Research interests

Genetic Analysis of Cerebellar Development and Function

The focus of our laboratory is the elucidation of the molecular mechanisms underlying the development and maintenance of neurons in the cerebellum, which coordinate motor function. The cerebellar cortex has two principal neuronal cell types, the granule cell interneuron and the Purkinje cell, which is the sole output neuron of the cortex. These cell types are found in distinct layers that arise from independently controlled migrations during development. Furthermore, distinct genes control the maintenance of these neurons in the adult cerebellum.

Granule Cell Migration and Survival

Mice homozygous for mutations in the rostral cerebellar malformation gene (rcm, now known to be an allele of Unc5c) exhibit cerebellar and midbrain defects. The cerebellum of mutants is reduced in both size and number of folia, and ectopic cerebellar cells are present in midbrain regions by 3 days after birth. We found that the rcm 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 movement of cells away from the netrin ligand, which is encoded by the unc-6 gene.

Our studies of aggregation chimeras demonstrated that in the mouse, the UNC5C protein is necessary for recognition of the anterior border of the cerebellum 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. These results identify a novel chemorepulsive mechanism underlying the formation of boundaries of the midbrain/hindbrain and the inner granule cell layer. In addition to neuronal migration abnormalities, we have found corticospinal tract (CST) defects in both Unc5c^rcm/Unc5c^rcm 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 have observed that Unc5c mutants on an inbred genetic background die shortly after birth, apparently due to respiratory problems. In contrast, mutant mice on a segregating background live a normal life span. Analysis of the inbred mice revealed that UNC5C is necessary for phrenic nerve guidance on this genetic background, uncovering a novel role for netrins and their receptors. We are currently analyzing F2 mice in an effort to genetically define the modifier genes that influence guidance of these axons.

To identify the molecular mechanisms for granule cell survival, we are studying the spontaneous mutant, harlequin (Hq). Adult Hq mutant mice develop progressive ataxia and retinal degeneration. Cell loss in the cerebellum and retina peaks at 5-7 months. Large numbers of mature retinal neurons and cerebellar granule cells, many of which are undergoing apoptosis, are in the S phase of the cell cycle in these mice.

Using a positional cloning approach, we have identified the Hq mutation as a proviral insertion in the apoptosis-inducing factor (Aif also called Pdcd8) gene, causing an approximate 80% reduction in AIF expression. AIF is a mitochondrial oxidoreductase that contains a pyridine nucleotide-disulphide oxidoreductase subunit similar to regulators of plant and bacterial hydrogen peroxide scavengers. Although the function of AIF within the mitochondria is unknown, AIF has been previously shown to cause apoptosis when it translocates to the nucleus.

Our studies show that reduced levels of AIF lead to oxidative stress in the retina and cerebellum. Further, we demonstrate that it is these stressed neurons that re-enter the cell cycle. Thus these data demonstrate that oxidative stress can mediate neuronal cell cycle re-entry and subsequent neuronal apoptosis in the adult central nervous system (CNS). We are currently evaluating signaling pathways that are regulated by oxidative stress and may result in this aberrant re-entry into the cell cycle and subsequent apoptosis.

Purkinje Cell Migration and Survival

Mice homozygous for the cerebellar deficient folia (cdf) mutation are ataxic, and display cerebellar hypoplasia and abnormal lobulation of the cerebellum. In the cdf mutant cerebellum, approximately 40% of Purkinje cells are ectopically located within the white matter and the inner granule cell layer (IGL). These ectopic cells constitute a specific subset of Purkinje cells, demonstrating that migrations of classes of Purkinje cells are regulated independently during cerebellar development. By analysis of aggregation chimeras from mutant and wild-type cells, we have demonstrated that the cdf mutation acts intrinsically to Purkinje cells. In addition to cerebellar defects, many pyramidal cells of the hippocampus are scattered in the plexiform layers with the remaining cells less densely packed than wild-type. Fear conditioning, a form of associative learning, and prepulse inhibition of the startle response, a measure of sensorimotor gating, are also disrupted in cdf mutant mice.

We have identified a deletion on Chromosome 6 that removes approximately 150 kb of DNA in the cdf critical region, including part of the gene encoding αN-catenin (Catna2), the protein that links the classical cadherins to the neuronal cytoskeleton. Normal cerebellar and hippocampal morphology was restored in cdf mutant mice expressing an αN-catenin cDNA transgene, demonstrating that that disruption of αN-catenin is responsible for cdf mutant phenotypes. Our data demonstrate that the catenin/cadherin cell adhesion complexes play an important role in the lamination of the cerebellum and hippocampus, and in the control of startle modulation. Analysis of the role of other catenins in cerebellar development is under way.

In addition, our lab is studying several mutant strains with adult onset Purkinje cell loss. These strains provide a means of identifying potentially novel molecules that are important for the survival and integrity of neurons in the adult CNS. For example, mice homozygous for the sticky (sti) mutation develop progressive ataxia as adults. The sti mutants lose a specific subpopulation of Purkinje cells at 1-2 months of age, followed by rostral granule cell loss at 4-6 months of age. We mapped the sti mutation to a 0.17-cM region on Chromosome 8 and have completed analysis of candidate genes from this interval, identifying a polymorphism in one gene in sti mutant mice. Transgenic rescue studies and functional tests of this sticky gene candidate are under way.

Study of neurodegenerative mutant strains can also yield genetic modifiers that modulate neuron loss. Identification of these genes can provide additional entry points into molecular pathways necessary for neuron survival and potential therapeutic targets. We have demonstrated the presence of a CAST/EiJ derived allele on Chromosome 2 that suppresses sti-mediated neurodegeneration, even in animals over 1 year old. We have fine-mapped this region and recently produced transgenic animals with a CAST/EiJ BAC from this region. Analysis of these animals demonstrates that this BAC contains the sti suppressor (Stis). We are currently molecularly analyzing the genes contained on this BAC.

Publications

• (2002 - present)
• Finger JH, Bronson RT, Harris B, Johnson K, Przyborski SA, Ackerman SL. 2002. The Netrin1 receptors Unc5h3 and Dcc are necessary at multiple choice points for the guidance of corticospinal tract axons. J Neurosci 22:10346-10356.
• Klein JA, Longo-Guess CM, Rossmann MP, Seburn KL, Hurd RE, Frankel WN, Bronson RT, Ackerman SL. 2002. The harlequin mouse mutation downregulates apoptosis-inducing factor. Nature 419:367-374.
• Park C, Falls W, Edgar JH, Longo-Guess CM, Ackerman SL. 2002. Deletion in Catna2, encoding αN-catenin, causes cerebellar and hippocampal lamination defects and impaired startle modulation. Nat Genet 31:279-284.
• Park C, Finger JH, Cooper JA, Ackerman SL. 2002. The cerebellar deficient folia (cdf) gene acts intrinsically in Purkinje cell migrations. Genesis 32:32-41.
• Klein JA, Ackerman SL. 2003. Oxidative stress, cell cycle, and neurodegeneration. J Clin Invest 111:785-793.
• Herrup K, Neve R, Ackerman SL, Copani A. 2004. Divide and die: Cell cycle events as triggers of nerve cell death. J Neurosci 24:9232-9239.
• Munroe RJ, Ackerman SL, Schimenti JC. 2004. Genomewide two-generation screens for recessive mutations by ES cell mutagenesis. Mamm Genome 15:960-965.
• Schwarting GA, Raitcheva D, Bless EP, Ackerman SL, Tobet S. 2004. Netrin 1 mediated chemoattraction regulates the migratory pathway of LHRH neurons. Eur J Neurosci 19:11-20.
• 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.

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