Journal of Molecular Neuroscience, 2016 Apr; 58(4): 411-5.
mRNA Levels of ACh-related enzymes in the hippocampus of THY-Tau22 mouse: a model of human tauopathy with no signs of motor disturbance
Beatriz E. García-Gómez1, Francisco J. Fernández-Gómez2, Encarnación Muñoz-Delgado1, Luc Buée2, David Blum2, Cecilio J. Vidal1*.
1Departamento de Bioquímica y Biología Molecular-A, Universidad de Murcia, IMIB-Arrixaca, Regional Campus of International Excellence “Campus Mare Nostrum”, Murcia, Spain.
2INSERM U837 Alzheimer and Tauopathies, & Université Lille Nord de France, Institute of Predictive Medicine and Therapeutic Research, France.
Cecilio J. Vidal, Ph.D. Member of the International Advisory Board of Cholinesterases
Departamento de Bioquímica y Biología Molecular‑A, Edificio de Veterinaria.
Universidad de Murcia, Apdo. 4021, E-30071, Espinardo, Murcia, Spain.
The microtubule-associated protein Tau tends to form aggregates in neurodegenerative disorders referred to as tauopathies. The tauopathy model transgenic (Tg) THY-Tau22 (Tau22) mouse shows disturbed septo-hippocampal transmission, memory deficits and no signs of motor dysfunction. The reported hippocampal down-regulation of choline acetyltransferase (ChAT) in SAMP8 mice, a model of aging, and hippocampal up‑regulation of acetylcholinesterase (AChE) in Tg-VLW mice, a model of FTDP17 tauopathy, may lead to think that the supply of ACh to the hippocampus may be seen compromised as aging or Tau pathology progress. The above was tested by comparing the mRNA levels for ACh-related enzymes in hippocampuses of wild-type and Tau22 mice at ages when the neuropathological signs are debuting (3-4 months), moderate (6-7 months) and extensive (>9 months). Tau22 and wild-type mice hippocampuses showed similar ChAT, AChE‑T, BChE and PRiMA mRNA levels, any change most likely arising from ACh homeostasis. The almost unchanging hippocampal levels of AChE‑T mRNA and AChE activity observed in Tau22 mice, expressing G272V-P301S hTau, differed from the reported increase in the levels of AChE-T mRNA and AChE activity in Tg‑VLW mice, expressing G272V-P301L-R406W hTau. The difference supports the idea that AChE up-regulation may proceed or not depending on the particular Tau mutation, which would dictate Tau folding, the accessibility/affinity to kinases and phosphatases, and P-Tau aggregation with itself and protein partners, transcription factors included.
Alzheimer’s disease (AD) is characterized by the production in the brain of senile plaques made of amyloid-beta, and fibrillary tangles made of Tau abnormally phosphorylated. These tangles may be the starters of the neurofibrillary degenerative process, whose development in the brain seems to be causally related to AD pathology .
The availability of ACh in the brain relies on the coordinate actions of choline acetyltransferase (ChAT), the enzyme that controls the synthesis of ACh, and the cholinesterases (ChEs) acetyl- (AChE) and butyrylcholinesterase (BChE), two proteins that hydrolyze ACh with high efficiency. The down-regulation of ChAT or up-regulation of AChE or BChE would decrease the level of synaptic ACh, a deficiency exacerbated in AD owing to the loss of ACh-forming neurons in the brain areas involved in cognition . To attenuate the ACh deficiency, about 100 million patients afflicted of AD has been treated with ChE inhibitors since 1986 and, despite clinical stabilization of up to one year in 20% of them AD effects persist .
Whilst the BChE gene generates a single protein type, the splicing of the AChE pre-mRNA at the 3’-end leads to three distinct mRNAs, which translate into tailed (synaptic) AChE‑T (AChE-S), hydrophobic (erythrocytic) AChE-H (AChE-E), and readthrough AChE-R variants. In mammals, this range of AChE mRNAs increases due to the presence in the pre-mRNA of various E1 exons . AChE-T organizes into tetramers, which with four catalytic sites have high ACh-hydrolyzing activity, and hydrophilic (G4H) or amphiphilic (G4A) properties according to the lack or linkage of PRiMA (Proline‑Rich Membrane Anchor). The PRiMA-bound AChE tetramers are abundant in the brain, nerve and muscle, and the lack of PRiMA in non-neural cells justifies the synthesis and secretion of G4H AChE . Our results showing decreased levels of both PRiMA mRNA and G4A AChE and BChE components in nerve and muscle of mice afflicted of muscular dystrophy  highlight the relevance of PRiMA for nerve and muscle functioning.
Mice that express mutated hTau display the pathological symptoms of AD. The Tau22 mouse exhibits spatial memory impairment, but, contrary to most Tg‑Tau mice that show motor defects due to the transgene expression in the spinal cord, Tau22 mice do not exhibit motor defects, which makes them a reliable model to study pathogenic aspects of neurofibrillary degeneration . The reports showing decreased hippocampal ChAT activity in SAMP8 mice, an aging model , and increased hippocampal expression of AChE in Tg-VLW mice, an FTDP17 tauopathy model , prompted us to compare 3‑, 7- and 12-month old wild-type and Tau22 mice for their hippocampal levels of ChAT, ChEs and PRiMA mRNAs.
Real-time PCR results (Fig. 1) showed wild-type mice and Tau22 mice hippocampuses supplied with scanty ChAT mRNA, which agreed with the few ChAT-labelled neurons identified in the rat hippocampus and with the absence from the mouse hippocampus of a clear hybridization signal for ChAT mRNA .
Fig. 1. Hippocampal mRNAs levels for ACh-related proteins in control and Tau22 mice. Mean values ± SD of five separate hippocampuses.
In spite of previous observations made in hippocampuses of Tg-VLW mice  ChE assays showed nearly equal levels of AChE (and BChE) activity in hippocampuses of wild-type and Tau22 mice. In addition, only minor changes between the hippocampuses of wild-type and Tau22 mice were observed in the levels of AChE-T, AChE-H, AChE-R, and BChE mRNAs (Fig. 1), in the content of E1c‑ and E1e-including AChE mRNAs, and in the abundance of PRiMA-II and PRiMA-I mRNAs (Fig. 1). The lack of great changes in the levels of ChAT mRNA, ChE activity and AChE, BChE and PRiMA mRNAs made very improbable an unbalance of available ACh as the leading cause for the cognition impairment observed in Tau22 mice. Despite the reliable demonstration of an up-regulation of AChE in the Tg‑VLW mouse hippocampus , our results do not confirm such an over-expression. Thus, the small age-dependent changes in the mRNA levels for ACh-related enzymes observed in wild-type mice and in Tau22 mice hippocampuses (Fig. 1), besides the absence from them of major differences in the content of AChE‑T and PRiMA mRNAs (Fig. 1), and the nearly equal hippocampal AChE (and BChE) activity in control and Tau22 mice (activity values not shown) are all observations which do not fit in the well assessed up-regulation of AChE in the hippocampus.
The discrepancy may arise from the notable conformational flexibility of Tau protein and the specific folding of separate Tau mutants. Up to 32 mutations have been identified in over 100 families, about half of the mutations with effects for Tau folding . The tight structure‑function relationship of proteins and the predictable unequal folding of G272V-P301S Tau and G272V-P301L-R406W Tau make it possible that what happens in Tg‑VLW mice may not occur in Tau22 mice. In support of this idea are: 1) the varying extent to which separate Tau mutants bind with protein-phosphatase 2A ; 2) the unequal effects of Tau mutants on Xenopus oocyte maturation ; 3) the lack of motor disturbances in mice expressing G272V-P301S hTau or K257T-P301S hTau and their occurrence in mice making G272V-P301L-R406W hTau ; 4) the differences in motor impairment and cognitive deficit that exhibit the Tg mice line 66 and line 1, the former expressing P301S‑G335D hTau and the latter a truncated 3-repeat hTau fragment ; 5) the distinct extent to which Ser404 is phosphorylated by Cdk5 kinase depending on whether Ser404 occurs in full R406W hTau or in a peptide fragment ; and 6) the distinct effects of single-point Tau mutations on the distribution of fibril conformers .
Summarising, the subtle age-related changes observed in hippocampuses of control and Tau22 mice with respect to the mRNA levels for ACh-related proteins likely reflect the normal compensatory inputs involved in ACh homeostasis. The almost invariable level of AChE-T mRNA in hippocampuses of Tau22 mice and the increased mRNA level in hippocampuses of Tg-VLW mice reinforce the idea that individual Tau mutations differ in their effects, which calls to caution when comparing data gathered from distinct Tau mutants.
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Research entirely supported by the University of Murcia.