The accumulation of aggregated ␣-synuclein is thought to contribute to the pathophysiology of Parkinson's disease, but the mechanism of toxicity is poorly understood. Recent studies suggest that aggregated proteins cause toxicity by inhibiting the ubiquitin-dependent proteasomal system. In the present study, we explore how ␣-synuclein interacts with the proteasome. The proteasome exists as a 26 S and a 20 S species. The 26 S proteasome is composed of the 19 S cap and the 20 S core. Aggregated ␣-synuclein strongly inhibited the function of the 26 S proteasome. The IC 50 of aggregated ␣-synuclein for ubiquitin-independent 26 S proteasomal activity was 1 nM. Aggregated ␣-synuclein also inhibited 26 S ubiquitin-dependent proteasomal activity at a dose of 500 nM. In contrast, the IC 50 of aggregated ␣-synuclein for 20 S proteasomal activity was > 1 M. This suggests that aggregated ␣-synuclein selectively interacts with the 19 S cap. Monomeric ␣-synuclein also inhibited proteasomal activity but with lower affinity and less potency. Recombinant monomeric ␣-synuclein inhibited the activity of the 20 S proteasomal core with an IC 50 > 10 M, exhibited no inhibition of 26 S ubiquitin-dependent proteasomal activity at doses up to 5 M, and exhibited only partial inhibition (50%) of the 26 S ubiquitinindependent proteasomal activity at doses up to 10 mM. Binding studies demonstrate that both aggregated and monomeric ␣-synuclein selectively bind to the proteasomal protein S6, a subunit of the 19 S cap. These studies suggest that proteasomal inhibition by aggregated ␣-synuclein could be mediated by interaction with S6.
Cholesterol is eliminated from neurons by oxidization, which generates oxysterols. Cholesterol oxidation is mediated by the enzymes cholesterol 24-hydroxylase (CYP46A1) and cholesterol 27-hydroxylase (CYP27A1). Immunocytochemical studies show that CYP46A1 and CYP27A1 are expressed in neurons and some astrocytes in the normal brain, and CYP27A1 is present in oligodendrocytes. In Alzheimer's disease (AD), CYP46A1 shows prominent expression in astrocytes and around amyloid plaques, whereas CYP27A1 expression decreases in neurons and is not apparent around amyloid plaques but increases in oligodendrocytes. Although previous studies have examined the effects of synthetic oxysterols on the processing of amyloid precursor protein (APP), the actions of the naturally occurring oxysterols have yet to be examined. To understand the role of cholesterol oxidation in AD, we compared the effects of 24(S)-and 27-hydroxycholesterol on the processing of APP and analyzed the cell-specific expression patterns of the two cholesterol hydroxylases in the human brain. Both oxysterols inhibited production of A in neurons, but 24(S)-hydroxycholesterol was ϳ1000-fold more potent than 27-hydroxycholesterol. The IC 50 of 24(S)-hydroxycholesterol for inhibiting A secretion was ϳ1 nM. Both oxysterols induced ABCA1 expression with IC 50 values similar to that for inhibition of A secretion, suggesting the involvement of liver X receptor. Oxysterols also inhibited protein kinase C activity and APP secretion following stimulation of protein kinase C. The selective expression of CYP46A1 around neuritic plaques and the potent inhibition of APP processing in neurons by 24(S)-hydroxycholesterol suggests that CYP46A1 affects the pathophysiology of AD and provides insight into how polymorphisms in the CYP46A1 gene might influence the pathophysiology of this prevalent disease.
Multiple studies implicate metals in the pathophysiology of neurodegenerative diseases. Disturbances in brain iron metabolism are linked with synucleinopathies. For example, in Parkinson's disease, iron levels are increased and magnesium levels are reduced in the brains of patients. To understand how changes in iron and magnesium might affect the pathophysiology of Parkinson's disease, we investigated binding of iron to ␣-synuclein, which accumulates in Lewy bodies. Using fluorescence of the four tyrosines in ␣-synuclein as indicators of metal-related conformational changes in ␣-synuclein, we show that iron and magnesium both interact with ␣-synuclein. ␣-Synuclein exhibits fluorescence peaks at 310 and 375 nm. Iron lowers both fluorescence peaks, while magnesium increases the fluorescence peak only at 375 nm, which suggests that magnesium affects the conformation of ␣-synuclein differently than iron. Consistent with this hypothesis, we also observe that magnesium inhibits ␣-synuclein aggregation, measured by immunoblot, cellulose acetate filtration, or thioflavine-T fluorescence. In each of these studies, iron increases ␣-synuclein aggregation, while magnesium at concentrations >0.75 mM inhibits the aggregation of ␣-synuclein induced either spontaneously or by incubation with iron. These data suggest that the conformation of ␣-synuclein can be modulated by metals, with iron promoting aggregation and magnesium inhibiting aggregation. Parkinson's disease (PD)1 is a common motor disorder that affects about 1% of population over the age of 65 (1). The disease is characterized by progressive neurodegeneration predominantly affecting dopaminergic neurons in the nigrostriatal system (2). The degenerating neurons develop intracellular inclusions, termed Lewy bodies, which are composed of a dense core of filamentous and granular material (3). Recent studies indicate that ␣-synuclein is a major filamentous component of Lewy bodies (3,4). Genetic studies suggest that ␣-synuclein plays a key role in the pathophysiology of PD, because mutations in ␣-synuclein, at A53T or A30P, are associated with early-onset familial PD (5, 6).The accumulation of aggregated protein underlies the pathophysiology of many neurodegenerative disorders, and increasing evidence suggests that aggregated ␣-synuclein plays a key role in the pathophysiology of PD. ␣-Synuclein has a strong tendency to aggregate and does so spontaneously in vitro at a slow rate (7-9). Both the A53T and the A30P mutations in PD increase the tendency of ␣-synuclein to aggregate. Many studies in cultured neurons, and some studies in transgenic animals, suggest that ␣-synuclein aggregation is linked to cellular toxicity and neurodegeneration (10 -12). In cell culture, formation of ␣-synuclein aggregates correlates with cell injury (10). Overexpressing ␣-synuclein in Drosophila leads to an age-dependent accumulation of aggregated ␣-synuclein and associated neurodegeneration (12). Masliah and colleagues also observed that aggregated ␣-synuclein is associated with loss of marke...
Homeobox (HOX) genes play a definitive role in determination of cell fate during embryogenesis and hematopoiesis. MLL-related leukemia is coincident with increased expression of a subset of HOX genes, including HOXA9. MLL functions to maintain, rather than initiate, expression of its target genes. However, the mechanism of MLL maintenance of target gene expression is not understood. Here, we demonstrate that Mll binds to specific clusters of CpG residues within the Hoxa9 locus and regulates expression of multiple transcripts. The presence of Mll at these clusters provides protection from DNA methylation. shRNA knock-down of Mll reverses the methylation protection status at the previously protected CpG clusters; methylation at these CpG residues is similar to that observed in Mll null cells. Furthermore, reconstituting MLL expression in Mll null cells can reverse DNA methylation of the same CpG residues, demonstrating a dominant effect of MLL in protecting this specific region from DNA methylation. Intriguingly, an oncogenic MLL-AF4 fusion can also reverse DNA methylation, but only for a subset of these CpGs. This method of transcriptional regulation suggests a mechanism that explains the role of Mll in transcriptional maintenance, but it may extend to other CpG DNA binding proteins. Protection from methylation may be an important mechanism of epigenetic inheritance by regulating the function of both de novo and maintenance DNA methyltransferases.homeodomain ͉ leukemia ͉ maintenance
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