A TREM2-activating antibody with a blood-brain barrier transport vehicle enhances microglial metabolism in Alzheimer's disease models.

Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood-brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.

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.

TorsinB overexpression prevents abnormal twisting in DYT1 dystonia mouse models.

Genetic redundancy can be exploited to identify therapeutic targets for inherited disorders. We explored this possibility in DYT1 dystonia, a neurodevelopmental movement disorder caused by a loss-of-function (LOF) mutation in the TOR1A gene encoding torsinA. Prior work demonstrates that torsinA and its paralog torsinB have conserved functions at the nuclear envelope. This work established that low neuronal levels of torsinB dictate the neuronal selective phenotype of nuclear membrane budding. Here, we examined whether torsinB expression levels impact the onset or severity of abnormal movements or neuropathological features in DYT1 mouse models. We demonstrate that torsinB levels bidirectionally regulate these phenotypes. Reducing torsinB levels causes a dose-dependent worsening whereas torsinB overexpression rescues torsinA LOF-mediated abnormal movements and neurodegeneration. These findings identify torsinB as a potent modifier of torsinA LOF phenotypes and suggest that augmentation of torsinB expression may retard or prevent symptom development in DYT1 dystonia.

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.

TorsinA dysfunction causes persistent neuronal nuclear pore defects.

A critical challenge to deciphering the pathophysiology of neurodevelopmental disease is identifying which of the myriad abnormalities that emerge during CNS maturation persist to contribute to long-term brain dysfunction. Childhood-onset dystonia caused by a loss-of-function mutation in the AAA+ protein torsinA exemplifies this challenge. Neurons lacking torsinA develop transient nuclear envelope (NE) malformations during CNS maturation, but no NE defects are described in mature torsinA null neurons. We find that during postnatal CNS maturation torsinA null neurons develop mislocalized and dysfunctional nuclear pore complexes (NPC) that lack NUP358, normally added late in NPC biogenesis. SUN1, a torsinA-related molecule implicated in interphase NPC biogenesis, also exhibits localization abnormalities. Whereas SUN1 and associated nuclear membrane abnormalities resolve in juvenile mice, NPC defects persist into adulthood. These findings support a role for torsinA function in NPC biogenesis during neuronal maturation and implicate altered NPC function in dystonia pathophysiology.

The DYT6 Dystonia Protein THAP1 Regulates Myelination within the Oligodendrocyte Lineage.

The childhood-onset motor disorder DYT6 dystonia is caused by loss-of-function mutations in the transcription factor THAP1, but the neurodevelopmental processes in which THAP1 participates are unknown. We find that THAP1 is essential for the timing of myelination initiation during CNS maturation. Conditional deletion of THAP1 in the CNS retards maturation of the oligodendrocyte (OL) lineage, delaying myelination and causing persistent motor deficits. The CNS myelination defect results from a cell-autonomous requirement for THAP1 in the OL lineage and is recapitulated in developmental assays performed on OL progenitor cells purified from Thap1 null mice. Loss of THAP1 function disrupts a core set of OL maturation genes and reduces the DNA occupancy of YY1, a transcription factor required for OL maturation. These studies establish a role for THAP1 transcriptional regulation at the inception of myelination and implicate abnormal timing of myelination in the pathogenesis of childhood-onset dystonia.

Sensorimotor tests unmask a phenotype in the DYT1 knock-in mouse model of dystonia.

Hereditary generalized dystonia is often caused by a GAG deletion in TOR1A (DYT1) that encodes for the protein torsinA. Although mutation carriers show alterations in neuronal connectivity and sensorimotor deficits, only 30% develop dystonia. Uncovering the factors triggering the dystonic symptoms and underlying pathophysiology would greatly benefit the development of more effective therapies. In DYT1 knock-in (KI) mice, the expression of torsinA mutant alters the connectivity of neurons and the function of striatal cholinergic interneurons. We aimed to determine if heterozygous DYT1 KI mice exhibit deficits in behavioural tests that explore the connectivity of the sensory and motor system. DYT1 KI mice were tested in cognitive tests and challenging motor paradigms, followed by the adhesive removal test and the adaptive rotating beam test which both require sensorimotor integration. DYT1 KI mice did not exhibit cognitive deficits and were able to perform similarly to wild type mice even in challenging motor tests with relatively stable sensory input. Conversely, DYT1 KI mice spent more time on sensing and removing an adhesive sticker from the back of the nose; they exhibited difficulty to traverse rotating rods, especially if the surface was smooth and the diameter small. Our observations further support a role of sensorimotor integration in manifestation of this movement disorder. Future studies in DYT1 KI mice will explore the involved neurocircuitry and underlying molecular mechanisms.

Neuronal Nuclear Membrane Budding Occurs during a Developmental Window Modulated by Torsin Paralogs.

DYT1 dystonia is a neurodevelopmental disease that manifests during a discrete period of childhood. The disease is caused by impaired function of torsinA, a protein linked to nuclear membrane budding. The relationship of NE budding to neural development and CNS function is unclear, however, obscuring its potential role in dystonia pathogenesis. We find NE budding begins and resolves during a discrete neurodevelopmental window in torsinA null neurons in vivo. The developmental resolution of NE budding corresponds to increased torsinB protein, while ablating torsinB from torsinA null neurons prevents budding resolution and causes lethal neural dysfunction. Developmental changes in torsinB also correlate with NE bud formation in differentiating DYT1 embryonic stem cells, and overexpression of torsinA or torsinB rescues NE bud formation in this system. These findings identify a torsinA neurodevelopmental window that is essential for normal CNS function and have important implications for dystonia pathogenesis and therapeutics.

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.

TorsinA hypofunction causes abnormal twisting movements and sensorimotor circuit neurodegeneration.

Lack of a preclinical model of primary dystonia that exhibits dystonic-like twisting movements has stymied identification of the cellular and molecular underpinnings of the disease. The classical familial form of primary dystonia is caused by the DYT1 (ΔE) mutation in TOR1A, which encodes torsinA, AAA⁺ ATPase resident in the lumen of the endoplasmic reticular/nuclear envelope. Here, we found that conditional deletion of Tor1a in the CNS (nestin-Cre Tor1a(flox/-)) or isolated CNS expression of DYT1 mutant torsinA (nestin-Cre Tor1a(flox/ΔE)) causes striking abnormal twisting movements. These animals developed perinuclear accumulation of ubiquitin and the E3 ubiquitin ligase HRD1 in discrete sensorimotor regions, followed by neurodegeneration that was substantially milder in nestin-Cre Tor1a(flox/ΔE) compared with nestin-Cre Tor1a(flox/-) animals. Similar to the neurodevelopmental onset of DYT1 dystonia in humans, the behavioral and histopathological abnormalities emerged and became fixed during CNS maturation in the murine models. Our results establish a genetic model of primary dystonia that is overtly symptomatic, and link torsinA hypofunction to neurodegeneration and abnormal twisting movements. These findings provide a cellular and molecular framework for how impaired torsinA function selectively disrupts neural circuits and raise the possibility that discrete foci of neurodegeneration may contribute to the pathogenesis of DYT1 dystonia.

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.

Neural-specific deletion of FIP200 leads to cerebellar degeneration caused by increased neuronal death and axon degeneration.

FIP200 (FAK family-interacting protein of 200 kDa) is a conserved protein recently identified as a potential mammalian counterpart of yeast autophagy protein Atg17. However, it remains unknown whether mammalian FIP200 regulates autophagy in vivo. Here we show that neural-specific deletion of FIP200 resulted in cerebellar degeneration accompanied by progressive neuronal loss, spongiosis, and neurite degeneration in the cerebellum. Furthermore, deletion of FIP200 led to increased apoptosis in cerebellum as well as accumulation of ubiquitinated protein aggregates without any deficiency in proteasome catalytic functions. We also observed an increased p62/SQSTM1 accumulation in the cerebellum and reduced autophagosome formation as well as accumulation of damaged mitochondria in the mutant mice. Lastly, analysis of cerebellar neurons in vitro showed reduced JNK activation and increased susceptibility to serum deprivation-induced apoptosis in cerebellar neurons from the mutant mice. Taken together, these results provide strong genetic evidence for a role of FIP200 in the regulation of neuronal homeostasis through its function in autophagy in vivo.

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.

In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.

The in vitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro. The basic steps involve creating a "scratch" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells. Compared to other methods, the in vitro scratch assay is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration, mimic cell migration during wound healing in vivo and are compatible with imaging of live cells during migration to monitor intracellular events if desired. Besides monitoring migration of homogenous cell populations, this method has also been adopted to measure migration of individual cells in the leading edge of the scratch. Not taking into account the time for transfection of cells, in vitro scratch assay per se usually takes from several hours to overnight.

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.