Protection of dopamine neurons by CDNF and neurturin variant N4 against MPP+ in dissociated cultures from rat mesencephalon

Parkinson's disease is associated with the loss of dopamine (DA) neurons in ventral mesencephalon. We have previously reported that no single neurotrophic factor we tested protected DA neurons from the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+) in dissociated cultures isolated from the P0 rat substantia nigra, but that a combination of five neurotrophic factors was protective. We now report that cerebral DA neurotrophic factor (CDNF) and a variant of neurturin (NRTN), N4, were also not protective when provided alone but were protective when added together. In cultures isolated from the substantia nigra, MPP+ (10 mu M) decreased tyrosine hydroxylase-positive cells to 41.7 +/- 5.4% of vehicle control. Although treatment of cultures with 100 ng/ml of either CDNF or N4 individually before and after toxin exposure did not significantly increase survival in MPP+-treated cultures, when the two trophic factors were added together at 100 ng/ml each, survival of cells was increased 28.2 +/- 6.1% above the effect of MPP+ alone. In cultures isolated from the ventral tegmental area, another DA rich area, a higher dose of MPP+ (1 mM) was required to produce an EC50 in TH-positive cells but, as in the substantia nigra, only the combination of CDNF and N4 (100 ng/ml each) was successful at increasing the survival of these cells compared to MPP+ alone (by 22.5 +/- 3.5%). These data support previous findings that CDNF and N4 may be of therapeutic value for treatment of PD, but suggest that they may need to be administered together.Peer reviewe

CDNF Protein Therapy in Parkinson’s Disease

Neurotrophic factors (NTF) are a subgroup of growth factors that promote survival and differentiation of neurons. Due to their neuroprotective and neurorestorative properties, their therapeutic potential has been tested in various neurodegenerative diseases. Bioavailability of NTFs in the target tissue remains a major challenge for NTF-based therapies. Various intracerebral delivery approaches, both protein and gene transfer-based, have been tested with varying outcomes. Three growth factors, glial cell-line derived neurotrophic factor (GDNF), neurturin (NRTN) and platelet-derived growth factor (PDGF-BB) have been tested in clinical trials in Parkinson?s Disease (PD) during the past 20 years. A new protein can now be added to this list, as cerebral dopamine neurotrophic factor (CDNF) has recently entered clinical trials. Despite their misleading names, CDNF, together with its closest relative mesencephalic astrocyte-derived neurotrophic factor (MANF), form a novel family of unconventional NTF that are both structurally and mechanistically distinct from other growth factors. CDNF and MANF are localized mainly to the lumen of endoplasmic reticulum (ER) and their primary function appears to be modulation of the unfolded protein response (UPR) pathway. Prolonged ER stress, via the UPR signaling pathways, contributes to the pathogenesis in a number of chronic degenerative diseases, and is an important target for therapeutic modulation. Intraputamenally administered recombinant human CDNF has shown robust neurorestorative effects in a number of small and large animal models of PD, and had a good safety profile in preclinical toxicology studies. Intermittent monthly bilateral intraputamenal infusions of CDNF are currently being tested in a randomized placebo-controlled phase I?II clinical study in moderately advanced PD patients. Here, we review the history of growth factor-based clinical trials in PD, and discuss how CDNF differs from the previously tested growth factors.Peer reviewe

Genetic components of grey cattle in Estonia as revealed by microsatellite analysis using two Bayesian clustering methods

<p>Abstract</p> <p>Background</p> <p>It was recently postulated that a few individual grey cattle still found in Estonia might be a relict of the old native cattle stock. Genotypes at 17 microsatellite loci from a total of 243 cattle from North European breeds and 11 grey cattle in Estonia were used in an attempt to clarify the genetic composition of the grey cattle.</p> <p>Findings</p> <p>We characterize the genetic components of 11 examples of the grey cattle in Estonia at the population and individual levels. Our results show that the grey cattle in Estonia are most genetically similar to the Holstein-Friesian breed and secondarily to the Estonian Red cattle.</p> <p>Conclusions</p> <p>Both Bayesian approaches gave similar results in terms of the identification of numbers of clusters and the estimation of proportions of genetic components. This study suggested that the Estonian grey cattle included in the analysis are a genetic composite resulting from cross-breeding of European dairy breeds.</p

Augmenting hematoma-scavenging capacity of innate immune cells by CDNF reduces brain injury and promotes functional recovery after intracerebral hemorrhage

During intracerebral hemorrhage (ICH), hematoma formation at the site of blood vessel damage results in local mechanical injury. Subsequently, erythrocytes lyse to release hemoglobin and heme, which act as neurotoxins and induce inflammation and secondary brain injury, resulting in severe neurological deficits. Accelerating hematoma resorption and mitigating hematoma-induced brain edema by modulating immune cells has potential as a novel therapeutic strategy for functional recovery after ICH. Here, we show that intracerebroventricular administration of recombinant human cerebral dopamine neurotrophic factor (rhCDNF) accelerates hemorrhagic lesion resolution, reduces peri-focal edema, and improves neurological outcomes in an animal model of collagenase-induced ICH. We demonstrate that CDNF acts on microglia/macrophages in the hemorrhagic striatum by promoting scavenger receptor expression, enhancing erythrophagocytosis and increasing anti-inflammatory mediators while suppressing the production of pro-inflammatory cytokines. Administration of rhCDNF results in upregulation of the Nrf2-HO-1 pathway, but alleviation of oxidative stress and unfolded protein responses in the perihematomal area. Finally, we demonstrate that intravenous delivery of rhCDNF has beneficial effects in an animal model of ICH and that systemic application promotes scavenging by the brain's myeloid cells for the treatment of ICH.Peer reviewe

The Echinococcus canadensis (G7) genome: A key knowledge of parasitic platyhelminth human diseases

Background: The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. Results: We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. Conclusions: This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.Fil: Maldonado, Lucas Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Assis, Juliana. Fundación Oswaldo Cruz; BrasilFil: Gomes Araújo, Flávio M.. Fundación Oswaldo Cruz; BrasilFil: Salim, Anna C. M.. Fundación Oswaldo Cruz; BrasilFil: Macchiaroli, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Cucher, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Camicia, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Fox, Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Oliveira, Guilherme. Instituto Tecnológico Vale; Brasil. Fundación Oswaldo Cruz; BrasilFil: Kamenetzky, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Three Thousand Years of Continuity in the Maternal Lineages of Ancient Sheep (Ovis aries) in Estonia

lthough sheep (Ovis aries) have been one of the most exploited domestic animals in Estonia since the Late Bronze Age, relatively little is known about their genetic history. Here, we explore temporal changes in Estonian sheep populations and their mitochondrial genetic diversity over the last 3000 years. We target a 558 base pair fragment of the mitochondrial hypervariable region in 115 ancient sheep from 71 sites in Estonia (c. 1200 BC – AD 1900s), 19 ancient samples from Latvia, Russia, Poland and Greece (6800 BC – AD 1700), as well as 44 samples of modern Kihnu native sheep breed. Our analyses revealed: (1) 49 mitochondrial haplotypes, associated with sheep haplogroups A and B; (2) high haplotype diversity in Estonian ancient sheep; (3) continuity in mtDNA haplotypes through time; (4) possible population expansion during the first centuries of the Middle Ages (associated with the establishment of the new power regime related to 13th century crusades); (5) significant difference in genetic diversity between ancient populations and modern native sheep, in agreement with the beginning of large-scale breeding in the 19th century and population decline in local sheep. Overall, our results suggest that in spite of the observed fluctuations in ancient sheep populations, and changes in the natural and historical conditions, the utilisation of local sheep has been constant in the territory of Estonia, displaying matrilineal continuity from the Middle Bronze Age through the Modern Period, and into modern native sheep

GDNF and Parkinson's Disease : Where Next? A Summary from a Recent Workshop

The concept of repairing the brain with growth factors has been pursued for many years in a variety of neurodegenerative diseases including primarily Parkinson's disease (PD) using glial cell line-derived neurotrophic factor (GDNF). This neurotrophic factor was discovered in 1993 and shown to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. These observations led to a series of clinical trials in PD patients including using infusions or gene delivery of GDNF or the related growth factor, neurturin (NRTN). Initial studies, some of which were open label, suggested that this approach could be of value in PD when the agent was injected into the putamen rather than the cerebral ventricles. In subsequent double-blind, placebo-controlled trials, the most recent reporting in 2019, treatment with GDNF did not achieve its primary end point. As a result, there has been uncertainty as to whether GDNF (and by extrapolation, related GDNF family neurotrophic factors) has merit in the future treatment of PD. To critically appraise the existing work and its future, a special workshop was held to discuss and debate this issue. This paper is a summary of that meeting with recommendations on whether there is a future for this therapeutic approach and also what any future PD trial involving GDNF and other GDNF family neurotrophic factors should consider in its design.Peer reviewe

The Effect of Inappropriate Calibration: Three Case Studies in Molecular Ecology

Time-scales estimated from sequence data play an important role in molecular ecology. They can be used to draw correlations between evolutionary and palaeoclimatic events, to measure the tempo of speciation, and to study the demographic history of an endangered species. In all of these studies, it is paramount to have accurate estimates of time-scales and substitution rates. Molecular ecological studies typically focus on intraspecific data that have evolved on genealogical scales, but often these studies inappropriately employ deep fossil calibrations or canonical substitution rates (e.g., 1% per million years for birds and mammals) for calibrating estimates of divergence times. These approaches can yield misleading estimates of molecular time-scales, with significant impacts on subsequent evolutionary and ecological inferences. We illustrate this calibration problem using three case studies: avian speciation in the late Pleistocene, the demographic history of bowhead whales, and the Pleistocene biogeography of brown bears. For each data set, we compare the date estimates that are obtained using internal and external calibration points. In all three cases, the conclusions are significantly altered by the application of revised, internally-calibrated substitution rates. Collectively, the results emphasise the importance of judicious selection of calibrations for analyses of recent evolutionary events

PEDF and GDNF are key regulators of photoreceptor development and retinal neurogenesis in reaggregates from chick embryonic retina

Here, role(s) of pigment epithelial-derived factor (PEDF) and glial-derived neurotrophic factor (GDNF) on photoreceptor development in three-dimensional reaggregates from the retinae of the E6 chick embryo (rosetted spheroids) was investigated. Fully dispersed cells were reaggregated under serum-reduced conditions and supplemented with 50 ng/ml PEDF alone or in combination with 50 ng/ml GDNF. The spheroids were analyzed for cell growth, differentiation, and death using proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, and other immunocytochemical stainings and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) methods. PEDF strongly promoted synthesis of the messenger RNAs for blue and violet cone opsins and to a lesser extent on the red and green cone opsins. This correlated with an increase in the number of cone photoreceptors, as determined by the cone cell marker CERN906. Likewise, PEDF nearly completely inhibited rod differentiation, as detected by immunostaining with anti-rho4D2 and RT-PCR. Furthermore, PEDF accelerated proliferation of cells in the spheroids and inhibited apoptosis. As negative effects, PEDF inhibited the normal histotypic tissue formation of retinal aggregates and reduced the frequency of photoreceptor rosettes and IPL-like areas. Noticeably, supplementation of PEDF-treated cultures with GDNF reversed the effects of PEDF on spheroid morphology and on rod differentiation. This study establishes that PEDF strongly affects three-dimensional retinogenesis in vitro, most notably by inhibiting rod development and supporting proliferation and differentiation of cones, effects which are partially counteracted by GDNF