Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%–4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

The current standard of treatment against tuberculosis consists of a cocktail of first-line drugs, including isoniazid and pyrazinamide. Although these drugs are known to be bactericidal, contribution of host cell responses in the context of antimycobacterial chemotherapy, if any, remains unknown. We demonstrate that isoniazid and pyrazinamide promote autophagy activation and phagosomal maturation in Mycobacterium tuberculosis (Mtb)-infected host cells. Treatment of Mtb-infected macrophages with isoniazid or pyrazinamide caused significant activation of cellular and mitochondrial reactive oxygen species and autophagy, which was triggered by bacterial hydroxyl radical generation. Mycobacterium marinum-infected autophagy-defective, atg7 mutant Drosophila exhibited decreased survival rates, which could not be rescued by antimycobacterial treatment, indicating that autophagy is required for effective antimycobacterial drug action in vivo. Moreover, activation of autophagy by antibiotic treatment dampened Mtb-induced proinflammatory responses in macrophages. Together, these findings underscore the importance of host autophagy in orchestrating successful antimicrobial responses to mycobacteria during chemotherapy.

Parkin, an E3 ubiquitin ligase, has been found to be responsible for autosomal recessive juvenile parkinsonism characterized primarily by selective loss of dopaminergic neurons with subsequent defects in movements. However, the molecular mechanisms underlying this neuron loss remain elusive. Here, we characterized Drosophila parkin loss-of-function mutants, which exhibit shrinkage of dopaminergic neurons with decreased tyrosine hydroxylase level and impaired locomotion. The behavioral defect of parkin mutant flies was partially restored by administering L-DOPA, and the dopamine level in the brains of parkin mutant flies was highly decreased. Intriguingly, we found that c-Jun N-terminal kinase (JNK) is strongly activated in the dopaminergic neurons of parkin mutants and that impaired dopaminergic neuron phenotypes are dependent on the activation of the JNK signaling pathway. In consistent with this, our epistatic analysis and mammalian cell studies showed that Parkin inhibits the JNK signaling pathway in an E3 activity-dependent manner. These results suggest that loss of Parkin function up-regulates the JNK signaling pathway, which may contribute to the vulnerability of dopaminergic neurons in Drosophila parkin mutants and perhaps autosomal recessive juvenile parkinsonism patients.Parkinson's disease ͉ ubiquitination ͉ tyrosine hydroxylase ͉ degeneration ͉ apoptosis P arkinson's disease (PD) is the second most common neurodegenerative disease in North America, affecting Ͼ1 million people. The major symptoms of PD include rigidity, tremor, bradykinesia of the limbs, and postural instability. These symptoms result primarily from a deficiency of dopamine caused by selective degeneration of dopaminergic neurons in two regions of the brain, the substantia nigra pars compacta and striatum. Another pathological feature of this disease is the presence of inclusion bodies, called Lewy bodies, in those surviving neurons (1). The average onset of the disease is at the age of 60, but the pervasion of PD is not restricted to aging individuals; the onset of autosomal recessive juvenile parkinsonism (AR-JP) is usually between the age of 20 and 40 (2).Even though PD is largely a sporadic disorder, mutations in the genes responsible for PD, either autosomal dominant or recessive, have been found in a number of affected families. To date, six genes, namely, ␣-synuclein (PARK1) (3), parkin (PARK2) (4), UCH-L1 (PARK5) (5), PINK1 (PARK6) (6), DJ-1 (PARK7) (7), and the most recently identified, LRRK2͞dardarin (PARK8) (8, 9), have been isolated as pathological candidates for PD by family-based linkage analyses and positional cloning. Among them, mutations in parkin, PINK1, and DJ-1 were found to be associated with earlyonset autosomal recessive parkinsonism (4, 6, 7).In particular, Parkin is an E3 ubiquitin ligase, encoded by parkin, the most common gene mutated in familial PD. Although its exact in vivo function is not clearly revealed, the structure of Parkin gives significant clues to its possible function in the ubiquitination path...

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