Susan L. Ackerman

The harlequin mouse mutation downregulates apoptosis-inducing factor

Harlequin (Hq) mutant mice have progressive degeneration of terminally differentiated cerebellar and retinal neurons. We have identified the Hq mutation as a proviral insertion in the apoptosis-inducing factor (Aif) gene, causing about an 80% reduction in AIF expression. Mutant cerebellar granule cells are susceptible to exogenous and endogenous peroxide-mediated apoptosis, but can be rescued by AIF expression. Overexpression of AIF in wild-type granule cells further decreases peroxide-mediated cell death, suggesting that AIF serves as a free radical scavenger. In agreement, dying neurons in aged Hq mutant mice show oxidative stress. In addition, neurons damaged by oxidative stress in both the cerebellum and retina of Hq mutant mice re-enter the cell cycle before undergoing apoptosis. Our results provide a genetic model of oxidative stress-mediated neurodegeneration and demonstrate a direct connection between cell cycle re-entry and oxidative stress in the ageing central nervous system.

Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.

Oxidative stress, cell cycle, and neurodegeneration.

While numerous studies have examined the existence of increased reactive oxygen species (ROS) in later-onset neurodegenerative disorders, the mechanism by which neurons die under conditions of oxidative stress remains largely unknown. Fairly recent evidence has suggested that one mechanism linked to the death of terminally differentiated neurons is aberrant reentry into the cell cycle. This phenomenon has been reported in Alzheimer disease (AD) patients (1), Down syndrome patients (2), and several mouse neurodegenerative models (3–5). We will discuss recent findings regarding the influence of oxidative stress on neurodegeneration and possible connections between oxidative stress and unscheduled cell cycle reentry, the understanding of which could lead to new strategies in the development of therapeutic agents for neurodegenerative disorders.

Divide and Die: Cell Cycle Events as Triggers of Nerve Cell Death

For over a decade, evidence has mounted that nerve cell death in the CNS is often intimately linked to a process of cell division. Mitotic markers appear in neurons at risk for death in a variety of neurodegenerative conditions, in mouse and in humans. Beyond correlation, studies have shown that

Protein accumulation and neurodegeneration in the woozy mutant mouse is caused by disruption of SIL1, a cochaperone of BiP.

Endoplasmic reticulum (ER) chaperones and ER stress have been implicated in the pathogenesis of neurodegenerative disorders, such as Alzheimer and Parkinson diseases, but their contribution to neuron death remains uncertain. In this study, we establish a direct in vivo link between ER dysfunction and neurodegeneration. Mice homozygous with respect to the woozy (wz) mutation develop adult-onset ataxia with cerebellar Purkinje cell loss. Affected cells have intracellular protein accumulations reminiscent of protein inclusions in both the ER and the nucleus. In addition, upregulation of the unfolded protein response, suggestive of ER stress, occurs in mutant Purkinje cells. We report that the wz mutation disrupts the gene Sil1 that encodes an adenine nucleotide exchange factor of BiP, a crucial ER chaperone. These findings provide evidence that perturbation of ER chaperone function in terminally differentiated neurons leads to protein accumulation, ER stress and subsequent neurodegeneration.

The Netrin 1 Receptors Unc5h3 and Dcc Are Necessary at Multiple Choice Points for the Guidance of Corticospinal Tract Axons

Migrating axons require the correct presentation of guidance molecules, often at multiple choice points, to find their target. Netrin 1, a bifunctional cue involved in both attracting and repelling axons, is involved in many cell migration and axon pathfinding processes in the CNS. The netrin 1 receptor DCC and itsCaenorhabditis elegans homolog UNC-40 have been implicated in directing the guidance of axons toward netrin sources, whereas the C. elegans UNC-6 receptor, UNC-5 is necessary for migrations away from UNC-6. However, a role of vertebrate UNC-5 homologs in axonal migration has not been demonstrated. We demonstrate that the Unc5h3 gene product, shown previously to regulate cerebellar granule cell migrations, also controls the guidance of the corticospinal tract, the major tract responsible for coordination of limb movements. Furthermore, we show that corticospinal tract fibers respond differently to loss of UNC5H3. In addition, we observe corticospinal tract defects in mice homozygous for a spontaneous mutation that truncates the Dcctranscript. Postnatal day 0 netrin 1 mutant mice also demonstrate corticospinal tract abnormalities. Last, interactions between the Dcc and Unc5h3 mutations were observed in gene dosage experiments. This is the first evidence of an involvement in axon guidance for any member of the vertebrateunc-5 family and confirms that both the cellular and axonal guidance functions of C. elegans unc-5 have been conserved in vertebrates.

Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration

Problems making proteins kills nerve cells Neurodegeneration is associated with a variety of different diseases, but its cellular roots are often obscure. Ishimura et al. find that mutant mice whose brain cells start to die rapidly soon after birth have lost the function of two vital cellular components (see the Perspective by Darnell). The first is a protein that releases stalled ribosomes stuck on messenger RNA (mRNA); the second is a transfer RNA (tRNA), which reads the code for arginine in the mRNA. This tRNA is expressed predominantly in the central nervous system. The lack of the tRNA leads to increased ribosomal stalling at arginine codons, which, when left uncorrected, blocks protein synthesis and proves fatal. Science, this issue p. 455; see also p. 378 Mutations in a transfer RNA expressed in the nervous system stall ribosomes and can cause cell death if ribosome recycling fails. [Also see Perspective by Darnell] In higher eukaryotes, transfer RNAs (tRNAs) with the same anticodon are encoded by multiple nuclear genes, and little is known about how mutations in these genes affect translation and cellular homeostasis. Similarly, the surveillance systems that respond to such defects in higher eukaryotes are not clear. Here, we discover that loss of GTPBP2, a novel binding partner of the ribosome recycling protein Pelota, in mice with a mutation in a tRNA gene that is specifically expressed in the central nervous system causes ribosome stalling and widespread neurodegeneration. Our results not only define GTPBP2 as a ribosome rescue factor but also unmask the disease potential of mutations in nuclear-encoded tRNA genes.

Downregulation of Apoptosis-Inducing Factor in Harlequin Mutant Mice Sensitizes the Myocardium to Oxidative Stress-Related Cell Death and Pressure Overload-Induced Decompensation

Apoptosis-inducing factor (AIF), or programmed cell death 8 (Pdcd8), is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal apoptosis induced by oxidative stress. Conversely, in vitro, AIF has been demonstrated to have a proapoptotic role when, on induction of the mitochondrial death pathway, AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. To determine the role of AIF in myocardial apoptotic processes, we examined cardiomyocytes from an AIF-deficient mouse mutant, Harlequin (Hq). Hq mutant cardiomyocytes demonstrated increased sensitivity to H2O2-induced cell death. Further, Hq hearts subjected to ischemia/reperfusion revealed more cardiac damage and, unlike wild-type mice, the amount of damage increased with the age of the animal. Aortic banding caused enhanced hypertrophy, increased cardiomyocyte apoptotic and necrotic cell death, and accelerated progression toward maladaptive left ventricular remodeling in Hq mutant mice compared with wild-type counterparts. These findings correlated with a reduced capacity of subsarcolemmal mitochondria from Hq mutant hearts to scavenge free radicals. Together, these data demonstrate a critical role for AIF as a cardiac antioxidant in the protection against oxidative stress–induced cell death and development of heart failure induced by pressure overload.

Plexin-A2 and its ligand, Sema6A, control nucleus-centrosome coupling in migrating granule cells

During their migration, cerebellar granule cells switch from a tangential to a radial mode of migration. We have previously demonstrated that this involves the transmembrane semaphorin Sema6A. We show here that plexin-A2 is the receptor that controls Sema6A function in migrating granule cells. In plexin-A2–deficient (Plxna2−/−) mice, which were generated by homologous recombination, many granule cells remained in the molecular layer, as we saw in Sema6a mutants. A similar phenotype was observed in mutant mice that were generated by mutagenesis with N-ethyl-N-nitrosourea and had a single amino-acid substitution in the semaphorin domain of plexin-A2. We found that this mutation abolished the ability of Sema6A to bind to plexin-A2. Mouse chimera studies further suggested that plexin-A2 acts in a cell-autonomous manner. We also provide genetic evidence for a ligand-receptor relationship between Sema6A and plexin-A2 in this system. Using time-lapse video microscopy, we found that centrosome-nucleus coupling and coordinated motility were strongly perturbed in Sema6a−/− and Plxna2−/− granule cells. This suggests that semaphorin-plexin signaling modulates cell migration by controlling centrosome positioning.

Phosphatidylinositol transfer protein-|[alpha]| in netrin-1-induced PLC signalling and neurite outgrowth

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-α (PITPα) in neurite extension in response to netrin-1, an extracellular guidance cue. PITPα interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PITPα binding to DCC and to phosphatidylinositol (5) phosphate [PI(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PIP2). In addition, the stimulated PIP2 hydrolysis requires PITPα. Furthermore, cortical explants of PITPα mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PITPα mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PITPα expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PITPα in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PITPα regulation.