ABSTRACT
The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.
Subject(s)
Drosophila Proteins/metabolism , Nuclear Pore/metabolism , Phosphoprotein Phosphatases/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster , Fat Body/cytology , Fat Body/metabolism , Lipid Metabolism , Membrane Fusion , Phosphoprotein Phosphatases/geneticsABSTRACT
TOR1A/TorsinA mutations cause two incurable diseases: a recessive congenital syndrome that can be lethal, and a dominantly-inherited childhood-onset dystonia (DYT-TOR1A). TorsinA has been linked to phosphatidic acid lipid metabolism in Drosophila melanogaster. Here we evaluate the role of phosphatidic acid phosphatase (PAP) enzymes in TOR1A diseases using induced pluripotent stem cell-derived neurons from patients, and mouse models of recessive Tor1a disease. We find that Lipin PAP enzyme activity is abnormally elevated in human DYT-TOR1A dystonia patient cells and in the brains of four different Tor1a mouse models. Its severity also correlated with the dosage of Tor1a/TOR1A mutation. We assessed the role of excess Lipin activity in the neurological dysfunction of Tor1a disease mouse models by interbreeding these with Lpin1 knock-out mice. Genetic reduction of Lpin1 improved the survival of recessive Tor1a disease-model mice, alongside suppressing neurodegeneration, motor dysfunction, and nuclear membrane pathology. These data establish that TOR1A disease mutations cause abnormal phosphatidic acid metabolism, and suggest that approaches that suppress Lipin PAP enzyme activity could be therapeutically useful for TOR1A diseases.
Subject(s)
Molecular Chaperones/metabolism , Phosphatidate Phosphatase/metabolism , Animals , Brain/pathology , Disease Models, Animal , Dystonia/genetics , Dystonia/metabolism , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Chaperones/genetics , Mutation , Neurons/metabolism , Phosphatidate Phosphatase/genetics , Phosphatidate Phosphatase/physiologyABSTRACT
Dystonia is a neurological movement disorder characterized by sustained or intermittent involuntary muscle contractions. Loss-of-function mutations in the GNAL gene have been identified to be the cause of "isolated" dystonia DYT25. The GNAL gene encodes for the guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf), which is mainly expressed in the olfactory bulb and the striatum and functions as a modulator during neurotransmission coupling with D1R and A2AR. Previously, heterozygous Gαolf -deficient mice (Gnal+/-) have been generated and showed a mild phenotype at basal condition. In contrast, homozygous deletion of Gnal in mice (Gnal-/-) resulted in a significantly reduced survival rate. In this study, using the CRISPR-Cas9 system we generated and characterized heterozygous Gnal knockout rats (Gnal+/-) with a 13 base pair deletion in the first exon of the rat Gnal splicing variant 2, a major isoform in both human and rat striatum. Gnal+/- rats showed early-onset phenotypes associated with impaired dopamine transmission, including reduction in locomotor activity, deficits in rotarod performance and an abnormal motor skill learning ability. At cellular and molecular level, we found down-regulated Arc expression, increased cell surface distribution of AMPA receptors, and the loss of D2R-dependent corticostriatal long-term depression (LTD) in Gnal+/- rats. Based on the evidence that D2R activity is normally inhibited by adenosine A2ARs, co-localized on the same population of striatal neurons, we show that blockade of A2ARs restores physiological LTD. This animal model may be a valuable tool for investigating Gαolf function and finding a suitable treatment for dystonia associated with deficient dopamine transmission.
Subject(s)
Adenosine/metabolism , Disease Models, Animal , Dopamine/metabolism , Dystonia , Long-Term Synaptic Depression/physiology , Animals , Dystonia/metabolism , Dystonia/physiopathology , GTP-Binding Protein alpha Subunits/genetics , Gene Knockout Techniques , Male , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2A/metabolism , Signal Transduction/physiologyABSTRACT
Heterozygosity for the TOR1A-Δgag mutation causes semi-penetrant childhood-onset dystonia (OMIM #128100). More recently, homozygous TOR1A mutations were shown to cause severe neurological dysfunction in infants. However, there is little known about the recessive cases, including whether existing reports define the full spectrum of recessive TOR1A disease. Here we describe abnormal brain morphogenesis in â¼30% of Tor1a-/- mouse embryos while, in contrast, this is not found in Tor1aΔgag/Δgag mice. The abnormal Tor1a-/- brains contain excess neural tissue, as well as proliferative zone cytoarchitectural defects related to radial glial cell polarity and cytoskeletal organization. In cultured cells torsinA effects the linker of nucleoskeleton and cytoskeleton (LINC) complex that couples the nucleus and cytoskeleton. Here we identify that torsinA loss elevates LINC complex levels in the proliferative zone, and that genetic reduction of LINC complexes prevents abnormal brain morphogenesis in Tor1a-/- embryos. These data show that Tor1a affects radial glial cells via a LINC complex mediated mechanism. They also predict human TOR1A disease will include incompletely penetrant defects in embryonic brain morphogenesis in cases where mutations ablate TOR1A function.
Subject(s)
Dystonia/genetics , Molecular Chaperones/genetics , Morphogenesis/genetics , Neurogenesis/genetics , Animals , Brain/metabolism , Brain/pathology , Cells, Cultured , Disease Models, Animal , Dystonia/physiopathology , Heterozygote , Homozygote , Humans , Mice , Mice, Knockout , Neurons , Nuclear Matrix/geneticsABSTRACT
Endoplasmic reticulum (ER)-localized enzymes synthesize the vast majority of cellular lipids. The ER therefore has a major influence on cellular lipid biomass and balances the production of different lipid categories, classes, and species. Signals from outside and inside the cell are directed to ER-localized enzymes, and lipid enzyme activities are defined by the integration of internal, homeostatic, and external information. This allows ER-localized lipid synthesis to provide the cell with membrane lipids for growth, proliferation, and differentiation-based changes in morphology and structure, and to maintain membrane homeostasis across the cell. ER enzymes also respond to physiological signals to drive carbohydrates and nutritionally derived lipids into energy-storing triglycerides. In this review, we highlight some key regulatory mechanisms that control ER-localized enzyme activities in animal cells. We also discuss how they act in concert to maintain cellular lipid homeostasis, as well as how their dysregulation contributes to human disease.
Subject(s)
Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Lipids/biosynthesis , Lipogenesis/genetics , Triglycerides/biosynthesis , Animals , Cell Membrane/chemistry , Endoplasmic Reticulum/ultrastructure , Eukaryotic Cells/cytology , Eukaryotic Cells/metabolism , Feedback, Physiological , Gene Expression Regulation , Homeostasis/genetics , Humans , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Sterol Regulatory Element Binding Proteins/genetics , Sterol Regulatory Element Binding Proteins/metabolism , Unfolded Protein ResponseABSTRACT
Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM.IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress and implicates LINC complex integrity in nonconventional nuclear membrane trafficking.
Subject(s)
Active Transport, Cell Nucleus/physiology , Cytoskeleton/metabolism , Herpesviridae/growth & development , Nuclear Envelope/metabolism , Nuclear Matrix/metabolism , Active Transport, Cell Nucleus/genetics , Animals , Cell Line , Herpesviridae/metabolism , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , Nuclear Proteins/metabolism , Rabbits , Swine , Virus Assembly/physiology , Virus Release/physiologyABSTRACT
Striatal dysfunction is implicated in many movement disorders. However, the precise nature of defects often remains uncharacterized, which hinders therapy development. Here we examined striatal function in a mouse model of the incurable movement disorder, myoclonus dystonia, caused by SGCE mutations. Using RNAseq we found surprisingly normal gene expression, including normal levels of neuronal subclass markers to strongly suggest that striatal microcircuitry is spared by the disease insult. We then functionally characterized Sgce mutant medium spiny projection neurons (MSNs) and cholinergic interneurons (ChIs). This revealed normal intrinsic electrophysiological properties and normal responses to basic excitatory and inhibitory neurotransmission. Nevertheless, high-frequency stimulation in Sgce mutants failed to induce normal long-term depression (LTD) at corticostriatal glutamatergic synapses. We also found that pharmacological manipulation of MSNs by inhibiting adenosine 2A receptors (A2AR) restores LTD, again pointing to structurally intact striatal circuitry. The fact that Sgce loss specifically inhibits LTD implicates this neurophysiological defect in myoclonus dystonia, and emphasizes that neurophysiological changes can occur in the absence of broad striatal dysfunction. Further, the positive effect of A2AR antagonists indicates that this drug class be tested in DYT11/SGCE dystonia.
Subject(s)
Adenosine A2 Receptor Antagonists/pharmacology , Corpus Striatum/drug effects , Dystonic Disorders/drug therapy , Neuronal Plasticity/drug effects , Animals , Corpus Striatum/physiopathology , Disease Models, Animal , Dystonic Disorders/physiopathology , Female , Glutamic Acid/metabolism , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Neuronal Plasticity/physiology , Neurons/drug effects , Neurons/physiology , Patch-Clamp Techniques , RNA, Messenger/metabolism , Receptor, Adenosine A2A/metabolism , Sarcoglycans/genetics , Sarcoglycans/metabolism , Tissue Culture TechniquesABSTRACT
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.
Subject(s)
Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Nuclear Envelope/metabolism , 3T3 Cells , Adenosine Triphosphatases/metabolism , Animals , CHO Cells , Carrier Proteins/genetics , Cell Line , Cricetulus , Membrane Proteins/genetics , Mice , Multiprotein Complexes/genetics , Nuclear Proteins/metabolism , Protein BindingABSTRACT
Heterozygosity for a 3-base pair deletion (ΔGAG) in TOR1A/torsinA is one of the most common causes of hereditary dystonia. In this review, we highlight current understanding of how this mutation causes disease from research spanning structural biochemistry, cell science, neurobiology, and several model organisms. We now know that homozygosity for ΔGAG has the same effects as Tor1aKO , implicating a partial loss of function mechanism in the ΔGAG/+ disease state. In addition, torsinA loss specifically affects neurons in mice, even though the gene is broadly expressed, apparently because of differential expression of homologous torsinB. Furthermore, certain neuronal subtypes are more severely affected by torsinA loss. Interestingly, these include striatal cholinergic interneurons that display abnormal responses to dopamine in several Tor1a animal models. There is also progress on understanding torsinA molecular cell biology. The structural basis of how ΔGAG inhibits torsinA ATPase activity is defined, although mutant torsinAΔGAG protein also displays some characteristics suggesting it contributes to dystonia by a gain-of-function mechanism. Furthermore, a consistent relationship is emerging between torsin dysfunction and membrane biology, including an evolutionarily conserved regulation of lipid metabolism. Considered together, these findings provide major advances toward understanding the molecular, cellular, and neurobiological pathologies of DYT1/TOR1A dystonia that can hopefully be exploited for new approaches to treat this disease. © 2016 International Parkinson and Movement Disorder Society.
Subject(s)
Dystonia Musculorum Deformans/metabolism , Molecular Chaperones/metabolism , Animals , Dystonia Musculorum Deformans/genetics , Humans , Molecular Chaperones/geneticsABSTRACT
A three base-pair deletion in the widely expressed TOR1A gene causes the childhood onset, neurological disease of DYT1 dystonia. Mouse Tor1a gene knockout also specifically affects the developing nervous system. However, in both cases, the basis of neuronal tissue specificity is unknown. TorsinA is one of four predicted mammalian torsin ATPases associated with assorted cellular activities (AAA+) proteins, raising the possibility that expression of a functionally homologous torsin compensates for torsinA loss in non-neuronal tissues. We find that all four mammalian torsins are endoplasmic reticulum resident glycoproteins. TorsinA, torsinB and torsin2 are all present in large M(r) complexes, which suggests that each assembles into an oligomeric AAA+ enzyme. Introducing a mutation (WB(EQ)) that typically stabilizes AAA+ proteins in a substrate-bound state causes torsinA and torsinB to associate with a shared nuclear envelope (NE) binding partner and this NE localization requires the torsinA interacting protein, lamina associated polypeptide 1. Although torsin proteins are widely expressed in the adult mouse, we identified that embryonic neuronal tissues contain relatively low torsinB levels. Therefore, our results reveal that torsinB expression inversely correlates with the cell and developmental requirement for torsinA. In conclusion, multiple cell types appear to utilize torsin AAA+ proteins and differential expression of torsinB may contribute to both the neuronal specific importance of torsinA and the symptom specificity of DYT1 dystonia.
Subject(s)
Carrier Proteins/genetics , Molecular Chaperones/genetics , Neurons/metabolism , Animals , Carrier Proteins/metabolism , Cells, Cultured , Dystonic Disorders/genetics , Dystonic Disorders/metabolism , HeLa Cells , Humans , Mice , Molecular Chaperones/metabolism , NIH 3T3 CellsABSTRACT
BACKGROUND: DYT1 dystonia is an autosomal dominant neurological condition caused by a mutation that removes a single glutamic acid residue (ΔE) from the torsinA (torA) AAA+ protein. TorA appears to possess a nuclear envelope (NE) localized activity that requires Lamina-Associated-Polypeptide 1 (LAP1), which is an inner nuclear membrane localized torA-binding partner. Although hypoactive, the DYT1 dystonia torA-ΔE isoform often concentrates in the NE, suggesting that torA-ΔE also interacts with an NE-localized binding partner. RESULTS: We confirm that NE-localized torA-ΔE does not co-immunoprecipitate with LAP1, and find that torA-ΔE continues to concentrate in the NE of cells that lack LAP1. Instead, we find that variability in torA-ΔE localization correlates with the presence of the SUN-domain and Nesprin proteins that assemble into the LINC complex. We also find that siRNA depletion of SUN1, but not other LINC complex components, removes torA-ΔE from the NE. In contrast, the LAP1-dependent NE-accumulation of an ATP-locked torA mutant is unaffected by loss of LINC complex proteins. This SUN1 dependent torA-ΔE localization requires the torA membrane association domain, as well as a putative substrate-interaction residue, Y147, neither of which are required for torA interaction with LAP1. We also find that mutation of these motifs, or depletion of SUN1, decreases the amount of torA-WT that colocalizes with NE markers, indicating that each also underlies a normal NE-localized torA binding interaction. CONCLUSIONS: These data suggest that the disease causing ΔE mutation promotes an association between torA and SUN1 that is distinct to the interaction between LAP1 and ATP-bound torA. This evidence for two NE-localized binding partners suggests that torA may act on multiple substrates and/or possesses regulatory co-factor partners. In addition, finding that the DYT1 mutation causes abnormal association with SUN1 implicates LINC complex dysfunction in DYT1 dystonia pathogenesis, and suggests a gain-of-function activity contributes to this dominantly inherited disease.
Subject(s)
Microtubule-Associated Proteins/metabolism , Molecular Chaperones/analysis , Nuclear Envelope/metabolism , Animals , Cell Nucleus/metabolism , Dystonia/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , NIH 3T3 Cells , Protein Interaction Domains and Motifs , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.
ABSTRACT
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.
Subject(s)
Endoplasmic Reticulum/metabolism , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Proteins/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/metabolism , Cell Line , Conserved Sequence , Cricetinae , Endoplasmic Reticulum/chemistry , Fluorescence Recovery After Photobleaching , Green Fluorescent Proteins/metabolism , HSC70 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , HeLa Cells , Humans , Kinetics , Membrane Proteins/metabolism , Mice , Microscopy, Confocal , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Nuclear Envelope/metabolism , Precipitin Tests , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Sequence Deletion , Sequence Homology, Amino Acid , TransfectionABSTRACT
An enigmatic feature of many genetic diseases is that mutations in widely expressed genes cause tissue-specific illness. One example is DYT1 dystonia, a neurodevelopmental disease caused by an in-frame deletion (Deltagag) in the gene encoding torsinA. Here we show that neurons from both torsinA null (Tor1a(-/-)) and homozygous disease mutant "knockin" mice (Tor1a(Deltagag/Deltagag)) contain severely abnormal nuclear membranes, although non-neuronal cell types appear normal. These membrane abnormalities develop in postmigratory embryonic neurons and subsequently worsen with further neuronal maturation, a finding evocative of the developmental dependence of DYT1 dystonia. These observations demonstrate that neurons have a unique requirement for nuclear envelope localized torsinA function and suggest that loss of this activity is a key molecular event in the pathogenesis of DYT1 dystonia.
Subject(s)
Brain/abnormalities , Brain/metabolism , Dystonia Musculorum Deformans/metabolism , Molecular Chaperones/genetics , Neurons/metabolism , Nuclear Envelope/metabolism , Animals , Brain/physiopathology , Carrier Proteins/metabolism , Cell Differentiation/genetics , Cell Movement/genetics , Cells, Cultured , Disease Models, Animal , Dystonia Musculorum Deformans/genetics , Dystonia Musculorum Deformans/physiopathology , Genetic Predisposition to Disease/genetics , HSC70 Heat-Shock Proteins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Microscopy, Electron, Transmission , Mutation/genetics , Neurons/pathology , Neurons/ultrastructure , Nuclear Envelope/pathology , Nuclear Envelope/ultrastructureABSTRACT
Neuregulin-1 (Nrg1)/erbB signaling regulates neuronal development, migration, myelination, and synaptic maintenance. The Nrg1 gene is a schizophrenia susceptibility gene. To understand the contribution of Nrg1 signaling to adult brain structure and behaviors, we studied the regulation of type III Nrg1 expression and evaluated the effect of decreased expression of the type III Nrg1 isoforms. Type III Nrg1 is transcribed by a promoter distinct from those for other Nrg1 isoforms and, in the adult brain, is expressed in the medial prefrontal cortex, ventral hippocampus, and ventral subiculum, regions involved in the regulation of sensorimotor gating and short-term memory. Adult heterozygous mutant mice with a targeted disruption for type III Nrg1 (Nrg1(tm1.1Lwr+/-)) have enlarged lateral ventricles and decreased dendritic spine density on subicular pyramidal neurons. Magnetic resonance imaging of type III Nrg1 heterozygous mice revealed hypofunction in the medial prefrontal cortex and the hippocampal CA1 and subiculum regions. Type III Nrg1 heterozygous mice also have impaired performance on delayed alternation memory tasks, and deficits in prepulse inhibition (PPI). Chronic nicotine treatment eliminated differences in PPI between type III Nrg1 heterozygous mice and their wild-type littermates. Our findings demonstrate a role of type III Nrg1 signaling in the maintenance of corticostriatal components and in the neural circuits involved in sensorimotor gating and short-term memory.
Subject(s)
Corpus Striatum/abnormalities , Hippocampus/abnormalities , Memory Disorders/genetics , Nerve Tissue Proteins/genetics , Prefrontal Cortex/abnormalities , Sensation Disorders/genetics , Animals , Atrophy/genetics , Atrophy/metabolism , Atrophy/physiopathology , Corpus Striatum/metabolism , Corpus Striatum/physiopathology , Disease Models, Animal , Gene Expression Regulation, Developmental/genetics , Heterozygote , Hippocampus/metabolism , Hippocampus/physiopathology , Lateral Ventricles/abnormalities , Memory Disorders/metabolism , Memory Disorders/physiopathology , Memory, Short-Term/physiology , Mice , Mice, Knockout , Mice, Transgenic , Nervous System Malformations/genetics , Nervous System Malformations/metabolism , Nervous System Malformations/physiopathology , Neural Inhibition/genetics , Neural Pathways/abnormalities , Neural Pathways/metabolism , Neural Pathways/physiopathology , Neuregulin-1 , Nicotinic Agonists/pharmacology , Prefrontal Cortex/metabolism , Prefrontal Cortex/physiopathology , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sensation Disorders/metabolism , Sensation Disorders/physiopathologyABSTRACT
Dopamine D2 receptor signaling is central for striatal function and movement, while abnormal activity is associated with neurological disorders including the severe early-onset DYT1 dystonia. Nevertheless, the mechanisms that regulate D2 receptor signaling in health and disease remain poorly understood. Here, we identify a reduced D2 receptor binding, paralleled by an abrupt reduction in receptor protein level, in the striatum of juvenile Dyt1 mice. This occurs through increased lysosomal degradation, controlled by competition between ß-arrestin 2 and D2 receptor binding proteins. Accordingly, we found lower levels of striatal RGS9-2 and spinophilin. Further, we show that genetic depletion of RGS9-2 mimics the D2 receptor loss of DYT1 dystonia striatum, whereas RGS9-2 overexpression rescues both receptor levels and electrophysiological responses in Dyt1 striatal neurons. This work uncovers the molecular mechanism underlying D2 receptor downregulation in Dyt1 mice and in turn explains why dopaminergic drugs lack efficacy in DYT1 patients despite significant evidence for striatal D2 receptor dysfunction. Our data also open up novel avenues for disease-modifying therapeutics to this incurable neurological disorder.
Subject(s)
Corpus Striatum/pathology , Dystonia Musculorum Deformans/pathology , Dystonia Musculorum Deformans/physiopathology , Molecular Chaperones/genetics , RGS Proteins/analysis , Receptors, Dopamine D2/analysis , Signal Transduction , Animals , Disease Models, Animal , Gene Expression , Gene Knockdown Techniques , Mice, Inbred C57BL , Microfilament Proteins/analysis , Nerve Tissue Proteins/analysis , RGS Proteins/geneticsABSTRACT
The nucleopore is an essential structure of the eukaryotic cell, regulating passage between the nucleus and cytoplasm. While individual functions of core nucleopore proteins have been identified, the role of other components, such as Nup210, are poorly defined. Here, through the use of an unbiased ENU mutagenesis screen for mutations effecting the peripheral T cell compartment, we identified a Nup210 mutation in a mouse strain with altered CD4/CD8 T cell ratios. Through the generation of Nup210 knockout mice we identified Nup210 as having a T cell-intrinsic function in the peripheral homeostasis of T cells. Remarkably, despite the deep evolutionary conservation of this key nucleopore complex member, no other major phenotypes developed, with viable and healthy knockout mice. These results identify Nup210 as an important nucleopore complex component for peripheral T cells, and raise further questions of why this nucleopore component shows deep evolutionary conservation despite seemingly redundant functions in most cell types.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Nuclear Pore Complex Proteins/genetics , Animals , Female , Homeostasis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Mutation , Nuclear Pore/metabolism , Statistics, Nonparametric , Th1 Cells/metabolism , Th2 Cells/metabolism , Thymocytes/metabolism , Thymus Gland/cytologyABSTRACT
Lipids are the most abundant organic compounds in the brain. The brain has a unique lipidome, and changes in lipid concentration, organization, and metabolism are associated with many neuronal diseases. Here, we discuss recent advances in understanding presynaptic membrane lipid organization, centered on illustrative examples of how the lipids themselves regulate membrane trafficking and control protein activity. This insight highlights that presynaptic terminals are membrane-remodeling machines and that cooperation between lipid and protein molecules underlies presynaptic activity.
Subject(s)
Membrane Lipids/metabolism , Membrane Proteins/metabolism , Neurotransmitter Agents/metabolism , Presynaptic Terminals/metabolism , Protein Transport , Synaptic Transmission , Synaptic Vesicles/metabolism , Animals , Bipolar Disorder/genetics , Bipolar Disorder/metabolism , Endocytosis , Exocytosis , Humans , Ion Channels/metabolism , Lipid Metabolism/genetics , Parkinson Disease/genetics , Parkinson Disease/metabolism , Phosphatidylinositols/metabolism , SNARE Proteins/metabolism , Schizophrenia/genetics , Schizophrenia/metabolismABSTRACT
Torsins are developmentally essential AAA+ proteins, and mutation of human torsinA causes the neurological disease DYT1 dystonia. They localize in the ER membranes, but their cellular function remains unclear. We now show that dTorsin is required in Drosophila adipose tissue, where it suppresses triglyceride levels, promotes cell growth, and elevates membrane lipid content. We also see that human torsinA at the inner nuclear membrane is associated with membrane expansion and elevated cellular lipid content. Furthermore, the key lipid metabolizing enzyme, lipin, is mislocalized in dTorsin-KO cells, and dTorsin increases levels of the lipin substrate, phosphatidate, and reduces the product, diacylglycerol. Finally, genetic suppression of dLipin rescues dTorsin-KO defects, including adipose cell size, animal growth, and survival. These findings identify that torsins are essential regulators of cellular lipid metabolism and implicate disturbed lipid biology in childhood-onset DYT1 dystonia.