Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia.

BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.

Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region.

Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease-associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full-length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α-secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α-secretase-mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho-SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid ß-peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9-bound. Moreover, in a mouse model for Alzheimer's disease-related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease-associated state.

Nuclear envelope-localized torsinA-LAP1 complex regulates hepatic VLDL secretion and steatosis.

Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Recent evidence suggests that the nuclear envelope is a site of regulation of lipid metabolism but there is limited appreciation of the responsible mechanisms and molecular components within this organelle. We showed that conditional hepatocyte deletion of the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1) caused defective VLDL secretion and steatosis, including intranuclear lipid accumulation. LAP1 binds to and activates torsinA, an AAA+ ATPase that resides in the perinuclear space and continuous main ER. Deletion of torsinA from mouse hepatocytes caused even greater reductions in VLDL secretion and profound steatosis. Both of these mutant mouse lines developed hepatic steatosis and subsequent steatohepatitis on a regular chow diet in the absence of whole-body insulin resistance or obesity. Our results establish an essential role for the nuclear envelope-localized torsinA-LAP1 complex in hepatic VLDL secretion and suggest that the torsinA pathway participates in the pathophysiology of nonalcoholic fatty liver disease.

Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model.

Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582-585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo.

Access of torsinA to the inner nuclear membrane is activity dependent and regulated in the endoplasmic reticulum.

TorsinA (also known as torsin-1A) is a membrane-embedded AAA+ ATPase that has an important role in the nuclear envelope lumen. However, most torsinA is localized in the peripheral endoplasmic reticulum (ER) lumen where it has a slow mobility that is incompatible with free equilibration between ER subdomains. We now find that nuclear-envelope-localized torsinA is present on the inner nuclear membrane (INM) and ask how torsinA reaches this subdomain. The ER system contains two transmembrane proteins, LAP1 and LULL1 (also known as TOR1AIP1 and TOR1AIP2, respectively), that reversibly co-assemble with and activate torsinA. Whereas LAP1 localizes on the INM, we show that LULL1 is in the peripheral ER and does not enter the INM. Paradoxically, interaction between torsinA and LULL1 in the ER targets torsinA to the INM. Native gel electrophoresis reveals torsinA oligomeric complexes that are destabilized by LULL1. Mutations in torsinA or LULL1 that inhibit ATPase activity reduce the access of torsinA to the INM. Furthermore, although LULL1 binds torsinA in the ER lumen, its effect on torsinA localization requires cytosolic-domain-mediated oligomerization. These data suggest that LULL1 oligomerizes to engage and transiently disassemble torsinA oligomers, and is thereby positioned to transduce cytoplasmic signals to the INM through torsinA.

FIP200 is required for maintenance and differentiation of postnatal neural stem cells.

Despite recent studies showing that inhibition of autophagy depletes the hematopoietic stem cell pool and increases intracellular reactive oxygen species (ROS), it remains unknown whether autophagy is essential in the maintenance of other stem cells. Moreover, it is unclear whether and how the aberrant ROS increase causes depletion of stem cells. Here we report that ablation of FIP200 (also known as Rb1cc1), a gene essential for autophagy induction in mammalian cells, results in a progressive loss of neural stem cells (NSCs) and impairment in neuronal differentiation specifically in the postnatal brain, but not the embryonic brain, in mice. The defect in maintaining the postnatal NSC pool was caused by p53-dependent apoptotic responses and cell cycle arrest. However, the impaired neuronal differentiation was rescued by treatment with the antioxidant N-acetylcysteine but not by p53 inactivation. These data reveal that FIP200-mediated autophagy contributes to the maintenance and functions of NSCs through regulation of oxidative state.

Tyrosine phosphorylation of growth factor receptor-bound protein-7 by focal adhesion kinase in the regulation of cell migration, proliferation, and tumorigenesis.

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK*Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phosphorylated tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phosphorylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phosphorylation-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phosphorylation-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phosphorylation of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK*Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK*Grb7 complexes and Grb7 phosphorylation by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK*Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK*Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.

Mammary epithelial-specific deletion of the focal adhesion kinase gene leads to severe lobulo-alveolar hypoplasia and secretory immaturity of the murine mammary gland.

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.

Synergistic effect of focal adhesion kinase overexpression and hepatocyte growth factor stimulation on cell transformation.

Although an elevated level of focal adhesion kinase (FAK) has been observed in a variety of invasive human tumors, forced expression of FAK alone in cultured cells does not cause them to exhibit transformed phenotypes. Therefore, the role of FAK in oncogenic transformation remains unclear. In this study, we have demonstrated that FAK overexpression in Madin-Darby canine kidney epithelial cells rendered them susceptible to transformation by hepatocyte growth factor (HGF). Using various FAK mutants, we found that the simultaneous bindings of Src and p130(cas) were required for FAK to potentiate cell transformation. Expression of FAK-related nonkinase, kinase-deficient Src, or the Src homology 3 domain of p130(cas), which respectively serve as dominant negative versions of FAK, Src, and p130(cas), apparently reversed the transformed phenotypes of FAK-overexpressed cells upon HGF stimulation. Moreover, FAK overexpression was able to enhance HGF-elicited signals, leading to sustained activation of ERK, JNK, and AKT, which could be prevented by the expression of the Src homology 3 domain of p130(cas). Taken together, our results indicate that the synergistic effect of FAK overexpression and HGF stimulation leads to cell transformation and implicate a critical role of p130(cas) in this process.