Dottorato in NEUROSCIENZE CLINICO-SPERIMENTALI E PSICHIATRIA

Titolo della tesi: Neuroimmunity in Neurodevelopmental Disorders: Antibodies detection to neuronal surface antigens in Chronic Tic Disorders

Summary The role of immunological processes in the etiopathogenesis of neurodevelopmental disorders is of increasing scientific interest, with recent data amount. Indeed, a multifactorial model has been postulated, in which both polygenic vulnerability and environmental factors are involved in these disorders, including immunologic changes. According to the “immune theory” of neurodevelopmental disorders, an immune system trigger (i.e. toxins, infections or maternal immune molecules), acting in different stages of growth (from prenatal to postnatal periods) will transfer in a genetic vulnerable individual, causing alterations in neural circuits that lead to the neurodevelopmental disorder. Several antigenic targets in the central nervous system (CNS), as CASPR2, LGI1 and NMDAR, have been candidates as antibody-mediated neurodevelopment disorder, given their relevance in normal brain development. Tourette Syndrome (TS) represents a valid model for the study of neurodevelopmental disorders because of clinic and pathophysiological overlaps with other neurodevelopment conditions (i.e. autism; adhd; obsessive-compulsive disorders). In TS a role for autoantibodies directed against neuronal proteins has long been suspected, particularly involving deregulations in the dopamine system, but so far results are still inconsistent. This doctoral thesis is divided into two parts. In the first part I have reviewed the state of the art of neurodevelopmental disorders (Austism; ADHD; OCD; Tourette/tic diosrders) associated with neuro-immune alterations (cytokines; microglia activation, surface-antibodies), and the role of the most relevant antigenic targets in the CNS, as CASPR2, LGI1 and NMDAR, during several stage of the neurodevelopment. The second part is constituted by two-experimental studies, aimed to look for antibodies to specific or undefined neuronal proteins that could be involved in the aetiology of TS/Tic disorders (TDs), as part of the EMTICS project, a prospective European longitudinal observational study for tic disorders in children. The results of these studies have been published (Addabbo F et al., 2020; Baglioni V. et al., 2019). The first experimental research was focused on D2DR antibodies (D2DR-Ab) detection in 137 patients during symptoms exacerbation, carried on in collaboration with the University of Bari, under the supervision of Professor Davide Martino and Maura Buttiglione. The second experimental research was on 218 EMTICS participants sera, resulted D2DR-Ab negative, looking for those antibodies that had already been described in autoimmune neurological diseases (NMDAR; CASPR2; LGI1, AMPAR, GABAAR), and aimed to search for evidence of possible unknown antibodies in TS/TD in different disease stages (preclinic; clinic; chronic). I personally led on the second study and laboratory examinations during one-year internship period in the Nuffield Department of Clinical Neuroscience of Oxford University, under the supervision of professor Angela Vincent. Serum anti-D2R antibodies were evaluated using a cell-based assay, as for the presence of specific antibodies to NMDAR, CASPR2, LGI1, AMPAR and GABAAR. Presence of antibodies binding to rodent brain tissue was assessed by immunohistology on rat brain sections and by immunofluorescent staining of live hippocampal neurons. In the D2R antibodies study, 11 (8%) participants became anti-D2R-positive at exacerbation (‘early peri- exacerbation seroconverters’), and nine (6.6%) became anti-D2R-positive at post-exacerbation (‘late peri-exacerbation seroconverters’). The anti-D2R antibodies were significantly associated with exacerbations when compared to baseline (McNemar’s odds ratio=11, p=0.003) and conditional logistic regression confirmed this association (Z=3.49, p<0.001) after adjustment for demographic and clinical data and use of psychotropic drugs. In the second study, for the research of know and unknown surface-neuroantibodies, in D2R-antibodies negative participants, immunohistology indicated evidence of antibodies reactive with brain tissue, binding mainly to the hippocampus, the basal ganglia or the cerebellum in 26/218 (12%), with 8% of the preclinical or onset sera binding to the dentate gyrus/CA3 region or cerebellum. Only two individuals (one pre-clinical, one chronic) had antibodies binding the NMDAR and the binding was only weakly positive. No other specific antibodies were detected. There is a potential association between immune mechanisms and the severity course of tics in adolescents with TS/TDs. To the best of our knowledge, this is the first study exploring the association between anti-D2R antibodies and the clinical course of chronic TDs. The observed association between anti-D2R antibodies and tic exacerbations remains an important finding with potential, albeit still to verify, pathophysiological implications, regardless of the relative frequency of these antibodies in the tic disorder population. Findings cannot support the use of these antibodies as a novel clinical biomarker, but expand the existing evidence of immune dysregulation in these conditions, supported also by the reported neuronal reactivity towards rodent brain tissue sections in a limited number of samples.