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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. [ABSTRACT FROM AUTHOR]

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 timelapse 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. [ABSTRACT FROM AUTHOR]

How many RBPMS+ retinal ganglion cells (RGCs) does a standard C57BL/6 laboratory mouse have on average and is this number substrain- or sex-dependent? Do RGCs of (European) C57BL/6J and -N mice show a different intrinsic vulnerability upon glaucomatous injury? Global RGC numbers and densities of common laboratory mice were previously determined via axon counts, retrograde tracing or BRN3A immunohistochemistry. Here, we report the global RGC number and density by exploiting the freely available tool RGCode to automatically count RGC numbers and densities on entire retinal wholemounts immunostained for the pan-RGC marker RBPMS. The intrinsic vulnerability of RGCs from different substrains to glaucomatous injury was evaluated upon introduction of the microbead occlusion model, followed by RBPMS counts, retrograde tracing and electroretinography five weeks post-injury. We demonstrate that the global RGC number and density varies between substrains, yet is not sex-dependent. C57BL/6J mice have on average 46K ± 2K RBPMS+ RGCs per retina, representing a global RGC density of 3268 ± 177 RGCs/mm2. C57BL/6N mice, on the other hand, have on average less RBPMS+ RGCs (41K ± 3K RGCs) and a lower density (3018 ± 189 RGCs/mm2). The vulnerability of the RGC population of the two C57BL/6 substrains to glaucomatous injury did, however, not differ in any of the interrogated parameters. [ABSTRACT FROM AUTHOR]

The [gamma]-secretase complex plays a role in Alzheimer's disease and cancer progression. The development of clinically useful inhibitors, however, is complicated by the role of the [gamma]-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different [gamma]-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B [gamma]-secretase in a mouse Alzheimer's disease model red to improvements of Alzheimer's disease-relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total [gamma]-secretase activity in the human brain, and thus specific targeting of Aph1B-containing [gamma]-secretase complexes may help generate less toxic therapies for Alzheimer's disease.

The mechanisms of BM hematopoietic stem/progenitor cell (HSPC) adhesion, engraftment, and mobilization remain incompletely identified. Here, using WT and transgenic mice, we have shown that membrane-anchored plasminogen activator, urokinase receptor ([.sup.M]uPAR) marks a subset of HSPCs and promotes the preservation of the size of this pool of cells in the BM. Loss or inhibition of [.sup.M]uPAR increased HSPC proliferation and impaired their homing, engraftment, and adhesion to the BM microenvironment. During mobilization, [.sup.M]uPAR was inactivated by plasmin via proteolytic cleavage. Cell-autonomous loss of the gene encoding [.sup.M]uPAR also impaired long-term engraftment and multilineage repopulation in primary and secondary recipient mice. These findings identify [.sup.M]uPAR and plasmin as regulators of the proliferation, marrow pool size, homing, engraftment, and mobilization of HSPCs and possibly also of HSCs., Introduction Hematopoietic stem/progenitor cells (HSPCs) refer to a heterogenous population of HSCs and slightly more committed hematopoietic progenitor cells (HPCs). BM HSPCs express various cell surface receptors, such as Sca-1, [...]

One important facet of glaucoma pathophysiology is axonal damage, which ultimately disrupts the connection between the retina and its postsynaptic brain targets. The concurrent loss of retrograde support interferes with the functionality and survival of the retinal ganglion cells (RGCs). Previous research has shown that stimulation of neuronal activity in a primary retinal target area—i.e., the superior colliculus—promotes RGC survival in an acute mouse model of glaucoma. To build further on this observation, we applied repeated chemogenetics in the superior colliculus of a more chronic murine glaucoma model—i.e., the microbead occlusion model—and performed bulk RNA sequencing on collicular lysates and isolated RGCs. Our study revealed that chronic target stimulation upon glaucomatous injury phenocopies the a priori expected molecular response: growth factors were pinpointed as essential transcriptional regulators both in the locally stimulated tissue and in distant, unstimulated RGCs. Strikingly, and although the RGC transcriptome revealed a partial reversal of the glaucomatous signature and an enrichment of pro-survival signaling pathways, functional rescue of injured RGCs was not achieved. By postulating various explanations for the lack of RGC neuroprotection, we aim to warrant researchers and drug developers for the complexity of chronic neuromodulation and growth factor signaling. [ABSTRACT FROM AUTHOR]

Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of technologies that enable detection of gene expression in the whole organ or embryo is essential. Here, we describe an optimized protocol of whole mount multiplexed RNA in situ hybridization chain reaction version 3.0 (HCR v3.0) in combination with immunohistochemistry (IHC), followed by fructose-glycerol clearing and light sheet fluorescence microscopy (LSFM) imaging on Octopus vulgaris embryos. We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent in situ mRNA visualization. As proof of concept, neuronal (Ov-elav) and glial (Ov-apolpp) markers were used for multiplexed HCR v3.0. Neural progenitor (Ov-ascl1) and precursor (Ov-neuroD) markers were combined with immunostaining for phosphorylated-histone H3, a marker for mitosis. After comparing several tissue clearing methods, fructose-glycerol clearing was found optimal in preserving the fluorescent signal of HCR v3.0. The expression that was observed in whole mount octopus embryos matched with the previous expression data gathered from paraffin-embedded transverse sections. Three-dimensional reconstruction revealed additional spatial organization that had not been discovered using two-dimensional methods. [ABSTRACT FROM AUTHOR]