PEDF-derived peptide protects against Amyloid-ß toxicity in vitro and prevents retinal dysfunction in rats.

Amyloid-beta (Aß), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aß42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aß42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aß-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aß42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aß, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aß42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aß42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aß42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aß42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aß42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aß42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aß42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aß42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aß42 in the retina and suggest concepts on the molecular mechanism of Aß retinal pathogenicity.

RBL2 bi-allelic truncating variants cause severe motor and cognitive impairment without evidence for abnormalities in DNA methylation or telomeric function.

RBL2/p130, a member of the retinoblastoma family of proteins, is a key regulator of cell division and propagates irreversible senescence. RBL2/p130 is also involved in neuronal differentiation and survival, and eliminating Rbl2 in certain mouse strains leads to embryonic lethality accompanied by an abnormal central nervous system (CNS) phenotype. Conflicting reports exist regarding a role of RBL2/p130 in transcriptional regulation of DNA methyltransferases (DNMTs), as well as the control of telomere length. Here we describe the phenotype of three patients carrying bi-allelic RBL2-truncating variants. All presented with infantile hypotonia, severe developmental delay and microcephaly. Malignancies were not reported in carriers or patients. Previous studies carried out on mice and human cultured cells, associated RBL2 loss to DNA methylation and telomere length dysregulation. Here, we investigated whether patient cells lacking RBL2 display related abnormalities. The study of primary patient fibroblasts did not detect abnormalities in expression of DNMTs. Furthermore, methylation levels of whole genome DNA, and specifically of pericentromeric repeats and subtelomeric regions, were unperturbed. RBL2-null fibroblasts show no evidence for abnormal elongation by telomeric recombination. Finally, gradual telomere shortening, and normal onset of senescence were observed following continuous culturing of RBL2-mutated fibroblasts. Thus, this study resolves uncertainties regarding a potential non-redundant role for RBL2 in DNA methylation and telomere length regulation, and indicates that loss of function variants in RBL2 cause a severe autosomal recessive neurodevelopmental disorder in humans.

Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.

The retinal toxicity profile towards assemblies of Amyloid-ß indicate the predominant pathophysiological activity of oligomeric species.

Amyloid-ß (Aß), reported as a significant constituent of drusen, was implicated in the pathophysiology of age-related macular degeneration (AMD), yet the identity of the major pathogenic Aß species in the retina has remained hitherto unclear. Here, we examined the in-vivo retinal impact of distinct supramolecular assemblies of Aß. Fibrillar (Aß40, Aß42) and oligomeric (Aß42) preparations showed clear biophysical hallmarks of amyloid assemblies. Measures of retinal structure and function were studied longitudinally following intravitreal administration of the various Aß assemblies in rats. Electroretinography (ERG) delineated differential retinal neurotoxicity of Aß species. Oligomeric Aß42 inflicted the major toxic effect, exerting diminished ERG responses through 30 days post injection. A lesser degree of retinal dysfunction was noted following treatment with fibrillar Aß42, whereas no retinal compromise was recorded in response to Aß40 fibrils. The toxic effect of Aß42 architectures was further reflected by retinal glial response. Fluorescence labelling of Aß42 species was used to detect their accumulation into the retinal tissue. These results provide conceptual evidence of the differential toxicity of particular Aß species in-vivo, and promote the mechanistic understanding of their retinal pathogenicity. Stratifying the impact of pathological Aß aggregation in the retina may merit further investigation to decipher the pathophysiological relevance of processes of molecular self-assembly in retinal disorders.

ERK Activity in Immature Leukemic Cells Drives Clonal Selection during Induction Therapy for Acute Myeloid Leukemia.

Selection of resistant clones following intensive chemotherapy is a common obstacle for cure in many cancers, particularly in acute myeloid leukemia (AML). In AML, clone-specific sensitivity to chemotherapy varies even within the same patient. Multiple mutations and genetic aberrations are associated with clones surviving chemotherapy. The current study explored the role of activated signaling pathways in chemoresistance as a function of cell maturation, reflected by CD34 expression. In-vitro, Kasumi-1 leukemic cell line, sorted by CD34 expression, showed increased apoptosis only in the CD34- subpopulation after exposure to cytosine arabinoside (Ara-C) or daunorubicin. The resistant CD34+ subset demonstrated higher expression of ERK1/2 and BCL-2 proteins than CD34- cells. MEK1/2 inhibition elevated Ara-C ability to induce apoptosis in CD34+ cells, suggesting that MEK1/2-ERK1/2 is surviving signaling, which correlates to cell maturation levels and plays a role in chemoresistance. Deep sequencing of sorted CD34+/- populations, both derived from the same patient samples, demonstrated various subclonal distribution of NPM1, DNMT3A and FLT3-ITD mutations. Interestingly, in these samples, p-ERK levels and apoptosis rates following chemotherapy exposure significantly differed between CD34+/- populations. Hence, clones may be selected due to their ability to escape apoptosis rather than a direct effect of chemotherapy on a specific mutated clone.