Intersectin 1 Research Articles

Abstract 3249: Intersectin 1 is required for neuroblastoma tumorigenesis

Abstract Neuroblastoma (NB) is a malignant tumor deriving from the neural crest and is responsible for 13% of cancer-related deaths in children. Phosphoinositide 3-kinases (PI3Ks) play a central role in NB tumorigenesis. PI3Ks activate AKT, which regulates survival and stabilizes the MYCN oncogene. Both MYCN and AKT are markers for poor prognosis in NBs. Inhibiting PI3Ks with pan-inhibitors blocks malignant progression in vivo; however, these inhibitors have high toxicity that limits their use in NB treatment. Though there are three classes of PI3Ks, only Class I isoforms have been implicated in NB tumorigenesis. We have previously found that the scaffold protein intersectin 1 (ITSN1) interacts with PI3KC2α and regulates its activation as well as AKT activation in neuronal cell lines. In addition, ITSN1 overexpression is transforming in rodent fibroblasts. Thus, we examined the role of the ITSN1-PI3KC2α pathway in human NB tumorigenesis. ITSN1 and PI3KC2α are highly expressed in primary human neuroblastoma tumors of varying stage and MYCN amplification status. In addition, both ITSN1 and PI3KC2α are expressed in NB tumor cell lines derived from high grade NB tumors. Silencing ITSN1 dramatically inhibited the anchorage-independent growth of NB tumor cells in vitro and tumor formation in xenograft assays in vivo independent of MYCN status. Conversely, heterologous overexpression of ITSN1 increased the formation of soft agar colonies. These results suggest that ITSN1 functions as an oncogene in NB cells. Furthermore, PI3KC2α overexpression restores anchorage-independent growth of ITSN1-depleted NB cells demonstrating that PI3KC2α mediates ITSN1's tumorigenic properties. This study reveals a new pathway, i.e., ITSN1-PI3KC2α, involved in regulating the pathogenesis of NBs and suggests that this pathway may represent a new target for therapeutic intervention in the treatment of NB patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3249. doi:1538-7445.AM2012-3249

MironRNA‐194 (mir‐194) regulates ROMk channel activity by down‐regulation of intersectin 1 (ITSN1)‐WNK pathway

The aim of the present study is to test whether miR‐194 plays a role in mediating the effect of high K (HK) intake on ROMK channels through inhibiting ITSN1‐dependent endocytosis of ROMK channels. Northern blot showed that a HK intake increased the miR‐194 expression in the mouse kidney. The qPCR further demonstrated that a HK intake increased the miR‐194 transcription. Moreover, a HK intake decreased the expression of ITSN1. The role of miR‐194 in regulating ITSN1 expression is further confirmed by Luciferase Assay. Moreover, down‐regulation of endogenous miR‐194 by the antagomer increased ITSN1 protein expression in HEK cells. The whole‐cell recording and biotin labeling technique further showed that expression of miR‐194 increased ROMK channel activity in HEK cells transfected with GFP‐ROMK1, and effect was abolished by co‐expression of ITSN1. We conclude that miR‐194 regulates ROMK channel activity by modulating ITSN1 translation and that HK‐induced increase in miR‐194 is involved in mediating the effect of a HK intake on ROMK channel activity.

Intersectin 1 is required for Neuroblastoma tumorigenesis

Neuroblastoma (NB) is a malignant tumor deriving from the neural crest that is responsible for 13% of cancer‐related deaths in children. Phosphoinositide 3‐kinases (PI3Ks) play a central role in NB tumorigenesis. PI3Ks activate AKT, which regulates survival and stabilizes the MYCN oncogene. Both MYCN and AKT are markers for poor prognosis in NBs. Inhibiting PI3Ks with pan‐inhibitors blocks malignant progression in vivo. These inhibitors have high toxicity that limits their use in NB treatment. Though there are three classes of PI3Ks, only Class I has been implicated in NB. Class 2 beta PI3K (PI3KC2β) is overexpressed in cancer and involved in adherent and anchorage‐independent growth of cancer cells. We found that the scaffold protein intersectin 1 (ITSN1) interacts with PI3KC2β and regulates its activation and Akt activation in mouse NB lines. Thus, we examined the role of the ITSN1‐PI3KC2β pathway in human NB tumorigenesis. We found that stable silencing of ITSN1 in human NB cells attenuates their growth both in soft agar assays in vitro and in xenograft tumor assays in vivo. Furthermore, PI3KC2β overexpression restores anchorage‐independent growth of ITSN1‐depleted NB cells suggesting that PI3KC2β mediates ITSN1's tumorigenic phenotype. This study reveals a new pathway, i.e., ITSN1‐ PI3KC2β is involved in regulating the pathogenesis of NBs.

Intersectin 1 Enhances Cbl Ubiquitylation of Epidermal Growth Factor Receptor through Regulation of Sprouty2-Cbl Interaction

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.

Intersectin 1 is required for neuroblastoma tumorigenesis

Intersectin 1 (ITSN1) is a scaffold protein that regulates diverse cellular pathways including endocytosis and several signal transduction pathways including phosphotidylinositol 3-kinase, Class IIβ (PI3K-C2β). ITSN1's transforming potential in vitro suggests that this scaffold protein may be involved in human tumorigenesis. Herein, we demonstrate that ITSN1 is expressed in primary human neuroblastoma tumors and tumor cell lines and is necessary for their in vitro and in vivo tumorigenic properties. Silencing ITSN1 dramatically inhibits the anchorage independent growth of tumor cells in vitro and tumor formation in xenograft assays independent of MYCN status. Overexpression of the ITSN1 target, PI3K-C2β, rescues the soft agar growth of ITSN1-silenced cells demonstrating the importance of the ITSN1-PI3K-C2β pathway in NB tumorigenesis. These findings represent the first demonstration that the ITSN1-PI3K-C2β pathway plays a requisite role in human cancer, specifically neuroblastomas.

Intersectin 1 contributes to phenotypes in vivo

Intersectin 1 (ITSN1) is a human chromosome 21 (HSA21) gene product encoding a multidomain scaffold protein that functions in endocytosis, signal transduction, and is implicated in Down's syndrome, Alzheimer's Disease, and potentially other neurodegenerative diseases through activation of c-Jun N-terminal kinase. We report for the first time that ITSN1 proteins are elevated in individuals with Down's syndrome of varying ages. However, ITSN1 levels decreased in aged cases with Down's syndrome with Alzheimer's disease-like neuropathology. Analysis of a novel ITSN1 transgenic mouse reveals that ITSN1 overexpression results in a sex-dependent decrease in locomotor activity. This study reveals a link between overexpression of specific ITSN1 isoforms and behavioral phenotypes and has implications for human neurodegenerative diseases such as Down's syndrome and Alzheimer's disease.

Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein engaged in clathrin-mediated endocytosis, cell signaling and actin cytoskeleton rearrangements. Two major ITSN1 isoforms were initially described, the ubiquitous short isoform (ITSN1-s) and the long isoform (ITSN1-l) expressed predominantly in neurons. Numerous alternative splicing events for ITSN1 pre-mRNA were later identified. Here we describe a novel isoform ITSN1-22a with an alternative C-terminus encoded by exon 22a. This exon is only found in placental mammals. The transcript of ITSN1-22a is detected in a wide range of human and mouse tissues. We show here that two alternative splicing events affect the coding sequence of the ITSN1-22a isoform. Moreover, alternative polyadenylation of these transcripts was demonstrated in human tissues. The protein encoded by the ITSN1-22a transcript possesses two EH domains, a coiled-coil region, an SH3A domain and a specific C-terminal domain (CTD) but lacks four SH3 domains in comparison with ITSN1-s. The level of ITSN1-22a protein varies in different mouse tissues and human cell lines. The highest amounts of this isoform occur in mouse brain, spleen, lung and the human B cell line DG75. ITSN1-22a binds via its CTD to the SH3 domain of the endocytic protein amphiphysin 1 and the SH3A domain of ITSN1. Furthermore association in vivo and codistribution of ITSN1-22a and ITSN1-s were demonstrated suggesting that these isoforms could function in concert. We have revealed differential binding of ITSN1-s and ITSN1-22a to the ubiquitin ligase Cbl. Both isoforms possess the SH3A domain capable of binding to Cbl; however ITSN1-22a in contrast to ITSN1-s did not interact with Cbl in vivo. In vitro binding experiments demonstrated that the CTD of ITSN1-22a negatively regulated its binding to Cbl; at the same time interaction with another partner, dynamin 1 was not affected by the presence of the CTD. These data suggest that intramolecular interaction within ITSN1-22a could specifically regulate its binding to protein partners. Thus, this novel mammalian ITSN1 isoform possesses a significantly altered domain structure and performs specific protein–protein interactions.

Intersectin1‐s is involved in migration and invasion of human glioma cells

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1-l and ITSN1-s. In this study, we show that siRNA-mediated down regulation of ITSN1-s inhibited migration and invasion of glioma cells. In addition, we demonstrate the possible mechanisms by which ITSN1-s functions in migration and invasion. Several key proteins, including cofilin, LIMK, PAK, FAK, integrin β1, and MMP-9, which are critical for cells migration and invasion, were probably involved in ITSN1-s signaling pathways. These results suggest that ITSN1-s contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP-9. Our results indicate that ITSN1-s is a critical factor in gliomas invasion and identify that ITSN1-s is a new potentially antiinvasion target for therapeutic intervention in gliomas.

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 ( Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 ( RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR ( Bin/ amphiphysin/ Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.

Intersectin 1: a versatile actor in the synaptic vesicle cycle

During neurotransmitter release, SVs (synaptic vesicles) fuse at the active zone and are recovered predominantly via clathrin-mediated endocytosis at the presynaptic compartment surrounding the site of release, referred to as the periactive zone. Exo- and endo-cytosis in synapses are tightly temporarily and spatially coupled to sustain synaptic transmission. The molecular mechanisms linking these two cellular events, which take place in separate compartments of the nerve terminal, remain largely enigmatic. Several lines of evidence indicate that multiple factors may be involved in exocytic-endocytic coupling including SV integral membrane proteins, SV membrane lipids and the membrane-associated actin cytoskeleton. A number of recent studies also indicate that multimodular adaptor proteins shuttling between the active and periactive zones aid the dynamic assembly of macromolecular protein complexes that execute the exo- and endo-cytic limbs of the SV cycle. Here, we discuss recent evidence implicating the multidomain scaffolding and adaptor protein ITSN1 (intersectin 1) as a central regulator of SV cycling.

Structural diversity and differential expression of novel human intersectin 1 isoforms

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling and cytoskeleton rearrangements. The ITSN1 gene encodes two main isoforms: a short form (ITSN1-s), which is ubiquitously expressed and consists of two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains, and a long form (ITSN1-l), which is predominantly expressed in the brain and contains three additional domains, a Dbl homology (DH) domain, a Pleckstrin homology (PH) domain and a C2 domain. Using computational analysis of the EST database and 3' RACE we determined the length of the 3' untranslated region of ITSN1-l and demonstrated that the polyadenylation site is located 11,559 nt downstream of the stop codon of the ITSN1-l mRNA. Recently, additional splicing events affecting ITSN1 transcripts were reported, but full-length transcriptional isoforms with different combinations of alternatively spliced exons remained unknown. Here we report the identification of fifteen novel transcriptional isoforms of the human ITSN1 gene with full-length coding sequences that are the result of different combinations of the alternatively spliced exons 5, 6/6', 20, 23, 25, 26, 26a and 35. The isoforms identified differ in domain organization and expression level in different tissues and more likely contribute to the modulation of many complex protein interactions in which ITSN1 participates.

ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells

Aim. Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which precisely regulate biological outcome. These processes are tightly controlled by coordinated action of a plethora of proteins – enzymes, scaffolds and inhibitory molecules. An example of an endocytic accessory protein that also functions in cell signaling is provided by intersectin 1 (ITSN1). Previously we have shown that ITSN1 forms a complex with adaptor protein Ruk/CIN85 and ubiquitin ligase Cbl-b, which are implicated in down regulation of RTK. The present study aimed to determine the subcellular localization of ITSN1-Ruk/CIN85 complexes relatively to clathrin light chain and Cbl-b. Methods. Transient transfection of MCF-7 breast adenocarcinoma cells with the constructs containing Omni-tagged intersectin 1 and clathrin light chain fused with mCherry fluorescent protein was utilized to determine subcellular localization by direct or indirect immunofluorescence. Results. We found that Ruk/CIN85-ITSN1 complexes partially colocalized with Cbl-b and clathrin light chain in MCF-7 cells. Conclusions. In our report we provide experimental evidence that ITSN1-Ruk/CIN85 complexes exist in pre-assembled state with Cbl-b and are targeted to clathrin-coated pits in MCF-7 cells.

Intersectin 1 forms a complex with adaptor protein Ruk/CIN85 in vivo independently of epidermal growth factor stimulation

Intersectin 1 (ITSN1) is an adaptor protein involved in clathrin-mediated endocytosis, apoptosis, signal transduction and cytoskeleton organization. Here, we show that ITSN1 forms a complex with adaptor protein Ruk/CIN85, implicated in downregulation of receptor tyrosine kinases. The interaction is mediated by the SH3A domain of ITSN1 and the third or fourth proline-rich blocks of Ruk/CIN85, and does not depend on epidermal growth factor stimulation, suggesting a constitutive association of ITSN1 with Ruk/CIN85. Moreover, both proteins colocalize in MCF-7 cells with their common binding partner, the ubiquitin ligase c-Cbl. The possible biological role of the interaction between ITSN1 and Ruk/CIN85 is discussed.

Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities

Enlarged early endosomes in the neurons of young Down syndrome (DS) and pre-Alzheimer's disease (AD) brains suggest that a disturbance in endocytosis is one of the earliest hallmarks of AD pathogenesis in both conditions. We identified a chromosome 21 gene, Intersectin-1 (ITSN1) that is up-regulated in DS brains and has a putative function in endocytosis and vesicle trafficking. To elucidate the function of ITSN1 and assess its contribution to endocytic defects associated with DS and AD, we generated Itsn1 null mice. In knockout mice we found alterations in a number of parameters associated with endocytic and vesicle trafficking events. We found a reduced number of exocytosis events in chromaffin cells and a slowing of endocytosis in neurons. Endosome size was increased in neurons and NGF levels were reduced in the septal region of the brain. Our data is the first indication that Itsn1 has a role in endocytosis in an in vivo mammalian model, and that a disruption in Itsn1 expression causes a disturbance in vesicle trafficking and endocytic function in the brain. These results imply a role for ITSN1 in the early endocytic anomalies reported in DS brains which may have ramifications for the onset of AD.

Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons

Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in endocytosis, regulation of actin cytoskeleton rearrangements and mitogenic signaling. Its expression is characterized by multiple alternative splicing. Here we show neuron-specific expression of ITSN1 isoforms containing exon 20, which encodes five amino acid residues in the first SH3 domain (SH3A). In vitro binding experiments demonstrated that inclusion of exon 20 changes the binding properties of the SH3A domain. Endocytic proteins dynamin 1 and synaptojanin 1 as well as GTPase-activating protein CdGAP bound the neuron-specific variant of the SH3A domain with higher affinity than ubiquitously expressed SH3A. In contrast, SOS1, a guanine nucleotide exchange factor for Ras, and the ubiquitin ligase Cbl mainly interact with the ubiquitously expressed isoform. These results demonstrate that alternative splicing leads to the formation of two pools of ITSN1 with potentially different properties in neurons, affecting ITSN1 function as adaptor protein.

Intersectin (ITSN) regulation of Rab family GTPases

ITSN is a multi‐domain scaffolding protein that regulates endocytosis and signal transduction. In addition, ITSN enhances receptor tyrosine kinase (RTK) trafficking in part through regulation of RTK ubiquitylation. We now report that ITSN regulates Rab‐mediated trafficking of RTKs. ITSN and Rab5 co‐localize on early endosomes. To examine the relationship of ITSN with various Rab family GTPases, we used bimolecular fluorescence complementation (BiFC) in which a YFP variant, Venus, is split into amino (VN)‐ and carboxyl (VC)‐terminal fragments, neither of which is fluorescent. When these fragments are fused to proteins that interact, fluorescence is reconstituted thus allowing for temporal and spatial monitoring of protein:protein interactions in vivo. Using BiFC, we shown that ITSN complexes with Rab5 and Rab7, but not with the dominant negative forms of these GTPases indicating that ITSN preferentially associates with active Rab5 and Rab7. In addition, we have identified a new component of the Rab pathway that forms a complex with ITSN and Rab5/Rab7 suggesting a role for this protein in Rab‐mediated trafficking. The localization of ITSN to vesicles as well as ITSN's interaction with the Rab pathway supports the model that ITSN regulates intracellular trafficking.K.A.W. supported in part by NIH PSTP Training Grant (5T32GM070388)

Intersectin (ITSN) regulates Ras activation of PI3K Class2β

Intersectin (ITSN) is an evolutionarily conserved multi‐domain scaffolding protein that regulates endocytosis as well as a number of signal proteins including Ras, Cbl, and PI3K. Although ITSN activates Ras on intracellular vesicles, this pool of Ras did not activate classical downstream pathways such as ERK or JNK MAPKs. We now report the identification of the downstream target for ITSN‐Ras. We discovered that ITSN interacts with and activates a novel PI3K isoform, PI3K Class 2β (PI3K‐C2β), which is necessary for survival of differentiating neurons. PI3K‐C2β possesses a Ras binding domain (RBD) suggesting a potential interaction with Ras. We now demonstrate that Ras interacts with PI3K‐C2β and ITSN. This interaction requires the RBD of PI3K‐C2β, and ITSN interaction with PI3K requires both the PRD and RBD of PI3K suggesting that Ras is necessary for ITSN‐PI3K interaction. In support of this model, we demonstrate that ITSN, Ras, and PI3K‐C2β are part of a trimolecular complex on vesicles. Finally, we demonstrate that dominant‐negative Ras inhibits ITSN activation of AKT. These data support a model whereby ITSN activates Ras on vesicles leading to recruitment and activation PI3K‐C2β to stimulate cell survival.

Structure of the Eps15–stonin2 complex provides a molecular explanation for EH-domain ligand specificity

Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH-NPF interactions described thus far are of very low affinity and specificity. Here, we identify the presynaptic endocytic sorting adaptor stonin2 as a high-affinity ligand for the second EH domain (EH2) of the clathrin accessory protein Eps15. Calorimetric data indicate that both NPF motifs within stonin2 interact with EH2 simultaneously and with sub-micromolar affinity. The solution structure of this complex reveals that the first NPF motif binds to the conserved site on the EH domain, whereas the second motif inserts into a novel hydrophobic pocket. Our data show how combination of two EH-attachment sites provides a means for modulating specificity and allows discrimination from a large pool of potential binding partners containing NPF motifs.

Regulation of Neuron Survival through an Intersectin-Phosphoinositide 3′-Kinase C2β-AKT Pathway

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.

Intersectin enhances huntingtin aggregation and neurodegeneration through activation of c-Jun-NH2-terminal kinase

Huntingon's disease is a progressive neurodegenerative disease arising from expansion of a polyglutamine (polyQ) tract in the protein huntingtin (Htt) resulting in aggregation of mutant Htt into nuclear and/or cytosolic inclusions in neurons. Mutant Htt affects multiple processes including protein degradation, transcription, signal transduction, fast axonal transport and endocytosis [reviewed in Ross, C.A. and Poirier, M.A. (2005) Opinion: what is the role of protein aggregation in neurodegeneration? Nat. Rev. Mol. Cell. Biol., 6, 891-898]. Here, we report that the endocytic and signal transduction scaffold intersectin (ITSN) increased aggregate formation by mutant Htt through activation of the c-Jun-NH(2)-terminal kinase (JNK)-MAPK pathway. Conversely, silencing ITSN or inhibiting JNK attenuated aggregate formation. Using a Drosophila model for polyQ repeat disease, we observed that ITSN enhanced polyQ-mediated neurotoxicity. A reciprocal relationship was observed between ITSN and Htt. While ITSN enhanced Htt aggregation and toxicity, Htt, in turn, inhibited the cooperativity between ITSN and the epidermal growth factor receptor signal transduction pathway. Finally, we observed that ITSN overexpression enhanced aggregation of polyQ-expanded androgen receptor (AR) as well as wild-type versions of both Htt and AR suggesting a broader involvement of ITSN in neurodegenerative diseases through destabilization of polyQ-containing proteins.