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Identification of cellular identities and characterization of the STAT3 signalling pathway in Purkinje cell layer regeneration

Periodic Reporting for period 1 - PURKINJEREG (Identification of cellular identities and characterization of the STAT3 signalling pathway in Purkinje cell layer regeneration)

Reporting period: 2016-07-01 to 2018-06-30

An increasing share of the human population suffers from various neurodegenerative diseases characterized by the loss of neuronal tissue. Understanding the molecular nature of regeneration in organisms that can invoke special programs to replenish lost structures is not only interesting to understand mechanisms of regeneration, but can also be instrumental for devising therapeutic applications for humans. Different from mammals, with only very limited or no existing neurogenic regeneration in adults, the zebrafish possesses a high regeneration capacity in many areas of the central nervous system throughout its entire lifespan. Moreover, the fast development and transparency of zebrafish larvae allow the analysis of regeneration via non-invasive bio-imaging.
In focus of our research is the cerebellum (CB), that is involved in the integration of sensory-motor information, body balance, motor learning, and cognitive behavior. Neurodegenerative diseases affecting the CB include the spinocerebellar ataxia, characterized by severe locomotor symptoms. Furthermore, cerebellar hypoplasia is highly correlated with the autism spectrum disorder. From the evolutionary point of view, the CB is a highly conserved structure, showing the same main cell types and layers, both in zebrafish and mammalians.

Classical studies on degeneration/regeneration are based on local mechanical damage, where the acute injury targets a limited area but -unlike in neurodegenerative diseases- affects several cell types within this region. Moreover, regeneration after mechanical damage can be influenced by compensatory mechanisms of residual cells from the same neuronal population or undesired side effects. Therefore, a non-invasive cell-type specific ablation system would be suitable to study molecular and cellular mechanisms of neuronal regeneration in more detail. For this purpose, the host research group designed the PC-ATTAC system (Purkinje Cell Apoptosis Through Targeted Activation of Caspase by tamoxifen), a new approach that genetically induces cell death specifically of Purkinje cells (PCs), the main output neuron population of the CB. The transgenic PC-ATTAC RFP zebrafish reporter line also allows controlled ablation of PCs at any time point of choice, that enables for example to study the input of aging for the regenerative response. Among the main objectives, we seek for functional deficits in the zebrafish CB as a consequence of PCs depletion, as well as the nature and time course of recovery programs to restore the CB function. Furthermore, we aim to reveal the cellular and molecular processes involved in the regeneration of PCs. Our combination of interdisciplinary methods, cellular and molecular biology, pharmacology, and in vivo imaging, represents a valuable experimental system to gain insight into mechanisms driving the regeneration of a specific neuronal population in the brain.
The PC-ATTAC system, designed by the host research group, allows for the genetically induced cell death of cerebellar Purkinje cells (PCs). The inducible genetic cell ablation system is based on tamoxifen-inducible Caspase 8 activity targeted to PCs leading to the apoptotic death of this cell population. At first, the efficiency of cell ablation was tested by analyzing specific markers for PCs, confirming that the vast majority of PCs were deleted. Subsequently, the time course of degeneration and recovery of the PC layer was investigated. Due to the transparency and co-expression of the fluorescent red reporter of PC-ATTAC larvae, the recovery of the PC layer can be monitored in living animals.
In order to reveal functional deficits in the zebrafish, as a consequence of losing the main type of output neurons of the cerebellum, behavioral analyses are carried out, such as the optokinetic response, a visuo-motor test. Additional locomotor tests are currently ongoing.
Regarding the cellular characterization of the PC regeneration, specific questions addressed were: which cell type provide the progenitors for regenerating PCs, and what is the time course of their proliferation/differentiation for this putative progenitor pool. For this purpose, the general cell proliferation profile of the cerebellar cells was analyzed during the time course of PC degeneration/recovery. To identify the source, we crossed the PC-ATTAC transgenic line with transgenic reporter lines, specific for cerebellar cell types such as neuronal precursors and glial cells. With respect to molecular aspects of the PC regeneration program, we started to analyze the response of several signaling pathways known to be involved in the regulation of regeneration, such as Stat3, Wnt or Notch signaling.
The findings of the current project were presented in oral presentations at three international conferences. Additionally, at the host university, the project was introduced in an institute seminar where all research groups of the Zoological Institute were present, as well as to several international guest researchers, who visited our department. A manuscript, containing the main results obtained so far is in preparation and will be published in open access. In addition, in a joint approach of the research group primary cell culture from adult brains was established and published recently.
The current project reinforces the high potential of the zebrafish as a model organism for regenerative studies, to unravel cellular and molecular mechanisms controlling neuronal regeneration, largely nonexistent in mammalians. The PC-ATTAC system allows the genetically induced ablation of a unique neuronal cell population, which provides a very useful research tool to study neurodegenerative diseases, affecting specific neuronal populations. This approach differs from classic regenerative studies, based on local mechanical damage, whose main drawback is that they affect several cell types in a certain brain area, and may trigger of plasticity by remaining cells, not damaged by this procedure, rather than regeneration. Elucidating the potential source as well as the activation process of progenitors for regenerating PCs will have a major impact to understand cellular control mechanisms underlying neuronal regeneration. Another valuable advantage of our genetically inducible in vivo system is that it also offers the possibility of selectively ablate PCs at a time point of choice, important for studies of age-related limitations of regeneration. Regarding the analysis of molecular mechanisms controlling the regeneration of the PCs, the identification of signaling pathways involved in this process will be possible. Moreover, chemical compound treatments will reveal putative mechanisms that may induce a delay or speed-up of the regenerative process. In summary, the PC-ATTAC represents an innovative tool for regenerative studies, and to address therapeutic approaches against cerebellar neurodegenerative diseases. Furthermore, the findings obtained from this project could be compared to respective mechanisms involved in the regenerative processes in other vertebrates, which might provide valuable knowledge of the mechanisms lost through evolution, entailing the limited neuro-regeneration capacities in mammalians. Additionally, this ground breaking approach may encourage the scientific community to transfer the proposed approach to study regeneration of specific cell types in other areas of the brain.
Purkinje neurons in zebrafish cerebellum