Amelioration of Tau and ApoE4-linked glial lipid accumulation and neurodegeneration with an LXR agonist.

Apolipoprotein E (APOE) is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). APOE4 increases and APOE2 decreases risk relative to APOE3. In the P301S mouse model of tauopathy, ApoE4 increases tau pathology and neurodegeneration when compared with ApoE3 or the absence of ApoE. However, the role of ApoE isoforms and lipid metabolism in contributing to tau-mediated degeneration is unknown. We demonstrate that in P301S tau mice, ApoE4 strongly promotes glial lipid accumulation and perturbations in cholesterol metabolism and lysosomal function. Increasing lipid efflux in glia via an LXR agonist or Abca1 overexpression strongly attenuates tau pathology and neurodegeneration in P301S/ApoE4 mice. We also demonstrate reductions in reactive astrocytes and microglia, as well as changes in cholesterol biosynthesis and metabolism in glia of tauopathy mice in response to LXR activation. These data suggest that promoting efflux of glial lipids may serve as a therapeutic approach to ameliorate tau and ApoE4-linked neurodegeneration.

APOE3ch alters microglial response and suppresses Aß-induced tau seeding and spread.

A recent case report described an individual who was a homozygous carrier of the APOE3 Christchurch (APOE3ch) mutation and resistant to autosomal dominant Alzheimer's Disease (AD) caused by a PSEN1-E280A mutation. Whether APOE3ch contributed to the protective effect remains unclear. We generated a humanized APOE3ch knock-in mouse and crossed it to an amyloid-ß (Aß) plaque-depositing model. We injected AD-tau brain extract to investigate tau seeding and spreading in the presence or absence of amyloid. Similar to the case report, APOE3ch expression resulted in peripheral dyslipidemia and a marked reduction in plaque-associated tau pathology. Additionally, we observed decreased amyloid response and enhanced microglial response around plaques. We also demonstrate increased myeloid cell phagocytosis and degradation of tau aggregates linked to weaker APOE3ch binding to heparin sulfate proteoglycans. APOE3ch influences the microglial response to Aß plaques, which suppresses Aß-induced tau seeding and spreading. The results reveal new possibilities to target Aß-induced tauopathy.

Current status of amyloid-targeting immunotherapies for Alzheimer's disease.

Anti-amyloid antibodies are moving from clinical trials into patients to treat early clinical stages of Alzheimer's disease.

Microglial REV-ERBα regulates inflammation and lipid droplet formation to drive tauopathy in male mice.

Alzheimer's disease, the most common age-related neurodegenerative disease, is characterized by tau aggregation and associated with disrupted circadian rhythms and dampened clock gene expression. REV-ERBα is a core circadian clock protein which also serves as a nuclear receptor and transcriptional repressor involved in lipid metabolism and macrophage function. Global REV-ERBα deletion has been shown to promote microglial activation and mitigate amyloid plaque formation. However, the cell-autonomous effects of microglial REV-ERBα in healthy brain and in tauopathy are unexplored. Here, we show that microglial REV-ERBα deletion enhances inflammatory signaling, disrupts lipid metabolism, and causes lipid droplet (LD) accumulation specifically in male microglia. These events impair microglial tau phagocytosis, which can be partially rescued by blockage of LD formation. In vivo, microglial REV-ERBα deletion exacerbates tau aggregation and neuroinflammation in two mouse tauopathy models, specifically in male mice. These data demonstrate the importance of microglial lipid droplets in tau accumulation and reveal REV-ERBα as a therapeutically accessible, sex-dependent regulator of microglial inflammatory signaling, lipid metabolism, and tauopathy.

TREM2 inhibition triggers antitumor cell activity of myeloid cells in glioblastoma.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays important roles in brain microglial function in neurodegenerative diseases, but the role of TREM2 in the GBM TME has not been examined. Here, we found that TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors and that high TREM2 expression correlates with poor prognosis in patients with GBM. TREM2 loss of function in human macrophages and mouse myeloid cells increased interferon-γ-induced immunoactivation, proinflammatory polarization, and tumoricidal capacity. In orthotopic mouse GBM models, mice with chronic and acute Trem2 loss of function exhibited decreased tumor growth and increased survival. Trem2 inhibition reprogrammed myeloid phenotypes and increased programmed cell death protein 1 (PD-1)+CD8+ T cells in the TME. Last, Trem2 deficiency enhanced the effectiveness of anti-PD-1 treatment, which may represent a therapeutic strategy for patients with GBM.

Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility.

Voltage-gated K+ channels function in macromolecular complexes with accessory subunits to regulate brain function. Here, we describe a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)-dependent mechanism that regulates the association of the A-type K+ channel subunit Kv4.2 with its auxiliary subunit dipeptidyl peptidase 6 (DPP6), and thereby modulates neuronal excitability and cognitive flexibility. We show that activity-induced Kv4.2 phosphorylation triggers Pin1 binding to, and isomerization of, Kv4.2 at the pThr607-Pro motif, leading to the dissociation of the Kv4.2-DPP6 complex. We generated a novel mouse line harboring a knock-in Thr607 to Ala (Kv4.2TA) mutation that abolished dynamic Pin1 binding to Kv4.2. CA1 pyramidal neurons of the hippocampus from these mice exhibited altered Kv4.2-DPP6 interaction, increased A-type K+ current, and reduced neuronal excitability. Behaviorally, Kv4.2TA mice displayed normal initial learning but improved reversal learning in both Morris water maze and lever press paradigms. These findings reveal a Pin1-mediated mechanism regulating reversal learning and provide potential targets for the treatment of neuropsychiatric disorders characterized by cognitive inflexibility.

Lack of hepatic apoE does not influence early Aß deposition: observations from a new APOE knock-in model.

BACKGROUND: The apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer disease (AD). ApoE is produced by both astrocytes and microglia in the brain, whereas hepatocytes produce the majority of apoE found in the periphery. Studies using APOE knock-in and transgenic mice have demonstrated a strong isoform-dependent effect of apoE on the accumulation of amyloid-ß (Aß) deposition in the brain in the form of both Aß-containing amyloid plaques and cerebral amyloid angiopathy. However, the specific contributions of different apoE pools to AD pathogenesis remain unknown. METHODS: We have begun to address these questions by generating new lines of APOE knock-in (APOE-KI) mice (ε2/ε2, ε3/ε3, and ε4/ε4) where the exons in the coding region of APOE are flanked by loxP sites, allowing for cell type-specific manipulation of gene expression. We assessed these mice both alone and after crossing them with mice with amyloid deposition in the brain. Using biochemical and histological methods. We also investigated how removal of APOE expression from hepatocytes affected cerebral amyloid deposition. RESULTS: As in other APOE knock-in mice, apoE protein was present predominantly in astrocytes in the brain under basal conditions and was also detected in reactive microglia surrounding amyloid plaques. Primary cultured astrocytes and microglia from the APOE-KI mice secreted apoE in lipoprotein particles of distinct size distribution upon native gel analysis with microglial particles being substantially smaller than the HDL-like particles secreted by astrocytes. Crossing of APP/PS1 transgenic mice to the different APOE-KI mice recapitulated the previously described isoform-specific effect (ε4 > ε3) on amyloid plaque and Aß accumulation. Deletion of APOE in hepatocytes did not alter brain apoE levels but did lead to a marked decrease in plasma apoE levels and changes in plasma lipid profile. Despite these changes in peripheral apoE and on plasma lipids, cerebral accumulation of amyloid plaques in APP/PS1 mice was not affected. CONCLUSIONS: Altogether, these new knock-in strains offer a novel and dynamic tool to study the role of APOE in AD pathogenesis in a spatially and temporally controlled manner.

Disruption of GpI mGluR-Dependent Cav2.3 Translation in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is an inherited intellectual impairment that results from the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates mRNA translation at synapses. The absence of FMRP leads to neuronal and circuit-level hyperexcitability that is thought to arise from the aberrant expression and activity of voltage-gated ion channels, although the identification and characterization of these ion channels have been limited. Here, we show that FMRP binds the mRNA of the R-type voltage-gated calcium channel Cav2.3 in mouse brain synaptoneurosomes and represses Cav2.3 translation under basal conditions. Consequently, in hippocampal neurons from male and female FMRP KO mice, we find enhanced Cav2.3 protein expression by western blotting and abnormally large R currents in whole-cell voltage-clamp recordings. In agreement with previous studies showing that FMRP couples Group I metabotropic glutamate receptor (GpI mGluR) signaling to protein translation, we find that GpI mGluR stimulation results in increased Cav2.3 translation and R current in hippocampal neurons which is disrupted in FMRP KO mice. Thus, FMRP serves as a key translational regulator of Cav2.3 expression under basal conditions and in response to GpI mGluR stimulation. Loss of regulated Cav2.3 expression could underlie the neuronal hyperactivity and aberrant calcium spiking in FMRP KO mice and contribute to FXS, potentially serving as a novel target for future therapeutic strategies.SIGNIFICANCE STATEMENT Patients with fragile X syndrome (FXS) exhibit signs of neuronal and circuit hyperexcitability, including anxiety and hyperactive behavior, attention deficit disorder, and seizures. FXS is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein, and the neuronal hyperexcitability observed in the absence of FMRP likely results from its ability to regulate the expression and activity of voltage-gated ion channels. Here we find that FMRP serves as a key translational regulator of the voltage-gated calcium channel Cav2.3 under basal conditions and following activity. Cav2.3 impacts cellular excitability and calcium signaling, and the alterations in channel translation and expression observed in the absence of FMRP could contribute to the neuronal hyperactivity that underlies FXS.

A novel bungarotoxin binding site-tagged construct reveals MAPK-dependent Kv4.2 trafficking.

Kv4.2 voltage-gated K+ channel subunits, the primary source of the somatodendritic A-type K+ current in CA1 pyramidal neurons of the hippocampus, play important roles in regulating dendritic excitability and plasticity. To better study the trafficking and subcellular distribution of Kv4.2, we created and characterized a novel Kv4.2 construct encoding a bungarotoxin binding site in the extracellular S3-S4 linker region of the α-subunit. When expressed, this construct can be visualized in living cells after staining with rhodamine-conjugated bungarotoxin. We validated the utility of this construct by visualizing the spontaneous internalization and insertion of Kv4.2 in HEK 293T cells. We further report that Kv4.2 colocalized with several endosome markers in HEK 293T cells. In addition, Kv4.2 internalization is significantly impaired by mitogen-activated protein kinase (MAPK) inhibitors in transfected primary hippocampal neurons. Therefore, this newly developed BBS-Kv4.2 construct provides a novel and powerful tool for studying surface Kv4.2 channel localization and trafficking.

Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition.

BACKGROUND: The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity. RESULTS: We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls. CONCLUSIONS: Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

Comparison of LES of steady transitional flow in an idealized stenosed axisymmetric artery model with a RANS transitional model.

In this study, two different turbulence methodologies are investigated to predict transitional flow in a 75% stenosed axisymmetric experimental arterial model and in a slightly modified version of the model with an eccentric stenosis. Large eddy simulation (LES) and Reynolds-averaged Navier-Stokes (RANS) methods were applied; in the LES simulations eddy viscosity subgrid-scale models were employed (basic and dynamic Smagorinsky) while the RANS method involved the correlation-based transitional version of the hybrid k-ε/k-ω flow model. The RANS simulations used 410,000 and 820,000 element meshes for the axisymmetric and eccentric stenoses, respectively, with y(+) less than 2 viscous wall units for the boundary elements, while the LES used 1,200,000 elements with y(+) less than 1. Implicit filtering was used for LES, giving an overlap between the resolved and modeled eddies, ensuring accurate treatment of near wall turbulence structures. Flow analysis was carried out in terms of vorticity and eddy viscosity magnitudes, velocity, and turbulence intensity profiles and the results were compared both with established experimental data and with available direct numerical simulations (DNSs) from the literature. The simulation results demonstrated that the dynamic Smagorinsky LES and RANS transitional model predicted fairly comparable velocity and turbulence intensity profiles with the experimental data, although the dynamic Smagorinsky model gave the best overall agreement. The present study demonstrated the power of LES methods, although they were computationally more costly, and added further evidence of the promise of the RANS transition model used here, previously tested in pulsatile flow on a similar model. Both dynamic Smagorinsky LES and the RANS model captured the complex transition phenomena under physiological Reynolds numbers in steady flow, including separation and reattachment. In this respect, LES with dynamic Smagorinsky appeared more successful than DNS in replicating the axisymmetric experimental results, although inflow conditions, which are subject to caveats, may have differed. For the eccentric stenosis, LES with Smagorinsky coefficient of 0.13 gave the closest agreement with DNS despite the known shortcomings of fixed coefficients. The relaminarization as the flow escaped the influence of the stenosis was amply demonstrated in the simulations, graphically so in the case of LES.

Modeling flow in collecting lymphatic vessels: one-dimensional flow through a series of contractile elements.

The lymphatic system comprises a series of elements, lymphangions, separated by valves and possessed of active, contractile walls to pump interstitial fluid from its collection in the terminal lymphatics back to the main circulation. Despite its importance, there is a dearth of information on the fluid dynamics of the lymphatic system. In this article, we describe linked experimental and computational work aimed at elucidating the biomechanical properties of the individual lymphangions. We measure the static and dynamic mechanical properties of excised bovine collecting lymphatics and develop a one-dimensional computational model of the coupled fluid flow/wall motion. The computational model is able to reproduce the pumping behavior of the real vessel using a simple contraction function producing fast contraction pulses traveling in the retrograde direction to the flow.

A computational fluid dynamics study of inspiratory flow in orotracheal geometries.

Computational fluid dynamics (CFD) has been used to investigate the flow of air through the human orotracheal system. Results from an idealised geometry, and from a patient-specific geometry created from MRI scans were compared. The results showed a significant difference in the flow structures between the two geometries. Inert particles with diameters in the range 1-9 microm were tracked through the two geometries. Particle diameter has proved to be an important factor in defining the eventual destinations of inhaled particles. Results from our calculations match other experimental and computational results in the literature, and differences between the idealised and patient-specific geometries are less significant.

Genotypes A and B of Cadophora gregata Differ in Ability to Colonize Susceptible Soybean.

Growth chamber experiments were conducted to determine if extent of colonization of soybean stems by genotypes A and B of Cadophora gregata (Phialophora gregata), the causal agent of brown stem rot (BSR) of soybean, is similar in soybean plants resistant or susceptible to genotype A. Upon introduction of the two genotypes separately into the base of stems of 2-week-old seedlings, genotype A advanced with the growing tips of susceptible but not resistant genotypes. In contrast, genotype B did not advance with the growing tips of either resistant or susceptible soybean. In similar experiments, 5 weeks after introduction of genotype A, both mean percent stem length colonized by C. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. For genotype B, there was no or a slight difference between resistant and susceptible soybean genotypes in mean percent stem length colonized and no difference in mean percentage of symptomatic trifoliate leaflets 5 weeks after introduction of the pathogen. These results indicate that genotype A and genotype B differ not only in the severity of foliar symptoms they cause on genotype A-susceptible soybean plants, but also in how severely they colonize the stems of these soybean plants. In our experiments, genotype A and genotype B did not differ consistently in their ability to cause internal stem discoloration. The two genotypes of C. gregata can be distinguished based on how severely they colonize stems of genotype A-susceptible soybean. Thus, a BSR resistance screening method, which relies on assessment of stem colonization by C. gregata, works only for screening soybean lines resistance to genotype A. In light of these results, it is important to distinguish soybean resistance to genotype A versus genotype B of C. gregata. Whether genotype B causes yield loss and whether soybean plants can be distinguished as resistant or susceptible to genotype B needs to be investigated.

Soybean Stem Colonization by Genotypes A and B of Cadophora gregata Increases with Increasing Population Densities of Heterodera glycines.

Growth chamber experiments were conducted to investigate whether parasitism by increasing population densities of Heterodera glycines, the soybean cyst nematode, increases the incidence and severity of stem colonization by the aggressive genotype A and the mild genotype B of Cadophora gregata (Phialophora gregata), causal agents of brown stem rot of soybeans. Soybean genotypes with three combinations of resistance and susceptibility to H. glycines and genotype A of C. gregata were inoculated with each genotype of C. gregata alone or each genotype with two population densities of H. glycines eggs, 1,500 or 10,000 per 100 cm3 of soil. Stems of two H. glycines-susceptible soybeans were more colonized by both aggressive and mild genotypes of C. gregata in the presence of high than in the presence of low H. glycines population density.

A New Greenhouse Method to Assay Soybean Resistance to Brown Stem Rot.

Greenhouse, growth chamber, and field experiments were conducted to develop a method to assess resistance of soybeans to Cadophora gregata (Phialophora gregata), causal agent of brown stem rot (BSR). In the new method, C. gregata is introduced at the base of the stems of 2-week-old soybeans, and the presence of the fungus is assessed in the tips of the stems 5 weeks later. To test the effectiveness of the method, two populations of soybeans and 10 checks were inoculated at the stem base and then assayed for fungal colonization of the stem tips, percentage of symptomatic leaflets, and percent internal stem length discolored. The lines also were planted in naturally infested fields to assess for percent internal stem length discolored, and were tested for the presence/absence of a BSR-resistant molecular marker. Greenhouse, field, and molecular marker data were compared. Linear regression analysis suggested that percentage of plants with colonized stem tips explained 41 to 64% of the variability (P < 0.0001) in percent stem length discolored in the field and 58 to 85% of the variability (P < 0.0001) in molecular marker data for BSR resistance. Percent stem length discolored assessed in the greenhouse had the lowest correlation with percent stem length discolored in the field and with the molecular marker. Of three incubation temperatures tested, 22°C was the most conducive for distinguishing resistant/susceptible soybeans using the colonization method.

Resistance to Phialophora gregata Is Expressed in the Stems of Resistant Soybeans.

Growth chamber experiments were conducted to determine if resistance to Phialophora gregata, the causal agent of brown stem rot (BSR) of soybean, is expressed in the stems of resistant soybean genotypes. Upon introduction of the pathogen into the base of stems of 2-week-old seedlings, the fungus advanced with the growing tips of plants of susceptible genotypes but lagged behind in resistant genotypes. Five weeks after introduction of the pathogen, both mean percent stem length colonized by P. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. These results indicate that resistance can be expressed in the stems of resistant soybean plants and suggest that stem inoculation methods may be useful for assessing resistance to P. gregata. Also, in our experiments, internal stem discoloration was not as useful as colonization and foliar symptoms in discriminating resistant from susceptible genotypes.

Heterodera glycines Infection Increases Incidence and Severity of Brown Stem Rot in Both Resistant and Susceptible Soybean.

Growth chamber experiments were conducted to investigate whether parasitism by Heterodera glycines, the soybean cyst nematode, increases incidence and severity of brown stem rot (BSR) of soybean, caused by Phialophora gregata, in both resistant and susceptible soybean cultivars. Soybean genotypes with various combinations of resistance and susceptibility to both pathogens were inoculated with P. gregata alone or P. gregata plus H. glycines. In most tests of H. glycines-susceptible genotypes, incidence and severity of internal stem discoloration, characteristic of BSR, was greater in the presence than in the absence of H. glycines, regardless of susceptibility or resistance to BSR. There was less of an increasing effect of H. glycines on stem symptoms in genotypes resistant to both BSR and H. glycines; however, P. gregata colonization of these genotypes was increased. Stems of both a BSR-resistant and a BSR-susceptible genotype were colonized earlier by P. gregata in the presence than in the absence of H. glycines. Our findings indicate that H. glycines can increase the incidence and severity of BSR in soybean regardless of resistance or susceptibility to either pathogen.