Impaired copper transport slows prion-induced neurodegeneration
Prions cause fatal infectious neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (i.e. Mad Cow Disease) in cattle, scrapie in sheep, and chronic wasting disease in cervids (e.g. deer, caribou, elk, and moose). Prion disease is caused by the misfolding and subsequent aggregation of the copper binding cellular prion protein (PrP) into a disease-associated form (PrPres) (1-4). Chelation studies have suggested involvement of copper in prion disease pathogenesis, but ambiguously so, with a slower course observed in some models and exaggeration of disease in others (5-7). Human patients with Menkes disease, an inherited neurological disorder, are copper-deficient as a result of mutations within the X chromosome-linked ATP7A gene (8-11). Copper is an essential nutrient that assists in several cellular processes including pigmentation, immune responses to skin wounds, formation of connective tissue, and the generation of neurotransmitters within the nervous system (12;13). An excess of copper, however, can be toxic to cells. To maintain copper homeostasis, the P1B-type ATPase Atp7a assists in the transport of copper. In the course of our studies, we identified a mouse with a mutation in the Atp7a gene termed brown (14). Brown mice have a relatively mild Atp7a-associated phenotype: affected males exhibit a brown coat, and there is no visible phenotype in female heterozygotes (14). Other mutations in Atp7a cause total, or near-total, ablation of transporter function leading to profound hypopigmentation in affected males and mottling in heterozygous females due to tissue mosaicism; affected males die shortly after birth (15-17). By using the fully viable brown hemizygous males, we sought to determine whether there is a link between Atp7a-mediated copper transport and prion disease pathogenesis. Copper content in the brains of the brown mice was approximately 40% of that measured in the brains of wild-type mice, and a significant delay in the appearance of clinical signs was observed following intracerebral inoculation of scrapie (14). Furthermore, the amount of the PrPres protein was reduced in the brains of scrapie-inoculated brown animals compared to inoculated wild-type animals (14). These findings confirm that copper facilitates the conversion of PrP to PrPres and establishes the brown mouse as a model with which to study the impact of copper deficiency on chronic disease states.
- Brown, D. R., Qin, K., Herms, J. W., Madlung, A., Manson, J., Strome, R., Fraser, P. E., Kruck, T., von Bohlen, A., Schulz-Schaeffer, W., Giese, A., Westaway, D., and Kretzschmar, H. (1997) The Cellular Prion Protein Binds Copper in Vivo. Nature. 390, 684-687.
- Zidar, J., Pirc, E. T., Hodoscek, M., and Bukovec, P. (2008) Copper(II) Ion Binding to Cellular Prion Protein. J. Chem. Inf. Model. 48, 283-287.
- Varela-Nallar, L., Toledo, E. M., Larrondo, L. F., Cabral, A. L., Martins, V. R., and Inestrosa, N. C. (2006) Induction of Cellular Prion Protein Gene Expression by Copper in Neurons. Am. J. Physiol. Cell. Physiol. 290, C271-81.
- Armendariz, A. D., Gonzalez, M., Loguinov, A. V., and Vulpe, C. D. (2004) Gene Expression Profiling in Chronic Copper Overload Reveals Upregulation of Prnp and App. Physiol. Genomics. 20, 45-54.
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- Mitteregger, G., Korte, S., Shakarami, M., Herms, J., and Kretzschmar, H. A. (2009) Role of Copper and Manganese in Prion Disease Progression. Brain Res. 1292, 155-164.
- Sigurdsson, E. M., Brown, D. R., Alim, M. A., Scholtzova, H., Carp, R., Meeker, H. C., Prelli, F., Frangione, B., and Wisniewski, T. (2003) Copper Chelation Delays the Onset of Prion Disease. J. Biol. Chem. 278, 46199-46202.
- Danks, D. M., Campbell, P. E., Stevens, B. J., Mayne, V., and Cartwright, E. (1972) Menkes's Kinky Hair Syndrome. an Inherited Defect in Copper Absorption with Widespread Effects. Pediatrics. 50, 188-201.
- Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993) Isolation of a Candidate Gene for Menkes Disease and Evidence that it Encodes a Copper-Transporting ATPase. Nat. Genet. 3, 7-13.
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- Kim, H. W., Chan, Q., Afton, S. E., Caruso, J. A., Lai, B., Weintraub, N. L., and Qin, Z. (2012) Human Macrophage ATP7A is Localized in the Trans-Golgi Apparatus, Controls Intracellular Copper Levels, and Mediates Macrophage Responses to Dermal Wounds. Inflammation. 35, 167-175.
- de Bie, P., Muller, P., Wijmenga, C., and Klomp, L. W. (2007) Molecular Pathogenesis of Wilson and Menkes Disease: Correlation of Mutations with Molecular Defects and Disease Phenotypes. J. Med. Genet. 44, 673-688.
- Siggs, O. M., Cruite, J. T., Du, X., Rutschmann, S., Masliah, E., Beutler, B., and Oldstone, M. B. (2012) Disruption of Copper Homeostasis due to a Mutation of Atp7a Delays the Onset of Prion Disease. Proc. Natl. Acad. Sci. U. S. A.
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Yipf6 is needed for vesicle formation in cells of the intestinal tract
Inflammatory bowel disease (IBD), a chronic disease associated with inflammation of the digestive tract, affects approximately 0.4% of the population worldwide (1). Most IBD falls into two diagnostic groups: ulcerative colitis or Crohn’s disease. Ulcerative colitis causes long-lasting inflammation of the lining of the large intestine and/or rectum; Crohn’s disease may affect any part of the intestinal tract. Both ailments are thought to be caused by aberrant immune responses to enteric bacteria. We recently identified a new strain of mice called Klein-Zschocher based on their susceptibility to experimentally induced colitis and a propensity to develop spontaneous colitis (2). Susceptibility was traced to a mutation within a splice acceptor site in the X chromosome-linked gene Yip-1 domain family member 6 (Yipf6). The mutation leads to alterations in the coding sequence and the subsequent coding of a premature stop codon. The protein Yipf6 is a member of the Yip1 domain family, which is known to be involved in the vesicular transport of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi apparatus in cells. In Klein-Zschocher mice, granules within two specialized cell types of the intestine, Paneth and goblet cells, failed to form normally. This would be expected to limit the production of antimicrobial peptides and mucin, both protective proteins that prevent invasive bacteria from gaining a foothold within tissues of the intestinal tract. Interestingly, other tissues were unaffected by the mutation, suggesting that Yipf6 is specifically needed for the formation, transport, and secretion of granules from cells of the intestine but not other organs. YIPF6, the corresponding human gene, should be examined for mutations when considering possible causes of IBD in humans.
- Lakatos, P. L. (2006) Recent Trends in the Epidemiology of Inflammatory Bowel Diseases: Up Or Down? World J. Gastroenterol. 12, 6102-6108.
- Brandl, K., Tomisato, W., Li, X., Neppl, C., Pirie, E., Falk, W., Xia, Y., Moresco, E. M., Baccala, R., Theofilopoulos, A. N., Schnabl, B., and Beutler, B. (2012) Yip1 Domain Family, Member 6 (Yipf6) Mutation Induces Spontaneous Intestinal Inflammation in Mice. Proc. Natl. Acad. Sci. U. S. A. 109, 12650-12655.
iRhom2 is essential for the secretion of TNFα during an immune response
Toll-like receptor (TLR) signaling is essential to mount an innate immune response to many types of infection. The TLR family recognizes diverse ligands such as viral RNA and DNA, bacterial DNA, and proteins, glycolipids, and glycopeptides expressed by bacteria. Once a ligand binds its cognate TLR, a signaling cascade is initiated that will subsequently induce the expression and secretion of cytokines such as tumor necrosis factor-α (TNF-α), summoning inflammatory cells to the site of infection. One of the goals of the Beutler laboratory is to identify new regulators of TLR-induced TNF-α secretion. In the course of this study, the sinecure mouse was identified and characterized. These mice exhibit reduced TNF-α secretion in response to the ligands for all of the TLRs tested (TLR2/6, TLR9, TLR4, and TLR3) (1). However, the secretion of other TLR-induced cytokines was unaffected. DNA sequence analysis identified a mutation in the Rhbdf2 gene, which encodes iRhom2. iRhom2 is a proteolytically inactive member of the rhomboid protease family; active members of the rhomboid family cleave transmembrane proteins within the plane of biological membranes. Rhomboid proteases are involved in several processes within the cell including epidermal growth factor receptor (EGFR) signaling, protein export across membranes, apoptosis regulation, and mitochondrial morphology and function. But the function of proteolytically inactive family members was previously unknown. For TNF-α to be secreted as an active cytokine, a metalloprotease known as TNF-α converting enzyme (TACE) must cleave the membrane-bound TNF-α precursor, liberating the mature protein from the cell. iRhom2 appears to regulate TACE activity and subsequently, its encounter with TNF-α (1). Recent studies by other groups using Rhbdf2 knockout mice have proposed that iRhom2 is involved in the folding, the maturation of TACE in the ER, and/or the trafficking of TACE (2;3).
- Siggs, O. M., Xiao, N., Wang, Y., Shi, H., Tomisato, W., Li, X., Xia, Y., and Beutler, B. (2012) IRhom2 is Required for the Secretion of Mouse TNFalpha. Blood. 119, 5769-5771.
- McIlwain, D. R., Lang, P. A., Maretzky, T., Hamada, K., Ohishi, K., Maney, S. K., Berger, T., Murthy, A., Duncan, G., Xu, H. C., Lang, K. S., Haussinger, D., Wakeham, A., Itie-Youten, A., Khokha, R., Ohashi, P. S., Blobel, C. P., and Mak, T. W. (2012) IRhom2 Regulation of TACE Controls TNF-Mediated Protection Against Listeria and Responses to LPS. Science. 335, 229-232.
- Adrain, C., Zettl, M., Christova, Y., Taylor, N., and Freeman, M. (2012) Tumor Necrosis Factor Signaling Requires iRhom2 to Promote Trafficking and Activation of TACE. Science. 335, 225-228.
Site 1 protease (S1P): an enzyme with diverse and essential functions
The woodrat mouse was identified because of its hypopigmented coat, which develops some weeks after birth (1). Interestingly, if skin from a wild type mouse is transplanted onto a woodrat recipient, the hair becomes hypopigmented. And if skin from a woodrat mouse is transplanted onto a wild type recipient, hypopigmentation is maintained. Positional cloning and bacterial artificial chromosome (BAC) complementation studies identified the causative mutation in Mbtps1, a gene that encodes Site 1 protease (S1P), an enzyme that cleaves several substrates, including transcription factors of the bZIP family (2). Curiously, while homozygous woodrat mothers can give birth to heterozygous carriers of the woodrat allele, they cannot give birth to homozygous offspring (1). This is known as maternal-zygotic effect lethality. Only one other example of maternal-zygotic effect lethality is known in mammals, caused by mutations of the gene Zfp57, which encodes a KRAM zinc finger transcription factor (3).
Further studies identified woodrat as a model for colitis susceptibility (4). One of the substrates of S1P is activating transcription factor 6 (ATF6), a latent transcription factor that is activated by ER stress. ER stress is a condition caused by the accumulation of unfolded or misfolded proteins in the ER. It induces the unfolded protein response (UPR), which assists in the proper folding and processing—or alternatively degradation—of misfolded ER proteins. The UPR is initiated, in part, when ATF6 translocates to the Golgi and is cleaved by S1P. The cleaved protein subsequently traffics to the nucleus, binds to specific sequences within target genes, and activates transcription. Some ATF6-dependent genes encode chaperones, which traffic to the ER and alleviate ER stress by assisting the misfolded proteins to properly fold and exit the ER and/or to promote their degradation. ER stress has been documented as one of the causes of IBD. ER chaperones GRP78 and GRP94 were reduced in the colons of woodrat mice with experimentally induced colitis. The woodrat mouse provides evidence that S1P-initiated ATF6 cleavage and UPR induction are required for the prevention of IBD.
Lymphocytic choromeningitis virus (LCMV) is a pathogenic arenavirus, and it depends on S1P to cleave its envelope glycoprotein. Without S1P, the virus cannot replicate (5). Following infection with LCMV clone 13, which causes persistent infection in wild-type mice, woodrat animals were found to resist persistent infection. Therefore, S1P blockade could potentially be a therapeutic option for the treatment of persistent arenavirus infections.
S1P is perhaps best known for its crucial role in processing sterol regulatory element binding proteins (SREBPs), bZIP transcription factors involved in steroid biosynthesis (6-8). Data developed from studies with the woodrat mouse indicate that S1P is essential for diverse biological processes, at all stages of life, in many different tissues. The hypomorphic woodrat mutation has opened a window into these other important roles of the enzyme.
- Rutschmann, S., Crozat, K., Li, X., Du, X., Hanselman, J. C., Shigeoka, A. A., Brandl, K., Popkin, D. L., McKay, D. B., Xia, Y., Moresco, E. M., and Beutler, B. (2012) Hypopigmentation and Maternal-Zygotic Embryonic Lethality Caused by a Hypomorphic mbtps1 Mutation in Mice. G3 (Bethesda). 2, 499-504.
- Stirling, J., and O'hare, P. (2006) CREB4, a Transmembrane bZip Transcription Factor and Potential New Substrate for Regulation and Cleavage by S1P. Mol. Biol. Cell. 17, 413-426.
- Li, X., Ito, M., Zhou, F., Youngson, N., Zuo, X., Leder, P., and Ferguson-Smith, A. C. (2008) A Maternal-Zygotic Effect Gene, Zfp57, Maintains both Maternal and Paternal Imprints. Dev. Cell. 15, 547-557.
- Brandl, K., Rutschmann, S., Li, X., Du, X., Xiao, N., Schnabl, B., Brenner, D. A., and Beutler, B. (2009) Enhanced Sensitivity to DSS Colitis Caused by a Hypomorphic Mbtps1 Mutation Disrupting the ATF6-Driven Unfolded Protein Response. Proc. Natl. Acad. Sci. U. S. A. 106, 3300-3305.
- Popkin, D. L., Teijaro, J. R., Sullivan, B. M., Urata, S., Rutschmann, S., de la Torre, J. C., Kunz, S., Beutler, B., and Oldstone, M. (2011) Hypomorphic Mutation in the Site-1 Protease Mbtps1 Endows Resistance to Persistent Viral Infection in a Cell-Specific Manner. Cell. Host Microbe. 9, 212-222.
- Sakai, J., Rawson, R. B., Espenshade, P. J., Cheng, D., Seegmiller, A. C., Goldstein, J. L., and Brown, M. S. (1998) Molecular Identification of the Sterol-Regulated Luminal Protease that Cleaves SREBPs and Controls Lipid Composition of Animal Cells. Mol. Cell. 2, 505-514.
- DeBose-Boyd, R. A., Brown, M. S., Li, W. P., Nohturfft, A., Goldstein, J. L., and Espenshade, P. J. (1999) Transport-Dependent Proteolysis of SREBP: Relocation of Site-1 Protease from Golgi to ER Obviates the Need for SREBP Transport to Golgi. Cell. 99, 703-712.
- Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999) Secreted Site-1 Protease Cleaves Peptides Corresponding to Luminal Loop of Sterol Regulatory Element-Binding Proteins. J. Biol. Chem. 274, 22805-22812.
Possum: an unusual stimulus-dependent neurobehavioral abnormality
Proteins in the voltage-gated sodium channel (VGSC) family are essential for maintaining the activity of neurons, muscle, and the heart. Mutations in VGSCs can cause generalized epilepsy with febrile seizures (mutations in sodium channel, voltage-gated, type I, alpha (SCN1A) (1) and SCN, type II, alpha (SCN2A) (2-4)), long QT syndrome (mutations in SCN, type 5, alpha (SCN5A); (5;6)), Sudden Infant Death Syndrome (mutations in SCN5A (7;8)), and altered pain sensitivity (mutations in SCN1A (9;10) and SCN, type 9, alpha (SCN9A) (11;12)). In the course of our mutagenesis work, we identified a dominant mutation called Possum (13). Affected mice display temporary whole-body arrest of movement, pronounced slowing of the heart as measured by electrocardiography, and electroencephalographic changes consistent with a conscious alert state when the skin at the nape of the neck and shoulders is grasped (i.e. scruffed). After a few minutes of immobility, the mice return to normal activity with reversion of electrocardiographic and electroencephalographic changes; the mice do not exhibit residual effects. The mutation was mapped to Scn10a, which encodes Nav1.8, a voltage-gated sodium channel expressed chiefly in cells of the dorsal root ganglia (DRG), located just adjacent to the spinal cord. Nav1.8 currents in Possum DRG neurons are significantly larger than those in wild-type animals. In addition, Nav1.8 inactivation is slower in the Possum mice and the animals show an aversion to cold (Nav1.8 is known to mediate the sensation of pain associated with cold (14)). Only scruffing would induce the Possum reflex, no other noxious stimulus would do so. The reflex could not be abolished by analgesia nor could it be established by conditioning. Mysteriously, Nav1.8 does not seem to be produced within the central nervous system (CNS). Why do Possum mice show obvious CNS consequences due to a change that affects primarily the peripheral nervous system? Much remains to be understood.
- Escayg, A., MacDonald, B. T., Meisler, M. H., Baulac, S., Huberfeld, G., An-Gourfinkel, I., Brice, A., LeGuern, E., Moulard, B., Chaigne, D., Buresi, C., and Malafosse, A. (2000) Mutations of SCN1A, Encoding a Neuronal Sodium Channel, in Two Families with GEFS+2. Nat. Genet. 24, 343-345.
- Kohling, R. (2002) Voltage-Gated Sodium Channels in Epilepsy. Epilepsia. 43, 1278-1295.
- Kearney, J. A., Plummer, N. W., Smith, M. R., Kapur, J., Cummins, T. R., Waxman, S. G., Goldin, A. L., and Meisler, M. H. (2001) A Gain-of-Function Mutation in the Sodium Channel Gene Scn2a Results in Seizures and Behavioral Abnormalities. Neuroscience. 102, 307-317.
- Sugawara, T., Tsurubuchi, Y., Agarwala, K. L., Ito, M., Fukuma, G., Mazaki-Miyazaki, E., Nagafuji, H., Noda, M., Imoto, K., Wada, K., Mitsudome, A., Kaneko, S., Montal, M., Nagata, K., Hirose, S., and Yamakawa, K. (2001) A Missense Mutation of the Na+ Channel Alpha II Subunit Gene Na(v)1.2 in a Patient with Febrile and Afebrile Seizures Causes Channel Dysfunction. Proc. Natl. Acad. Sci. U. S. A. 98, 6384-6389.
- Splawski, I., Shen, J., Timothy, K. W., Lehmann, M. H., Priori, S., Robinson, J. L., Moss, A. J., Schwartz, P. J., Towbin, J. A., Vincent, G. M., and Keating, M. T. (2000) Spectrum of Mutations in Long-QT Syndrome Genes. KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Circulation. 102, 1178-1185.
- Baroudi, G., and Chahine, M. (2000) Biophysical Phenotypes of SCN5A Mutations Causing Long QT and Brugada Syndromes. FEBS Lett. 487, 224-228.
- Ackerman, M. J., Siu, B. L., Sturner, W. Q., Tester, D. J., Valdivia, C. R., Makielski, J. C., and Towbin, J. A. (2001) Postmortem Molecular Analysis of SCN5A Defects in Sudden Infant Death Syndrome. JAMA. 286, 2264-2269.
- Turillazzi, E., La Rocca, G., Anzalone, R., Corrao, S., Neri, M., Pomara, C., Riezzo, I., Karch, S. B., and Fineschi, V. (2008) Heterozygous Nonsense SCN5A Mutation W822X Explains a Simultaneous Sudden Infant Death Syndrome. Virchows Arch. 453, 209-216.
- Kahlig, K. M., Rhodes, T. H., Pusch, M., Freilinger, T., Pereira-Monteiro, J. M., Ferrari, M. D., van den Maagdenberg, A. M., Dichgans, M., and George, A. L.,Jr. (2008) Divergent Sodium Channel Defects in Familial Hemiplegic Migraine. Proc. Natl. Acad. Sci. U. S. A. 105, 9799-9804.
- de Vries, B., Frants, R. R., Ferrari, M. D., and van den Maagdenberg, A. M. (2009) Molecular Genetics of Migraine. Hum. Genet. 126, 115-132.
- Drenth, J. P., and Waxman, S. G. (2007) Mutations in Sodium-Channel Gene SCN9A Cause a Spectrum of Human Genetic Pain Disorders. J. Clin. Invest. 117, 3603-3609.
- Nassar, M. A., Stirling, L. C., Forlani, G., Baker, M. D., Matthews, E. A., Dickenson, A. H., and Wood, J. N. (2004) Nociceptor-Specific Gene Deletion Reveals a Major Role for Nav1.7 (PN1) in Acute and Inflammatory Pain. Proc. Natl. Acad. Sci. U. S. A. 101, 12706-12711.
- Blasius, A. L., Dubin, A. E., Petrus, M. J., Lim, B. K., Narezkina, A., Criado, J. R., Wills, D. N., Xia, Y., Moresco, E. M., Ehlers, C., Knowlton, K. U., Patapoutian, A., and Beutler, B. (2011) Hypermorphic Mutation of the Voltage-Gated Sodium Channel Encoding Gene Scn10a Causes a Dramatic Stimulus-Dependent Neurobehavioral Phenotype. Proc. Natl. Acad. Sci. U. S. A. 108, 19413-19418.
- Zimmermann, K., Leffler, A., Babes, A., Cendan, C. M., Carr, R. W., Kobayashi, J., Nau, C., Wood, J. N., and Reeh, P. W. (2007) Sensory Neuron Sodium Channel Nav1.8 is Essential for Pain at Low Temperatures. Nature. 447, 855-858.