The sodium‐coupled, hemicholinium‐3‐sensitive, high‐affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following ...acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts in both SH‐SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH‐SY5Y cells expressing rat CHT with filipin, methyl‐β‐cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol‐saturated MβC. Kinetic analysis of binding of 3Hhemicholinium‐3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol‐rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.
The sodium‐coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol‐rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.
The sodium‐coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol‐rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by synapse dysfunction and cognitive impairment. Understanding the development and progression of AD is challenging, ...as the disease is highly complex and multifactorial. Both environmental and genetic factors play a role in AD pathogenesis, highlighted by observations of complex DNA modifications at the single gene level, and by new evidence that also implicates changes in genome architecture in AD patients. The four-dimensional structure of chromatin in space and time is essential for context-dependent regulation of gene expression in post-mitotic neurons. Dysregulation of epigenetic processes have been observed in the aging brain and in patients with AD, though there is not yet agreement on the impact of these changes on transcription. New evidence shows that proteins involved in genome organization have altered expression and localization in the AD brain, suggesting that the genomic landscape may play a critical role in the development of AD. This review discusses the role of the chromatin organizers and epigenetic modifiers in post-mitotic cells, the aging brain, and in the development and progression of AD. How these new insights can be used to help determine disease risk and inform treatment strategies will also be discussed.
The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function
. Methods based on chromatin conformation capture have mapped chromatin structures ...in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi
. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation
, are invisible with such approaches
. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)
, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation
. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.
Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic ...syndrome (CMS). One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues
PKLP
PP
. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val
(V18M) enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID), co-immunoprecipitation and
proximity-ligation assay (PLA), we identified the heat shock proteins (HSPs) HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT) compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP
, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by pro-stabilizing HSPs and the pro-degradative co-chaperone p97/VCP that may have broader implications for ChAT function during cellular stress and disease.
Abstract Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline ...acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1–42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1–42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aβ. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aβ production, with this potentiating the decline in function of cholinergic neurons.
Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of ...isocitrate dehydrogenase (
-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the
promoter and increased
expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an
-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.
The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to ...oligomeric amyloid-β1-42 (Aβ1-42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1-42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1-42-exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1-42-induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1-42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ-exposure.
The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to ...oligomeric amyloid-β1-42 (Aβ1-42 ) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1-42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1-42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1-42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1-42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure.
The three-dimensional structure of chromatin is essential for context-dependent regulation of gene expression in post-mitotic neurons. Chromosomal rearrangements have been observed in the aging ...brain, and proteins involved in chromatin organization have altered expression and/or localization in Alzheimer’s disease (AD). A human- and primate-specific transcript of choline acetyltransferase produces an 82-kDa protein (82-kDa ChAT) that is localized to the nucleus of cholinergic neurons, but is found in the cytoplasm in individuals with mild cognitive impairment (MCI) and AD. The function of the 82-kDa ChAT protein is unknown, though recent evidence suggests it has a role in gene expression changes in response to cellular perturbations. In the present study, we explore whether 82-kDa ChAT is involved in global chromatin organization and an epigenetic response to cytotoxic amyloid-β(Aβ) exposure. We show that 82-kDa ChAT associates with chromatin in human SH-SY5Y neural cells using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), finding that acute exposure of cells to oligomeric Aβ1–42 increases 82-kDa ChAT associations with gene promoters and introns. Following Aβ1–42-exposure, 82-kDa ChAT co-localizes in nuclear aggregates with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 has similar increases in genic associations following Aβ1–42-exposure, and both SATB1 and 82-kDa ChAT associate with synapse-related genes. The 82-kDa ChAT and SATB1 proteins have patterned genomic associations at regions enriched with S/MAR binding motifs, preventing an Aβ1–42-induced increase in an isoform-specific APP mRNA transcript. Finally, we show that 82-kDa ChAT expression during cholinergic differentiation of SH-SY5Y cells increases the steady-state levels of proteins related to synapse formation, resulting in increased neurite complexity. These results demonstrate that 82-kDa ChAT and SATB1 regulate chromatin organization at S/MARs, resulting in context-dependent gene expression changes in cholinergic cells and increased expression of synapse formation-related proteins during cholinergic differentiation. Cholinergic synapse dysfunction and degeneration is observed early in AD progression and 82-kDa ChAT is mislocalized in AD, therefore the loss of both the epigenetic response to Aβ and gene expression changes related to synapse formation and maintenance may have implications for the etiology or progression of MCI and AD.
Cocaine exposure has been reported to alter central μ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be ...involved in this action of cocaine.
To assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin.
Both cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression.
In the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine's action on MOR expression in neurons.
PDF
