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Identification of Genes Controlling Neurodevelopment and Neuron Survival
Summary: Susan Ackerman is working to identify and analyze the genes, pathways, and networks involved in brain development and age-related death of neurons in the central nervous system.
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.
Mutations causing protein misfolding and neurodegeneration. 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. 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. These data suggest that the adenine nucleotide exchange function of Sil1 is necessary for the chaperone function of BiP, but not for the ER-stress function of BiP. Recently, Sil1 mutations were 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.
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's lab (Scripps Research Institute), we have 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 identified a dominant modifier gene that suppresses neurodegeneration in sti mutant mice. Experiments to delineate the protective role of this gene in sti/sti Purkinje cells are ongoing, as are tests to determine whether this gene protects against other misfolded protein–associated neurodegenerative disorders.
Oxidative stress and neurodegeneration. Although increased indices of reactive oxygen species are observed in postmortem brain tissues from patients with neurodegenerative disorders, the causal nature of oxidative stress in these disorders remains controversial. The harlequin (Hq) mouse is a spontaneous mutant with progressive ataxia and loss of vision due to death of cerebellar and retinal neurons. We demonstrated that the Hq mutation is a hypomorphic mutation in the apoptosis-inducing factor (Aif) gene, which encodes a mitochondrial protein thought to be an effector molecule of caspase-independent apoptosis. When AIF is down-regulated, oxidative stress occurs months before the onset of neurodegeneration.
Although terminally differentiated neurons are postmitotic, several studies on tissue from Alzheimer's disease, amyotrophic lateral sclerosis, and stroke patients have demonstrated that neuron death is associated with aberrant cell cycle reentry. A causal association is indicated by demonstrations that experimentally driving neurons into the cell cycle causes death, not cell division, and blocking the cell cycle initiation can prevent neuron death. Although the importance of cell cycle control in neuronal death is becoming apparent, the events that cause lethal cell cycle events in the aging brain are unknown. Our studies with Hq mutant mice demonstrate that oxidative stress can trigger neuronal cell cycle reentry and subsequent apoptosis, providing a novel mechanism for late-onset neurodegeneration.
Genes Controlling Cerebellar Development
In addition to our studies on neurodegeneration, we are also identifying genes that control CNS development, particularly that of the cerebellum. These studies have yielded insights into the molecular control of the developmental migrations of cerebellar granule cells and Purkinje cells and specific CNS axonal tracts. Mice homozygous for mutations in the rostral cerebellar malformation gene (rcm, now renamed Unc5c) exhibit cerebellar and midbrain defects. The cerebellum of mutants is reduced in both size and number of folia, and the midbrain and brainstem both contain ectopic cerebellar neurons.
We found that the Unc5c cDNA encodes a transmembrane receptor of the immunoglobulin superfamily that is highly similar to the Caenorhabditis elegans protein UNC-5, which is essential for dorsal guidance of pioneer axons. UNC-5 is also necessary for migration of cells away from the netrin ligand, which is encoded by the unc-6 gene. Our chimera studies have shown that this netrin receptor is necessary for the recognition of the cerebellum's anterior border by migrating granule cell precursors during embryogenesis, and for recognition of the ventral boundary of the inner granule cell layer in the lateral regions of the cerebellum by radially migrating postnatal granule cells. In addition to neuronal migration abnormalities, we have found corticospinal tract defects in both Unc5c/Unc5c mice and mice homozygous for a mutation in the netrin receptor, deleted in colorectal carcinoma (Dcc), demonstrating a role for these receptors in navigations of these axons.
Recently, we observed that Unc5c mutants on an inbred genetic background die shortly after birth, of apparent respiratory problems. In contrast, mutant mice on a segregating background live a normal life span. Analysis of mutant embryos on this inbred background revealed that Unc5c is necessary for guidance of both the phrenic nerve, which normally innervates the diaphragm muscle, and trochlear motor axons. Our results define a novel role for mammalian netrins and their receptors in motor axon guidance and also illustrate the importance of modifier genes on axonal guidance.
Portions of these projects were supported by grants from the National Institutes of Health.
Last updated: March 3, 2008
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