Hydromethylthionine rescues synaptic SNARE proteins in a mouse model of tauopathies: Interference by cholinesterase inhibitors.

In clinical trials for Alzheimer's disease (AD), hydromethylthionine mesylate (HMTM) showed reduced efficacy when administered as an add-on to symptomatic treatments, while it produced a significant improvement of cognitive function when taken as monotherapy. Interference of cholinesterase inhibition with HMTM was observed also in a tau transgenic mouse model, where rivastigmine reduced the pharmacological activity of HMTM at multiple brain levels including hippocampal acetylcholine release, synaptosomal glutamate release and mitochondrial activity. Here, we examined the effect of HMTM, given alone or in combination with the acetylcholinesterase inhibitor, rivastigmine, at the level of expression of selected pre-synaptic proteins (syntaxin-1; SNAP-25, VAMP-2, synaptophysin-1, synapsin-1, α-synuclein) in brain tissue harvested from tau-transgenic Line 1 (L1) and wild-type mice using immunohistochemistry. L1 mice overexpress the tau-core unit that induces tau aggregation and results in an AD-like phenotype. Synaptic proteins were lower in hippocampus and cortex but greater in basal forebrain regions in L1 compared to wild-type mice. HMTM partially normalised the expression pattern of several of these proteins in basal forebrain. This effect was diminished when HMTM was administered in combination with rivastigmine, where mean protein expression seemed supressed. This was further confirmed by group-based correlation network analyses where important levels of co-expression correlations in basal forebrain regions were lost in L1 mice and partially re-established when HMTM was given alone but not in combination with rivastigmine. These data indicate a reduction in pharmacological activity of HMTM when given as an add-on therapy, a result that is consistent with the responses observed in the clinic. Attenuation of the therapeutic effects of HMTM by cholinergic treatments may have important implications for other potential AD therapies.

Neuroprotection of Cholinergic Neurons with a Tau Aggregation Inhibitor and Rivastigmine in an Alzheimer's-like Tauopathy Mouse Model.

Basal forebrain cholinergic dysfunction, most likely linked with tau protein aggregation, is a characteristic feature of Alzheimer's disease (AD). Recent evidence suggests that tau protein is a putative target for the treatment of dementia, and the tau aggregation inhibitor, hydromethylthionine mesylate (HMTM), has emerged as a potential disease-modifying treatment. However, its efficacy was diminished in patients already receiving approved acetylcholinesterase inhibitors. In this study, we ask whether this negative interaction can also be mimicked in experimental tau models of AD and whether the underlying mechanism can be understood. From a previous age profiling study, 6-month-old line 1 (L1) tau transgenic mice were characterized by a severe reduction in several cholinergic markers. We therefore assessed whether long-term pre-exposure with the acetylcholinesterase inhibitor rivastigmine alone and in conjunction with the tau aggregation inhibitor HMTM can reverse cholinergic deficits in L1. Rivastigmine and HMTM, and combinations of the two compounds were administered orally for 11 weeks to both L1 and wild-type mice. The brains were sectioned with a focus on the basal forebrain, motor cortex and hippocampus. Immunohistochemical staining and quantification of choline acetyltransferase (ChAT), tyrosine kinase A (TrkA)-positive neurons and relative optical intensity (ROI) for vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) reactivity confirmed reversal of the diminished cholinergic phenotype of interneurons (nucleus accumbens, striatum) and projection neurons (medial septum, nucleus basalis magnocellularis) by HMTM, to a greater extent than by rivastigmine alone in L1 mice. Combined administration did not yield additivity but, in most proxies, led to antagonistic effects in which rivastigmine decreased the benefits shown with HMTM alone. Local markers (VAChT and AChE) in target structures of the basal forebrain, motor cortex and hippocampal CA3 seemed to be normalized by HMTM, but not by rivastigmine or the combination of both drugs. HMTM, which was developed as a tau aggregation inhibitor, strongly decreased the tau load in L1 mice, however, not in combination with rivastigmine. Taken together, these data confirm a cholinergic phenotype in L1 tau transgenic mice that resembles the deficits observed in AD patients. This phenotype is reversible by HMTM, but at the same time appears to be subject to a homeostatic regulation induced by chronic pre-treatment with an acetylcholinesterase inhibitor, which interferes with the efficacy of HMTM. The strongest phenotypic reversal coincided with a normalization of the tau load in the cortex and hippocampus of L1, suggesting that tau accumulation underpins the loss of cholinergic markers in the basal forebrain and its projection targets.

The Biology and Pathobiology of Glutamatergic, Cholinergic, and Dopaminergic Signaling in the Aging Brain.

The elderly population is growing worldwide, with important health and socioeconomic implications. Clinical and experimental studies on aging have uncovered numerous changes in the brain, such as decreased neurogenesis, increased synaptic defects, greater metabolic stress, and enhanced inflammation. These changes are associated with cognitive decline and neurobehavioral deficits. Although aging is not a disease, it is a significant risk factor for functional worsening, affective impairment, disease exaggeration, dementia, and general disease susceptibility. Conversely, life events related to mental stress and trauma can also lead to accelerated age-associated disorders and dementia. Here, we review human studies and studies on mice and rats, such as those modeling human neurodegenerative diseases, that have helped elucidate (1) the dynamics and mechanisms underlying the biological and pathological aging of the main projecting systems in the brain (glutamatergic, cholinergic, and dopaminergic) and (2) the effect of defective glutamatergic, cholinergic, and dopaminergic projection on disabilities associated with aging and neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Detailed knowledge of the mechanisms of age-related diseases can be an important element in the development of effective ways of treatment. In this context, we briefly analyze which adverse changes associated with neurodegenerative diseases in the cholinergic, glutaminergic and dopaminergic systems could be targeted by therapeutic strategies developed as a result of our better understanding of these damaging mechanisms.

Cholinergic and inflammatory phenotypes in transgenic tau mouse models of Alzheimer's disease and frontotemporal lobar degeneration.

An early and sizeable loss of basal forebrain cholinergic neurons is a well-characterized feature associated with measurable deficits in spatial learning and cognitive impairment in patients with Alzheimer's disease. In addition, pro-inflammatory glial cells such as astrocytes and microglia may play a key role in the neurodegenerative cascade of Alzheimer's disease and tauopathies. We recently presented two mouse models: Line 1, expressing the truncated tau fragment identified as the core of the Alzheimer's paired helical filament, and Line 66, expressing full-length human tau carrying a double mutation (P301S and G335D). Line 1 mice have a pathology that is akin to Alzheimer's, whilst Line 66 resembles frontotemporal lobar degeneration. However, their cholinergic and inflammatory phenotypes remain elusive. We performed histological evaluation of choline acetyltransferase, acetylcholinesterase, p75 neurotrophin receptor, microglial ionized calcium binding adaptor molecule 1 and astrocytic glial fibrillary acidic protein in the basal forebrain, hippocampus and cortex of these models. A significant lowering of choline acetyltransferase-positive neurons and p75-positive neurons in the basal forebrain of Line 1 at 3, 6 and 9 months was observed in two independent studies, alongside a significant decrease in acetylcholinesterase staining in the cortex and hippocampus. The reductions in choline acetyltransferase positivity varied between 30% and 50% at an age when Line 1 mice show spatial learning impairments. Furthermore, an increase in microglial ionized calcium binding adaptor molecule 1 staining was observed in the basal forebrain, hippocampus and entorhinal cortex of Line 1 at 6 months. Line 66 mice displayed an intact cholinergic basal forebrain, and no difference in p75-positive neurons at 3 or 9 months. In addition, Line 66 exhibited significant microglial ionized calcium binding adaptor molecule 1 increase in the basal forebrain and hippocampus, suggesting a prominent neuroinflammatory profile. Increased concentrations of microglial interleukin-1ß and astrocytic complement 3 were also seen in the hippocampus of both Line 1 and Line 66. The cholinergic deficit in Line 1 mice confirms the Alzheimer's disease-like phenotype in Line 1 mice, whilst Line 66 revealed no measurable change in total cholinergic expression, a phenotypic trait of frontotemporal lobar degeneration. These two transgenic lines are therefore suitable for discriminating mechanistic underpinnings between the Alzheimer's and frontotemporal lobar degeneration-like phenotypes of these mice.

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

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.