Protein aggregation, ageing and neurodegeneration in the emerging model Nothobranchius furzeri

The teleost Nothobranchius furzeri is an annual fish and is the shortest-lived vertebrate that can be cultured in captivity. The longer-lived strains of N. furzeri show a median lifespan of 40 weeks making it a unique model organism for ageing research. N. furzeri recapitulates the major features of mammalian brain ageing, such as gliosis, lipofuscin and iron accumulation and also more global changes at transcriptomic- and proteomic-level. Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Fronto-Temporal Dementia (FTD), are the prevalent causes of dementia and are currently viewed as cerebral proteopathies, having age as the major risk factor and in which the accumulation of specific misfolded proteins is the most relevant causative factor. In the initial part of my thesis, I detected age-dependent protein aggregation, and in particular ribosome aggregation, in the brain of N. furzeri during physiological ageing. This observation is key to suggest this organism as a model for observing and studying neurodegeneration. In my thesis work, I laid the groundwork for future studies in this direction, and, in particular, I focused my attention on studying PD- related and TDP43-related protein aggregation and neurodegeneration. Parkinson’s disease is the second most common age-related neurodegenerative disorder after Alzheimer’s disease with an estimated 7-10 million persons affected worldwide. Its main neuropathological feature is the aggregation of the phosphorylated form of the protein α-Synuclein (α-Syn) creating characteristic formations called Lewy bodies in the soma and Lewy neurites in neuronal processes. This aggregation is considered the major cause of the neurodegeneration of Tyrosine Hydroxylase (TH) enzyme containing central neurons belonging to the Noradrenergic and Dopaminergic system in the Substantia Nigra and in the Locus coeruleus nuclei, respectively. To assess possible PD-related neurodegeneration in N. furzeri, I first analyzed the expression of TH in Nothobranchius furzeri brain and realized a whole mount 3D map of the monoaminergic system in clarified brains. Secondly, I compared the amount of TH positive cells between young- and old-animals in the Locus coeruleus and in the hypothalamic posterior tuberculum (the teleost homolog to the mammalian Substantia Nigra) to investigate markers of neurodegeneration. I analyzed animals at 5, 12 and 37 weeks of age and found a statistically significant age-dependent reduction of TH+ neurons in the Locus coeruleus but not in the posterior tuberculum. Immunoreactivity for phospho-Synuclein (pSyn) in the soma of Locus coeruleus neurons was detected already at 5 weeks of age, indicating initial aggregation early in life, with the signal increasing in older samples. No signs of immunoreactivity for phospho-Synuclein were detected in the somata of TH+ hypothalamic neurons at any age. This observation supports the link between the accumulation of somatic phospho-Synuclein and neurodegeneration that is restricted to Locus coeruleus neurons. This observation is also in line with Braak’s staging of human PD development according to α-Syn immunoreactivity: Braak hypothesizes a caudo-rostral spreading of aggregation and neurodegeneration starting in the vagal nuclei, then spreading to the Locus coeruleus and only in later stages involving the Substantia nigra. Staining for aggregated proteins in Locus coeruleus cells also revealed presence of aggresomes localized mainly in vacuolized areas of cytoplasm devoid of TH staining in old samples. Overall, this data point to N. furzeri showing a pre-symptomatic like form of PD-related neurodegeneration during normal ageing. FTD is a neurodegenerative disease involving aggregation and mislocalization of TDP-43. TDP-43 is normally localized into neuronal nuclei, but in FTD patients it localizes in the cytoplasm near the nuclear envelope forming aggregates. To assess TDP-43 localization in N. furzeri brain during ageing, I performed immunofluorescence labelling on both sections and whole mount brains for TDP-43 in young and old animals and I detected abnormal TDP-43 localization in old brains: the protein concentrated in proximity of the nuclear envelope. I was also able to detect protein aggregates stained by aggresome dye associated with cells showing an abnormal TDP-43 staining, supporting the idea of the correlation between abnormal TDP-43 localization and protein aggregation. Moreover, I was also able to observe localization of TDP-43 staining overlapping with G3BP in stress granules (SG) in accordance with the known role of TDP-43 in regulating SG formation. Finally, I also developed organotypic slice culture of Nothobranchius furzeri to exploit the fast ageing of this model for in vitro studies. I report culturing of thick brain slices for at least five weeks. I could also demonstrate persistence of adult neurogenesis and survival of TH+ cells in these cultures