Dynamic regulation of hepatic lipid metabolism by torsinA and its activators.

Depletion of torsinA from hepatocytes leads to reduced liver triglyceride secretion and marked hepatic steatosis. TorsinA is an atypical ATPase that lacks intrinsic activity unless it is bound to its activator, lamina-associated polypeptide 1 (LAP1) or luminal domain-like LAP1 (LULL1). We previously demonstrated that depletion of LAP1 from hepatocytes has more modest effects on liver triglyceride secretion and steatosis development than depletion of torsinA. We now show that depletion of LULL1 alone does not significantly decrease triglyceride secretion or cause steatosis. However, simultaneous depletion of both LAP1 and LULL1 leads to defective triglyceride secretion and marked steatosis similar to that observed with depletion of torsinA. Depletion of both LAP1 and torsinA from hepatocytes generated phenotypes similar to those observed with only torsinA depletion, implying that the 2 proteins act in the same pathway in liver lipid metabolism. Our results demonstrate that torsinA and its activators dynamically regulate hepatic lipid metabolism.

Functional interaction of torsinA and its activators in liver lipid metabolism.

TorsinA is an atypical ATPase that lacks intrinsic activity unless it is bound to its activators lamina-associated polypeptide 1 (LAP1) in the perinuclear space or luminal domain-like LAP1 (LULL1) throughout the endoplasmic reticulum. However, the interaction of torsinA with LAP1 and LULL1 has not yet been shown to modulate a defined physiological process in mammals in vivo . We previously demonstrated that depletion of torsinA from mouse hepatocytes leads to reduced liver triglyceride secretion and marked steatosis, whereas depletion of LAP1 had more modest similar effects. We now show that depletion of LULL1 alone does not significantly decrease liver triglyceride secretion or cause steatosis. However, simultaneous depletion of both LAP1 and LULL1 from hepatocytes leads to defective triglyceride secretion and marked steatosis similar to that observed with depletion of torsinA. Our results demonstrate that torsinA and its activators dynamically regulate a physiological process in mammals in vivo .

Hepatocytes Deficient in Nuclear Envelope Protein Lamina-associated Polypeptide 1 are an Ideal Mammalian System to Study Intranuclear Lipid Droplets.

Lipid droplets (LDs) are generally considered to be synthesized in the ER and utilized in the cytoplasm. However, LDs have been observed inside nuclei in some cells, although recent research on nuclear LDs has focused on cultured cell lines. To better understand nuclear LDs that occur in vivo, here we examined LDs in primary hepatocytes from mice following depletion of the nuclear envelope protein lamina-associated polypeptide 1 (LAP1). Microscopic image analysis showed that LAP1-depleted hepatocytes contain frequent nuclear LDs, which differ from cytoplasmic LDs in their associated proteins. We found type 1 nucleoplasmic reticula, which are invaginations of the inner nuclear membrane, are often associated with nuclear LDs in these hepatocytes. Furthermore, in vivo depletion of the nuclear envelope proteins lamin A and C from mouse hepatocytes led to severely abnormal nuclear morphology, but significantly fewer nuclear LDs than were observed upon depletion of LAP1. In addition, we show both high-fat diet feeding and fasting of mice increased cytoplasmic lipids in LAP1-depleted hepatocytes but reduced nuclear LDs, demonstrating a relationship of LD formation with nutritional state. Finally, depletion of microsomal triglyceride transfer protein did not change the frequency of nuclear LDs in LAP1-depleted hepatocytes, suggesting that it is not required for the biogenesis of nuclear LDs in these cells. Together, these data show that LAP1-depleted hepatocytes represent an ideal mammalian system to investigate the biogenesis of nuclear LDs and their partitioning between the nucleus and cytoplasm in response to changes in nutritional state and cellular metabolism in vivo.

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 controls TAN line assembly and the retrograde flow of dorsal perinuclear actin cables during rearward nuclear movement.

The nucleus is positioned toward the rear of most migratory cells. In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, resulting in the orientation of the centrosome in the direction of migration. In this study, we report that the nuclear envelope-localized AAA+ (ATPase associated with various cellular activities) torsinA (TA) and its activator, the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1), are required for rearward nuclear movement during centrosome orientation in migrating fibroblasts. Both TA and LAP1 contributed to the assembly of transmembrane actin-associated nuclear (TAN) lines, which couple the nucleus to dorsal perinuclear actin cables undergoing retrograde flow. In addition, TA localized to TAN lines and was necessary for the proper mobility of EGFP-mini-nesprin-2G, a functional TAN line reporter construct, within the nuclear envelope. Furthermore, TA and LAP1 were indispensable for the retrograde flow of dorsal perinuclear actin cables, supporting the recently proposed function for the nucleus in spatially organizing actin flow and cytoplasmic polarity. Collectively, these results identify TA as a key regulator of actin-dependent rearward nuclear movement during centrosome orientation.

Lamina-associated polypeptide 1 is dispensable for embryonic myogenesis but required for postnatal skeletal muscle growth.

Lamina-associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that has been implicated in striated muscle maintenance. Mutations in its gene have been linked to muscular dystrophy and cardiomyopathy. As germline deletion of the gene encoding LAP1 is perinatal lethal, we explored its potential role in myogenic differentiation and development by generating a conditional knockout mouse in which the protein is depleted from muscle progenitors at embryonic day 8.5 (Myf5-Lap1CKO mice). Although cultured myoblasts lacking LAP1 demonstrated defective terminal differentiation and altered expression of muscle regulatory factors, embryonic myogenesis and formation of skeletal muscle occurred in both mice with a Lap1 germline deletion and Myf5-Lap1CKO mice. However, skeletal muscle fibres were hypotrophic and their nuclei were morphologically abnormal with a wider perinuclear space than normal myonuclei. Myf5-Lap1CKO mouse skeletal muscle contained fewer satellite cells than normal and these cells had evidence of reduced myogenic potential. Abnormalities in signalling pathways required for postnatal hypertrophic growth were also observed in skeletal muscles of these mice. Our results demonstrate that early embryonic depletion of LAP1 does not impair myogenesis but that it is necessary for postnatal skeletal muscle growth.

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.

α-Synuclein-independent histopathological and motor deficits in mice lacking the endolysosomal Parkinsonism protein Atp13a2.

Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.

A novel function for the Caenorhabditis elegans torsin OOC-5 in nucleoporin localization and nuclear import.

Torsin proteins are AAA+ ATPases that localize to the endoplasmic reticular/nuclear envelope (ER/NE) lumen. A mutation that markedly impairs torsinA function causes the CNS disorder DYT1 dystonia. Abnormalities of NE membranes have been linked to torsinA loss of function and the pathogenesis of DYT1 dystonia, leading us to investigate the role of the Caenorhabditis elegans torsinA homologue OOC-5 at the NE. We report a novel role for torsin in nuclear pore biology. In ooc-5-mutant germ cell nuclei, nucleoporins (Nups) were mislocalized in large plaques beginning at meiotic entry and persisted throughout meiosis. Moreover, the KASH protein ZYG-12 was mislocalized in ooc-5 gonads. Nups were mislocalized in adult intestinal nuclei and in embryos from mutant mothers. EM analysis revealed vesicle-like structures in the perinuclear space of intestinal and germ cell nuclei, similar to defects reported in torsin-mutant flies and mice. Consistent with a functional disruption of Nups, ooc-5-mutant embryos displayed impaired nuclear import kinetics, although the nuclear pore-size exclusion barrier was maintained. Our data are the first to demonstrate a requirement for a torsin for normal Nup localization and function and suggest that these functions are likely conserved.

Depletion of lamina-associated polypeptide 1 from cardiomyocytes causes cardiac dysfunction in mice.

We previously showed that striated muscle-selective depletion of lamina-associated polypeptide 1 (LAP1), an integral inner nuclear membrane protein, leads to profound muscular dystrophy with premature death in mice. As LAP1 is also depleted in hearts of these mice, we examined their cardiac phenotype. Striated muscle-selective LAP1 knockout mice display ventricular systolic dysfunction with abnormal induction of genes encoding cardiomyopathy related proteins. To eliminate possible confounding effects due to skeletal muscle pathology, we generated a new mouse line in which LAP1 is deleted in a cardiomyocyte-selective manner. These mice had no skeletal muscle pathology and appeared overtly normal at 20 weeks of age. However, cardiac echocardiography revealed that they developed left ventricular systolic dysfunction and cardiac gene expression analysis revealed abnormal induction of cardiomyopathy-related genes. Our results demonstrate that LAP1 expression in cardiomyocytes is required for normal left ventricular function, consistent with a report of cardiomyopathy in a human subject with mutation in the gene encoding LAP1.

Lamina-associated polypeptide 1: protein interactions and tissue-selective functions.

Mutations in genes encoding widely expressed nuclear envelope proteins often lead to diseases that manifest in specific tissues. Lamina-associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is expressed in most cells and tissues. Within the nuclear envelope, LAP1 interacts physically with lamins, torsinA and emerin, suggesting it may serve as a key node for transducing signals across the inner nuclear membrane. Indeed, recent in vivo studies in genetically modified mice strongly support functional links between LAP1 and both torsinA (in neurons) and emerin (in muscle). These studies suggest that tissue-selective diseases caused by mutations in genes encoding nuclear envelope proteins may result, at least in part, from the selective disruption of discrete nuclear envelope protein complexes.

The nuclear envelope protein, LAP1B, is a novel protein phosphatase 1 substrate.

Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases.

Lamina-associated polypeptide-1 interacts with the muscular dystrophy protein emerin and is essential for skeletal muscle maintenance.

X-linked Emery-Dreifuss muscular dystrophy is caused by loss of function of emerin, an integral protein of the inner nuclear membrane. Yet emerin null mice are essentially normal, suggesting the existence of a critical compensating factor. We show that the lamina-associated polypeptide1 (LAP1) interacts with emerin. Conditional deletion of LAP1 from striated muscle causes muscular dystrophy; this pathology is worsened in the absence of emerin. LAP1 levels are significantly higher in mouse than human skeletal muscle, and reducing LAP1 by approximately half in mice also induces muscle abnormalities in emerin null mice. Conditional deletion of LAP1 from hepatocytes yields mice that exhibit normal liver function and are indistinguishable from littermate controls. These results establish that LAP1 interacts physically and functionally with emerin and plays an essential and selective role in skeletal muscle maintenance. They also highlight how dissecting differences between mouse and human phenotypes can provide fundamental insights into disease mechanisms.

Targeted disruption of neuronal 19S proteasome subunits induces the formation of ubiquitinated inclusions in the absence of cell death.

Proteasome-mediated proteolysis is a major protein degradation mechanism in cells and its dysfunction has been implicated in the pathogenesis of several neurodegenerative diseases, each with the common features of neuronal death and formation of ubiquitinated inclusions found within neurites, the cell body, or nucleus. Previous models of proteasome dysfunction have employed pharmacological inhibition of the catalytic subunits of the 20S proteasome core, or the genetic manipulation of specific subunits resulting in altered proteasome assembly. In this study, we report the use of dominant negative subunits of the 19S regulatory proteasome complex that mediate the recognition of ubiquitinated substrates as well as the removal of the poly-ubiquitin chain. Interestingly, while each mutant subunit-induced inclusion formation, like that seen with pharmacological inhibition of the 20S proteasome, none was able to induce apoptotic death, or trigger activation of macroautophagy, in either dopaminergic cell lines or primary cortical neurons. This finding highlights the dissociation between the mechanisms of neuronal inclusion formation and the induction of cell death, and represents a novel cellular model for Lewy body-like inclusion formation in neurons.

A molecular mechanism underlying the neural-specific defect in torsinA mutant mice.

A striking but poorly understood feature of many diseases is the unique involvement of neural tissue. One example is the CNS-specific disorder DYT1 dystonia, caused by a 3-bp deletion ("DeltaE") in the widely expressed gene TOR1A. Disease mutant knockin mice (Tor1a(DeltaE/DeltaE)) exhibit disrupted nuclear membranes selectively in neurons, mimicking the tissue specificity of the human disease and providing a model system in which to dissect the mechanisms underlying neural selectivity. Our in vivo studies demonstrate that lamina-associated polypeptide 1 (LAP1) and torsinB function with torsinA to maintain normal nuclear membrane morphology. Moreover, we show that nonneuronal cells express dramatically higher levels of torsinB and that RNAi-mediated depletion of torsinB (but not other torsin family members) causes nuclear membrane abnormalities in Tor1a(DeltaE/DeltaE) nonneuronal cells. The Tor1a(DeltaE/DeltaE) neural selective phenotype therefore arises because high levels of torsinB protect nonneuronal cells from the consequences of torsinA dysfunction, demonstrating how tissue specificity may result from differential susceptibility of cell types to insults that disrupt ubiquitous biological pathways.

The AAA+ protein torsinA interacts with a conserved domain present in LAP1 and a novel ER protein.

A glutamic acid deletion (DeltaE) in the AAA+ protein torsinA causes DYT1 dystonia. Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the DeltaE mutation enhances this interaction. Using a novel cell-based screen, we identify lamina-associated polypeptide 1 (LAP1) as a torsinA-interacting protein. LAP1 may be a torsinA substrate, as expression of the isolated lumenal domain of LAP1 inhibits the NE localization of "substrate trap" EQ-torsinA and EQ-torsinA coimmunoprecipitates with LAP1 to a greater extent than wild-type torsinA. Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA. Interestingly, LULL1 resides in the main ER. Consequently, torsinA interacts directly or indirectly with a novel class of transmembrane proteins that are localized in different subdomains of the ER system, either or both of which may play a role in the pathogenesis of DYT1 dystonia.

alpha-synuclein is required for the fibrillar nature of ubiquitinated inclusions induced by proteasomal inhibition in primary neurons.

Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for alpha-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. alpha-Synuclein is thought to be a central component of Lewy bodies. Whether alpha-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether alpha-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from alpha-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of alpha-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in alpha-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although alpha-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in alpha-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model.