Age-related dysregulation of the retinal transcriptome in African turquoise killifish.

Age-related vision loss caused by retinal neurodegenerative pathologies is becoming more prevalent in our ageing society. To understand the physiological and molecular impact of ageing on retinal homeostasis, we used the short-lived African turquoise killifish, a model known to naturally develop central nervous system (CNS) ageing hallmarks and vision loss. Bulk and single-cell RNA-sequencing (scRNAseq) of three age groups (6-, 12-, and 18-week-old) identified transcriptional ageing fingerprints in the killifish retina, unveiling pathways also identified in the aged brain, including oxidative stress, gliosis, and inflammageing. These findings were comparable to observations in the ageing mouse retina. Additionally, transcriptional changes in genes related to retinal diseases, such as glaucoma and age-related macular degeneration, were observed. The cellular heterogeneity in the killifish retina was characterized, confirming the presence of all typical vertebrate retinal cell types. Data integration from age-matched samples between the bulk and scRNAseq experiments revealed a loss of cellular specificity in gene expression upon ageing, suggesting potential disruption in transcriptional homeostasis. Differential expression analysis within the identified cell types highlighted the role of glial/immune cells as important stress regulators during ageing. Our work emphasizes the value of the fast-ageing killifish in elucidating molecular signatures in age-associated retinal disease and vision decline. This study contributes to the understanding of how age-related changes in molecular pathways may impact CNS health, providing insights that may inform future therapeutic strategies for age-related pathologies.

Age-related dysregulation of the retinal transcriptome in African turquoise killifish.

Age-related vision loss caused by retinal neurodegenerative pathologies is becoming more prevalent in our ageing society. To understand the physiological and molecular impact of ageing on retinal homeostasis, we used the short-lived African turquoise killifish, a model known to naturally develop central nervous system (CNS) ageing hallmarks and vision loss. Bulk and single-cell RNA-sequencing (scRNA-seq) of three age groups (6-, 12-, and 18-week-old) identified transcriptional ageing fingerprints in the killifish retina, unveiling pathways also identified in the aged brain, including oxidative stress, gliosis, and inflammageing. These findings were comparable to observations in ageing mouse retina. Additionally, transcriptional changes in genes related to retinal diseases, such as glaucoma and age-related macular degeneration, were observed. The cellular heterogeneity in the killifish retina was characterised, confirming the presence of all typical vertebrate retinal cell types. Data integration from age-matched samples between the bulk and scRNA-seq experiments revealed a loss of cellular specificity in gene expression upon ageing, suggesting potential disruption in transcriptional homeostasis. Differential expression analysis within the identified cell types highlighted the role of glial/immune cells as important stress regulators during ageing. Our work emphasises the value of the fast-ageing killifish in elucidating molecular signatures in age-associated retinal disease and vision decline. This study contributes to the understanding of how age-related changes in molecular pathways may impact CNS health, providing insights that may inform future therapeutic strategies for age-related pathologies.

A review on neurodegeneration in the fast-ageing killifish, the first animal model to study the natural occurrence of neuronal cell loss.

Thanks to medical and technological improvements, our world population has become ever-greying. In consequence, the incidence and prevalence of age-related central nervous system neuropathies, such as Alzheimer's (AD) and Parkinson's disease (PD), are increasing tremendously. Despite many research efforts, the precise aetiology of these age-related neurodegenerative disorders remains elusive, highlighting the urgent need for more effective treatments. Current preclinical research mainly uses animal models that do not fully recapitulate the complex cellular context in which these diseases occur, thereby lacking good construct validity. Indeed, most investigations are performed using relatively young animals, thereby ignoring the ageing environment in which neurodegenerative diseases manifest. This points out a major hiatus in current research: a vertebrate model organism that combines the complex disease context (onset, spreading and further manifestation into functional impairment) with an ageing environment. In recent years, the African turquoise killifish has emerged as a promising novel animal model to study age-related neurodegenerative disorders that combines these essential features. In this review, we bundle all reported findings up till now and provide a detailed overview of the neurodegenerative events within the central nervous system of this teleost fish, with a focus on PD.

A new microfluidic model to study dendritic remodeling and mitochondrial dynamics during axonal regeneration of adult zebrafish retinal neurons.

Unlike mammals, adult zebrafish are able to fully regenerate axons and functionally recover from neuronal damage in the mature central nervous system (CNS). Decades of research have tried to identify the mechanisms behind their spontaneous regenerative capacity, but the exact underlying pathways and molecular drivers remain to be fully elucidated. By studying optic nerve injury-induced axonal regrowth of adult zebrafish retinal ganglion cells (RGCs), we previously reported transient dendritic shrinkage and changes in the distribution and morphology of mitochondria in the different neuronal compartments throughout the regenerative process. These data suggest that dendrite remodeling and temporary changes in mitochondrial dynamics contribute to effective axonal and dendritic repair upon optic nerve injury. To further elucidate these interactions, we here present a novel adult zebrafish microfluidic model in which we can demonstrate compartment-specific alterations in resource allocation in real-time at single neuron level. First, we developed a pioneering method that enables to isolate and culture adult zebrafish retinal neurons in a microfluidic setup. Notably, with this protocol, we report on a long-term adult primary neuronal culture with a high number of surviving and spontaneously outgrowing mature neurons, which was thus far only very limitedly described in literature. By performing time-lapse live cell imaging and kymographic analyses in this setup, we can explore changes in dendritic remodeling and mitochondrial motility during spontaneous axonal regeneration. This innovative model system will enable to discover how redirecting intraneuronal energy resources supports successful regeneration in the adult zebrafish CNS, and might facilitate the discovery of new therapeutic targets to promote neuronal repair in humans.

Immune stimulation recruits a subset of pro-regenerative macrophages to the retina that promotes axonal regrowth of injured neurons.

The multifaceted nature of neuroinflammation is highlighted by its ability to both aggravate and promote neuronal health. While in mammals retinal ganglion cells (RGCs) are unable to regenerate following injury, acute inflammation can induce axonal regrowth. However, the nature of the cells, cellular states and signalling pathways that drive this inflammation-induced regeneration have remained elusive. Here, we investigated the functional significance of macrophages during RGC de- and regeneration, by characterizing the inflammatory cascade evoked by optic nerve crush (ONC) injury, with or without local inflammatory stimulation in the vitreous. By combining single-cell RNA sequencing and fate mapping approaches, we elucidated the response of retinal microglia and recruited monocyte-derived macrophages (MDMs) to RGC injury. Importantly, inflammatory stimulation recruited large numbers of MDMs to the retina, which exhibited long-term engraftment and promoted axonal regrowth. Ligand-receptor analysis highlighted a subset of recruited macrophages that exhibited expression of pro-regenerative secreted factors, which were able to promote axon regrowth via paracrine signalling. Our work reveals how inflammation may promote CNS regeneration by modulating innate immune responses, providing a rationale for macrophage-centred strategies for driving neuronal repair following injury and disease.

Analysis of Axonal Regrowth and Dendritic Remodeling After Optic Nerve Crush in Adult Zebrafish.

Neurodegenerative diseases and central nervous system (CNS) injuries are frequently characterized by axonal damage, as well as dendritic pathology. In contrast to mammals, adult zebrafish show a robust regeneration capacity after CNS injury and form the ideal model organism to further unravel the underlying mechanisms for both axonal and dendritic regrowth upon CNS damage. Here, we first describe an optic nerve crush injury model in adult zebrafish, an injury paradigm that inflicts de- and regeneration of the axons of retinal ganglion cells (RGCs), but also triggers RGC dendrite disintegration and subsequent recovery in a stereotyped and timed process. Next, we outline protocols for quantifying axonal regeneration and synaptic recovery in the brain, using retro- and anterograde tracing experiments and an immunofluorescent staining for presynaptic compartments, respectively. Finally, methods to analyze RGC dendrite retraction and subsequent regrowth in the retina are delineated, using morphological measurements and immunofluorescent staining for dendritic and synaptic markers.

Analysis of Visual Recovery After Optic Nerve Crush in Adult Zebrafish.

Zebrafish can successfully regenerate axons after optic nerve crush (ONC). Here, we describe two different behavioral tests to map visual recovery: the dorsal light reflex (DLR) test and the optokinetic response (OKR) test. The DLR is based on the tendency of fish to orient their back to a light source, and it can be tested by rotating a flashlight around the dorsolateral axis of the animal or by measuring the angle between the left/right body axis and the horizon. The OKR, in contrast, consists of reflexive eye movements triggered by motion in the visual field of the subject and is measured by placing the fish in a drum on which rotating black-and-white stripes are projected.

Visually Driven Behavior Assays to Study Functionality and Recovery after Damage of the Aged African Turquoise Killifish Visual System.

Loss of vision is a prominent feature of aging and vision is considered by many to be the most valuable sense to be lost. In our graying society, we are increasingly challenged by age-related deterioration of the central nervous system (CNS), as well as by age-associated neurodegenerative diseases and brain injuries, all often affecting the visual system and thus its performance. Here, we describe two visually driven behavior assays to evaluate visual performance upon aging or CNS damage in the fast-aging killifish. The first test, the optokinetic response (OKR), measures the reflexive eye movement triggered by motion in the visual field and allows assessment of visual acuity. The second assay, the dorsal light reflex (DLR), evaluates the swimming angle based on input of light coming from above. The OKR can be used to study the effect of aging on visual acuity as well as visual improvement and recovery after rejuvenation therapy or visual system injury or disease, whereas the DLR is best used to assess functional repair after a unilateral optic nerve crush.

Morphological Analysis of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As the number of elderly individuals is increasing in modern society, the need for a relevant gerontology model is higher than ever before. Aging can be defined by specific cellular hallmarks, described by López-Otín and colleagues, who provided a map which can be used to scavenge the aging tissue environment. As revealing the presence of individual hallmarks does not necessarily indicate aging, here we provide different (immuno)histochemical approaches that can be used to investigate several aging hallmarks-namely, genomic damage, mitochondrial dysfunction/oxidative stress, cellular senescence, stem cell exhaustion, and altered intercellular communication-in the killifish retina, optic tectum, and/or telencephalon at a morphological level. In combination with molecular and biochemical analysis of these aging hallmarks, this protocol offers the opportunity to fully characterize the aged killifish central nervous system.

The Optic Nerve Crush Injury Paradigm in African Turquoise Killifish to Study Axonal Regeneration in an Aged Environment.

In our graying world population, we are increasingly facing brain injuries and age-associated neurodegenerative diseases, which are often characterized by axonal pathology. Here, we propose the killifish visual/retinotectal system as a model for investigating central nervous system repair, more specifically axonal regeneration, in an aging context. We first describe an optic nerve crush (ONC) injury paradigm in killifish to induce and study both de- and regeneration of retinal ganglion cells (RGCs) and their axons. Subsequently, we summarize several methods for mapping different steps of the regenerative process-namely, axonal regrowth and synapse reformation-using retro- and anterograde tracing methods, (immuno)histochemistry, and morphometrical analyses.

Tissue stretching is a confounding factor for the evaluation of neurodegeneration in the fast-ageing killifish.

The fast-ageing killifish has gained increasing attention as a promising gerontology model to study age-related processes and neurodegeneration. Interestingly, it is the first vertebrate model organism that shows physiological neuron loss at old age in its central nervous system (CNS), including its brain and retina. However, the fact that the killifish brain and retina are ever-growing tissues complicates studying neurodegenerative events in aged fish. Indeed, recent studies showed that the method of tissue sampling, either using sections or whole-organs, has a large effect on the observed cell densities in the fast-expanding CNS. Here, we elaborated on how these two sampling methods affect neuronal counts in the senescent retina and how this tissue grows upon ageing. Analysis of the different retinal layers in cryosections revealed age-dependent reduction in cellular density but evaluation of whole-mount retinas did not detect any neuron loss, as a result of an extremely fast retinal expansion with age. Using BrdU pulse-chase experiments, we showed that the young adult killifish retina mainly grows by cell addition. However, with increasing age, the neurogenic potency of the retina declines while the tissue keeps on growing. Further histological analyses revealed tissue stretching, including cell size increase, as the main driver of retinal growth at old age. Indeed, both cell size and inter-neuronal distance augment with ageing, thereby decreasing neuronal density. All in all, our findings urge the 'ageing science' community to consider cell quantification bias and employ tissue-wide counting methods to reliably quantify neuronal numbers in this unique gerontology model.

Molecular and Biochemical Analyses of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As modern society is graying, aging research and biogerontology models, in which the aging process can be studied, are becoming increasingly important. A proper aging model can be defined as one that displays many of the aging hallmarks. Here, we provide two different practical approaches-namely, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting-that can be used to investigate cellular senescence (RT-qPCR for p21 and p27), altered intercellular communication/inflammaging (RT-qPCR for il-10, sirt-1, il-6, il-1b, il-8, and tnf), and oxidative stress (western blotting for 4-HNE) in the killifish central nervous system, and, more specifically, in the retina, optic tectum, and telencephalon. These molecular and biochemical analyses are a first step in confirming the aging characteristics but should preferably be combined with morphological analyses.

Optic nerve injury-induced regeneration in the adult zebrafish is accompanied by spatiotemporal changes in mitochondrial dynamics.

Axonal regeneration in the central nervous system is an energy-intensive process. In contrast to mammals, adult zebrafish can functionally recover from neuronal injury. This raises the question of how zebrafish can cope with this high energy demand. We previously showed that in adult zebrafish, subjected to an optic nerve crush, an antagonistic axon-dendrite interplay exists wherein the retraction of retinal ganglion cell dendrites is a prerequisite for effective axonal repair. We postulate a 'dendrites for regeneration' paradigm that might be linked to intraneuronal mitochondrial reshuffling, as ganglion cells likely have insufficient resources to maintain dendrites and restore axons simultaneously. Here, we characterized both mitochondrial distribution and mitochondrial dynamics within the different ganglion cell compartments (dendrites, somas, and axons) during the regenerative process. Optic nerve crush resulted in a reduction of mitochondria in the dendrites during dendritic retraction, whereafter enlarged mitochondria appeared in the optic nerve/tract during axonal regrowth. Upon dendritic regrowth in the retina, mitochondrial density inside the retinal dendrites returned to baseline levels. Moreover, a transient increase in mitochondrial fission and biogenesis was observed in retinal ganglion cell somas after optic nerve damage. Taken together, these findings suggest that during optic nerve injury-induced regeneration, mitochondria shift from the dendrites to the axons and back again and that temporary changes in mitochondrial dynamics support axonal and dendritic regrowth after optic nerve crush.

The age factor in optic nerve regeneration: Intrinsic and extrinsic barriers hinder successful recovery in the short-living killifish.

As the mammalian central nervous system matures, its regenerative ability decreases, leading to incomplete or non-recovery from the neurodegenerative diseases and central nervous system insults that we are increasingly facing in our aging world population. Current neuroregenerative research is largely directed toward identifying the molecular and cellular players that underlie central nervous system repair, yet it repeatedly ignores the aging context in which many of these diseases appear. Using an optic nerve crush model in a novel biogerontology model, that is, the short-living African turquoise killifish, the impact of aging on injury-induced optic nerve repair was investigated. This work reveals an age-related decline in axonal regeneration in female killifish, with different phases of the repair process being affected depending on the age. Interestingly, as in mammals, both a reduced intrinsic growth potential and a non-supportive cellular environment seem to lie at the basis of this impairment. Overall, we introduce the killifish visual system and its age-dependent regenerative ability as a model to identify new targets for neurorepair in non-regenerating individuals, thereby also considering the effects of aging on neurorepair.

MMP2 Modulates Inflammatory Response during Axonal Regeneration in the Murine Visual System.

Neuroinflammation has been put forward as a mechanism triggering axonal regrowth in the mammalian central nervous system (CNS), yet little is known about the underlying cellular and molecular players connecting these two processes. In this study, we provide evidence that MMP2 is an essential factor linking inflammation to axonal regeneration by using an in vivo mouse model of inflammation-induced axonal regeneration in the optic nerve. We show that infiltrating myeloid cells abundantly express MMP2 and that MMP2 deficiency results in reduced long-distance axonal regeneration. However, this phenotype can be rescued by restoring MMP2 expression in myeloid cells via a heterologous bone marrow transplantation. Furthermore, while MMP2 deficiency does not affect the number of infiltrating myeloid cells, it does determine the coordinated expression of pro- and anti-inflammatory molecules. Altogether, in addition to its role in axonal regeneration via resolution of the glial scar, here, we reveal a new mechanism via which MMP2 facilitates axonal regeneration, namely orchestrating the expression of pro- and anti-inflammatory molecules by infiltrating innate immune cells.

The killifish visual system as an in vivo model to study brain aging and rejuvenation.

Worldwide, people are getting older, and this prolonged lifespan unfortunately also results in an increased prevalence of age-related neurodegenerative diseases, contributing to a diminished life quality of elderly. Age-associated neuropathies typically include diseases leading to dementia (Alzheimer's and Parkinson's disease), as well as eye diseases such as glaucoma and age-related macular degeneration. Despite many research attempts aiming to unravel aging processes and their involvement in neurodegeneration and functional decline, achieving healthy brain aging remains a challenge. The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived reported vertebrate that can be bred in captivity and displays many of the aging hallmarks that have been described for human aging, which makes it a very promising biogerontology model. As vision decline is an important hallmark of aging as well as a manifestation of many neurodegenerative diseases, we performed a comprehensive characterization of this fish's aging visual system. Our work reveals several aging hallmarks in the killifish retina and brain that eventually result in a diminished visual performance. Moreover, we found evidence for the occurrence of neurodegenerative events in the old killifish retina. Altogether, we introduce the visual system of the fast-aging killifish as a valuable model to understand the cellular and molecular mechanisms underlying aging in the vertebrate central nervous system. These findings put forward the killifish for target validation as well as drug discovery for rejuvenating or neuroprotective therapies ensuring healthy aging.

Injury-induced Autophagy Delays Axonal Regeneration after Optic Nerve Damage in Adult Zebrafish.

Optic neuropathies comprise a group of disorders in which the axons of retinal ganglion cells (RGCs), the retinal projection neurons conveying visual information to the brain, are damaged. This results in visual impairment or even blindness, which is irreversible as adult mammals lack the capacity to repair or replace injured or lost neurons. Despite intensive research, no efficient treatment to induce axonal regeneration in the central nervous system (CNS) is available yet. Autophagy, the cellular recycling response, was shown repeatedly to be elevated in animal models of optic nerve injury, and both beneficial and detrimental effects have been reported. In this study, we subjected spontaneously regenerating adult zebrafish to optic nerve damage (ONC) and revealed that autophagy is enhanced after optic nerve damage in zebrafish, both in RGC axons and somas, as well as in macroglial cells of the retina, the optic nerve and the visual target areas in the brain. Interestingly, the pattern of the autophagic response in the axons followed the spatiotemporal window of axonal regrowth, which suggests that autophagy is ongoing at the growth cones. Pharmacological inhibition of the recycling pathway resulted in accelerated RGC target reinnervation, possibly linked to increased mechanistic target of rapamycin (mTOR) activity, known to stimulate axonal regrowth. Taken together, these intriguing findings underline that further research is warranted to decipher if modulation of autophagy could be an effective therapeutic method to induce CNS regeneration.

A novel retinal ganglion cell quantification tool based on deep learning.

Glaucoma is a disease associated with the loss of retinal ganglion cells (RGCs), and remains one of the primary causes of blindness worldwide. Major research efforts are presently directed towards the understanding of disease pathogenesis and the development of new therapies, with the help of rodent models as an important preclinical research tool. The ultimate goal is reaching neuroprotection of the RGCs, which requires a tool to reliably quantify RGC survival. Hence, we demonstrate a novel deep learning pipeline that enables fully automated RGC quantification in the entire murine retina. This software, called RGCode (Retinal Ganglion Cell quantification based On DEep learning), provides a user-friendly interface that requires the input of RBPMS-immunostained flatmounts and returns the total RGC count, retinal area and density, together with output images showing the computed counts and isodensity maps. The counting model was trained on RBPMS-stained healthy and glaucomatous retinas, obtained from mice subjected to microbead-induced ocular hypertension and optic nerve crush injury paradigms. RGCode demonstrates excellent performance in RGC quantification as compared to manual counts. Furthermore, we convincingly show that RGCode has potential for wider application, by retraining the model with a minimal set of training data to count FluoroGold-traced RGCs.