Chun-Chi Liang

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 (App^SAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-β pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous App^SAA 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 Aβ 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 Aβ content. The App^SAA 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 Aβ 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.

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.

Genetic redundancy can be exploited to identify therapeutic targets for inherited disorders. An example is 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 dosedependent 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 level may retard or prevent symptom development in DYT1 dystonia.

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 is a membrane-embedded AAA+ ATPase important in the nuclear envelope (NE) lumen. However, most torsinA is localized in the peripheral endoplasmic reticulum (ER) lumen with slow mobility incompatible with free equilibration between ER subdomains. We now find that NE-localized torsinA is on the inner nuclear membrane (INM) and ask how torsinA reaches this subdomain. The ER system contains two transmembrane proteins, LAP1 and LULL1, 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 decreased by LULL1. Mutations in torsinA or LULL1 that inhibit ATPase activity reduce torsinA access 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 via torsinA.

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.

Wang et al. find that deletion of FIP200, a protein essential for autophagy, leads to deficits in the maintenance and differentiation of postnatal neural stem cells (NSC). These effects are rescued by an antioxidant treatment, suggesting that FIP200-mediated autophagy regulates neurogenesis by controlling the oxidative state of NSC. 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.