Mechanisms of Anticholinesterase Interference with Tau Aggregation Inhibitor Activity in a Tau-Transgenic Mouse Model

Abstract

Background: Symptomatic treatments of Alzheimer's Disease (AD) with cholinesterase inhibitors and/or memantine are relatively ineffective and there is a need for new treatments targeting the underlying pathology of AD. In most of the failed disease-modifying trials, patients have been allowed to continue taking symptomatic treatments at stable doses, under the assumption that they do not impair efficacy. In recently completed Phase 3 trials testing the tau aggregation inhibitor leuco-methylthioninium bis (hydromethanesulfonate) (LMTM), we found significant differences in treatment response according to whether patients were taking LMTM either as monotherapy or as an add-on to symptomatic treatments.

Methods: We have examined the effect of either LMTM alone or chronic rivastigmine prior to LMTM treatment of tau transgenic mice expressing the short tau fragment that constitutes the tangle filaments of AD. We have measured acetylcholine levels, synaptosomal glutamate release, synaptic proteins, mitochondrial complex IV activity, tau pathology and Choline Acetyltransferase (ChAT) immunoreactivity.

Results: LMTM given alone increased hippocampal Acetylcholine (ACh) levels, glutamate release from synaptosomal preparations, synaptophysin levels in multiple brain regions and mitochondrial complex IV activity, reduced tau pathology, partially restored ChAT immunoreactivity in the basal forebrain and reversed deficits in spatial learning. Chronic pretreatment with rivastigmine was found to reduce or eliminate almost all these effects, apart from a reduction in tau aggregation pathology. LMTM effects on hippocampal ACh and synaptophysin levels were also reduced in wild-type mice.

Conclusion: The interference with the pharmacological activity of LMTM by a cholinesterase inhibitor can be reproduced in a tau transgenic mouse model and, to a lesser extent, in wild-type mice. Long-term pretreatment with a symptomatic drug alters a broad range of brain responses to LMTM across different transmitter systems and cellular compartments at multiple levels of brain function. There is, therefore, no single locus for the negative interaction. Rather, the chronic neuronal activation induced by reducing cholinesterase function produces compensatory homeostatic downregulation in multiple neuronal systems. This reduces a broad range of treatment responses to LMTM associated with a reduction in tau aggregation pathology. Since the interference is dictated by homeostatic responses to prior symptomatic treatment, it is likely that there would be similar interference with other drugs tested as add-on to the existing symptomatic treatment, regardless of the intended therapeutic target or mode of action. The present findings outline key results that now provide a working model to explain interference by symptomatic treatment.

Keywords: Alzheimer’s disease; Tau aggregation inhibitor; acetylcholinesterase inhibitor (AChEI); drug interaction; hydromethylthionine; mouse model; synaptic proteins; tauopathy..

Fig. (1)

Treatment regime outline used in this study to mimic previous clinical trials. While there has been no inclusion of placebo control in the clinical trials, we have included a vehicle group gavaged with saline throughout. In addition, a 5-week monotherapy was typically followed by a 6-week treatment of either vehicle +LMTM or rivastigmine +LMTM followed by tissue harvest. Drugs were administered as a daily oral cocktail. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (2)

Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in wild-type mice. Hippocampal levels of ACh (A), measured by high performance liquid chromatography as described previously [44], or synaptophysin levels for the hippocampus, visual cortex, diagonal band of Broca and septum (B) are shown (total n = 25). Data expressed as mean values + SE (**, p< 0.01; ***, p< 0.001). Photomicrographs (C) show representative images of synaptophysin labelling in hippocampal CA1 in respective groups. Scale bar, 100 µm. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (3)

Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in tau transgenic L1 mice. Photomicrographs of hippocampal CA1 sections for labelled for SNAP25 as one of the SNARE proteins (A) and for α-synuclein (B) in respective treatment groups. Scale bar, 100 µm. Quantitative data for (C) SNARE complex proteins (SNAP25, syntaxin and VAMP2) and (D) α-synuclein expressed as the mean + SE for the hippocampus, visual cortex, diagonal band of Broca and septum. (*, p< 0.05; ***, p< 0.001; ****, p< 0.0001). (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (4)

Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in tau transgenic L1 mice on complex IV activity. Data normalized to citrate synthase activity and expressed as mean + SE. (*, p< 0.05). (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (5)

Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in tau transgenic L1 mice. Values are compared with vehicle-treated wild-type mice for levels of tau immunoreactivity (relative optical intensity, ROI) (A) and number of neurons immunoreactive for choline acetyltransferase (B) in vertical diagonal band of Broca. Data are expressed as mean + SE. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (6)

Summary schema of likely treatment effects of LMT that are subject to dynamic modulation by chronic pretreatment with rivastigmine (riva). Particular attention is afforded to changes in mitochondrial metabolism, presynaptic proteins and to tau aggregation inhibitor activity. Combined treatment with AChEI does not impair LMT effects on tau aggregation pathology (blue arrows). By contrast, the combination prevents the increases in synaptic proteins, glutamate (Glu) release and increased complex IV activity within the mitochondrial electron transport chain (ETC) that are observed following treatment with LMTM alone (white arrows). LMT also induces mitochondrial biogenesis and activates Nrf2-mediated oxidative stress response elements that can protect against damaging reactive oxygen species (ROS) (green and red arrows). (A higher resolution / colour version of this figure is available in the electronic copy of the article).