Deciphering brain ageing using the visual system of the short-lived killifish

Many people underestimate the impact ageing can have on their life. Most often those persons only have some of the typical macroscopic characteristics of ageing, such as wrinkles, grey hair, a slower reaction time, and minor functional loss of their sight and/or hearing. Those are, however, the few lucky ones. Indeed, within the greying society, an increasing number of seniors are succumbing to age-related diseases. Many organs are at risk to develop such disorders, but the central nervous system (CNS) is particularly vulnerable as it comprises billions of high-energy-demanding neurons that are tremendously susceptible to ageing. The fact that the adult CNS is irreparable only accentuates the challenges associated with age-related damage and its accumulation. Elderly individuals, affected by age-related CNS deficits, often present with sensory deficiencies, diminished cognition and impaired locomotor function. These deficits may manifest regardless of whether they are caused by typical age-related neurodegenerative diseases such as dementia, Alzheimer's (AD) and Parkinson's (PD) disease, glaucoma, and age-related macular degeneration (AMD). To ensure a higher quality of life at old age, there is a pressing need for gerontological research focused on studying brain ageing. A golden standard framework has been designed to study human ageing, which delineates 12 cellular and molecular hallmarks of ageing, including genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. These defined hallmarks enable a standardized assessment of aged tissues. An accumulation of these ageing indicators in the CNS can lead to its previously described functional deterioration. Unfortunately, many of the canonical vertebrate model organisms, such as zebrafish, rats, and mice, have a relatively long lifespan, hampering gerontological studies. Higher-throughput ageing research can be performed in an emerging non-canonical animal model, the African turquoise killifish. This teleost is the shortest-lived vertebrate that can be kept in captivity with a median lifespan of a few months. It rapidly accumulates ageing hallmarks and presents subsequently with pathological phenotypes. However, in-depth knowledge on how the killifish CNS ages remains largely elusive. To further expand the insights on brain ageing, the killifish visual system seems to be a valuable tool. Indeed, the visual pathway, an integral part of the CNS, is highly conserved in both structure and function across vertebrates, allowing for translation between teleost and mammals. Using (immuno)histological and molecular techniques, the occurrence of several ageing hallmarks was investigated in the visual system. To gain insights into the timely emergence of these hallmarks, experiments were conducted across four distinct age groups of female fish: young adults (6 weeks, w), middle-aged (12w), old (18w), and very old (24w). Several ageing hallmarks, such as mitochondrial dysfunction, stem cell exhaustion, and altered intercellular communication, could already be noticed rather early in life, i.e., at middle-age, while other hallmarks, such as genomic damage, chronic inflammation, and cellular senescence, only manifested in old and very old fish. To evaluate whether the accumulation of these hallmarks resulted in a functional phenotype, both morphological approaches and behavioural assays were used. Interestingly, the aged retina clearly exhibited a degenerative phenotype on spatially pre-sampled preparations, characterised by thinning of the retinal layers, and a reduction in the number of cells in the ganglion cell and inner nuclear layer. Additionally, using the optokinetic response test, a significant age-related decrease in visual acuity could be observed. Overall, these data confirmed that the aged killifish visual system is a valuable tool to study CNS ageing. To delve deeper into the molecular signature of the ageing killifish retina and potentially discover novel drivers of ageing, we investigated the transcriptional dynamics of retinal tissue from aged fish. By employing bulk and single-cell RNA sequencing (scRNA-seq) on young adult, middle-aged and old female killifish, many of the previously identified ageing hallmarks, including stem cell exhaustion, inflammageing, gliosis, and oxidative stress, were confirmed. Interestingly, the aged killifish retina showed transcriptomic signs of known (age-related) retinal pathologies, such as glaucoma, AMD and Usher syndrome. Also, a clear upregulation of the gene encoding alpha-synuclein (αSYN) was observed. The scRNA-seq dataset revealed the cellular heterogeneity of the killifish retina, in which all major retinal cell types were detected and spatially validated using in situ hybridisation chain reaction. Integration of the bulk and scRNA-seq datasets revealed a global dysregulation of transcription in the aged retina. Indeed, retinal cell types started to express or increased expression of transcripts for which they normally do not show enrichment. Neurons, for example, upregulated glial stress response genes. The aggregate of upregulated genes detected by bulk RNA-seq showed, however, to be most highly expressed in glial and innate immune cells. As our data provided evidence for potential neurodegeneration in the aged killifish retina, we conducted a study assessing retinal cell loss in relation to its growth. Prior studies in zebrafish have indicated that retinal growth is not solely dependent on cell addition but is also facilitated by tissue stretching. The latter is known to be a confounding factor when evaluating cell numbers on spatially pre-sampled preparations. Our investigations revealed that the killifish retina has two distinct growth phases. During the exponential expansion of the tissue, at young age, cell addition is the main driver of growth. As the fish ages and the neurogenic niche of the retina, the ciliary marginal zone, becomes depleted, growth transitions from being primarily supported by cell addition to being facilitated by tissue stretching. Indeed, the old killifish retina exhibits an increase in intercellular distance as well as a rise in cell size. Therefore, cell numbers must be evaluated tissue-wide. Automated cell counts on retinal whole-mounts for retinal ganglion cells, dopaminergic and cholinergic amacrines no longer confirmed the previously observed degenerative phenotype. However, these observations do not entirely rule out the presence of neurodegeneration. Most likely there is a nuanced interplay between cell addition, tissue stretching and neurodegeneration that potentially masks the rather restricted neurodegenerative events in the retina. The killifish brain, however, does show a clear degenerative phenotype, as was extensively reviewed within this dissertation. Certainly, a Parkinson-like phenotype emerges within the aged killifish brain. In addition to the aggregation and toxic post-translational modification of αSYN in the ageing brain, also loss of dopaminergic and noradrenergic neurons could be detected in specific brain areas. There are even indications suggesting a prion-like propagation of pathology, as described in patients with PD. Beside the presence of this Parkinsonian phenotype, indications for the development of AD and amyotrophic lateral sclerosis/frontotemporal dementia were observed, as evidenced by the detection of amyloid β-plaques and TAR DNA binding protein 43 aggregates in the brain. In summary, I strongly believe that the findings presented in this thesis offer the ageing field with significant insights and provide evidence that the killifish is a valuable model to study brain ageing. Further research focussing on retinal degeneration and specific retinal diseases, such as glaucoma and AMD, would greatly benefit the ageing and neurodegeneration communities. I believe that one day, research in the killifish will contribute to development of novel therapeutic interventions targeting (retinal) degeneration, healthy brain ageing, and rejuvenation, thereby improving the quality of life for the elderly in our greying society.

Publication year:2024

Accessibility:Embargoed