Search Results (12)

Background/Objectives: The normal cellular prion protein (PrP^C) is a cell-surface glycoprotein, mainly localised in neurons of the central nervous system (CNS). The human PRNP gene encodes 253 amino acid residues of precursor PrP^C. Several studies that investigated the role of PRNP and PrP^C in placental mammals, such as humans and mice, failed to reveal its exact function. Methods: In this study, we sequenced and characterised the PRNP gene and PrP^C of the marsupial, P. calura, as a strategy to gain molecular insights into its structure and physicochemical properties. Placentals are separated from marsupials by approximately 125 million years of independent evolution. Results: Standard Western blotting analysis of PrP^C phascogale displayed the typical un-, mono-, and di-glycosylated bands recognized in placentals. Furthermore, we showed that phascogale PRNP gene has two exons, similar to all the marsupials and placentals of the PRNP genes studied. Of note, the phascogale PRNP gene contained distinctive repeats in the PrP^C tail region comparable to the closely related Tasmanian devil (Sarcophilus harrisii) and more distantly related to the grey short-tailed opossum (Monodelphis domestica), common wombat (Vombatus ursinus), and Tammar wallaby (Macropus eugenii); however, its specific composition and numbers were different from placentals. Of importance, comparisons of the phascogale’s PrP^C physicochemical properties with other monotremes, marsupials, and placentals confirmed the Monotremata–Marsupialia–Placentalia evolutionary distance. We found that the protein instability index, a method used to predict the stability of a protein in vivo (Stable: <40; Instable >40), showed that the PrP^C of all marsupials tested, including phascogale, were highly stable compared with the birds, reptiles, amphibians, and fish that were shown to be highly unstable. However, the instability index predicted that all placental species, including human (Homo sapiens), mouse (Mus musculus), bank vole (Myodes glareolus), rhinoceros (Rhinocerotidae), dog (Canis lupus familiaris), flying fox (Pteropus vampyrus), whale (Physeter catodon), cattle (Bos taurus), and sheep (Ovis aries), were either slightly unstable or nearly unstable. Further, our analysis revealed that despite their predicted high PrP^C stability, P. calura exhibited substantial N-terminal disorder (53.76%), while species with highly unstable PrP^Cs based on their instability index, such as Danio rerio, Oryzias latipes, and Astyanax mexicanus, displayed even higher levels of N-terminal disorder (up to 75.84%). These findings highlight a discrepancy between overall predicted stability and N-terminal disorder, suggesting a potential compensatory role of disorder in modulating prion protein stability and function. Conclusions: These results suggest that the high stability of marsupial prion proteins indicates a vital role in maintaining protein homeostasis; however more work is warranted to further depict the exact function. Full article

The Zika virus (ZIKV) epidemic elicited a rapid commitment to the development of animal models for ZIKV research. Non-human primates (NHPs) and mice have made significant contributions to this research, but NHPs are expensive, have a long gestation period, and are available only in small numbers; non-genetically modified mice are resistant to infection. To address these deficiencies, we have established the laboratory opossum, Monodelphis domestica, as a small animal model that complements the mouse and monkey models. We developed and validated an indirect ELISA for measuring antibodies to ZIKV in opossums, as well as an immunohistochemistry (IHC) method to detect ZIKV NS1 protein in tissue samples. Opossum pups inoculated intracerebrally as embryos, juveniles inoculated by several routes, and mothers that cannibalized inoculated pups became persistently infected with ZIKV. The virus spread to multiple organs and persisted for up to 38 weeks (the latest endpoint of the experiments). A robust humoral immune response was mounted, and high titers of antibodies also persisted for 38 weeks. The results establish M. domestica as a natural, non-genetically modified animal model in which ZIKV persists long-term after experimental exposure and as a unique animal model for research on the immune response to ZIKV. Full article

In marsupials, upper-layer cortical neurons derived from the progenitors of the subventricular zone of the lateral ventricle (SVZ) mature morphologically and send their axons to form interhemispheric connections through the anterior commissure. In contrast, eutherians have evolved a new extra callosal pathway, the corpus callosum, that interconnects both hemispheres. In this study, we aimed to examine neurogenesis during the formation of cortical upper layers, including their morphological maturation in a marsupial species, namely the opossum (Monodelphis domestica). Furthermore, we studied how the axons of upper layers neurons pass through the anterior commissure of the opossum, which connects neocortical areas. We showed that upper-layer II/III neurons were generated within at least seven days in the opossum neocortex. Surprisingly, these neurons expressed special AT-rich sequence binding protein 2 (Satb2) and neuropilin 1 interacting protein (Nrp1), which are proteins known to be essential for the formation of the corpus callosum in eutherians. This indicates that extrinsic, but not intrinsic, cues could be key players in guiding the axons of newly generated cortical neurons in the opossum. Although oligodendrocyte precursor cells were present in the neocortex and anterior commissure, newly generated upper-layer neurons sent unmyelinated axons to the anterior commissure. We also found numerous GFAP-expressing progenitor cells in both brain structures, the neocortex and the anterior commissure. However, at P12–P17 in the opossums, a small population of astrocytes was observed only in the midline area of the anterior commissure. We postulate that in the opossum, midline astrocytes allow neocortical axons to be guided to cross the midline, as this structure resembles the glial wedge required by fibers to cross the midline area of the corpus callosum in the rodent. Full article

(1) Background: Central nervous system (CNS) development is characterized by dynamic changes in cell proliferation and differentiation. Key regulators of these transitions are the transcription factors such as SOX2 and SOX9. SOX2 is involved in the maintenance of progenitor cell state and neural stem cell multipotency, while SOX9, expressed in neurogenic niches, plays an important role in neuron/glia switch with predominant expression in astrocytes in the adult brain. (2) Methods: To validate SOX2 and SOX9 expression patterns in developing opossum (Monodelphis domestica) cortex, we used immunohistochemistry (IHC) and the isotropic fractionator method on fixed cortical tissue from comparable postnatal ages, as well as dissociated primary neuronal cultures. (3) Results: Neurons positive for both neuronal (TUJ1 or NeuN) and stem cell (SOX2) markers were identified, and their presence was confirmed with all methods and postnatal age groups (P4-6, P6-18, and P30) analyzed. SOX9 showed exclusive staining in non-neuronal cells, and it was coexpressed with SOX2. (4) Conclusions: The persistence of SOX2 expression in developing cortical neurons of M. domestica during the first postnatal month implies the functional role of SOX2 during neuronal differentiation and maturation, which was not previously reported in opossums. Full article

Monodelphis domestica (the laboratory opossum) is a marsupial native to South America. At birth, these animals are developmentally equivalent to human embryos at approximately 5 weeks of gestation, which, when coupled with other characteristics including the size of the animals, the development of a robust immune system during juvenile development, and the relative ease of experimental manipulation, have made M. domestica a valuable model in many areas of biomedical research. However, their suitability as models for infectious diseases, especially neurotropic viruses such as Zika virus (ZIKV), is currently unknown. Here, we describe the replicative effects of ZIKV using a fetal intra-cerebral model of inoculation. Using immunohistology and in situ hybridization, we found that opossum embryos and fetuses are susceptible to infection by ZIKV administered intra-cerebrally, that the infection persists, and that viral replication results in neural pathology and may occasionally result in global growth restriction. These results demonstrate the utility of M. domestica as a new animal model for investigating ZIKV infection in vivo and facilitate further inquiry into viral pathogenesis, particularly for those viruses that are neurotropic, that require a host with the ability to sustain sustained viremia, and/or that may require intra-cerebral inoculations of large numbers of embryos or fetuses. Full article

Marsupials have been a powerful comparative model to understand mammalian biology. However, because of the unique characteristics of their embryology, marsupial pluripotency architecture remains to be fully understood, and nobody has succeeded in developing embryonic stem cells (ESCs) from any marsupial species. We have developed an integration-free iPSC reprogramming method and established validated iPSCs from two inbred strains of a marsupial, Monodelphis domestica. The monoiPSCs showed a significant (6181 DE-genes) and highly uniform (r² [95% CI] = 0.973 ± 0.007) resetting of the cellular transcriptome and were similar to eutherian ESCs and iPSCs in their overall transcriptomic profiles. However, monoiPSCs showed unique regulatory architecture of the core pluripotency transcription factors and were more like marsupial epiblasts. Our results suggest that POU5F1 and the splice-variant-specific expression of POU5F3 synergistically regulate the opossum pluripotency gene network. It is plausible that POU5F1, POU5F3 splice variant XM_016427856.1, and SOX2 form a self-regulatory network. NANOG expression, however, was specific to monoiPSCs and epiblasts. Furthermore, POU5F1 was highly expressed in trophectoderm cells, whereas all other pluripotency transcription factors were significantly downregulated, suggesting that the regulatory architecture of core pluripotency genes of marsupials may be distinct from that of eutherians. Full article

Kinetoplastids include species economically important in agriculture, livestock, and human health. We evaluated the richness of kinetoplastids that infect small mammals in patches of unflooded forests in the Pantanal biome, an area where we hypothesize that its diversity is higher than currently recognized. Hemocultures (HC) and Next Generation Sequencing (NGS) targeting the 18S rDNA gene were employed for the detection of kinetoplastids. We grouped the positive samples into pools for each small mammal species (Monodelphis domestica, Thylamys macrurus, Oecomys mamorae, Thrichomys fosteri, Clyomys laticeps, and Holochilus chacarius). Eight parasite species were identified: Leishmania amazonensis, L. infantum; Trypanosoma cascavelli (HC + NGS), T. cruzi, T. lainsoni, T. rangeli (HC + NGS), Trypanosoma sp. DID, and Neobodo sp. The use of a tool as sensitive as NGS has increased our awareness of the diversity of kinetoplastids, as well as their host range, with emphasis on the species O. mamorae (seven kinetoplastid species, excepting T. cascavelli in a pool of nine individuals) and T. macrurus (four kinetoplastid species in a single individual). Furthermore, L. infantum and L. amazonensis infections were described in small mammals from this region for the first time. These findings make it mandatory to revisit the kinetoplastids/host associations proposed so far. Full article

Neurodegenerative diseases are one of the greatest medical burdens of the modern age, being mostly incurable and with limited prognostic and diagnostic tools. Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the loss of motoneurons, with a complex etiology, combining genetic, epigenetic, and environmental causes. The neuroprotective therapeutic approaches are very limited, while the diagnostics rely on clinical examination and the exclusion of other diseases. The recent advancement in the discovery of molecular pathways and gene mutations involved in ALS has deepened the understanding of the disease pathology and opened the possibility for new treatments and diagnostic procedures. Recently, 15 risk loci with distinct genetic architectures and neuron-specific biology were identified as linked to ALS through common and rare variant association analyses. Interestingly, the quantity of related proteins to these genes has been found to change during early postnatal development in mammalian spinal cord tissue (opossum Monodelphis domestica) at the particular time when neuroregeneration stops being possible. Here, we discuss the possibility that the ALS-related genes/proteins could be connected to neuroregeneration and development. Moreover, since the regulation of gene expression in developmental checkpoints is frequently regulated by non-coding RNAs, we propose that studying the changes in the composition and quantity of non-coding RNA molecules, both in ALS patients and in the developing central nervous (CNS) system of the opossum at the time when neuroregeneration ceases, could reveal potential biomarkers useful in ALS prognosis and diagnosis. Full article

In mammals, neurogenesis occurs during both embryonic and postnatal development. In eutherians, most brain structures develop embryonically; conversely, in marsupials, a number of brain structures develop after birth. The exception is the generation of granule cells in the dentate gyrus, olfactory bulb, and cerebellum of eutherian species. The formation of these structures starts during embryogenesis and continues postnatally. In both eutherians and marsupials, neurogenesis continues in the subventricular zone of the lateral ventricle (SVZ) and the dentate gyrus of the hippocampal formation throughout life. The majority of proliferated cells from the SVZ migrate to the olfactory bulb, whereas, in the dentate gyrus, cells reside within this structure after division and differentiation into neurons. A key aim of this review is to evaluate advances in understanding developmental neurogenesis that occurs postnatally in both marsupials and eutherians, with a particular emphasis on the generation of granule cells during the formation of the olfactory bulb, dentate gyrus, and cerebellum. We debate the significance of immature neurons in the piriform cortex of young mammals. We also synthesize the knowledge of adult neurogenesis in the olfactory bulb and the dentate gyrus of marsupials by considering whether adult-born neurons are essential for the functioning of a given area. Full article

The genus Bartonella (Rhizobiales: Bartonellaceae) encompasses facultative intracellular Gram-negative alphaproteobacteria that parasitize mainly erythrocytes and endothelial cells, as well as macrophages, monocytes and dendritic cells. Although they can infect numerous mammal species and arthropod vectors worldwide, reports of Bartonella infections in marsupials are scarce. In fact, such agents have only been detected in marsupials and/or associated ectoparasites in Australia and the United States of America until the present moment. The present study aimed to isolate and characterize molecularly, morphologically and phenotypically Bartonella infecting free-living marsupials sampled in the Brazilian Pantanal, the largest wetland in South America. Two marsupials were captured in December 2018 and six marsupials in February 2019, totaling eight small mammals sampled: five (62.5%) Thylamys macrurus and three (37.5%) Monodelphis domestica. All blood samples were submitted to qPCR for Bartonella spp. based on the nuoG gene, a pre-enrichment liquid culture and a chocolate agar solid culture. Bartonella sp. was isolated from 3 T. macrurus and one M. domestica. One Bartonella isolate obtained from a T. macrurus blood sample (strain 117A) that showed to be closely related to the Bartonella vinsonii complex and Bartonella machadoae was selected for whole genome sequencing using a hybrid approach based on Illumina NovaSeq and Nanopore sequencing platforms. This strain showed a genome of 2.35 Mbp, with an average C + G content of 38.8%, coding for 2013 genes, and a 29 kb plasmid with an average C + G content of 34.5%. In addition, this strain exhibited an average nucleotide identity (ANI) of 85% with Bartonella species belonging to the B. vinsonii group and 91% with B. machadoae. Phylogenomic analysis based on 291 protein coding genes shared by the genomes of 53 Bartonella species positioned this strain closely to B. machadoae. This new isolated species was named Bartonella harrusi sp. nov., which was characterized as having small capnophilic, microaerophilic and aerobic rods with an absence of pili and flagella. In conclusion, the present work describes the biochemical, phenotypic and genomic characteristics of Bartonella harrusi, a new species isolated from the T. macrurus blood samples of the Brazilian Pantanal. Finally, a review of the taxonomic classification of members of the genus Bartonella is proposed, based on the ANI values accessed by whole genome sequencing analyses. Full article

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding (CREB) family, is upregulated by various intracellular and extracellular signals such as injury and signals related to cell proliferation. ATF3 also belongs to the regeneration-associated genes (RAG) group of transcription factors. RAG and ATF/CREB transcription factors that play an important role in embryonic neuronal development and PNS regeneration may also be involved in postnatal neuronal differentiation and development, as well as in the regeneration of the injured CNS. Here we investigated the effect of ATF3 in differentiation, neural outgrowth, network formation, and regeneration after injury using postnatal dissociated cortical neurons derived from neonatal opossums (Monodelphis domestica). Our results show that RAG and ATF genes are differentially expressed in early differentiated neurons versus undifferentiated neurospheres and that many members of those families, ATF3 in particular, are upregulated in cortical cultures obtained from younger animals that have the ability to fully functionally regenerate spinal cord after injury. In addition, we observed different intracellular localization of ATF3 that shifts from nuclear (in neuronal progenitors) to cytoplasmic (in more mature neurons) during neuronal differentiation. The ATF3 inhibition, pharmacological or by specific antibody, reduced the neurite outgrowth and differentiation and caused increased cell death in early differentiating cortical neuronal cultures, suggesting the importance of ATF3 in the CNS development of neonatal opossums. Finally, we investigated the regeneration capacity of primary cortical cultures after mechanical injury using the scratch assay. Remarkably, neonatal opossum-derived cultures retain their capacity to regenerate for up to 1 month in vitro. Inhibition of ATF3 correlates with reduced neurite outgrowth and regeneration after injury. These results indicate that ATF3, and possibly other members of RAG and ATF/CREB family of transcription factors, have an important role both during cortical postnatal development and in response after injury. Full article

Males have evolved species-specifical sperm morphology and swimming patterns to adapt to different fertilization environments. In eutherians, only a small fraction of the sperm overcome the diverse obstacles in the female reproductive tract and successfully migrate to the fertilizing site. Sperm arriving at the fertilizing site show hyperactivated motility, a unique motility pattern displaying asymmetric beating of sperm flagella with increased amplitude. This motility change is triggered by Ca²⁺ influx through the sperm-specific ion channel, CatSper. However, the current understanding of the CatSper function and its molecular regulation is limited in eutherians. Here, we report molecular evolution and conservation of the CatSper channel in the genome throughout eutherians and marsupials. Sequence analyses reveal that CatSper proteins are slowly evolved in marsupials. Using an American marsupial, gray short-tailed opossum (Monodelphis domestica), we demonstrate the expression of CatSper in testes and its function in hyperactivation and unpairing of sperm. We demonstrate that a conserved IQ-like motif in CatSperζ is required for CatSperζ interaction with the pH-tuned Ca²⁺ sensor, EFCAB9, for regulating CatSper activity. Recombinant opossum EFCAB9 can interact with mouse CatSperζ despite high sequence divergence of CatSperζ among CatSper subunits in therians. Our finding suggests that molecular characteristics and functions of CatSper are evolutionarily conserved in gray short-tailed opossum, unraveling the significance of sperm hyperactivation and fertilization in marsupials for the first time. Full article