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Background Genetic variation at the PTK2B locus encoding the protein Pyk2 influences Alzheimer’s disease risk. Neurons express Pyk2 and the protein is required for Amyloid-β (Aβ) peptide driven deficits of synaptic function and memory in mouse models, but Pyk2 deletion has minimal effect on neuro-inflammation. Previous in vitro data suggested that Pyk2 activity might enhance GSK3β-dependent Tau phosphorylation and be required for tauopathy. Here, we examine the influence of Pyk2 on Tau phosphorylation and associated pathology. Methods The effect of Pyk2 on Tau phosphorylation was examined in cultured Hek cells through protein over-expression and in iPSC-derived human neurons through pharmacological Pyk2 inhibition. PS19 mice overexpressing the P301S mutant of human Tau were employed as an in vivo model of tauopathy. Phenotypes of PS19 mice with a targeted deletion of Pyk2 expression were compared with PS19 mice with intact Pyk2 expression. Phenotypes examined included Tau phosphorylation, Tau accumulation, synapse loss, gliosis, proteomic profiling and behavior. Results Over-expression experiments from Hek293T cells indicated that Pyk2 contributed to Tau phosphorylation, while iPSC-derived human neuronal cultures with endogenous protein levels supported the opposite conclusion. In vivo, multiple phenotypes of PS19 were exacerbated by Pyk2 deletion. In Pyk2-null PS19 mice, Tau phosphorylation and accumulation increased, mouse survival decreased, spatial memory was impaired and hippocampal C1q deposition increased relative to PS19 littermate controls. Proteomic profiles of Pyk2-null mouse brain revealed that several protein kinases known to interact with Tau are regulated by Pyk2. Endogenous Pyk2 suppresses LKB1 and p38 MAPK activity, validating one potential pathway contributing to increased Tau pathology. Conclusions The absence of Pyk2 results in greater mutant Tau-dependent phenotypes in PS19 mice, in part via increased LKB1 and MAPK activity. These data suggest that in AD, while Pyk2 activity mediates Aβ-driven deficits, Pyk2 suppresses Tau-related phenotypes.

ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

Amyloid-beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aβo binding site in the amino-terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Potency of AZ59 was evaluated by binding to PrPC, blockade of Aβo interaction and interruption of Aβo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5-7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose-dependent and persist for at least 1 month after the last dose. Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease-modifying therapeutic potential.

Objective Amyloid‐beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aβo binding site in the amino‐terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Methods Potency of AZ59 was evaluated by binding to PrPC, blockade of Aβo interaction and interruption of Aβo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. Results AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5–7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose‐dependent and persist for at least 1 month after the last dose. Interpretation Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease‐modifying therapeutic potential.

Genetic variation at the PTK2B locus encoding the protein Pyk2 influences Alzheimer's disease risk. Neurons express Pyk2 and the protein is required for Amyloid-[beta] (A[beta]) peptide driven deficits of synaptic function and memory in mouse models, but Pyk2 deletion has minimal effect on neuro-inflammation. Previous in vitro data suggested that Pyk2 activity might enhance GSK3[beta]-dependent Tau phosphorylation and be required for tauopathy. Here, we examine the influence of Pyk2 on Tau phosphorylation and associated pathology. The effect of Pyk2 on Tau phosphorylation was examined in cultured Hek cells through protein over-expression and in iPSC-derived human neurons through pharmacological Pyk2 inhibition. PS19 mice overexpressing the P301S mutant of human Tau were employed as an in vivo model of tauopathy. Phenotypes of PS19 mice with a targeted deletion of Pyk2 expression were compared with PS19 mice with intact Pyk2 expression. Phenotypes examined included Tau phosphorylation, Tau accumulation, synapse loss, gliosis, proteomic profiling and behavior. Over-expression experiments from Hek293T cells indicated that Pyk2 contributed to Tau phosphorylation, while iPSC-derived human neuronal cultures with endogenous protein levels supported the opposite conclusion. In vivo, multiple phenotypes of PS19 were exacerbated by Pyk2 deletion. In Pyk2-null PS19 mice, Tau phosphorylation and accumulation increased, mouse survival decreased, spatial memory was impaired and hippocampal C1q deposition increased relative to PS19 littermate controls. Proteomic profiles of Pyk2-null mouse brain revealed that several protein kinases known to interact with Tau are regulated by Pyk2. Endogenous Pyk2 suppresses LKB1 and p38 MAPK activity, validating one potential pathway contributing to increased Tau pathology. The absence of Pyk2 results in greater mutant Tau-dependent phenotypes in PS19 mice, in part via increased LKB1 and MAPK activity. These data suggest that in AD, while Pyk2 activity mediates A[beta]-driven deficits, Pyk2 suppresses Tau-related phenotypes.

Subgenomic flaviviral RNAs (sfRNAs) are virus-derived noncoding RNAs produced by pathogenic mosquito-borne flaviviruses (MBF) to counteract the host antiviral response. To date, the ability of non-pathogenic flaviviruses to produce and utilise sfRNAs remains largely unexplored, and it is unclear what role XRN1 resistance plays in flavivirus evolution and host adaptation. Herein the production of sfRNAs by several insect-specific flaviviruses (ISFs) that replicate exclusively in mosquitoes is shown, and the secondary structures of their complete 3’UTRs are determined. The xrRNAs responsible for the biogenesis of ISF sfRNAs are also identified, and the role of these sfRNAs in virus replication is demonstrated. We demonstrate that 3’UTRs of all classical ISFs, except Anopheles spp -asscoaited viruses, and of the dual-host associated ISF Binjari virus contain duplicated xrRNAs. We also reveal novel structural elements in the 3’UTRs of dual host-associated and Anopheles -associated classical ISFs. Structure-based phylogenetic analysis demonstrates that xrRNAs identified in Anopheles spp -associated ISF are likely ancestral to xrRNAs of ISFs and MBFs. In addition, our data provide evidence that duplicated xrRNAs are selected in the evolution of flaviviruses to provide functional redundancy, which preserves the production of sfRNAs if one of the structures is disabled by mutations or misfolding. Subgenomic flaviviral RNAs (sfRNAs) are produced by pathogenic flaviviruses to counteract the host antiviral response. sfRNAs are products of incomplete degradation of viral RNA by the host exoribonuclease XRN1 which stalls on XRN1-resistant structural elements (xrRNAs) in the 3‟UTR. Here, Slonchak et al. identify xrRNAs for mosquito-specific flaviviruses, characterize their ability to stall XRN1 and produce sfRNAs, and apply structure-based phylogenetic analysis to provide evidence for evolutionary selection of these elements.

Background In Cambodia, female entertainment workers (FEWs) are disproportionately affected by global and local disasters, such as the COVID-19 pandemic. To prevent the spread of COVID-19, the government imposed tight restrictions, including closures of entertainment venues, such as karaoke bars, beer gardens, nightclubs, or massage parlors, leading FEWs to face economic and social disruptions. This study aims to assess the relationship between income loss during the pandemic and gender-based violence (GBV) among FEWs in Cambodia to inform future disaster response programs. Methods We conducted a phone survey in August 2021 with 369 randomly sampled FEWs from a national organization’s email list. We used a structured questionnaire to ask the participants about job and income loss, food security, mental health, access to health services, and GBV. We fit a linear regression model to examine the differences in GBV experience between FEWs who lost all their income and those who lost partial income due to the COVID-19 pandemic. Key covariables comprised the number of dependents, smartphone ownership, internet access, food security, and mental health. Multivariable linear regression analysis was conducted. Results The mean age (31.6 vs. 30.6), years of formal education (6.3 vs. 6.3), marital status (24.2 vs. 23.8 never married), and the number of children (1.3 vs. 1.1) of women reporting having lost all income were not significantly different from those who experienced partial income loss. Overall, GBV experiences were significantly higher in FEWs who lost all income than in those who lost partial income (62.9% vs. 47.4%, p  = 0.03). Controlling for the number of dependents, smartphone ownership, and food security, the adjusted odds ratio for GBV was significant in the adjusted model (AOR = 1.23 (1.08–1.40), p  = 0.001) indicating that those who experienced total income loss were more likely to experience GBV than those who experienced partial income loss. In addition, they were significantly less likely to be food secure ( p  = 0.04), less likely to own a smartphone ( p  = 0.02), and had more dependents ( p  < 0.001). Conclusion Disaster response programs should consider the implications of safety measures and government support for both formal and informal workers regarding safety, food access, and mental health support. Food assistance programs should target the most vulnerable informal sector workers during crises.

Previous attempts to culture mouse alveolar type II (ATII) cells have been hampered by limited purity and cell recovery. We have now obtained culturable ATII cells from female C57BL/6 mice at a purity of 92% +/- 3 (mean +/- SD; n = 20), with viabilities of 96% +/- 2 and total yields of 5.1 +/- 0.7 X 10(6) cells per mouse. Crude lung cell suspensions were prepared by intratracheal instillation of Dispase and agarose followed by mechanical disaggregation of the lungs. Crude cell suspensions were purified by negative selection using a biotinylated-antibody, streptavidin-coated biomagnetic particle system. Cell purities were determined by Pap staining and confirmed ultrastructurally. Purified ATII cells were cultured on fibronectin-coated chamber slides and maintained for up to 5 days in DMEM with 10% fetal bovine serum. Cultures exhibited minimal contamination by Clara cells, mesenchymal cells, or endothelial cells, and the epithelial nature of the cultures was confirmed by positive cytokeratin staining in at least 97% of the cells through day 5. Day 3 cultures demonstrated osmium tetroxide/tannic acid-stained granules consistent with lamellar bodies in 76% +/- 3.6 of the cells. The cultures displayed features distinct from those previously described for adult rat ATII cells, including irregularly-shaped cells and the formation of numerous cytoplasmic projections in direct contact with other cells. These studies indicate that excellent yields of highly purified, culturable ATII cells can be obtained from genetically defined mice. These techniques may provide powerful new models for the study of parenchymal lung disease in vitro.