The role of granulins in regulating adult zebrafish brain homeostasis and regeneration

Reduced neuronal functionality and poor neuronal recovery are among the most detrimental outcomes in aging individuals and patients with neurodegenerative diseases and/or traumatic brain injuries. Therapeutic interventions face the major problems of effectively enhancing the generation of new neurons and limiting the secondary tissue damage in the CNS. The presence of long-lasting glial scar and chronic neuroinflammation hinders the survival and proper integration of the limited pool of new neurons in the pre-existing circuitry of the mammalian CNS. In contrast to mammals, zebrafish possess numerous active stem cell niches during adulthood as constant source of neurogenesis and display extensive regenerative capacity in the CNS. The high regenerative potential correlate with the ability to inactivate the immune response in a timely manner, thus avoiding the long-lasting neuroinflammation observed in mammals. For these reasons, understanding the cellular and molecular mechanisms underlying the neurogenic potential and the regenerative capacity in the injured CNS of zebrafish may play a pivotal role in the development of new therapeutic approaches aimed to ameliorate the quality of life of aging individuals and patients with neurodegenerative diseases and/or with CNS injuries. To this goal, I first validated the relevance of zebrafish as model to study the development and progression of established age-associated hallmarks, including reduced neurogenesis, exacerbated neuroinflammation and telomere shortening. Furthermore, I demonstrated the role of granulins in regulating the aging kinetics of the adult zebrafish CNS. Granulin-deficient zebrafish showed premature aging in the brain, displaying typical age-related hallmarks already during young adulthood. Moreover, I extensively studied the regenerative response to a mild model of traumatic brain injury and characterized the activation and de-activation of microglial cells during the regenerative time course. I identified an injury-induced pro-regenerative microglial population that is initially beneficial for regeneration but needs to be inactivated in a timely manner to prevent long-lasting neuroinflammation and tissue scarring. The pro-regenerative microglial population was characterized by accumulation of lipid droplets and phase-separated TDP-43 that were promptly cleared to complete regeneration. Furthermore, I demonstrated that granulins play a pivotal role in the regulation of microglial de-activation, promoting the clearance of lipid droplets and phase separated TDP-43 in microglial cells, subsequently stimulating their transition back to homeostasis. The translational value of my research is strengthened by the presence of enhanced microglial reactivity associated with lipid droplets and TDP-43 condensates in human patients with stroke. Altogether, the core data I present in this thesis identified granulins as key regulators of aging kinetics and regeneration in the adult zebrafish CNS, making them valuable targets for the development of new therapeutic applications aimed to ameliorate age-associated hallmarks and pathological outcomes caused by traumatic brain injuries in human patients

Innate Immune Pathways Promote Oligodendrocyte Progenitor Cell Recruitment to the Injury Site in Adult Zebrafish Brain

The oligodendrocyte progenitors (OPCs) are at the front of the glial reaction to the traumatic brain injury. However, regulatory pathways steering the OPC reaction as well as the role of reactive OPCs remain largely unknown. Here, we compared a long-lasting, exacerbated reaction of OPCs to the adult zebrafish brain injury with a timely restricted OPC activation to identify the specific molecular mechanisms regulating OPC reactivity and their contribution to regeneration. We demonstrated that the influx of the cerebrospinal fluid into the brain parenchyma after injury simultaneously activates the toll-like receptor 2 (Tlr2) and the chemokine receptor 3 (Cxcr3) innate immunity pathways, leading to increased OPC proliferation and thereby exacerbated glial reactivity. These pathways were critical for long-lasting OPC accumulation even after the ablation of microglia and infiltrating monocytes. Importantly, interference with the Tlr1/2 and Cxcr3 pathways after injury alleviated reactive gliosis, increased new neuron recruitment, and improved tissue restoration

Granulins Regulate Aging Kinetics in the Adult Zebrafish Telencephalon

Granulins (GRN) are secreted factors that promote neuronal survival and regulate inflammation in various pathological conditions. However, their roles in physiological conditions in the brain remain poorly understood. To address this knowledge gap, we analysed the telencephalon in Grn-deficient zebrafish and identified morphological and transcriptional changes in microglial cells, indicative of a pro-inflammatory phenotype in the absence of any insult. Unexpectedly, activated mutant microglia shared part of their transcriptional signature with aged human microglia. Furthermore, transcriptome profiles of the entire telencephali isolated from young Grn-deficient animals showed remarkable similarities with the profiles of the telencephali isolated from aged wildtype animals. Additionally, 50% of differentially regulated genes during aging were regulated in the telencephalon of young Grn-deficient animals compared to their wildtype littermates. Importantly, the telencephalon transcriptome in young Grn-deficent animals changed only mildly with aging, further suggesting premature aging of Grn-deficient brain. Indeed, Grn loss led to decreased neurogenesis and oligodendrogenesis, and to shortening of telomeres at young ages, to an extent comparable to that observed during aging. Altogether, our data demonstrate a role of Grn in regulating aging kinetics in the zebrafish telencephalon, thus providing a valuable tool for the development of new therapeutic approaches to treat age-associated pathologies

The Surface Proteome of Adult Neural Stem Cells in Zebrafish Unveils Long-Range Cell-Cell Connections and Age-Related Changes in Responsiveness to IGF

Summary: In adult stem cell populations, recruitment into division is parsimonious and most cells maintain a quiescent state. How individual cells decide to enter the cell cycle and how they coordinate their activity remains an essential problem to be resolved. It is thus important to develop methods to elucidate the mechanisms of cell communication and recruitment into the cell cycle. We made use of the advantageous architecture of the adult zebrafish telencephalon to isolate the surface proteins of an intact neural stem cell (NSC) population. We identified the proteome of NSCs in young and old brains. The data revealed a group of proteins involved in filopodia, which we validated by a morphological analysis of single cells, showing apically located cellular extensions. We further identified an age-related decrease in insulin-like growth factor (IGF) receptors. Expressing IGF2b induced divisions in young brains but resulted in incomplete divisions in old brains, stressing the role of cell-intrinsic processes in stem cell behavior. : In this article, Chapouton and colleagues use the brain of the adult zebrafish to identify communication pathways in a native population of neural stem cells. They identify proteins expressed on the apical surfaces by a biotinylation technique and document the presence of filopodial extensions between cells. They further show the appearance of an abnormal mitotic response to IGF with age. Keywords: telencephalon, pallium, GFAP, radial glia, filopodia, lamellipodia, biotinylation, mass spectrometry, aging, neurogenesis, quiescenc