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Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and alpha-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.

CD4+CD25hi FOXP3+ regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

The airway epithelium is altered in respiratory disease and is thought to contribute to disease aetiology. A caveat to disease research is that the technique of isolation of bronchial epithelial cells from patients is invasive and cells have a limited lifespan. The aim of the current study was to extensively characterise the plasticity of primary human bronchial epithelial cells that have been engineered to delay cell senescence including the ability of these cells to differentiate. Cells were engineered to express BMI-1 or hTERT using viral vector systems. Cells were characterised at passage (p) early (p5), mid (p10) and late (p15) stage for; BMI-1, p16 and CK14 protein expression, viability and the ability to differentiate at air-liquid interface (ALI), using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), Scanning Electron Microscopy (SEM), (MUC5AC and beta tubulin (BTUB) staining). BMI-1 expressing cells maintained elevated levels of the BMI-1 protein and the epithelial marker CK14 and showed a suppression of p16. BMI-1 expressing cells had a viability advantage, differentiated at ALI and had a normal karyotype. In contrast hTERT expressing cells had a reduced viability, showed limited differentiation and had an abnormal karyotype. We therefore provide extensive characterisation of the plasticity of BMI-1 expression cells in the context of the ALI model. These cells retain properties of wild-type cells and may be useful to characterise respiratory disease mechanisms in vitro over sustained periods.

CD4+CD25hi FOXP3+ regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

The airway epithelium is altered in respiratory disease and is thought to contribute to disease aetiology. A caveat to disease research is that the technique of isolation of bronchial epithelial cells from patients is invasive and cells have a limited lifespan. The aim of the current study was to extensively characterise the plasticity of primary human bronchial epithelial cells that have been engineered to delay cell senescence including the ability of these cells to differentiate. Cells were engineered to express BMI-1 or hTERT using viral vector systems. Cells were characterised at passage (p) early (p5), mid (p10) and late (p15) stage for; BMI-1, p16 and CK14 protein expression, viability and the ability to differentiate at air-liquid interface (ALI), using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), Scanning Electron Microscopy (SEM), (MUC5AC and beta tubulin (BTUB) staining). BMI-1 expressing cells maintained elevated levels of the BMI-1 protein and the epithelial marker CK14 and showed a suppression of p16. BMI-1 expressing cells had a viability advantage, differentiated at ALI and had a normal karyotype. In contrast hTERT expressing cells had a reduced viability, showed limited differentiation and had an abnormal karyotype. We therefore provide extensive characterisation of the plasticity of BMI-1 expression cells in the context of the ALI model. These cells retain properties of wild-type cells and may be useful to characterise respiratory disease mechanisms in vitro over sustained periods.

CD4+CD25hi FOXP3+ regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

The airway epithelium is altered in respiratory disease and is thought to contribute to disease aetiology. A caveat to disease research is that the technique of isolation of bronchial epithelial cells from patients is invasive and cells have a limited lifespan. The aim of the current study was to extensively characterise the plasticity of primary human bronchial epithelial cells that have been engineered to delay cell senescence including the ability of these cells to differentiate. Cells were engineered to express BMI-1 or hTERT using viral vector systems. Cells were characterised at passage (p) early (p5), mid (p10) and late (p15) stage for; BMI-1, p16 and CK14 protein expression, viability and the ability to differentiate at air-liquid interface (ALI), using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), Scanning Electron Microscopy (SEM), (MUC5AC and beta tubulin (BTUB) staining). BMI-1 expressing cells maintained elevated levels of the BMI-1 protein and the epithelial marker CK14 and showed a suppression of p16. BMI-1 expressing cells had a viability advantage, differentiated at ALI and had a normal karyotype. In contrast hTERT expressing cells had a reduced viability, showed limited differentiation and had an abnormal karyotype. We therefore provide extensive characterisation of the plasticity of BMI-1 expression cells in the context of the ALI model. These cells retain properties of wild-type cells and may be useful to characterise respiratory disease mechanisms in vitro over sustained periods.

CD4+CD25hi FOXP3+ regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

The airway epithelium is altered in respiratory disease and is thought to contribute to disease aetiology. A caveat to disease research is that the technique of isolation of bronchial epithelial cells from patients is invasive and cells have a limited lifespan. The aim of the current study was to extensively characterise the plasticity of primary human bronchial epithelial cells that have been engineered to delay cell senescence including the ability of these cells to differentiate. Cells were engineered to express BMI-1 or hTERT using viral vector systems. Cells were characterised at passage (p) early (p5), mid (p10) and late (p15) stage for; BMI-1, p16 and CK14 protein expression, viability and the ability to differentiate at air-liquid interface (ALI), using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), Scanning Electron Microscopy (SEM), (MUC5AC and beta tubulin (BTUB) staining). BMI-1 expressing cells maintained elevated levels of the BMI-1 protein and the epithelial marker CK14 and showed a suppression of p16. BMI-1 expressing cells had a viability advantage, differentiated at ALI and had a normal karyotype. In contrast hTERT expressing cells had a reduced viability, showed limited differentiation and had an abnormal karyotype. We therefore provide extensive characterisation of the plasticity of BMI-1 expression cells in the context of the ALI model. These cells retain properties of wild-type cells and may be useful to characterise respiratory disease mechanisms in vitro over sustained periods.