We sought novel strategies to reduce levels of the polyglutamine androgen receptor (polyQ AR) and achieve therapeutic benefits in models of spinobulbar muscular atrophy (SBMA), a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the Hsp90/Hsp70-based chaperone machinery, but mechanisms regulating the protein’s turnover are incompletely understood. We demonstrate that overexpression of Hip, a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of SBMA. These findings highlight the therapeutic potential of allosteric regulators of Hsp70, and provide new insights into the role of the chaperone machinery in protein quality control.

The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. We focused on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with K ATP (ATPsensitive potassium) channels, which are involved in metabolic homeostasis. We provide experimental evidence that Tinman directly regulates dSUR expression in the developing heart. We identified a cis-regulatory element in the first intron of dSUR, which contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly's myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacinginduced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance.aging ͉ hypoxia ͉ sulfonylurea receptor

Inductive signaling is of pivotal importance for developmental patterns to form. In Drosophila, the transfer of TGFβ (Dpp) and Wnt (Wg) signaling information from the ectoderm to the underlying mesoderm induces cardiacspecific differentiation in the presence of Tinman, a mesoderm-specific homeobox transcription factor. We present evidence that the Gata transcription factor, Pannier, and its binding partner U-shaped, also a zincfinger protein, cooperate in the process of heart development. Loss-of-function and germ layer-specific rescue experiments suggest that pannier provides an essential function in the mesoderm for initiation of cardiacspecific expression of tinman and for specification of the heart primordium. u-shaped also promotes heart development, but unlike pannier, only by maintaining tinman expression in the cardiogenic region. By contrast, pan-mesodermal overexpression of pannier ectopically expands tinman expression, whereas overexpression of ushaped inhibits cardiogenesis. Both factors are also required for maintaining dpp expression after germ band retraction in the dorsal ectoderm. Thus, we propose that Pannier mediates as well as maintains the cardiogenic Dpp signal. In support, we find that manipulation of pannier activity in either germ layer affects cardiac specification, suggesting that its function is required in both the mesoderm and the ectoderm.Key words: Drosophila, Heart, Cardiogenesis, Mesoderm, pannier, u-shaped, tinman, dpp 3028 Evans, 1996;Morrisey et al., 1996), where they are thought to regulate cardiac-specific genes (Grepin et al., 1994;Ip et al., 1994; Durocher et al., 1997;Murphy et al., 1997) (reviewed by Molkentin, 2000). Gata4 is already expressed in the early cardiac crescent of the lateral plate mesoderm, and in mice deficient for Gata4, these heart primordia fail to migrate towards the midline where they normally fuse into the primitive heart tube (Molkentin et al., 1997;Kuo et al., 1997). Owing to these ventral closure defects, it has been difficult to discriminate between a direct role for Gata4 in heart formation and an indirect involvement via its function in ventral morphogenesis. Furthermore, Gata4, Gata5 and Gata6 may act in part redundantly, which may further occlude their cardiogenic potential. Consistent with the direct involvement of Gata4 in heart development is the congenital heart disease phenotype observed in individuals heterozygous for deletions of chromosome 8p23.1 region, which includes the GATA4 gene (Pehlivan et al., 1999;Bhatia et al., 1999).In vitro, Gata4 interacts with a wide array of proteins, including the Tinman homolog Nkx2.5, the bHLH protein Hand and the multiple zinc-finger protein Fog2 (Durocher et al., 1997;Sepulveda et al., 1998;Lee et al., 1998;Lu et al., 1999;Sepulveda et al., 2002;Svensson et al., 1999;Tevosian et al., 1999; Dai et al., 2002). Fog2 apparently modulates Gatamediated transcriptional regulation not only as a repressor, but also as an activator, depending on the promoter and on cell type (Lu et al., 1999). Fog2 is co-...

Mitogen-activated protein (MAP) kinase signaling cascades orchestrate diverse cellular activities with common molecular players. To achieve specific cellular outcomes in response to specific signals, scaffolding proteins play an important role. Here we investigate the role of the scaffolding protein JNK interacting protein-1 (JIP1) in neuronal signaling by a conserved axonal MAP kinase kinase kinase, known as Wallenda (Wnd) in Drosophila and dual leucine kinase (DLK) in vertebrates and Caenorhabditis elegans. Recent studies in multiple model organisms suggest that Wnd/DLK regulates both regenerative and degenerative responses to axonal injury. Here we report a new role for Wnd in regulating synaptic structure during development, which implies that Wnd is also active in uninjured neurons. This synaptic role of Wnd can be functionally separated from the role of Wnd in axonal regeneration and injury signaling by the requirement for the JIP1 scaffold and the p38b MAP kinase. JIP1 mediates the synaptic function of Wnd via p38, which is not required for injury signaling or new axonal growth after injury. Our results indicate that Wnd regulates multiple independent pathways in Drosophila motoneurons and that JIP1 scaffolds a specific downstream cascade required for the organization of presynaptic microtubules during synaptic development.