Cu, Fe, and Zn isotope ratios in murine Alzheimer's disease models suggest specific signatures of amyloidogenesis and tauopathy.

Alzheimer's disease (AD) is characterized by accumulation of tau and amyloid-beta in the brain, and recent evidence suggests a correlation between associated protein aggregates and trace elements, such as copper, iron, and zinc. In AD, a distorted brain redox homeostasis and complexation by amyloid-beta and hyperphosphorylated tau may alter the isotopic composition of essential mineral elements. Therefore, high-precision isotopic analysis may reveal changes in the homeostasis of these elements. We used inductively coupled plasma-mass spectrometry (ICP-MS)-based techniques to determine the total Cu, Fe, and Zn contents in the brain, as well as their isotopic compositions in both mouse brain and serum. Results for male transgenic tau (Line 66, L66) and amyloid/presenilin (5xFAD) mice were compared with those for the corresponding age- and sex-matched wild-type control mice (WT). Our data show that L66 brains showed significantly higher Fe levels than did those from the corresponding WT. Significantly less Cu, but more Zn was found in 5xFAD brains. We observed significantly lighter isotopic compositions of Fe (enrichment in the lighter isotopes) in the brain and serum of L66 mice compared with WT. For 5xFAD mice, Zn exhibited a trend toward a lighter isotopic composition in the brain and a heavier isotopic composition in serum compared with WT. Neither mouse model yielded differences in the isotopic composition of Cu. Our findings indicate significant pathology-specific alterations of Fe and Zn brain homeostasis in mouse models of AD. The associated changes in isotopic composition may serve as a marker for proteinopathies underlying AD and other types of dementia.

Differential compartmental processing and phosphorylation of pathogenic human tau and native mouse tau in the line 66 model of frontotemporal dementia.

Synapse loss is associated with motor and cognitive decline in multiple neurodegenerative disorders, and the cellular redistribution of tau is related to synaptic impairment in tauopathies, such as Alzheimer's disease and frontotemporal dementia. Here, we examined the cellular distribution of tau protein species in human tau overexpressing line 66 mice, a transgenic mouse model akin to genetic variants of frontotemporal dementia. Line 66 mice express intracellular tau aggregates in multiple brain regions and exhibit sensorimotor and motor learning deficiencies. Using a series of anti-tau antibodies, we observed, histologically, that nonphosphorylated transgenic human tau is enriched in synapses, whereas phosphorylated tau accumulates predominantly in cell bodies and axons. Subcellular fractionation confirmed that human tau is highly enriched in insoluble cytosolic and synaptosomal fractions, whereas endogenous mouse tau is virtually absent from synapses. Cytosolic tau was resistant to solubilization with urea and Triton X-100, indicating the formation of larger tau aggregates. By contrast, synaptic tau was partially soluble after Triton X-100 treatment and most likely represents aggregates of smaller size. MS corroborated that synaptosomal tau is nonphosphorylated. Tau enriched in the synapse of line 66 mice, therefore, appears to be in an oligomeric and nonphosphorylated state, and one that could have a direct impact on cognitive function.

Effects of oxidized and reduced forms of methylthioninium in two transgenic mouse tauopathy models.

Given the repeated failure of amyloid-based approaches in Alzheimer's disease, there is increasing interest in tau-based therapeutics. Although methylthioninium (MT) treatment was found to be beneficial in tau transgenic models, the brain concentrations required to inhibit tau aggregation in vivo are unknown. The comparative efficacy of methylthioninium chloride (MTC) and leucomethylthioninium salts (LMTX; 5-75 mg/kg; oral administration for 3-8 weeks) was assessed in two novel transgenic tau mouse lines. Behavioural (spatial water maze, RotaRod motor performance) and histopathological (tau load per brain region) proxies were applied. Both MTC and LMTX dose-dependently rescued the learning impairment and restored behavioural flexibility in a spatial problem-solving water maze task in Line 1 (minimum effective dose: 35 mg MT/kg for MTC, 9 mg MT/kg for LMTX) and corrected motor learning in Line 66 (effective doses: 4 mg MT/kg). Simultaneously, both drugs reduced the number of tau-reactive neurons, particularly in the hippocampus and entorhinal cortex in Line 1 and in a more widespread manner in Line 66. MT levels in the brain followed a sigmoidal concentration-response relationship over a 10-fold range (0.13-1.38 µmol/l). These data establish that diaminophenothiazine compounds, like MT, can reverse both spatial and motor learning deficits and reduce the underlying tau pathology, and therefore offer the potential for treatment of tauopathies.

Glucocorticoids regulate barrier function and claudin expression in intestinal epithelial cells via MKP-1.

Barrier dysfunction is pivotal to the pathogenesis of inflammatory bowel diseases (IBD) and collagenous colitis. Glucocorticoids restore barrier function in Crohn's disease, but whether this reflects attenuated inflammation or an epithelial-specific action has not yet been addressed. Using filter-grown Caco-2 monolayers as an in vitro model of the intestinal epithelial barrier, we observed that glucocorticoids induced a time- and dose-dependent increase in transepithelial electrical resistance (TEER) in a glucocorticoid receptor-dependent manner without altering flux of larger solutes or changing principal tight junction architecture. This was accompanied by reduced paracellular cation flux, reduced expression of the pore-forming tight junction component claudin-2, and upregulation of the sealing tight junction protein claudin-4. In contrast, expression of occludin, claudin-1, -7, or -8 was not altered. Dexamethasone increased expression and activity of MAPK phosphatase-1 and inhibition of this phosphatase prevented the glucocorticoid-induced changes in TEER and claudin expression, whereas inhibiting p38 or MEK1/2 was not sufficient to replicate the glucocorticoid effects. Upon exposure to IFN-γ, TNF-α, or IL-1ß, TEERs declined in dexamethasone-treated cells but remained consistently higher than in cells not receiving glucocorticoids. Treatment with IFN/TNF resulted in an upregulation of claudin-2 that was significantly attenuated by dexamethasone, whereas increased claudin-2 expression upon IL-1ß stimulation was not affected by glucocorticoids. Taken together, barrier augmentation might represent a previously unrecognized mechanism of action, potentially contributing to the therapeutic efficacy of glucocorticoids in IBD and collagenous colitis.

Solubility of α-synuclein species in the L62 mouse model of synucleinopathy.

The accumulation of α-synuclein (α-Syn) into Lewy bodies is a hallmark of synucleinopathies, a group of neurological disorders that include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Small oligomers as well as larger fibrils of α-Syn have been suggested to induce cell toxicity leading to a degenerative loss of neurones. A richer understanding of α-Syn aggregation in disease, however, requires the identification of the different α-Syn species and the characterisation of their biochemical properties. We here aimed at a more in-depth characterisation of the α-Syn transgenic mice, Line 62 (L62), and examined the deposition pattern and solubility of human and murine α-Syn in these mice using immunohistochemical and biochemical methods. Application of multiple antibodies confirmed mAb syn204 as the most discriminatory antibody for human α-Syn in L62. Syn204 revealed an intense and widespread immunohistochemical α-Syn labelling in parietal cortex and hippocampus, and to a lower level in basal forebrain and hindbrain regions. The labelled α-Syn represented somatic inclusions as well as processes and synaptic endings. Biochemical analysis revealed a Triton-resistant human α-Syn pool of large oligomers, a second pool of small oligomers that was not resistant to solubilization with urea/Triton. A third SDS-soluble pool of intermediate sized aggregates containing a mixture of both, human and mouse α-Syn was also present. These data suggest that several pools of α-Syn can exist in neurones, most likely in different cellular compartments. Information about these different pools is important for the development of novel disease modifying therapies aimed at α-Syn.

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.

LETC inhibits α-Syn aggregation and ameliorates motor deficiencies in the L62 mouse model of synucleinopathy.

Alpha-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). Here, we explored the efficacy of N,N,N',N'-tetraethyl-10H-phenothiazine-3,7-diamine dihydrochloride (LETC), a protein aggregation inhibitor, on α-Syn aggregation. In both cellular models and transgenic mice, α-Syn aggregation was achieved by the overexpression of full-length human α-Syn fused with a signal sequence peptide. α-Syn accumulated in transfected DH60.21 neuroblastoma cells and α-Syn aggregation was inhibited by LETC with an EC50 of 0.066 ± 0.047 µM. Full-length human α-Syn overexpressing Line 62 (L62) mice accumulated neuronal α-Syn that was associated with a decreased motor performance in the open field and automated home cage. LETC, administered orally for 6 weeks at 10 mg/kg significantly decreased α-Syn-positive neurons in multiple brain regions and this resulted in a rescue of movement deficits in the open field in these mice. LETC however, did not improve activity deficits of L62 mice in the home cage environment. The results suggest that LETC may provide a potential disease modification therapy in synucleinopathies through the inhibition of α-Syn aggregation.

The aldo-keto reductase AKR1B7 coexpresses with renin without influencing renin production and secretion.

On the basis of evidence that within the adult kidney, the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7, also known as mouse vas deferens protein, MVDP) is selectively expressed in renin-producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wild-type kidneys. Renin cells in AKR1B7-deficient kidneys showed normal morphology, numbers, and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7-deficient mice were normal at standard chow and were lowered by a high-salt diet directly comparable to wild-type mice. Treatment with a low-salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7-deficient and wild-type mice. Under this condition, we also observed a strong retrograde recruitment of renin-expressing cell along the preglomerular vessels, however, without a difference between AKR1B7-deficient and wild-type mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by ANG II and by perfusion pressure. Regarding these parameters, no differences were observed between AKR1B7-deficient and wild-type kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.

Adalimumab prevents barrier dysfunction and antagonizes distinct effects of TNF-α on tight junction proteins and signaling pathways in intestinal epithelial cells.

Intestinal barrier dysfunction is pivotal in the etiology of inflammatory bowel diseases. Combined clinical and endoscopic remission ("mucosal healing") in patients who received anti-TNF-α therapies suggests restitution of the intestinal barrier, but the mechanisms involved are largely unknown. We therefore investigated the impact of the anti-TNF-α antibody adalimumab on barrier function in two in vitro models. Combined stimulation of Caco-2 and T-84 cells with interferon-γ and TNF-α resulted in a significant decrease of transepithelial electrical resistance (TEER) within 6 h that was prevented by adalimumab in concentrations down to 100 ng/ml. Adalimumab furthermore antagonized the appearance of irregular membrane undulations and prevented internalization of tight junction proteins upon cytokine exposure. In addition, TNF-α induced a downregulation of claudin-1, claudin-2, claudin-4, and occludin as well as activation of phosphatidylinositol 3-kinase signaling in T-84 but not Caco-2 cells, which was reversed by adalimumab. At the signaling level, adalimumab prevented increased phosphorylation of myosin light chain as well as activation of p38 MAPK and NF-κB accompanying the decline in TEER in both model systems. Pharmacological inhibition of NF-κB signaling partially prevented the TNF-α-induced TEER loss, whereas inhibition of p38 worsened barrier dysfunction in Caco-2 but not T-84 cells. Taken together, these data demonstrate that adalimumab prevents barrier dysfunction induced by TNF-α both functionally and structurally as well as at the level of signal transduction. Barrier protection might therefore constitute a novel mechanism how anti-TNF-α therapy contributes to epithelial restitution and tissue repair in inflammatory bowel diseases.

Dietary genistein enhances phosphorylation of regulatory myosin light chain in the myocardium of ovariectomized mice.

There is evidence that isoflavones, such as genistein, can directly or indirectly improve lipid profile and lower blood pressure and hence exert cardiovascular protection. It is further believed, that genistein attenuates vascular contraction and thus vascular tone and blood pressure through altering the phosphorylation of the regulatory myosin light chain (MLC) probably via the myosin light chain kinase (MLCK) or the RhoA pathway. However, the direct role of genistein in the myocardium is poorly reviewed. In this study, we investigated the impact of genistein on the cardiac proteome in ovariectomized female mice using a 2DE-MS approach. Dietary genistein intake considerably changed the abundance of several cytoskeletal and contractile proteins and enhanced the phosphorylation of MLC. The MLC phosphorylation was mediated through increased abundance of MLCK and inhibition of myosin light chain phosphatase latest known to be inversely regulated by RhoA. Contrary to others, in our model genistein did neither inhibit the cardiac MLCK, nor the cardiac RhoA pathway in vivo.

Analysis of cardiac and renal endothelin receptors by in situ hybridization in mice.

BACKGROUND: Endothelin-1 (ET-1) is a multifunctional peptide, which is implicated in the renal and cardiac physicology as well as in many pathologies of these systems. ET-1's actions take place after the activation of two receptors: ET(A) and ET(B). The expression of these receptors may be modulated during the pathologic process. The analysis of the distribution and level of expression of the receptors in animal models is therefore crucial. METHODS: We developed a protocol for non-radioactive in situ hybridization for the mRNA of the two endothelin receptors on paraffin-embedded tissue using digoxigenin-labeled RNA probes. RESULTS: In heart and kidney, the staining was reliable and specific. In a mouse model for endothelin/nitric oxide imbalance, cardiac ET(B) expression was reduced. The distribution of the receptors was in accordance with the actual knowledge. Differences in cell specific expression are discussed. CONCLUSIONS: We developed a protocol for the in situ hybridization of the endothelin receptors in mice. Given that the endothelin system is implicated in the development of many diseases, we believe that this protocol may be useful for a number of future preclinical studies..

Glutamatergic transmission and receptor expression in the synucleinopathy h-α-synL62 mouse model: Effects of hydromethylthionine.

The accumulation of alpha-synuclein (α-Syn) into Lewy bodies in cortical and subcortical regions has been linked to the pathogenesis of synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). While there is a strong link between synuclein aggregates and the reduction in dopamine function in the emergence of PD, less is known about the consequences of α-Syn accumulation in glutamatergic neurons and how this could be exploited as a therapeutic target. Transgenic h-α-synL62 (L62) mice, in which synuclein aggregation is achieved through the expression of full-length human α-Syn fused with a signal sequence peptide, were used to characterise glutamatergic transmission using a combination of behavioural, immunoblotting, and histopathological approaches. The protein aggregation inhibitor hydromethylthionine mesylate (HMTM) alone, or in combination with the glutamatergic compounds 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine hydrochloride (MTEP) and memantine, was used to target α-Syn aggregation. We show that accumulation of α-Syn aggregates in glutamatergic synapses affected synaptic protein expression including metabotropic glutamate receptor 5 (mGLUR5) levels and ratio of N-methyl-d-aspartate (NMDA) receptor subunits GluN1/GluN2A. The ratio of NMDA receptor subunits and levels of mGLUR5 were both normalised by HMTM in L62 mice. These alterations, however, did not affect glutamate release in synaptosomes derived from L62 mice or behavioural endpoints following pharmacological manipulations of glutamate functions. Our results confirm that HMTM acts in the L62 mouse model of PD as an inhibitor of pathological aggregation of synuclein and show that HMTM treatment normalises both the ratio of NMDA receptor subunits and mGLUR5 levels. These findings support the potential utility of HMTM as a disease-modifying treatment for PD aiming to reduce synuclein aggregation pathology.

Dietary phytoestrogen supplementation induces sex differences in the myocardial protein pattern of mice: a comparative proteomics study.

Elevated cardiovascular risk in postmenopausal women and beneficial actions of estrogen replacement in animal models have been related to protective effects of estrogens. However, randomized trials of hormone replacement therapy with synthetic estrogens in humans failed confirmation and phytoestrogens, natural plant hormones with agonistic properties for estrogen receptors, could represent potential alternatives. The aim of the present study is to characterize an animal model for alternative hormone replacement with genistein as a natural estrogenic compound. We performed a 2-DE/ESI-LC-MS approach in order to identify protein species varying with genistein receipt and sex in their relative abundance in the healthy murine heart (http://www.mpiib-berlin.mpg.de/2D-PAGE). Oral genistein treatment revealed a substantial effect on the relative abundance of both estrogen receptors. Several enzymes of the fatty acid metabolism and their transcriptional regulators varied differentially in male and in female animals, at the transcript and/or the protein species level. Increased levels of enzyme species involved in the oxidative phosphorylation and generation of ROS were accompanied by decreased amounts of antioxidants in male mice receiving genistein compared with control males, which have been previously associated with various pathological conditions. Exposure of female animals to genistein provoked an increased abundance of two species of LIM domain-binding protein and one species of desmin. These proteins have been associated with cardiac hypertrophy and our data warrant caution for the use of them as molecular markers, since the animals did not exhibit any histological signs of cardiac hypertrophy.

Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart.

Disturbed energy metabolism with impaired fatty acid oxidation, ATP synthesis and changing levels of contractile proteins has been observed during the development and manifestation of cardiovascular diseases, with the latter showing sexual differences in terms of onset, manifestation and progress. Estrogenic compounds, such as estrogens and phytoestrogens, are known to exert beneficial effects on several cardiovascular parameters. However, global studies implying the normal, non-failing myocardium are rare. Thus, identifying and characterizing protein patterns involved in the maintenance of normal heart physiology at the protein species level will help understanding disease conditions. In this study, we performed an adapted 2-DE/MS approach in order to identify and characterize post-translational modified and truncated protein species from murine heart. Female and male animals of different age were receiving the phytoestrogen genistein and comparative analyses were performed to identify sex and genistein treatment-related effects. Selected 2-DE spots that exposed varying abundance between animal groups and identified as identical proteins were subject to multi-protease cleavage to generate an elevated sequence coverage enabling characterization of post-translational modifications and truncation loci via high-resolution MS. Several truncated, phosphorylated and acetylated species were identified for mitochondrial ATP synthase, malate dehydrogenase and trifunctional enzyme subunit alpha. However, confirmation of several of these modifications by manual spectra interpretation failed. Thus, our results warrant caution for the blind trust in software output. For the regulatory light chain of myosin, we identified an N-terminal processed species, which so far has been related to ischemic conditions only. We tried to unravel the information content of protein species separated by high-resolution 2-DE as an alternative to high-throughput proteomics, which mainly is interested in lists of protein names, ignoring the protein species identity.

Proteomic Analysis of Hydromethylthionine in the Line 66 Model of Frontotemporal Dementia Demonstrates Actions on Tau-Dependent and Tau-Independent Networks.

Abnormal aggregation of tau is the pathological hallmark of tauopathies including frontotemporal dementia (FTD). We have generated tau-transgenic mice that express the aggregation-prone P301S human tau (line 66). These mice present with early-onset, high tau load in brain and FTD-like behavioural deficiencies. Several of these behavioural phenotypes and tau pathology are reversed by treatment with hydromethylthionine but key pathways underlying these corrections remain elusive. In two proteomic experiments, line 66 mice were compared with wild-type mice and then vehicle and hydromethylthionine treatments of line 66 mice were compared. The brain proteome was investigated using two-dimensional electrophoresis and mass spectrometry to identify protein networks and pathways that were altered due to tau overexpression or modified by hydromethylthionine treatment. Overexpression of mutant tau induced metabolic/mitochondrial dysfunction, changes in synaptic transmission and in stress responses, and these functions were recovered by hydromethylthionine. Other pathways, such as NRF2, oxidative phosphorylation and protein ubiquitination were activated by hydromethylthionine, presumably independent of its function as a tau aggregation inhibitor. Our results suggest that hydromethylthionine recovers cellular activity in both a tau-dependent and a tau-independent fashion that could lead to a wide-spread improvement of homeostatic function in the FTD brain.

Multiplex LA-ICP-MS bio-imaging of brain tissue of a parkinsonian mouse model stained with metal-coded affinity-tagged antibodies and coated with indium-spiked commercial inks as internal standards.

BACKGROUND: Immunohistochemistry techniques represent a powerful tool to detect and quantify disease related proteins. Improvements were accomplished by tagged antibodies using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS). However, these approaches are effected by day-to-variations due to instrumental drift. NEW METHOD: Brain tissue from line 62, a Parkinson's disease model, and control mice were incubated with four antibodies relevant to the disease and standardized to three house-keeping proteins. In addition, a new standardization approach was developed and the results compared. This new approach consisted of coating specimens with gelatin and printing an indium-doped ink with a commercial ink jet printer. Furthermore, the method was evaluated for different ablation spot sizes with respect to resolution and signal-to-noise ratio. RESULTS: Normalization using house-keeping proteins led to high background signals even at high resolution. Normalization using indium-doped ink improved the signal-to-noise ratio even when small laser spot sizes were used and further improved by overlaying tissue specimen with gelatin. COMPARISON WITH EXISTING METHODS: Line 62 mice had more α-Synuclein and gliosis but decreased numbers of neurons, as found by conventional immunohistochemistry. These data are in line with the results obtained by LA-ICP-MS with indium standardization. However, differences between L62 and controls for tyrosine hydroxylase were only detected by LA-ICP-MS. CONCLUSIONS: Internal standardisation using indium-doped inks is an effective method to overcome day-to-day variations and instrumental drifts. The new approach results in an increased signal-to-noise ratio and only under these conditions small but significant changes were detected, as seen for tyrosine hydroxylase.

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.

Increasing the number of 5-HT(1A)-receptors in cortex and hippocampus does not induce mnemonic deficits in mice.

Even though the role of the serotonin1A (5-HT(1A))-receptor for cognitive processes is undisputed, the exact involvement of pre- and postsynaptic sites remains unexplained. Recently, we introduced a mouse line overexpressing the 5-HT(1A)-receptor in the hippocampus and cortex. In this study we investigated in comparison to wild-type mice their cognitive abilities using the Morris water-maze task and inhibitory avoidance test. Acute effects of pre- and posttraining administered 8-OH-DPAT (0.03-0.3 mg/kg i.p.) were examined in the inhibitory avoidance test. Additionally, habituation learning was studied in the hole-board test. Transgenic mice showed no overall learning deficit. Spatial learning and memory revealed in the Morris water-maze task was comparable to wild-type mice, and both genotypes habituated to the hole-board arena in a similar manner. Comparing the performance of both genotypes in the inhibitory avoidance test, cognitive functions of transgenic mice seemed to be slightly impaired. When 8-OH-DPAT was administered pretraining an amnesic effect was produced only in transgenic mice and only at the highest dose (0.3 mg/kg). Posttraining administered 0.3 mg/kg 8-OH-DPAT did not affect the performance of both genotypes. Overall, the cortical and hippocampal overexpression of the 5-HT(1A)-receptor had no major effect on cognitive functions in mice, suggesting that changes in the 5-HT(1A)-receptor density are not necessarily accompanied with alterations of learning and memory processes.

Increased Cholinergic Response in α-Synuclein Transgenic Mice (h-α-synL62).

Pathological accumulation of misfolded α-synuclein (α-syn) in the brain plays a key role in the pathogenesis of Parkinson's disease, leading to neuronal dysfunction and motor disorders. The underlying mechanisms linking α-syn aggregations with neurotransmitter disturbance in Parkinson's brains are not well characterized. In the present study, we investigated transgenic mice expressing an aggregation-prone form of full-length human α-syn (h-α-synL62) linked to a signal sequence. These mice display dopamine depletion and progressive motor deficits. We detected accumulation of α-syn in cholinergic interneurons where they are colocalized with choline acetyltransferase. Using microdialysis, we measured acetylcholine levels in the striatum at baseline and during stimulation in the open field and with scopolamine. While no difference between wild-type and transgenic mice was detected in 3 month old mice, striatal acetylcholine levels at 9 months of age were significantly higher in transgenic mice. Concomitantly, high-affinity choline uptake was also increased while choline acetyltransferase and acetylcholine esterase activities were unchanged. The results suggest a disinhibition of acetylcholine release in α-syn transgenic mice.