The role of mTOR in the cortical development has become clear to such a point that the pathologies caused by the various mutations in this pathway are known as "mTORopathies", being tuberous scleroris complex (TSC) and focal cortical dysplasia (FCD) the prototypes. TSC is a multi-system disorder (1/6000) caused by mutations in the Tsc1 or Tsc2 genes, while FCD shares many histopathological and clinical features with TSC such as autism, neuropsychiatric disorders and drug-resistant epileptic seizures that often oblige the patients to complex surgical interventions. Up to now, drugs that inhibit the mTOR pathway (the "rapalogs") have yielded sub-optimal results. Indeed, the amelioration of the neurological symptoms is flanked by unpleasant side-effects caused by the systemic drawbacks of mTOR blockade. Therefore, there is the need of a strategy which can target mechanisms located downstream this pathway. In these regards, a potential candidate is the neurotransmission: the excitation/inhibition balance is altered in various neurodevelopmental pathologies including the mTORopathies as TSC and FCD.
Our objective is to analyze the TSC and FCD's neurotransmission in samples with different mutations of mTOR pathway to outline an electrophysiological profile of each genetic mutation. This approach will involve next-generation sequencing, relative molecular validation on surgical TSC and FCD tissues, and electrophysiology in Xenopus oocytes transplanted with cortical membranes from the same tissues. The patch-clamp recordings on cortical slices from patients will complete the picture. Further experiments based on the results of transcriptome and molecular analysis, will test drugs able to modulate the altered neurotransmission in mTORopathies. The expected output is to gather deeper knowledge of the physiopathology of neurotransmission linked to mTOR alterations and to pave the road for future studies in order to bring this "synapse-targeted" approach to clinical practice.
The neurosurgical approaches for refractory epilepsies represent established treatment methods for a selected group of patients with malformations of cortical development, FCD and TSC. However, there is a significant proportion of patients for whom epilepsy surgery does not lead to the freedom from seizures. Moreover, some patients cannot undergo surgical interventions due to eloquent cortex involvement or multiple tubers driving epileptogenic network. In addition, surgery candidates are not free from a certain risk of surgical morbidity and mortality.
The mTOR inhibitors (e.g., rapamycin, everolimus) have been studied in mTORopathies treatment. In particular, everolimus has been approved by the FDA in 2018 for the adjunctive treatment of patients with TSC-associated partial-onset seizures (French et al., 2016, Curatolo et al., 2018). The randomized, Phase 3, placebo-controlled, double-blind clinical trials EXIST-1 and EXIST-2 demonstrated beneficial effects of everolimus on tumor growth, epilepsy and neuropsychiatric problems (particularly autism and neurocognitive deficits) in TSC, even if the use of these drugs induces reported adverse effects such as increased risk of infections, acne, amenorrhoea, disturbed wound healing, and laboratory abnormalities (Sadowski et al., 2016; Franz et al., 2015).
Therefore, preclinical research for new alternative/supplementary therapeutic options to treat neurological symptoms of mTORopathies is of high priority.
This project is focused on the investigation of physiopathological mechanisms involved in the pathogenesis of the mTORopathies that, even if partly studied, are not yet fully understood. This has not only a theoretic value, but also could pave the way for future researches aimed to the development of new therapeutic agents to treat these diseases.
Indeed, we propose a "synapse targeted"approach as a new strategy to rescue the aberrant electrophysiological phenotype found in mTORopathies.
As already mentioned, the PI has a long-standing collaboration with the group of prof. Eleonora Aronica (Department of Neuropathology, UMC, Amsterdam, The Netherlands). This collaboration already led to publications on the topic of rare diseases and mTORopathies thanks to the integration of diverse approaches and techniques of electrophysiology, molecular biology and immunohistochemistry.
The collaboration of neuropathologists and basic scientists already allowed us to achieve the aforementioned results and here, with the implementation of a highly technological method, such as the NGS we will lead to stronger conclusions correlating the transcriptome of the patients to their electrophysiological function, thus realizing a translation "from the bedside to the bench".
The decisive step towards a completely translational approach, from the bed to the lab bench and back to the clinical practice, will be the experimentation of new compounds able to modify the synaptic transmission in selected populations of patients, similarly to what observed in the past for mTOR inhibitors.
The innovation of our research is that it will be focused on the analysis of selected pathways as potential candidates to link mTOR mutations to an aberrant neurotransmission. Furthermore, the results of the NGS and molecular studies will allow to perform additional electrophysiological experiments targeting specific molecular determinants identified during this phase of the study. We aim to develop new "synapse-targeted" approach in order to, in the next future, to act on the symptoms, but also on the physiopathological alterations that are their cause, slowing down or preventing the progression of the pathology itself.
The possible progression of this experimental design will be to expand the neurotransmission analysis also to different in vitro systems , animal models and patients currently in treatment for their comorbidities linked to mTORopathies.
This is exactly what our group wants to do also by collaborating with other groups in the Department of Physiology and Pharmacology (Prof. C. Limatola's group) or with other international partners.
We are already building a possible network with Prof. J. Jaworski (Poland) and Prof. A.Becker (Germany) to perform experiments in cellular and animal models of mTORopathies.
In particular, in the next future, it will be possible to test the newly identified targets in two models: iPSC-derived NSCs from patients, and zebrafish lacking either Tsc1 and Tsc2 (Prof. J.Jaworski, Poland); and to study the structural consequences of TSC1 versus TSC2-ablation based focal brain malformations by intraventricular in-utero electroporation in mice at the single neuron level in vivo (Prof. A.Becker, Germany).
This network of new and pre-existing collaborations will strengthen the multi- and interdisciplinary nature of this proposal and strongly support the possibility of a future development of the project.