Age-related neurodegenerative diseases are highly debilitating and incurable pathologies that impinge a high socio-economic burden on our society (El-Hayek et al., 2019). They share a progressive degeneration of neurons, which results in loss of brain function and a heterogeneous array of incapacitating symptoms (Dugger & Dickson, 2017). Therapeutic strategies for brain restoration consist of compensating for neuronal loss by generating new neurons from the existing stem cell pools that can integrate into the existing circuitry. The capacity for neuroregeneration is naturally limited in the adult mammalian brain (Zhao et al., 2016). Neural stem cells

Palmitoylation has been recently identified as an important post-translational rheostat for controlling protein function in eukaryotes. However, the molecular machinery underlying palmitoylation remains unclear in the neglected tropical parasite, Leishmania donovani. Herein, we have identified a catalog of 20 novel palmitoyl acyltransferases (PATs) and characterized the promastigote-specific PAT (LdPAT4) containing the canonical Asp-His-His-Cys (DHHC) domain. Immunofluorescence analysis using in-house generated LdPAT4-specific antibody demonstrated distinct expression of LdPAT4 in the flagellar pocket of promastigotes. Using metabolic labeling-coupled click chemistry method, the functionality of this recombinant enzyme could be authenticated in E. coli strain expressing LdPAT4-DHHC domain. This was evident by the cellular uptake of palmitic acid analogs, which could be successfully inhibited by 2-BMP, a PAT-specific inhibitor. Using CSS-Palm based in-silico proteomic analysis, we could predict up to 23 palmitoylated sites per protein in the promastigotes, and further identify distinctive palmitoylated protein clusters involved in microtubule assembly, flagella motility and vesicular trafficking. To highlight, proteins such as Flagellar Member proteins (FLAM1, FLAM5), Intraflagellar Transport proteins (IFT88), and flagellar motor assembly proteins including the Dynein family were found to be enriched. Furthermore, analysis of global palmitoylation in promastigotes using Acyl-biotin exchange purification identified a set of S-palmitoylated proteins overlapping with the in-silico proteomics data. The attenuation of palmitoylation using 2-BMP demonstrated several phenotypic alterations in the promastigotes including distorted morphology, reduced motility (flagellar loss or slow flagellar beating), and inefficient invasion of promastigotes to host macrophages. These analyses confirm the essential role of palmitoylation in promastigotes. In summary, the findings suggest that LdPAT4 acts as a functional acyltransferase that can regulate palmitoylation of proteins involved in parasite motility and invasion, thus, can serve as a potential target for designing chemotherapeutics in Visceral Leishmaniasis.

Mature human erythrocytes contain a rich pool of microRNAs (miRNAs), which result from differentiation of the erythrocytes during the course of haematopoiesis. Recent studies have described the effect of erythrocytic miRNAs on the invasion and growth of the malaria parasite Plasmodium falciparum during the asexual blood stage of its life cycle. In this work, we have identified two erythrocytic miRNAs, miR-150-3p and miR-197-5p, that show favourable in silico hybridization with Plasmodium apicortin, a protein with putative microtubule-stabilizing properties. Co-expression of P. falciparum apicortin and these two miRNAs in a cell line model resulted in downregulation of apicortin at both the RNA and protein level. To create a disease model of erythrocytes containing miRNAs, chemically synthesized mimics of miR-150-3p and miR-197-5p were loaded into erythrocytes and subsequently used for invasion by the parasite. Growth of the parasite was hindered in miRNA-loaded erythrocytes, followed by impaired invasion; micronemal secretion was also reduced, especially in the case of miR-197-5p. Apicortin expression was found to be reduced in miRNA-loaded erythrocytes. To interpret the effect of downregulation of apicortin on parasite invasion to host erythrocytes, we investigated the secretion of the invasion-related microneme protein apical membrane antigen 1 (AMA1). AMA1 secretion was found to be reduced in miRNA-treated parasites. Overall, this study identifies apicortin as a novel target within the malaria parasite and establishes miR-197-5p as its miRNA inhibitor. This miRNA represents an unconventional nucleotide-based therapeutic and provides a new host factor-inspired strategy for the design of antimalarial molecular medicine. This article has an associated First Person interview with the first author of the paper.

Lipid‐based palmitoylation is a post‐translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, Plasmodium falciparum. P. falciparum palmitoylation is catalyzed by 12 putative palmitoyl acyl‐transferase enzymes containing the conserved DHHC‐CRD (DHHC motif within a cysteine‐rich domain) which can serve as a druggable target. However, the paucity of high‐throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of Escherichia coli, a PTM‐null system, to enforce ectopic expression of palmitoyl acyl‐transferase in order to study Plasmodium‐specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium‐specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl‐transferase activity via acyl‐biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC‐expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli proteins. Upon treatment with generic inhibitor 2‐bromopalmitate (2‐BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high‐throughput screening platform.

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 structureactivity relationship, which in turn helps to improve the therapeutic activity of lead compounds. K E Y W O R D Sbioisosterism, high-throughput kinase assay, imidazolidinones, leishmaniasis, target deconvolution

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.

The African turquoise killifish uniquely combines a short lifespan with vertebrate-specific features, including age-dependent loss of neuroregenerative capacity, that are missing from the currently used model organisms. In this study, we investigate the cellular landscape that shapes adult neuro- and gliogenesis using single-cell sequencing. Our analysis identifies seventeen cell types including neuronal cells (NC), and progenitors (PC) of glial and non-glial nature in the adult killifish telencephalon. PC subclustering unveils four radial glia types, one atypical non-glial progenitor (NGP) and two clusters representing transitioning states. NC subclustering classified neurons into immature and mature excitatory or inhibitory sub-clusters. Using lineage inference analysis, we discovered neuroepithelial-like radial glia to be the source of neuro- and gliogenesis, and a central role for NGP. Our findings are evidence for specialized progenitors within telencephalon and the data is accessible via an online database, providing a resource to understand normal brain function, as well as the role of cellular relationships in response to injury and disease.

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.