Extracellular and transmembrane region of a podocalyxin-like protein 1 fragment identified from colon cancer cell lines.

Regulated intramembrane proteolysis of membrane proteins has been shown to play an important role in cell differentiation and in the pathogenesis of diseases. The aim of the present study was to identify novel peptides generated by intramembrane proteolysis. The peptides were identified in serum-free cultured (SFC) media from various cell lines by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). A 2315-Da peptide found only in medium from SFC colon cancer cell lines was identified and shown to consist of a portion of both the extracellular and transmembrane regions of human podocalyxin-like 1. This protein fragment was not found in lung or pancreatic cancer cell lines by immunoprecipitation-SELDI tests using an antibody specific to this fragment, suggesting that this human podocalyxin-like protein 1 fragment may be unique to colon cancer cell lines.

Decreased expression of hypothalamic neuropeptides in Huntington disease transgenic mice with expanded polyglutamine-EGFP fluorescent aggregates.

Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the down-regulation of specific genes in this region of the brain.

Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease.

Inhibition of polyglutamine-induced protein aggregation could provide treatment options for polyglutamine diseases such as Huntington disease. Here we showed through in vitro screening studies that various disaccharides can inhibit polyglutamine-mediated protein aggregation. We also found that various disaccharides reduced polyglutamine aggregates and increased survival in a cellular model of Huntington disease. Oral administration of trehalose, the most effective of these disaccharides, decreased polyglutamine aggregates in cerebrum and liver, improved motor dysfunction and extended lifespan in a transgenic mouse model of Huntington disease. We suggest that these beneficial effects are the result of trehalose binding to expanded polyglutamines and stabilizing the partially unfolded polyglutamine-containing protein. Lack of toxicity and high solubility, coupled with efficacy upon oral administration, make trehalose promising as a therapeutic drug or lead compound for the treatment of polyglutamine diseases. The saccharide-polyglutamine interaction identified here thus provides a new therapeutic strategy for polyglutamine diseases.

Pro-apoptotic protein kinase C delta is associated with intranuclear inclusions in a transgenic model of Huntington's disease.

In order to investigate any effect of truncated mutant huntingtin (tNhtt) aggregation on protein kinase C (PKC) signaling in Huntington's disease (HD), we studied a possible association of PKC isoforms with the aggregates using cellular and transgenic models of HD. In this report we describe an association of mutant tNhtt with at least three PKC isoforms (alpha, delta, zeta), as revealed by co-immunoprecipitation assays and immunocytochemistry in a cellular model of HD (Neuro2a cells expressing tNhtt-150Q-EGFP), as well as a specific association of PKC delta with intranuclear aggregates in a transgenic model (R6/2 mice). Immunoblot analysis of isolated nuclear fractions shows an elevation of nuclear PKC delta in transgenic brain tissue. The observed elevation has a strong similarity with the apoptotic translocation of PKC delta detected in experiments with the mouse neuroblastoma Neuro2a cells. Using a Neuro2a cell line expressing tNhtt with the nuclear localization signal, we demonstrate the association of PKC delta with intranuclear aggregates and present evidence that accumulation of PKC delta in cell nuclei does not depend on mutant htt nuclear translocation. Our results suggest that the association of PKC delta with intranuclear htt-aggregates may affect its apoptotic function in a transgenic model of HD.

Purification of polyglutamine aggregates and identification of elongation factor-1alpha and heat shock protein 84 as aggregate-interacting proteins.

Aggregates of green fluorescent protein (GFP)-fused truncated N-terminal huntingtin containing abnormally long polyglutamine tracts (150 repeats of glutamine residue) were purified from an ecdysone-inducible mutant neuro2A cell line (HD150Q-28) by using a fluorescence-activated cell sorter. To analyze the aggregate-interacting proteins, we subjected the purified aggregates to SDS-PAGE; prominent protein bands in the gel were digested with Achromobactor lysyl endopeptidase, followed by a HPLC-mass spectrometry (MS) analysis. The resulting data of tandem MS analysis revealed that, in addition to ubiquitin and widely reported chaperone proteins such as heat shock cognate 70 (HSC70), human DNA J-1 (HDJ-1), and HDJ-2, the translational elongation factor-1alpha (EF-1alpha) and heat shock protein 84 (HSP84) also were recognized as aggregate-interacting proteins. Sequestration of these proteins to aggregates was confirmed further by several immunochemical methods. We confirmed that, in addition to the other known proteins, EF-1alpha and HSP84 also colocalized with the intracellular aggregates. An assay of the transient expression of EF-1alpha and HSP84 in HD150Q-28 cells revealed that both proteins improved cell viability. Moreover, the rate of aggregate formation decreased in both transfectants. Our study suggests that both EF-1alpha and HSP84 are involved in the neurodegenerative process of polyglutamine diseases.