Debra Toiber

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

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′ 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 mRNAs 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.

N-Acetylcholinesterase-Induced Apoptosis in Alzheimer's Disease

Background Alzheimers disease (AD) involves loss of cholinergic neurons and Tau protein hyper-phosphorylation. Here, we report that overexpression of an N-terminally extended “synaptic” acetylcholinesterase variant, N-AChE-S is causally involved in both these phenomena. Methodology and Principal Findings In transfected primary brain cultures, N-AChE-S induced cell death, morphological impairments and caspase 3 activation. Rapid internalization of fluorescently labeled fasciculin-2 to N-AChE-S transfected cells indicated membranal localization. In cultured cell lines, N-AChE-S transfection activated the Tau kinase GSK3, induced Tau hyper-phosphorylation and caused apoptosis. N-AChE-S-induced cell death was suppressible by inhibiting GSK3 or caspases, by enforced overexpression of the anti-apoptotic Bcl2 proteins, or by AChE inhibition or silencing. Moreover, inherent N-AChE-S was upregulated by stressors inducing protein misfolding and calcium imbalances, both characteristic of AD; and in cortical tissues from AD patients, N-AChE-S overexpression coincides with Tau hyper-phosphorylation. Conclusions Together, these findings attribute an apoptogenic role to N-AChE-S and outline a potential value to AChE inhibitor therapeutics in early AD.

Cellular stress reactions as putative cholinergic links in Alzheimer's disease.

Alzheimer’s disease involves normal cellular aging and chronic cellular stress events, leading to interrelated changes in gene expression and subsequent neurodegeneration. Premature death of cholinergic neurons and the formation of amyloid fibrils separately initiated the cholinergic and amyloid hypotheses of Alzheimer’s disease. Here, we present evidence to the fact that these two distinct phenomena both associate with specific changes in acetylcholinesterase (AChE) gene expression within cholinergic neurons. For example, calcium misregulation promotes aberrant transcription and pro-apoptotic events, as well as AChE-induced modifications in cellular signal cascades. These reciprocally intercept with AChE regulation at the Endoplasmic Reticulum, modifying AChE gene expression, folding and signaling. Altered AChE properties may reflect changes in the enzymatic and/or non-enzymatic features of the multiple AChE splice variants. Under chronic cellular stress, aberrant AChE regulation may thus facilitate apoptotic pathways, promoting plaque formation, cognitive impairments and degeneration of cholinergic nerve cells.

Pro-apoptotic protein–protein interactions of the extended N-AChE terminus

The N-terminally extended “synaptic” acetylcholinesterase variant N-AChE-S operates to promote apoptosis; however, the protein partners involved in this function remain unknown. Here, we report that when microinjected to fertilized mouse oocytes, N-AChE-S caused embryonic death as early as the zygotic stage. To identify the putative protein partners involved, we first tried yeast two hybrid screening, but this approach failed, probably because of the N-AChE-S-induced lethality. In contrast, sequence analysis and a corresponding peptide array revealed possible partners, which were validated by co-immunoprecipitation. These include the kinases GSK3, Aurora and GAK, the membrane integrin receptors, and the death receptor FAS. Each of these could potentially modulate N-AChE-S-induced apoptosis with possible therapeutic value for the treatment of Alzheimer’s disease.

Modulated splicing‐associated gene expression in P19 cells expressing distinct acetylcholinesterase splice variants

Alternative splicing configurations and acetylcholinesterase (AChE) gene expression are both modified in neurons under stress. However, it is unclear if these phenomena are functionally interrelated. Using a home‐made spotted microarray focused on splicing‐associated transcripts, we tested the effects of excess 3′ splice variants of human AChE on splicing‐related gene expression in semi‐differentiated neuronal P19 cells. Of the tested transcripts, 17.3% and 20.2% showed modified expression levels (log2 of the ratio < − 0.3 or > 0.3) in transfected P19 cells overexpressing the stress‐inducible AChE‐R variant or the synaptic AChE‐S protein, respectively. Multiple transcripts encoding serine‐arginine rich (SR) and SR‐related splicing regulators were suppressed in cells expressing either of these variants, whereas the gene groups including splicing‐related helicases and transcripts involved in apoptosis displayed variant‐specific changes. Our findings are compatible with the assumption that both neuronal overexpression and alternative splicing of pre‐AChE mRNA may be causally involved in initiating global changes in neuronal alternative splicing, causing subsequent modifications in the expression patterns of numerous target genes.

Acetylcholinesterase variants in Alzheimer's disease: from neuroprotection to programmed cell death.

Background: In Alzheimer’s disease (AD), cholinergic neurons are particularly vulnerable for as yet unclear reasons. Here, we report that modified composition, localization and properties of alternative splice variants encoding the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE) may be variably involved in disease progression or in systemic efforts to attenuate its progression. Objective: The purpose of this study was to explore the implications for AD of the cellular and biochemical properties of the various AChE proteins, differing in their N and C termini. Methods: We have used cell transfection with genetically engineered vectors as well as microinjection to overexpress specific AChE variants and explore the consequences to cellular well-being and survival. Additionally, we employed highly purified recombinant AChE-R and AChE-S to explore putative interactions with the AD β-amyloid peptide. Results: Our findings demonstrate distinct, and in certain cases inverse cell fate outcome under enforced expression of the human N- and C-terminally modified AChE variants, all of which have similar enzymatic activities. Conclusion: The N-terminal extension in conjunction with the primary helical C-terminal peptide of ‘tailed’ AChE-S facilitates, whereas the shorter, naturally unfolded C-terminus of the stress-induced AChE-R variant attenuates Alzheimer’s pathology.