An extensive protein–protein interaction network has been identified between proteins implicated in inherited ataxias. The protein sacsin, which is mutated in the early-onset neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay, is a node in this interactome. Here, we have established the neuronal expression of sacsin and functionally characterized domains of the 4579 amino acid protein. Sacsin is most highly expressed in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization in SH-SY5Y neuroblastoma cells was predominantly cytoplasmic with a mitochondrial component. We identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin and demonstrated an interaction with the proteasome. Furthermore, sacsin contains a predicted J-domain, the defining feature of DnaJ/Hsp40 proteins. Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional. The presence of both UbL and J-domains in sacsin suggests that it may integrate the ubiquitin–proteasome system and Hsp70 function to a specific cellular role. The Hsp70 chaperone machinery is an important component of the cellular response towards aggregation prone mutant proteins that are associated with neurodegenerative diseases. We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1. Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1. Thus, sacsin is an ataxia protein and a regulator of the Hsp70 chaperone machinery that is implicated in the processing of other ataxia-linked proteins.

Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.

Use of all-trans-retinoic acid (ATRA) in combinatorial differentiation therapy of acute promyelocytic leukemia (APL) results in exceptional cure rates. However, potent cell differentiation effects of ATRA are so far largely restricted to this disease and long-term survival rates in non-APL acute myelogeneous leukemia (AML) remain unacceptably poor, requiring development of novel therapeutic strategies. We demonstrate here that my- IntroductionRetinoids regulate growth and differentiation of normal and malignant cells. 1 In the hematopoietic system, all-trans retinoic acid (ATRA) has been shown to inhibit growth, induce differentiation of myelomonocytic progenitor cells, and to enhance selfrenewal of more immature multipotent stem cells. 2,3 In acute promyelocytic leukemia (APL) ATRA is a potent inducer of APL cell differentiation, and its use in anti-APL therapy markedly improved survival and prognosis of patients with this disease. 4 This ability of APL cells to respond to ATRA with terminal differentiation is likely due to the presence of RAR␣ gene translocation and expression of the RAR␣ chimeric proteins, which do not respond to physiologic levels of ATRA. Pharmacological doses of ATRA trigger the dissociation of nuclear receptor co-repressors from such "dominant-negative" RAR␣-fusion proteins. [5][6][7][8][9] These findings underscore the importance of ATRA signaling in myeloid cell differentiation. 3 Nevertheless, the use of ATRA as a single agent has not been successful in other types of acute myelogeneous leukemia (AML), where long-term survival rates remain low at 25% (http:// seer.cancer.gov/cgi-bin/csr/1975_2001/search.pl#results). Although ATRA by itself has not been effective in non-APL AML cell differentiation, potential use of ATRA in combination with other factors such as histone deacetylase inhibitors, 10 granulocyte colonystimulating factor (G-CSF), 11,12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) 13 has been evaluated in some studies. Recently, we have demonstrated that ATRA and G/GM-CSF cooperate to promote myelomonocytic differentiation in murine pluripotent myeloid progenitor cells. This differentiation induction was associated with increased expression of ATRAinducible RAR␣2 isoform. 14 Although these findings indicated a cross-talk between ATRA and myelomonocytic growth factors (GFs), the downstream signaling cascades that could be involved in the potential activation of RAR␣ expression by G/GM-CSF remain unknown. In order to further investigate the relationship between myelomonocytic GFs and ATRA, particularly in the context of human leukemic cell differentiation, we have used AML as a model. We demonstrate that through MAP kinase pathways G-and GM-CSF are able to potentiate transcriptional activities of liganded RAR␣1 and ␣2 isoforms and enhance ATRA-induced gene expression and myeloid differentiation of human leukemic cells. Convergence of these 2 major signaling pathways in myeloid differentiation reinforce the view that combinatorial use of retinoids with myelomon...

SummaryPrimary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.