Rapamycin pre-treatment protects against apoptosis.

Macroautophagy (generally referred to as autophagy) mediates the bulk degradation of cytoplasmic contents, including proteins and organelles, in lysosomes. Rapamycin, a lipophilic, macrolide antibiotic, induces autophagy by inactivating the protein mammalian target of rapamycin (mTOR). We previously showed that rapamycin protects against mutant huntingtin-induced neurodegeneration in cell, fly and mouse models of Huntington's disease [Ravikumar, B., Duden, R. and Rubinsztein, D.C. (2002) Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. Mol. Genet., 11, 1107-1117, Ravikumar, B., Vacher, C., Berger, Z., Davies, J.E., Luo, S., Oroz, L.G., Scaravilli, F., Easton, D.F., Duden, R., O'Kane, C.J. et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet., 36, 585-595]. This protective effect of rapamycin was attributed to enhanced clearance of the mutant protein via autophagy [Ravikumar, B., Duden, R. and Rubinsztein, D.C. (2002) Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. Mol. Genet., 11, 1107-1117, Ravikumar, B., Vacher, C., Berger, Z., Davies, J.E., Luo, S., Oroz, L.G., Scaravilli, F., Easton, D.F., Duden, R., O'Kane, C.J. et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet., 36, 585-595]. Here, we show that rapamycin may have additional cytoprotective effects--it protects cells against a range of subsequent pro-apoptotic insults and reduces paraquat toxicity in Drosophila. This protection can be accounted for by enhanced clearance of mitochondria by autophagy, thereby reducing cytosolic cytochrome c release and downstream caspase activation after pro-apoptotic insults. Thus, rapamycin (pro-autophagic) treatment may be useful in certain disease conditions (including various neurodegenerative diseases) where a slow but increased rate of apoptosis is evident, even if they are not associated with overt aggregate formation.

Huntington disease patients and transgenic mice have similar pro-catabolic serum metabolite profiles.

There has been considerable progress recently towards developing therapeutic strategies for Huntington's disease (HD), with several compounds showing beneficial effects in transgenic mouse models. However, human trials in HD are difficult, costly and time-consuming due to the slow disease course, insidious onset and patient-to-patient variability. Identification of molecular biomarkers associated with disease progression will aid the development of effective therapies by allowing further validation of animal models and by providing hopefully more sensitive measures of disease progression. Here, we apply metabolic profiling by gas chromatography-time-of-flight-mass spectrometry to serum samples from human HD patients and a transgenic mouse model in a hypothesis-generating search for disease biomarkers. We observed clear differences in metabolic profiles between transgenic mice and wild-type littermates, with a trend for similar differences in human patients and control subjects. Thus, the metabolites responsible for distinguishing transgenic mice also comprised a metabolic signature tentatively associated with the human disease. The candidate biomarkers composing this HD-associated metabolic signature in mouse and humans are indicative of a change to a pro-catabolic phenotype in early HD preceding symptom onset, with changes in various markers of fatty acid breakdown (including glycerol and malonate) and also in certain aliphatic amino acids. Our data raise the prospect of a robust molecular definition of progression of HD prior to symptom onset, and if validated in a genuinely prospective fashion these biomarker trajectories could facilitate the development of useful therapies for this disease.

Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington's disease.

Huntington's disease is a devastating neurodegenerative condition associated with the formation of intraneuronal aggregates by mutant huntingtin. Aggregate formation is a property shared by the nine related diseases caused by polyglutamine codon expansion mutations and also by other neurodegenerative conditions like Parkinsons's disease. The roles of aggregates and aggregation in these diseases have been a subject of heated controversy. Here, we have addressed the question in vivo by generating a new transgenic mouse overexpressing the yeast chaperone hsp104, as hsp104 overexpression reduced mutant huntingtin aggregation and toxicity in cell models. Hsp104 has no close mammalian orthologues and does not appear to have effects on mammalian cell death pathways. We crossed hsp104 transgenic mice with mice expressing the first 171 residues of mutant huntingtin. Hsp104 reduced aggregate formation and prolonged the lifespan of the HD mice by 20%. This protection may be mediated at the level of changing the conformation of a putative toxic monomer, reducing oligomerization or aggregation, reducing the levels of oligomeric species or aggregates or combinations of these non-mutually exclusive possibilities.

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.

Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH(2)-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH(2)-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.

Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice.

The muscular dystrophies are a heterogeneous group of disorders for which there are currently no cures. Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset, progressive disease that generally presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a (GCG)n trinucleotide repeat in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most individuals with OPMD this (GCG)6 repeat is expanded to (GCG)8-13, leading to a stretch of 12-17 alanines in mutant PABPN1. PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibers. We have developed a transgenic mouse model of OPMD that manifests progressive muscle weakness accompanied by intranuclear aggregates and TUNEL-stained nuclei in skeletal muscle fibers. The onset and severity of these abnormalities were substantially delayed and attenuated by doxycycline treatment, which may exert its therapeutic effect by reducing aggregates and by distinct antiapoptotic properties. Doxycycline may represent a safe and feasible therapeutic for this disease.

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.

Distribution of dopamine D2 receptor mRNAs in the brain and the pituitary of female rainbow trout: an in situ hybridization study.

The distribution of D(2)R (dopamine D(2) receptor) mRNAs was studied in the forebrain of maturing female rainbow trout by means of in situ hybridization using a (35)S-labeled riboprobe (810 bp) spanning the third intracytoplasmic loop. A hybridization signal was consistently obtained in the olfactory epithelium, the internal cell layer of the olfactory bulbs, the ventral and dorsal subdivisions of the ventral telencephalon, and most preoptic subdivisions, with the notable exception of the magnocellular preoptic nucleus, and the periventricular regions of the mediobasal hypothalamus, including the posterior tuberculum. In the pituitary, the signal was higher in the pars intermedia than in the proximal and the rostral pars distalis, but no obvious correspondence with a given cell type could be assigned. Labeled cells were also located in the thalamic region, some pretectal nuclei, the optic tectum, and the torus semicircularis. These results provide a morphologic basis for a better understanding on the functions and evolution of the dopaminergic systems in lower vertebrates.

Dopamine D2 receptors and secretion of FSH and LH: role of sexual steroids on the pituitary of the female rainbow trout.

The role of sexual steroids in the modulation of a dopaminergic inhibitory tone on FSH and LH release was studied in the rainbow trout. The experiments were performed on previtellogenic trout, implanted or not with estradiol (E(2)), and vitellogenic trout. E(2) implant increased the circulating levels of LH and decreased the circulating levels of FSH in previtellogenic fish. The catecholamine inhibitor alphaMPT increased the circulating levels of LH, implanted or not with E(2). AlphaMPT increased circulating levels of LH in vitellogenic fish. This increase could be prevented by the dopaminergic agonist bromocryptine. The dopaminergic drugs had no effect on the circulating levels of FSH in all groups. E(2) decreased mRNA levels of sGnRH1 and sGnRH2 in the telencephalon of previtellogenic fish. The dopaminergic treatments had no effect on mRNA levels of both forms of sGnRH in previtellogenic and vitellogenic fish. Primary cultures of pituitary cells were primed for three days with steroids (E(2) or 17alpha-hydroxy, 20beta-dihydroprogesterone (17alpha20betaP)) before treatment with increasing doses of bromocryptine, associated or not with sGnRH. E(2), but not 17alpha20betaP, potentiated the sGnRH-induced release of LH. Bromocryptine induced a slight dose-dependent decrease of sGnRH-induced release of LH. This decrease was potentiated by 17alpha20betaP. E(2) and 17alpha20betaP had no effect on the release of FSH, but bromocryptine decreased the 10(-8)M sGnRH-induced release of FSH. In conclusion, the development of the dopaminergic inhibitory tone on gonadotropin release, at the onset of vitellogenesis, requires factors other than estradiol. E(2) should contribute in part to decrease the release of FSH. At the end of the reproductive cycle, 17alpha20betaP should reinforce the dopaminergic inhibitory tone.

The bacterial chaperonin GroEL requires GroES to reduce aggregation and cell death in a COS-7 cell model of Huntington's disease.

Huntington's disease (HD) is caused by expansions of more than 35 CAG repeats in the HD gene. These repeats are translated into a long polyglutamine tract that confers a deleterious gain-of-function on the mutant protein. Intraneuronal inclusions comprising mutant huntingtin are found in HD patient brains. Here we show that the bacterial chaperonin GroEL can reduce aggregation of mutant huntingtin in COS-7 cells and requires GroES for efficient activity, analogous to what has been described in bacteria. The reduction in aggregation of mutant huntingtin by GroEL/GroES was associated with protection against polyglutamine-induced cell death.