Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease.     

Dynein mutations impair autophagic clearance of aggregate-prone proteins

Mutations that affect the dynein motor machinery are sufficient to cause motor neuron disease. It is not known why there are aggregates or inclusions in affected tissues in mice with such mutations and in most forms of human motor neuron disease. Here we identify a new mechanism of inclusion formation by showing that decreased dynein function impairs autophagic clearance of aggregate-prone proteins. We show that mutations of the dynein machinery enhanced the toxicity of the mutation that causes Huntington disease in fly and mouse models. Furthermore, loss of dynein function resulted in premature aggregate formation by mutant huntingtin and increased levels of the autophagosome marker LC3-II in both cell culture and mouse models, compatible with impaired autophagosome-lysosome fusion.     

Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity

Huntington disease (HD) is caused by expansion of a glutamine repeat in the amino-terminal region of huntingtin. Despite its widespread expression, mutant huntingtin induces selective neuronal loss in striatal neurons. Here we report that, in mutant mice expressing HD repeats, the production and aggregation of N-terminal huntingtin fragments preferentially occur in HD-affected neurons and their processes and axonal terminals. N-terminal fragments of mutant huntingtin form aggregates and induce neuritic degeneration in cultured striatal neurons. N-terminal mutant huntingtin also binds to synaptic vesicles and inhibits their glutamate uptake in vitro. The specific processing and accumulation of toxic fragments of N-terminal huntingtin in HD-affected striatal neurons, especially in their neuronal processes and axonal terminals, may contribute to the selective neuropathology of HD.     

Identification and characterization of rod-derived cone viability factor

Retinitis pigmentosa is an untreatable, inherited retinal disease that leads to blindness. The disease initiates with the loss of night vision due to rod photoreceptor degeneration, followed by irreversible, progressive loss of cone photoreceptor. Cone loss is responsible for the main visual handicap, as cones are essential for day and high-acuity vision. Their loss is indirect, as most genes associated with retinitis pigmentosa are not expressed by these cells. We previously showed that factors secreted from rods are essential for cone viability. Here we identified one such trophic factor by expression cloning and named it rod-derived cone viability factor (RdCVF). RdCVF is a truncated thioredoxin-like protein specifically expressed by photoreceptors. The identification of this protein offers new treatment possibilities for retinitis pigmentosa.     

Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70

At least eight inherited human neurodegenerative diseases are caused by expansion of a polyglutamine domain within the respective proteins. This confers dominant toxicity on the proteins, leading to dysfunction and loss of neurons. Expanded polyglutamine proteins form aggregates, including nuclear inclusions (NI), within neurons, possibly due to misfolding of the proteins. NI are ubiquitinated and sequester molecular chaperone proteins and proteasome components, suggesting that disease pathogenesis includes activation of cellular stress pathways to help refold, disaggregate or degrade the mutant disease proteins. Overexpression of specific chaperone proteins reduces polyglutamine aggregation in transfected cells, but whether this alters toxicity is unknown. Using a Drosophila melanogaster model of polyglutamine disease, we show that directed expression of the molecular chaperone HSP70 suppresses polyglutamine-induced neurodegeneration in vivo. Suppression by HSP70 occurred without a visible effect on NI formation, indicating that polyglutamine toxicity can be dissociated from formation of large aggregates. Our studies indicate that HSP70 or related molecular chaperones may provide a means of treating these and other neurodegenerative diseases associated with abnormal protein conformation and toxicity.     

Mutant dynactin in motor neuron disease

Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.     

TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis

Recently, TDP-43 was identified as a key component of ubiquitinated aggregates in amyotrophic lateral sclerosis (ALS), an adult-onset neurological disorder that leads to the degeneration of motor neurons. Here we report eight missense mutations in nine individuals--six from individuals with sporadic ALS (SALS) and three from those with familial ALS (FALS)--and a concurring increase of a smaller TDP-43 product. These findings further corroborate that TDP-43 is involved in ALS pathogenesis.     

Disruption of CREB function in brain leads to neurodegeneration

Control of cellular survival and proliferation is dependent on extracellular signals and is a prerequisite for ordered tissue development and maintenance. Activation of the cAMP responsive element binding protein (CREB) by phosphorylation has been implicated in the survival of mammalian cells. To define its roles in the mouse central nervous system, we disrupted Creb1 in brain of developing and adult mice using the Cre/loxP system. Mice with a Crem(-/-) background and lacking Creb in the central nervous system during development show extensive apoptosis of postmitotic neurons. By contrast, mice in which both Creb1 and Crem are disrupted in the postnatal forebrain show progressive neurodegeneration in the hippocampus and in the dorsolateral striatum. The striatal phenotype is reminiscent of Huntington disease and is consistent with the postulated role of CREB-mediated signaling in polyglutamine-triggered diseases.     

nagie oko, encoding a MAGUK-family protein, is essential for cellular patterning of the retina

A layered organization of cells is a common architectural feature of many neuronal formations. Mutations of the zebrafish gene nagie oko (nok) produce a severe disruption of retinal architecture, indicating a key role for this locus in neuronal patterning. We show that nok encodes a membrane-associated guanylate kinase-family scaffolding protein. Nok localizes to the vicinity of junctional complexes in retinal neuroepithelium and in the photoreceptor cell layer. Mosaic analysis indicates that the nok retinal patterning phenotype is not cell-autonomous. We propose that nok function in patterning of postmitotic neurons is mediated through neuroepithelial cells and is necessary for guiding neurons to their proper destinations in retinal laminae.     

A conserved sorting-associated protein is mutant in chorea-acanthocytosis

Chorea-acanthocytosis (CHAC, MIM 200150) is an autosomal recessive neurodegenerative disorder characterized by the gradual onset of hyperkinetic movements and abnormal erythrocyte morphology (acanthocytosis). Neurological findings closely resemble those observed in Huntington disease. We identified a gene in the CHAC critical region and found 16 different mutations in individuals with chorea-acanthocytosis. CHAC encodes an evolutionarily conserved protein that is probably involved in protein sorting.     

A null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa.

Mutations within the rhodopsin gene are known to give rise to autosomal dominant retinitis pigmentosa (RP), a common hereditary form of retinal degeneration. We now describe a patient with autosomal recessive RP who is homozygous for a nonsense mutation at codon 249 within exon 4 of the rhodopsin gene. This null mutation, the first gene defect identified in autosomal recessive retinitis pigmentosa, should result in a functionally inactive rhodopsin protein that is missing the sixth and seventh transmembrane domains including the 11-cis-retinal attachment site. We also found a different null mutation carried heterozygously by an unrelated unaffected individual. Heterozygous carriers of either mutation had normal ophthalmologic examinations but their electroretinograms revealed an abnormality in rod photoreceptor function.     

Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice

Inactivation of the mouse homologue of the Huntington disease gene (Hdh) results in early embryonic lethality. To investigate the normal function of Hdh in the adult and to evaluate current models for Huntington disease (HD), we have used the Cre/loxP site-specific recombination strategy to inactivate Hdh expression in the forebrain and testis, resulting in a progressive degenerative neuronal phenotype and sterility. On the basis of these results, we propose that huntingtin is required for neuronal function and survival in the brain and that a loss-of-function mechanism may contribute to HD pathogenesis.     

Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa

Mutation of a receptor tyrosine kinase gene, Mertk, in the Royal College of Surgeons (RCS) rat results in defective phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) and retinal degeneration. We screened the human orthologue, MERTK, located at 2q14.1 (ref. 10), in 328 DNA samples from individuals with various retinal dystrophies and found three mutations in three individuals with retinitis pigmentosa (RP). Our findings are the first conclusive evidence implicating the RPE phagocytosis pathway in human retinal disease.     

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12).

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31-q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.     

Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence in diabetes. FRDA is caused by severely reduced levels of frataxin, a mitochondrial protein of unknown function. Yeast knockout models as well as histological and biochemical data from heart biopsies or autopsies of FRDA patients have shown that frataxin defects cause a specific iron-sulfur protein deficiency and intramitochondrial iron accumulation. We have recently shown that complete absence of frataxin in the mouse leads to early embryonic lethality, demonstrating an important role for frataxin during mouse development. Through a conditional gene-targeting approach, we have generated in parallel a striated muscle frataxin-deficient line and a neuron/cardiac muscle frataxin-deficient line, which together reproduce important progressive pathophysiological and biochemical features of the human disease: cardiac hypertrophy without skeletal muscle involvement, large sensory neuron dysfunction without alteration of the small sensory and motor neurons, and deficient activities of complexes I-III of the respiratory chain and of the aconitases. Our models demonstrate time-dependent intramitochondrial iron accumulation in a frataxin-deficient mammal, which occurs after onset of the pathology and after inactivation of the Fe-S-dependent enzymes. These mutant mice represent the first mammalian models to evaluate treatment strategies for the human disease.     

Genetic inroads in familial ALS

Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disease causing cell death of motor neurons and progressive muscle weakness. The disease is familial in ten percent of cases, of which one-fifth are due to mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Two papers in this issue of Nature Genetics describe homozygous mutations in a new gene on chromosome 2q33 in 4 families of Arabian origin with a rare form of juvenile onset ALS (ALS2). The predicted protein structure has domains homologous to GTPase regulatory proteins, and both the types of mutation and the pattern of inheritance suggest that motor neuron degeneration is the result of a loss of function. Further work will determine the relevance of this breakthrough to other, more common forms of ALS.     

Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53

The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.