Trkb Hyperactivity Contributes to Brain Dysconnectivity, Epileptogenesis, and Anxiety in Zebrafish Model of Tuberous Sclerosis Complex
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TrkB hyperactivity contributes to brain dysconnectivity, epileptogenesis, and anxiety in zebrafish model of Tuberous Sclerosis Complex Magdalena Kedraa, Katarzyna Banasiaka, Katarzyna Kisielewskaa, Lidia Wolinska-Niziola, Jacek Jaworskia,1, and Justyna Zmorzynskaa,1 aLaboratory of Molecular and Cellular Neurobiology, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved December 16, 2019 (received for review June 29, 2019) Tuberous Sclerosis Complex (TSC) is a rare genetic disease that thology during embryonic development in utero when cortical manifests with early symptoms, including cortical malformations, malformations that lead to epileptogenesis arise (14). Thus, the childhood epilepsy, and TSC-associated neuropsychiatric disorders present study comprehensively examined brain development and (TANDs). Cortical malformations arise during embryonic develop- behavior in zebrafish, which provide an opportunity to study early ment and have been linked to childhood epilepsy before, but the neuronal development in vivo due to external development and underlying mechanisms of this relationship remain insufficiently body transparency. Although zebrafish do not contain a cortex per understood. Zebrafish have emerged as a convenient model to se, the homologous structures are present, the majority of which study elementary neurodevelopment; however, without in-depth are localized to the pallium (15–17). functional analysis, the Tsc2-deficient zebrafish line cannot be Previous studies investigated the utility of tsc2vu242 mutant used for studies of TANDs or new drug screening. In this study, we zebrafish as a model of TSC and reported abnormalities in pallial found that the lack of Tsc2 in zebrafish resulted in heterotopias WM organization (including the disruption of WM with ectopic cell and hyperactivation of the mTorC1 pathway in pallial regions, bodies), a decrease in locomotion, and abnormal brain activity in which are homologous to the mammalian cortex. We observed the optic tectum (18, 19). However, these studies did not elaborate commissural thinning that was responsible for brain dysconnec- potential disease mechanisms. Moreover, neurobehavioral changes tivity, recapitulating TSC pathology in human patients. The lack of that mimic symptoms of human TANDs were not investigated in NEUROSCIENCE Tsc2 also delayed axonal development and caused aberrant tract tsc2vu242/vu242 mutants. Therefore, in this study, we performed an in- fasciculation, corresponding to the abnormal expression of genes depth analysis of changes in brain connectivity during development. involved in axon navigation. The mutants underwent epilepto- We evaluated tsc2vu242 fish behavior and mapped seizure activity genesis that resulted in nonmotor seizures and exhibited increased and anxiety-like behavior to discrete parameters of locomotion. anxiety-like behavior. We further mapped discrete parameters of Finally, we tested the ability of drugs to rescue TSC-associated locomotor activity to epilepsy-like and anxiety-like behaviors, which phenotypes and found that reducing tyrosine receptor kinase B were rescued by reducing tyrosine receptor kinase B (TrkB) signaling. (TrkB) signaling reversed brain dysconnectivity, epileptogenesis, Moreover, in contrast to treatment with vigabatrin and rapamycin, TrkB inhibition rescued brain dysconnectivity and anxiety-like Significance behavior. These data reveal that commissural thinning results in the aberrant regulation of anxiety, providing a mechanistic link between brain anatomy and human TANDs. Our findings also Tuberous Sclerosis Complex (TSC) is a hereditary disease that implicate TrkB signaling in the complex pathology of TSC and reveal presents with early brain malformations, childhood epilepsy, a therapeutic target. and TSC-associated neuropsychiatric disorders (TANDs). Corti- cal malformations arise in utero and have been linked to Tuberous Sclerosis Complex | TANDs | brain dysconnectivity | anxiety | TrkB childhood epilepsy before. Externally developing zebrafish seem convenient to study elementary neurodevelopment; however, without the in-depth functional analysis, the Tsc2- uberous Sclerosis Complex (TSC) is an autosomal dominant deficient zebrafish cannot be used for studies of TANDs. Here, Tdisorder that is caused by loss-of-function mutations in the we found that Tsc2-deficient zebrafish recapitulated symptoms TSC1 or TSC2 genes (1), the products of which form a complex seen in TSC patients on anatomical and behavioral levels, in- that negatively regulates mechanistic/mammalian target of cluding aberrant brain morphology, thinning of brain connec- rapamycin (mTOR) complex 1 (mTORC1), a complex necessary tions, epileptogenesis, and increased anxiety-like behavior, for proper neuronal development (2). In TSC, epilepsy is the which was rescued by reducing TrkB signaling, revealing a most prominent neurological symptom, which begins in the first potential drug target. Moreover, we show that commissural year of life and often evolves into an intractable form (3). Epi- thinning cause aberrant regulation of anxiety, providing a link lepsy is accompanied by TSC-associated neuropsychiatric disor- between brain anatomy and emotion. ders (TANDs), including intellectual disability (ID) and anxiety (4). Epilepsy is thought to be caused by cortical malformations Author contributions: J.Z. designed research; J.J. and J.Z. secured funding; M.K., K.B., K.K., (5) that consist of heterotopias, tubers, and white matter (WM) L.W.-N., and J.Z. performed research; M.K. and J.Z. analyzed data; M.K, J.J., and J.Z. interpreted the data; and M.K., J.J., and J.Z. wrote the paper. dysconnectivity in TSC (4). Tubers are often diagnosed in utero, The authors declare no competing interest. suggesting that the pathology of TSC is acquired during embry- This article is a PNAS Direct Submission. onic development (6). This open access article is distributed under Creative Commons Attribution-NonCommercial- Cortical malformations result in childhood epilepsy and in- NoDerivatives License 4.0 (CC BY-NC-ND). tractable or treatment-refractory seizures in various other diseases, 1To whom correspondence may be addressed. Email: [email protected] or underscoring the importance of proper cortex development (5). [email protected]. The existing mammalian models of TSC mimic human disease in This article contains supporting information online at https://www.pnas.org/lookup/suppl/ many aspects, including cortical malformations and seizures (7–13). doi:10.1073/pnas.1910834117/-/DCSupplemental. However, these models present difficulties in studying TSC pa- www.pnas.org/cgi/doi/10.1073/pnas.1910834117 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 + + and anxiety-like behavior. The latter phenotype was also reversed thinner than in tsc2 / fish (Fig. 2A and SI Appendix,Fig.S2A). by inhibition of Rac1, which is potentially one of the downstream The postoptic commissure was also thinner in tsc2vu242/vu242 mu- + + targets of TrkB. tants, whereas the size of the lateral tracts was similar to tsc2 / fish (SI Appendix,Fig.S2B and C). The higher number of P-Rps6– Results positive cells negatively correlated with AC thickness, revealed by Lack of Tsc2 Leads to White Matter Disruption and Brain double immunostaining (Fig. 2 A and B). Dysconnectivity in Zebrafish. A disruption of WM organization is To evaluate axon development that may underlie WM thin- a common feature of TSC and was previously found in the tel- ning, we crossed the tsc2vu242 line into transgenic Tg(ptf1a:GFP) encephalon in tsc2vu242/vu242 mutants (19). Indeed, when we ex- vu242 background and examined them by live light-sheet imaging. amined the telencephalon in tsc2 fish, we confirmed the ptf1a:GFP-positive neurons in the posterior tuberculum extend presence of ectopic cell bodies in tsc2vu242/vu242 by two-dimensional their axons toward the posterior commissure, where they cross the (2D) brain section imaging (Fig. 1A) and three-dimensional (3D) brain midline and innervate the other hemisphere (Fig. 2C). In whole-brain imaging (SI Appendix,Fig.S1A). These cells pre- + + tsc2 / , ptf1a:GFP-positive axons from one hemisphere crossed sented higher levels of phosphorylated ribosomal protein s6 at vu242/vu242 serines 235 and 236 (P-Rps6; Fig. 1 B and C and SI Appendix, Fig. the brain boundary in one bundle. The same axons in tsc2 S1 B–E), representing the hyperactivation of mTorC1. The P- presented disturbances in tract fasciculation (Fig. 2D). We were Rps6–positive cells in the pallium of tsc2vu242/vu242 mutants had able to distinguish mild axonal phenotypes (e.g., one axon did higher intensity signals and larger cell bodies compared with pal- not cross the midline in the bundle) and severe disturbances in + + lial cells of tsc2 / fish (Fig. 1 D and E and SI Appendix,Fig.S1D axon bundling (e.g., axons crossed the midline stochastically; and E). We also measured the thickness of the main brain com- SI Appendix,Fig.S3A). Quantitative morphological analysis of vu242 missures because human TSC patients exhibit WM dysconnectivity ptf1a:GFP-positive axons of tsc2 confirmed the impaired tract that is associated with epileptic seizures and worse cognitive out- fasciculation in tsc2vu242/vu242 fish, reflected by a higher number of + + comes (12, 20–22). The tsc2vu242/vu242 anterior commissure (AC) intersections at the midline compared with tsc2 / (Fig.