Our laboratory is working to identify and analyze the genes, pathways, and networks involved in the age-related death of neurons in the central nervous system. We use a forward genetic approach to identify the molecular pathways associated with loss of neurons in the aging mammalian brain. Specifically, we study chemically induced and spontaneous mouse mutants with adult-onset neurodegeneration leading to progressive movement abnormalities associated with cerebellar ataxia. Because these mutants often have additional sites of neuron loss, pursuing this phenotype gives us access to genes affecting survival of multiple types of neurons.
Identification of the Molecular Mechanisms Underlying Neurodegeneration
Molecular Mechanisms of Neurodegeneration
We use a forward genetic approach to identify the molecular pathways associated with loss of neurons in the aging mammalian brain. Specifically, we study chemically induced and spontaneous mouse mutants with adult-onset neurodegeneration leading to progressive movement abnormalities associated with cerebellar ataxia. Because these mutants often have additional sites of neuron loss, pursuing this phenotype gives us access to genes affecting survival of multiple types of neurons. The advantage of this forward genetic approach is that it allows the identification, without a priori assumptions, of molecules critical to survival of terminally differentiated neurons in the mammalian central nervous system (CNS). Analysis of these mutants and determination of the underlying molecular defects, combined with identification of single-locus suppressor/enhancer genes of these mutations, will allow identification of the molecular mechanisms that underlie neuron death in the aging CNS and enhance progress toward prevention and development of effective therapies.
Oligomerization and the formation of aggregates of misfolded proteins are common to many genetic and sporadic forms of neurodegenerative diseases. Although some of these misfolded proteins are due to mutations directly within disease-related proteins, such as in the polyglutamine expansion diseases and some forms of familial Alzheimer's disease, the mechanisms underlying protein misfolding in many sporadic forms of neurodegenerative diseases remain unknown. Using a forward genetic approach, we have identified novel genes that, when their function is disrupted, cause the accumulation of misfolded proteins in neurons prior to their death.
ER stress and neurodegeneration
Mice homozygous for the woozy (wz) mutation develop ataxia between 3 and 4 months of age concomitant with Purkinje cell loss. Ubiquitinated protein accumulations are found in the endoplasmic reticulum (ER) and nucleus in these neurons prior to their degeneration. These abnormal protein accumulations induce the cellular response known as the unfolded protein response, which helps restore ER homeostasis. By positional cloning, we identified the wz mutation in the Sil1 gene, which encodes a cochaperone of the ER chaperone and ER-stress transducer, BiP. Like other HSP70 proteins, binding of substrates to BiP and their subsequent release is controlled by the cycle of ATP hydrolysis and exchange. In vivo alterations of genes that influence the BiP ATP/ADP cycle support that neurodegeneration in Sil1-/- mice is due to alterations in this cycle. Aggravation or suppression of neurodegeneration was observed in Sil1-deficient mice when the gene dosage of Hyou1, an atypical HSP70 protein which functions as a BiP nucleotide exchange factor, was reduced or transgenically overexpressed. Deletion of the Dnajc3 gene, which stimulates the ATPase activity of BiP, greatly attenuates neurodegeneration in Sil1-/- mice, consistent with the opposing functions of these genes. Sil1 mutations have now been found in several families with Marinesco-Sjögren syndrome, a disorder associated with cerebellar ataxia; thus the wz mutant mouse will be an excellent model for this syndrome.
Mistranslation and neurodegeneration
Purkinje cell loss in mice homozygous for the spontaneous sticky (sti) mutation is associated with accumulation of ubiquitinated proteins in the cytoplasm, ER, and nucleolus. We have determined that the sti molecular defect is a point mutation in the editing domain of alanyl transfer RNA (tRNA) synthetase (AlaRS). The aminoacyl tRNA synthetases establish the genetic code that links each amino acid to its cognate tRNA that bears the anticodon triplet of the code. The high accuracy of protein translation is largely due to the precision of these aminoacylation reactions, and much of this accuracy resides in the editing domains of these synthetases that clear misactivated amino acids or mischarged tRNAs. In collaboration with Paul Schimmel (Scripps Research Institute), we demonstrated that the sti mutation causes an increase in mischarged tRNAAla. This likely leads to random misincorporation of amino acids at Ala codons, ultimately causing production of unfolded, heterogeneous proteins. The loss of translational fidelity in sti mutant mice is an exciting new mechanism underlying neurodegeneration.
We have recently created a mouse with an AlaRS conditional knock-in mutation that relative to the sti mutation causes a more severe disruption of AlaRS editing. Using various Cre lines to induce expression of this defective molecule, we are examining the effects of mistranslation on different neuronal populations. In addition, by positional cloning we have identified a modifier gene that suppresses protein inclusion formation and neurodegeneration in sti/sti Purkinje cells in a gene dosage-dependent manner. Work to determine the function of this gene and its role in other neuron populations is ongoing.
In addition to these mutations that cause mistranslation, we have recently identified additional neurodegenerative mutations that disrupt other aspects of neuronal translation. We are investigating the role of these genes in neuron survival, as well as a novel modifier gene that suppresses neuron loss in these mutant strains.
Splicing and neurodegeneration
Mutations in RNA-binding proteins have been associated with familial and sporadic neurodegenerative disorders, and these mutations have been proposed to disrupt pre-mRNA splicing and other DNA and RNA metabolic functions.
We have recently identified an EMS-induced mutation in one member of the multigene snRNA family that results in degeneration of cerebellar and hippocampal neurons. Consistent with the essential role of these snRNAs in the spliceosome, ongoing work in the lab suggests that this mutation leads to global abnormalities of pre-mRNA splicing, demonstrating that disruptions in this process can indeed cause neurodegeneration. Our results also show that the genes encoding these RNAs may have important gene-dosage dependent functions that may act in a cell type specific manner.
Continued work on this project, as well as the investigation of other genes identified in our screen should continue to yield exciting insights into the mechanisms underlying neurodegeneration.
Postdoctoral Fellows: Hongjun Fu, Ph.D., Amy Hicks, Ph.D., Ye Liu, Ph.D., Mridu Kapur, Ph.D., Gabor Nagy, Ph.D.
Research Specialist II: Ryuta Ishimura, Ph.D., D.V.M.
Lab Manager: Thomas J. Jucius, B.S.
Research Assistant I: Krystal-Leigh Brown , B.A.
Research Administrative Assistant: Lauren Koncinsky, M.S.
van Empel VPM, Bertrand AT, van der Nagel R, Kostin S, Doevendans PA, Crijns HJ, de Wit E, Sluiter W, Ackerman SL, De Windt LJ. 2005. Downregulation of apoptosis-inducing factor in harlequin mutant mice sensitizes the myocardium to oxidative stress-related cell death and pressure overload-induced decompensation. Circ Res 96:92-101.
Xie Y, Ding Y-Q, Hong Y, Feng Z, Navarre S, Xi C-X, Zhu X-J, Wang C-L, Ackerman SL, Kozlowski D, Mei L, Xiong W-C. 2005. Phosphatidylinositol transfer protein-α in netrin-1-induced PLC signalling and neurite outgrowth. Nat Cell Biol 7:1124-1132.
Zhao L, Longo-Guess C, Harris BS, Lee JW, Ackerman SL. 2005. Protein accumulation and neurodegeneration in the woozy mutant mouse is caused by disruption of SIL1, a cochaperone of BiP. Nat Genet 37:974-979.
Bock NA, Kovacevic N, Lipina TV, Roder JC, Ackerman SL, Henkelman RM. 2006. In vivo magnetic resonance imaging and semiautomated image analysis extend the brain phenotype for cdf/cdf mice. J Neurosci 26:4455-4459.
Burgess RW, Jucius TJ, Ackerman SL. 2006. Motor axon guidance of the mammalian trochlear and phrenic nerves: Dependence on the netrin receptor Unc5c and modifier loci. J Neurosci 26:4455-4459.
Lee JW, Beebe K, Nangle LA, Jang J, Longo-Guess CM, Cook SA, Davisson MT, Sundburg JP, Schimmel P, Ackerman SL. 2006. Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443:50-55.
van Empel VPM, Bertrand AT, van Oort RJ, van der Nagel R, Engelen M, van Rijen HV, Doevendans PA, Crijns HJ, Ackerman SL, Sluiter W, De Windt LJ. 2006. EUK-8, a superoxide dismutase and catalase mimetic, reduces cardiac oxidative stress and ameliorates pressure overload-induced heart failure in the harlequin mouse mutant. J Am Coll Cardiol 48:824-832.
Watanabe K, Tamamaki N, Furuta T, Ackerman SL, Ikenaka K, Ono K. 2006. Dorsally derived netrin-1 provides an inhibitory cue and elaborates the 'waiting period' for primary sensory axons in the developing spinal cord. Development 133:1379-1387.
Xie Y, Hong Y, Ma X-Y, Ren XR, Ackerman S, Mei L, Xiong W-C. 2006. DCC-dependent phospholipase C signaling in netrin-1 induced neurite elongation. J Biol Chem 281:2605-2611.
Zhao L, Ackerman SL. 2006. ER stress in disease. Curr Opin Cel Biol 18:1-9.
Bernet A, Mazelin L, Coissieux MM, Gadot N, Ackerman SL, Scoazec JY, Mehlen P. 2007. Inactivation of the UNC5C Netrin-1 receptor is associated with tumor progression in colorectal malignancies. Gastroenterology 133:1840-1848.
Dillon AK, Jevince AR, Hinck L, Ackerman SL, Lu X, Tessier-Lavigne M, Kaprielian Z. 2007. UNC5C is required for spinal accessory motor neuron development. Mol Cell Neurosci. 35:482-489.
Ackerman SL, Cox GA. 2008. From ER to Eph receptors: new roles for VAP fragments. Cell 133:949-951. (Preview)
Hu Z, Shanker S, Maclean JA 2nd, Ackerman SL, Wilkinson MF. 2008. The RHOX5 homeodomain protein mediates transcriptional repression of the netrin-1 receptor gene Unc5c. J Biol Chem. 283:3866-3876.
Ishimura R, Martin GR, Ackerman SL. 2008. Loss of apoptosis inducing factor results in cell type-specific neurogenesis defects. J Neurosci. 28:4938-4948.
Renaud J, Kerjan G, Sumita I, Zagar Y, Georget V, Kim D, Fouquet C, Suda K, Sanbo M, Suto F, Ackerman SL, Mitchell KJ, Fujisawa H, Chédotal A. 2008. Plexin-A2 and its ligand Sema6A control nucleus-centosome coupling in migrating cerebellar granule cells. Nat Neurosci. 11:440-449.
Zhao L, Rosales C, Seburn K, Ron D, Ackerman SL. 2010. Alteration of the unfolded protein response modifies neurodegeneration in a mouse model of Marinesco-Sjšgren syndrome. Hum Mol Genet. 19:25-35.
Stum M, McLaughlin HM, Kleinbrink EL, Miers KE, Ackerman SL, Seburn KL, Antonellis A, Burgess RW. 2011. An assessment of mechanisms underlying peripheral axonal degeneration caused by aminoacyl-tRNA synthetase mutations. Mol Cell Neurosci 46:432-443.
Kim D, Ackerman SL. 2011. The Unc5c netrin receptor regulates dorsal guidance of mouse hindbrain axons. J Neurosci 31:2167-2179.
Zhao L, Spassieva SD, Jucius TJ, Schultz LD, Shick HE, Macklin WB, Hannun YA, Obeid LM, Ackerman SL. 2011. A deficiency of ceramide biosynthesis causes cerebellar Purkinje cell neurodegeneration and lipofuscin accumulation. PLoS Genet 7:e1002063.
Jia Y, Mu JC, Ackerman SL. 2012. Mutation of a U2 snRNA gene causes global disruption of alternative splicing and neurodegeneration. Cell 148:296-308.
Liu Y, Zaun H, Orlowski J, Ackerman SL. 2013. CHP1-mediated NHE1 biosynthetic maturation is required for Purkinje cell axon homeostasis. J Neurosci 33:12656-69
Natarajan S, Li Y, Miller EE, Shih DJ, Taylor MD, Stearns TM, Bronson RT, Ackerman, SL, Yoon J, Yun K. 2013. Notch1 induced brain tumor models the sonic hedgehog subgroup of human medulloblastoma. Cancer Res 73(17):5381-5390
Nagy G, Ackerman SL. 2013. Cholesterol metabolism and Rett syndrome pathogenesis. Nat Genet 45:965-967
Svirvatsa S, Parthasarathy S, Britanova O, Bormuth I, Donahoo AL, Ackerman SL, Richards L, Tarabykin V. 2014. Unc5C and DCC act downstream of Ctip2 and Satb2 to control corpus callosum formation. Nat Commun (in press)