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.

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.

Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional and/or genetic disruptions in homocysteine metabolism. The most common genetic cause of hyperhomocysteinemia is the 677C-->T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mild enzymatic deficiency, is associated with an increased risk for neural tube defects and pregnancy complications and with a decreased risk for colon cancer and leukemia. Although many studies have reported that this variant is also a risk factor for vascular disease, this area of investigation is still controversial. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. To investigate the in vivo pathogenetic mechanisms of MTHFR deficiency, we generated mice with a knockout of MTHFR: Plasma total homocysteine levels in heterozygous and homozygous knockout mice are 1.6- and 10-fold higher than those in wild-type littermates, respectively. Both heterozygous and homozygous knockouts have either significantly decreased S-adenosylmethionine levels or significantly increased S-adenosylhomocysteine levels, or both, with global DNA hypomethylation. The heterozygous knockout mice appear normal, whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology. Abnormal lipid deposition in the proximal portion of the aorta was observed in older heterozygotes and homozygotes, alluding to an atherogenic effect of hyperhomocysteinemia in these mice.

In higher eukaryotes, 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.

In the developing nervous system, migrating cells and axons are guided to their targets by cues in the extracellular environment. The netrins are a family of phylogenetically conserved guidance cues that can function as diffusible attractants and repellents for different classes of cells and axons. In vertebrates, insects and nematodes, members of the DCC subfamily of the immunoglobulin superfamily have been implicated as receptors that are involved in migration towards netrin sources. The mechanisms that direct migration away from netrin sources (presumed repulsions) are less well understood. In Caenorhabditis elegans, the transmembrane protein UNC-5 (ref. 14) has been implicated in these responses, as loss of unc-5 function causes migration defects and ectopic expression of unc-5 in some neurons can redirect their axons away from a netrin source. Whether UNC-5 is a netrin receptor or simply an accessory to such a receptor has not, however, been defined. We now report the identification of two vertebrate homologues of UNC-5 which, with UNC-5 and the product of the mouse rostral cerebellar malformation gene (rcm), define a new subfamily of the immunoglobulin superfamily, and whose messenger RNAs show prominent expression in various classes of differentiating neurons. We provide evidence that these two UNC-5 homologues, as well as the rcm gene product, are netrin-binding proteins, supporting the hypothesis that UNC-5 and its relatives are netrin receptors.

No abstract

Migration of neurons from proliferative zones to their functional sites is fundamental to the normal development of the central nervous system. Mice homozygous for the spontaneous rostral cerebellar malformation mutation (rcm(s)) or a newly identified transgenic insertion allele (rcm(tg)) exhibit cerebellar and midbrain defects, apparently as a result of abnormal neuronal migration. Laminar structure abnormalities in lateral regions of the rostral cerebellar cortex have been described in homozygous rcm(s) mice. We now demonstrate that the cerebellum of both rcm(s) and rcm(tg) homozygotes is smaller and has fewer folia than in the wild-type, ectopic cerebellar cells are present in midbrain regions by three days after birth, and there are abnormalities in postnatal cerebellar neuronal migration. We have cloned the rcm complementary DNA, which encodes a transmembrane receptor of the immunoglobulin superfamily. The sequence of the rcm protein (Rcm) is highly similar to that of UNC-5, a Caenorhabditis elegans protein that is essential for dorsal guidance of pioneer axons and for the movement of cells away from the netrin ligand, which is encoded by the unc-6 gene. As Rcm is a member of a newly described family of vertebrate homologues of UNC-5 which are netrin-binding proteins, our results indicate that UNC-5-like proteins may have a conserved function in mediating netrin-guided migration.

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 experimentally driving the cell cycle in a mature neuron leads to cell death rather than cell division, and blocking cell-cycle initiation can prevent many types of neuronal cell death. This unlikely linkage of cell cycle and cell death pathways is little appreciated among neuroscientists. As only one example, bromodeoxyuridine (BrdU) labeling is often uncritically accepted as proof of neurogenesis when it may well be attributable to a cell cycle-related cell death. This review is meant to enhance appreciation for the relevance of this phenomenon to development and neurodegenerative diseases, in particular the neurodegeneration found in Alzheimer's disease (AD). A brief overview of the participation of mitotic events in human Alzheimer's disease and its mouse models is presented. Against this background, we consider evidence that links various APP (amyloid precursor protein) binding proteins with the cell cycle in Alzheimer's disease. We also examine the role played by oxidative stress as a trigger for cell cycle-related neuronal death. Finally, we discuss the biochemical details of the lethal neuronal cell cycle events and present evidence that non-canonical pathways of DNA replication are probably involved. BackgroundSince the earliest days of neurobiology, the study of neuronal cell death has been a field full of surprises. For example, the very contention that the death of neurons might be a normal part of the developmental program of the brain was greeted initially with great skepticism, and its formal demonstration by Hamburger (1975) and others came as quite a surprise to most workers in the