Periodic Reporting for period 1 - INNERVATE (Investigating the functional impact of neurodegeneration-causing rare variants in the teleost model zebrafish)
Berichtszeitraum: 2020-05-01 bis 2022-04-30
Neurodevelopmental disorders with early-onset neurodegeneration are heterogenous rare diseases affecting small children and causing progressive loss of motor milestones and spasticity within the first years of life. Because of their rarity, the scarcity of specialized centers and pharmaceutical attention, the majority of these cases remain invisible and untreatable. A dramatic burden on patients, families and the healthcare system derives from it. A partial solution comes from the recent advancement of genetic sequencing that is unraveling potential genetic causes of previously unknown diseases. Several candidate genes involved in the control of the dynamic structure of the cellular architecture (cytoskeleton) are being associated to these diseases. Nevertheless, experimental approaches are needed which i) validate the pathogenic effect of the mutations and ii) study their deleterious impact on cellular and sub-cellular processes in the context of brain development. This is needed to impact on the search of targetable molecules and mechanisms likely generalizable also to other neurodegenerative conditions (i.e. those manifesting in adulthood). As proposed, my MSCA-IF project “INNERVATE” ( 844636) combined inter-sector expertise (i.e. molecular and developmental biology with clinical genetics) to generate disease models and advance our mechanistic understanding of the pathology. Our modeling in small fish embryo focused on a subset of these encephalopathies caused by gene perturbations recently identified within a larger sequencing effort of undiagnosed cases at the host institute (Ospedale pediatrico Bambino Gesù). Small and transparent fish (zebrafish) allow to observe embryogenesis (and its alterations) without need of invasive methods. By microscopy observation, molecular biology and biochemistry, their fast development allows us to determine morphogenesis, molecular and behavioral defects within short time. The data obtained so far establish the involvement of impaired motoneuron development and innervation in the disease. Neurobehavioral assays were established to quantitatively assess locomotor fitness decay and provide sharable resources that can be exploited for further research and pre-clinical applications.
We generated transient and stable animal models mimicking the loss of function patterns of TBCD and TBCE genes identified in a cohort of patients. In these genetic models we analyzed nervous system development, spinal neurons innervation and locomotor behavior. As planned, collaborative efforts allowed us also receive training and establish CRISPR/Cas-based models. Correct motor function depends on the proper development of neurons specialized in long range connections with muscle fibers and other (inter)neurons. Employing high-resolution microscopy, biochemistry and image analysis, we performed a first multi-level (subcellular, cellular and behavioral) characterization of the zebrafish mutants. This already established 1) a clear gene dosage effect on the disease traits, with partial protein loss involved in worsening of motor fitness; 2) a defective morphogenesis of the long motoneurons neurites required for correct innervation and 3) a downstream impairment in muscle innervation patterns, which could be alleviated by expression of the gene of interest. By probing the health of the cytoskeleton in developing motoneurons by employing microscopy and transgenic tools we began to 4) shed light on the sub-cellular alterations of the cytoskeleton in these specialized neurons. The results and the models obtained already represent a new resource for further research. They can be employed as pre-clinical tools to determine the effective developmental windows for possible intervention or to investigate further the patho-mechanisms. In this context, an important long-term goal of the proposed project consisted in the successful installation of collaborations with specialists in other fields and further independent exploitation of the results. Funding has been secured for collaborative efforts aimed to further analyze the models generated in comparison with alternative in vitro motoneurons preparations derived from patients’ stem cells and clinical assessment procedures of patients’ locomotor impairments. In agreement with the proposal, a good fraction of INNERVATE was dedicated to a) personnel mentoring, b) transfer of knowledge and c) dissemination and communication activities. During this time, I contributed to training on the specific methodologies young post-doctoral fellows and PhD students. I also actively share the approach and first results nationally and internationally with peers and experts in different fields. Thanks to the received MSCA-IF-funding, and despite the severe restrictions imposed by COVID-19 pandemics, the work was presented -and is planned to- to at least 5 scientific conferences. These include the International Zebrafish society and Zebrafish disease models annual meeting and the Italian Society of human genetics conference. The oral dissemination activities have so far received two awards and a nomination for best contribution 2022 (Italian Society of human genetics). Moreover, two peer reviewed publications another manuscripts in preparation result so far from the work. I also engaged into communications and outreach activities connected also to INNERVATE: 1. contribution to the periodical OPBG journal on platforms for research; 2. participating as researcher testimonial for “The European Researchers' Night, 2021- Horizon” at OPBG, producing video material (https://youtu.be/Zf4tWYZ-0Sg)(öffnet in neuem Fenster); 3. participating as invited instructor for scientific seminars on Animal Welfare and research ("La Sapienza" University, Italy).
In marked contrast to the overwhelming research progress achieved for common neurological pathologies, rare neurodevelopmental diseases displaying a early-onset neurodegenerative spectrum remain poorly investigated, leaving patients diagnostic and therapeutic care needs unmet. The work carried under this MSCA project contributed to generating a fast cross-disciplinary in vivo workflow for functional genomics that will be exploited beyond the diseases examined here. Bringing comparative embryology and neurobiology expertise to a hospital-type of research setting, the work has validated and investigated mechanistically the impact of genetic alterations recently identified in a group of patients with infantile neurodegeneration. Addressing the specifics of the pathological mechanisms and sharing the results offers differential diagnosis tools and stimulate the search for targeted cure. Adding to recent reports for a similar condition, we are among the first to clarify defective spinal motoneurons development and homeostasis as a major disease sign, which was so far hypothesized, maifested before the onset of locomotor disabilities. We began to correlate genotypes to phenotypes, showing a dosage effect in an organismal context, that provides insights to explain progressive motor deterioration. The data ultimately contribute to inform diagnosis with respect to life quality and therapeutic prospective. We expect to finalize soon the data collection for the preliminarily characterization of the stable zebrafish model of disease and for peer review publication. These will be made available within the scientific community for further exploitation, open-end hypothesis testing and drug screening.