS disease (Dobrowolny et al., 2018). Here we observed that PKCθ pharmacological inhibition can attenuate muscle metabolic changes occurring during ALS disease by counteracting the typical ALS fast-to-slow shift in ALS SOD1G93A mice at the terminal phase of the disease. Hypothalamus is the main central player in metabolic homeostasis and several works confirmed the strict relationship between hypothalamic damage and neurodegenerative diseases, including ALS (Dupuis et al., 2018). Hypothalamus can regulate energy expenditure through the melanocortin system, which is located in the region of arcuate nucleus. Therefore, to evaluate the metabolic homeostasis during ALS disease, we analyzed two main interactors of the melanocortin system (AgRP and POMC) in the hypothalamus of ALS mouse model at the terminal phase of the disease and we observed altered expression levels of both AgRP and POMC in this mouse model. Another important hypothalamic region, that is in communication with the arcuate nucleus, is the superchiasmatic nucleus, which controls circadian metabolic rhythms in mammals and is compromised in neurodegenerative diseases (Hood and Amir, 2017). We observed an altered circadian gene expression of the master interactors (Clock and BMAL) in both skeletal muscle and hypothalamic tissue in ALS SOD1G93A mice at the end stage of the disease. Furthermore, in order to elucidate whether the metabolic circadian alterations can precede clinical symptoms, we analyzed circadian gene expression of Clock and BMAL both in SOD1G93A mice at the pre-symptomatic phase of the disease and in the MLC/SOD1G93A adult mice. We observed altered gene expressions in both mice models, confirming that circadian metabolic alterations can occur independently and even before motor neuron degeneration. For this reason, circadian metabolic alterations can be considered a therapeutic tool in order to delay ALS onset and disease progression. Therefore based on evidence underlining timing of food and nutritional changes as some of the main entrainments of circadian clocks (Kohsaka, 2007; Sassone-Corsi 2013), ALS SOD1G93A mice were fed with a high fat diet with the attempt to develop a therapeutic approach that can delay disease onset and progression by “resynchronizing” the physiological circadian clock genes expression. We observed that under high fat diet condition, ALS mice model exhibit an improvement in clinical outcomes, in muscle and NMJ functionality and a resetting of the altered circadian clock genes expression compared to ALS SOD1G93A mice under control diet. Based on all these data, future perspectives of the project will be to combine PKCθ pharmacological inhibition and high fat diet nutritional approach in order to verify whether the recovery of the NMJ functionality together with the restoration of circadian metabolic homeostasis, could be able to delay ALS onset and disease progression improving quality of life. To translate our results about metabolic circadian alteration in ALS mice model into the clinical practice, we performed an array of circadian rhythm genes expression on peripheral blood mononuclear cells (PBMCs) of ALS patients and healthy individuals. We observed that in ALS patients there is a dysregulation, in terms of repression, of the circadian core clock’s regulators that are also strictly related to metabolic and inflammatory pathways, confirming the complexity of the pathology. In a future perspective this analysis could be useful to provide more details on ALS patients metabolic and circadian profiles, revealing new molecular targets and metabolic signaling involved in the disease, in order to guarantee a more personalized therapeutic approach in ALS patients.