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

No abstract

MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27 kip1 ) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias.leukemia ͉ acute lymphoblastic leukemias ͉ cell cycle M LL is involved in chromosomal translocations associated with leukemia. Remarkably, MLL is involved in translocations with Ͼ50 different genes (1, 2). MLL is specifically cleaved shortly after translation into two peptides that noncovalently associate with each other (3, 4). The amino-terminal portion of MLL contains a region with AT-hooks that binds DNA, as well as a region with transcriptional repression activity (5) that binds CpG-rich DNA (6) and recruits histone deacetylases, the corepressor CtBP1, and polycomb group proteins (7). The carboxyl-terminal portion contains a transcriptional activation domain (5), which interacts with CBP (2), and a SET domain, with histone methyltransferase activity (3,8). Different MLL fusion partners are associated with leukemias producing blast cells of various lineages. MLL-AF9 results mainly in acute myeloid leukemia (AML), whereas MLL-AF4 causes almost exclusively B-cell lineage acute lymphoblastic leukemias. These findings suggest that MLL chimeras affect the phenotype of the leukemia by influencing differentiation pathways of uncommitted cells or early progenitors. MLL-AF4, an MLL fusion protein that is associated with infant pro-B acute lymphoblastic leukemias, is the most prevalent of the numerous MLL fusion proteins (9), and it is usually associated with a poor prognosis (10). Numerous data show that MLL fu...

The human BCL6 gene on chromosome 3 band q27, which encodes a transcriptional repressor, is implicated in the pathogenesis of human lymphomas, especially the diffuse large B-cell type. We previously identified the human PDCD2 (programmed cell death-2) gene as a target of BCL6 repression. PDCD2 encodes a protein that is expressed in many human tissues, including lymphocytes, and is known to interact with corepressor complexes. We now show that BCL6 can bind directly to the PDCD2 promoter, repressing its transcription. Knockdown of endogenous BCL6 in a human B cell lymphoma line by introduction of small interfering RNA duplexes increases PDCD2 protein expression. Furthermore, there is an inverse relationship between the expression levels of the BCL6 and PDCD2 proteins in the lymphoid tissues of mice overexpressing human BCL6 (high BCL6 levels, minimal PDCD2) and controls (minimal BCL6, high PDCD2) as well as in tissues examined from some human B and T cell lymphomas. These data confirm PDCD2 as a target of BCL6 and support the concept that repression of PDCD2 by BCL6 is likely important in the pathogenesis of certain human lymphomas.human lymphomas ͉ target of BCL6 B CL6, a gene on chromosome 3, band q27, encodes a nuclear zinc finger protein that is a transcriptional repressor (1-3). This protein is expressed at high levels in human lymph node germinal center B cells, most cortical thymocytes, and some human B and T cell lymphomas (4). The BCL6 gene was identified (5-7) through its involvement in chromosomal translocations that occur in Ϸ40% of diffuse large-cell B cell lymphomas. In other lymphomas, mutations occur 5Ј to the BCL6 coding region (8). The BCL6 protein binds DNA in a sequencespecific fashion through its C-terminal zinc finger region (9, 10). It conveys transcriptional repression through an N-terminal POZ domain and a second domain that is more centrally located (1-3, 11) and interacts with a number of corepressors (12)(13)(14). It is thought that the BCL6-repressive effects are mediated through multiprotein repression complexes with histone deacetylase activity. A peptide that specifically binds BCL6 and blocks corepressor recruitment leads to apoptosis and cell-cycle arrest of BCL6-positive lymphoma cells (15). Mice overexpressing human (16) or murine (17) BCL6 develop lymphomas.We previously generated a dominant-negative cell system (18) with the use of a construct expressing the BCL6 zinc fingers, which compete with the binding of endogenous BCL6 in BJAB cells (an Epstein-Barr virus-negative Burkitt lymphoma cell line expressing high levels of BCL6) (19). Because BCL6 is a repressor, competition for binding of the full-length endogenous BCL6 protein by the exogenously transfected zinc fingers results in up-regulation of BCL6 target genes. We used subtractive hybridization techniques to amplify differentially expressed sequences. These studies led to the identification of the PDCD2 (programmed cell death-2) gene as a target of BCL6. Immunohistochemistry performed on human tonsil with antibodies sp...

The causes of late onset Alzheimer disease (AD) are poorly understood. Although beta-amyloid (Abeta) is thought to play a critical role in the pathophysiology of AD, no genetic evidence directly ties Abeta to late onset AD. This suggests that the accumulation of Abeta and neurodegeneration associated with AD might result from an abnormality that indirectly affects Abeta production or accumulation. Increasing evidence suggests that abnormalities in the metabolism of cholesterol and related molecules, such as cholseterol esters and 24(S) hydroxycholesterol might contribute to the pathophysiology of late onset AD by increasing production of Abeta. 24(S) Hydroxycholesterol is a member of a family of oxidized cholesterol catabolites, termed oxysterols, which function to regulate export of cholesterol from the cell and transcription of genes related to cholesterol metabolism. Cholesterol esters are cholesterol derivatives used for cholesterol storage. Levels of 24(S) hydroxycholesterol increase with AD. Polymorphisms in several different genes important for cholesterol physiology are associated with an increased load or level of Abeta in AD. These genes include apolipoprotein E, cholesterol 24 hydroxylase (Cyp46), acyl-CoA:cholesterol acetyltransferase (ACAT), and the cholesterol transporter ABCA1. Other studies show that levels of cholesterol, or its precursors, are elevated in subjects early in the course of AD. Finally, studies of the processing of amyloid precursor protein show that cholesterol and its catabolites modulate amyloid precursor protein processing and Abeta production. These lines of evidence raise the possibility that genetic abnormalities in cholesterol metabolism might contribute to the pathophysiology of AD.