Alternative Splicing and Neuritic mRNA Translocation Under Long-Term Neuronal Hypersensitivity

Meshorer, Eran; Erb, Christina; Gazit, Roi; Pavlovsky, Lev; Kaufer, Daniela; Friedman, Alon; Glick, David; Ben‐Arie, Nissim; Soreq, Hermona

doi:10.1126/science.1066752

Cited by 218 publications

(204 citation statements)

References 28 publications

Supporting

Mentioning

188

Contrasting

Unclassified

Order By: Relevance

“…Forty micrograms of crude protein lysates were electrophoresed on a 10% SDS-PAGE gel for immunoblot analysis. Primary anti-huAChE monoclonal antibody (Clone 46, BD Biosciences, Mississauga, ON) has previously been used to detect AChE in immunoblot, immunohistochemical, and immunocytochemical assays [Boudreau-Lariviere and Jasmin, 1999;Meshorer et al, 2002;Inkson et al, 2004]. Anti-huAChE was diluted 1:1,500 in blocking buffer (5% non-fat dehydrated milk in PBS/0.3% Tween-20) and secondary anti-mouse IgG conjugated to HRP (Cell Signaling Technology, Beverly, MA) was diluted 1:1,000 in blocking buffer.…”

Section: Immunoblot Analysis Of Achementioning

confidence: 99%

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Thullbery

Cox

Schule

et al. 2005

J of Cellular Biochemistry

View full text Add to dashboard Cite

Acetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH-SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non-neuronal (LLC-MK2, HeLa, THP-1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE-specific activity were higher in neuronal cells than in the non-neuronal cell types. CGN expressed significantly more AChE activity than SH-SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH-SY5Y, CGN, and nerve-growth factor-differentiated PC-12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC-MK2, fibroblasts, and undifferentiated PC-12 cells was significantly different than in neuronal cells-AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn't correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types. Keywords acetylcholinesterase; confocal microscopy; cerebellar granule neuron; neuroblastoma; SH-SY5Y; HeLa; PC-12 Acetylcholinesterase (AChE) belongs to the serine hydrolase family that contains the α/β hydrolase fold as a common structural element [Ollis et al., 1992]. The catalytic active site of AChE is made up of a precise arrangement of active site functional groups that catalyze the hydrolytic breakdown of esters that bear quaternary ammonium groups. The biological importance of AChE-catalyzed hydrolysis is manifested in the rapid termination of the neuronal impulse that occurs when acetylcholine (ACh) is released into the synaptic cleft. AChE rapidly hydrolyzes ACh into acetic acid and choline at extremely fast turnover rates [Taylor and Radic, 1994].

show abstract

Section: Immunoblot Analysis Of Achementioning

confidence: 99%

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Thullbery

Cox

Schule

et al. 2005

J of Cellular Biochemistry

View full text Add to dashboard Cite

show abstract

“…This predicted direct interaction between neuronal SC35 expression and the 3 0 splicing events forming AChE-R mRNA (Figure 4a). 13 In addition, previous tests in our laboratory demonstrated association of the primary AChE-S mRNA transcript with ASF/SF2. 36 Therefore, we explored possible association of neuronal SC35 and ASF/SF2 with AChE alternative splicing in vivo.…”

Section: Sc35 Shifts Ache's Alternative Splicing Towards Ache-r In Trmentioning

confidence: 81%

“…13 Orbital sinus blood of stressed mice served to quantify corticosterone levels following the second swim session as compared with naïve, nonstressed mice. Plasma corticosterone levels rose by ca.…”

Section: Resultsmentioning

confidence: 99%

“…13 Synthetic 2 0 -O-methyl 5 0 -biotin-labeled RNA probe (Microsynth, Balgach, Switzerland) for SC35 was as described. 29 For ASF/SF2, the probe 5 0 -TGCGACUCCUGCUGUUGCUUCUGCUACGGCUUCU GCUACGACUACGGCUU-3 0 was designed using Oligo software (Molecular Biology Insights, Cascade, CO, USA).…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

“…12,13 The stressand development-associated modulations in AChE gene expression involve facilitated transcription, altered promoter usage, modified splicing patterns of the 3 0 variants of the AChE pre-mRNA, and increased stability of the normally rare and unstable AChE-R mRNA transcript. 12,14 This renders AChE premRNA processing an appropriate model system for exploring the involvement of alternative splicing modulations in the delayed effects of acute stress in the adult and developing brain.…”

mentioning

confidence: 99%

See 2 more Smart Citations

SC35 promotes sustainable stress-induced alternative splicing of neuronal acetylcholinesterase mRNA

et al. 2005

View full text Add to dashboard Cite

Long-lasting alternative splicing of neuronal acetylcholinesterase (AChE) pre-mRNA occurs during neuronal development and following stress, altering synaptic properties. To explore the corresponding molecular events, we sought to identify mRNAs encoding for abundant splicing factors in the prefrontal cortex (PFC) following stress. Here we show elevated levels of the splicing factor SC35 in stressed as compared with naïve mice. In cotransfections of COS-1 and HEK293 cells with an AChE minigene allowing 3 0 splice variations, SC35 facilitated a shift from the primary AChE-S to the stress-induced AChE-R variant, while ASF/SF2 caused the opposite effect. Transfection with chimeric constructs comprising of SC35 and ASF/SF2 RRM/RS domains identified the SC35 RRM as responsible for AChE mRNA's alternative splicing. In poststress PFC neurons, increased SC35 mRNA and protein levels coincided with selective increase in AChE-R mRNA. In the developing mouse embryo, cortical progenitor cells in the ventricular zone displayed transient SC35 elevation concomitant with dominance of AChE-R over AChE-S mRNA. Finally, transgenic mice overexpressing human AChE-R, but not those overexpressing AChE-S, showed significant elevation in neuronal SC35 levels, suggesting a reciprocal reinforcement process. Together, these findings point to an interactive relationship of SC35 with cholinergic signals in the long-lasting consequences of stress on nervous system plasticity and development. Molecular Psychiatry (2005) 10, 985-997.

show abstract

Molecular Inhibition of Gene Expression in Hematopoietic Cells

Opalinska¹,

Gewirtz²

2003

Thomas' Hematopoietic Cell Transplantation

View full text Add to dashboard Cite

Alternative Splicing and Neuritic mRNA Translocation Under Long-Term Neuronal Hypersensitivity

Cited by 218 publications

References 28 publications

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

SC35 promotes sustainable stress-induced alternative splicing of neuronal acetylcholinesterase mRNA

Molecular Inhibition of Gene Expression in Hematopoietic Cells

Contact Info

Product

Resources

About