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.

Chemogenetic manipulation of astrocyte activity at the synapse- a gateway to manage brain disease.

Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability.

A short dasatinib and quercetin treatment is sufficient to reinstate potent adult neuroregenesis in the aged killifish.

The young African turquoise killifish has a high regenerative capacity, but loses it with advancing age, adopting several aspects of the limited form of mammalian regeneration. We deployed a proteomic strategy to identify pathways that underpin the loss of regenerative power caused by aging. Cellular senescence stood out as a potential brake on successful neurorepair. We applied the senolytic cocktail Dasatinib and Quercetin (D + Q) to test clearance of chronic senescent cells from the aged killifish central nervous system (CNS) as well as rebooting the neurogenic output. Our results show that the entire aged killifish telencephalon holds a very high senescent cell burden, including the parenchyma and the neurogenic niches, which could be diminished by a short-term, late-onset D + Q treatment. Reactive proliferation of non-glial progenitors increased substantially and lead to restorative neurogenesis after traumatic brain injury. Our results provide a cellular mechanism for age-related regeneration resilience and a proof-of-concept of a potential therapy to revive the neurogenic potential in an already aged or diseased CNS.

Aging impairs the essential contributions of non-glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri.

The aging central nervous system (CNS) of mammals displays progressive limited regenerative abilities. Recovery after loss of neurons is extremely restricted in the aged brain. Many research models fall short in recapitulating mammalian aging hallmarks or have an impractically long lifespan. We established a traumatic brain injury model in the African turquoise killifish (Nothobranchius furzeri), a regeneration-competent vertebrate that evolved to naturally age extremely fast. Stab-wound injury of the aged killifish dorsal telencephalon unveils an impaired and incomplete regeneration response when compared to young individuals. In the young adult killifish, brain regeneration is mainly supported by atypical non-glial progenitors, yet their proliferation capacity clearly declines with age. We identified a high inflammatory response and glial scarring to also underlie the hampered generation of new neurons in aged fish. These primary results will pave the way to unravel the factor age in relation to neurorepair, and to improve therapeutic strategies to restore the injured and/or diseased aged mammalian CNS.

Modeling Neuroregeneration and Neurorepair in an Aging Context: The Power of a Teleost Model.

Aging increases the risk for neurodegenerative disease and brain trauma, both leading to irreversible and multifaceted deficits that impose a clear societal and economic burden onto the growing world population. Despite tremendous research efforts, there are still no treatments available that can fully restore brain function, which would imply neuroregeneration. In the adult mammalian brain, neuroregeneration is naturally limited, even more so in an aging context. In view of the significant influence of aging on (late-onset) neurological disease, it is a critical factor in future research. This review discusses the use of a non-standard gerontology model, the teleost brain, for studying the impact of aging on neurorepair. Teleost fish share a vertebrate physiology with mammals, including mammalian-like aging, but in contrast to mammals have a high capacity for regeneration. Moreover, access to large mutagenesis screens empowers these teleost species to fill the gap between established invertebrate and rodent models. As such, we here highlight opportunities to decode the factor age in relation to neurorepair, and we propose the use of teleost fish, and in particular killifish, to fuel new research in the neuro-gerontology field.

Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence.

The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria.

A small bioactive glycoside inhibits epsilon toxin and prevents cell death.

Clostridium perfringens epsilon toxin (Etx) is categorized as the third most lethal bioterrorism agent by the Centers for Disease Control and Prevention (CDC), with no therapeutic counter measures available for humans. Here, we have developed a high-affinity inhibitory compound by synthesizing and evaluating the structure activity relationship (SAR) of a library of diverse glycosides (numbered 1-12). SAR of glycoside-Etx heptamers revealed exceptionally strong H-bond interactions of glycoside-4 with a druggable pocket in the oligomerization and ß-hairpin region of Etx. Analysis of its structure suggested that glycoside-4 might self-aggregate to form a robust micelle-like supra-molecular complex due to its linear side-chain architecture, which was authenticated by fluorescence spectroscopy. Further, this micelle hinders the Etx monomer-monomer interaction required for oligomerization, validated by both surface plasmon resonance (SPR) and immunoblotting. This phenomenon in turn leads to blockage of pore formation. Downstream evaluation revealed that glycoside-4 effectively blocked cell death of Etx-treated cultured primary cells and maintained cellular homeostasis via disrupting oligomerization, blocking pore formation, restoring calcium homeostasis, stabilizing the mitochondrial membrane and impairing high mobility group box 1 (HMGB1) translocation from nucleus to cytoplasm. Furthermore, a single dosage of glycoside-4 protected the Etx-challenged mice and restored normal function to multiple organs. This work reports for the first time a potent, nontoxic glycoside with strong ability to occlude toxin lethality, representing it as a bio-arm therapeutic against Etx-based biological threat.

Identification and functional characterization of a bacterial homologue of Zeta toxin in Leishmania donovani.

Zeta-toxin is a cognate toxin of epsilon antitoxin of prokaryotic Type II toxin-antitoxin system (TA) and play an important role in cell death. An orthologue of bacterial-zeta-toxin (BzT) was identified in Leishmania donovani with similar structural and functional features. Leishmania zeta-toxin (named Ld_ζ1) harboring similar UNAG and ATP-binding pockets showed UNAG kinase and ATP-binding activity. An active Ld_ζ1 was found to express in infective extracellular promastigotes stage of L. donovani and episomal overexpression of an active Ld_ζ1domain-triggered cell death. This study demonstrates the presence of prokaryotic-like-zeta-toxin in eukaryotic parasite Leishmania and its association with cell death. Conceivably, phosphorylated UNAG or analogues, the biochemical mimics of zeta-toxin function mediating cell death can act as a novel anti-leishmanial chemotherapeutics.

Design and synthesis of imidazolidinone derivatives as potent anti-leishmanial agents by bioisosterism.

Bioisosterism is a useful strategy in rational drug design to improve pharmacodynamic and pharmacokinetic properties of lead compounds. Imidazolidinones have been reported as potent kinase inhibitors and antileishmanial agents. In this study, bioisosteres of imidazolidinones (compounds 1-3) were evaluated for their antileishmanial properties. The modified imidazolidinones exhibited potent antileishmanial activity against extracellular as well as intracellular Leishmania donovani parasites in nanomolar concentrations. The selectivity index of these compounds on host cells was found to be more than 1000, emphasizing their specificity toward the parasite. Using SwissTargetPrediction software, we assessed the potential targets of these compounds and found MAPK as the most probable target. To in vitro validate, we developed a novel in vitro kinase assay that mimics the in vivo nature of the functional kinome. Compounds 1-3 displayed specific inhibition of parasite kinase activity accompanied by an increase in intracellular sodium levels in the parasites. This might be the effect of kinase inhibition that regulates sodium homeostasis through Na-ATPases. Finally, the compound-treated parasites underwent apoptosis-like death. This study represents bioisoterism as a novel approach for drug design to establish the structure-activity relationship, which in turn helps to improve the therapeutic activity of lead compounds.