The negative interference of treatments between the acetylcholinesterase inhibitor rivastigmine and the tau aggregation inhibitor hydromethylthionine mesylate (HMTM) has been reported in Line 1 tau-transgenic mice, which overexpress a truncated species of tau protein that is found in the core of paired helical filaments in Alzheimer´s disease (AD). However, little is known about whether such interactions could affect synapses in mice overexpressing tau carrying pathogenic mutations. Here, we have used Line 66 (L66) mice which overexpress full-length human tau carrying the P301S mutation as a model in which tau accumulates in synapses. We measured the abundance of tau and synaptic proteins (VAMP-2, SNAP-25, SNTX-1, SYNPY-1, SYN-1, A-SYN) immunohistochemically to reveal structural synaptic alterations in these mice. Tau and synaptic markers were also examined in L66 mice treated with hydromethylthionine mesylate (HMTM) (15mg/kg) and rivastigmine (0.5mg/kg) administered singly and in combination. Tau protein accumulated in L66 mouse brains, and the levels of synaptic proteins were also altered, most notably with decreased levels of SNAP-25 and SYN-1. A decrease in tau accumulation in L66 brains caused by HMTM was partially compromised by rivastigmine pretreatment. Differences in synaptic proteins induced by HMTM alone were not identical with those induced by HMTM pretreated with rivastigmine. The most prominent differences appeared in proteins of the SNARE complex (SNAP-25, VAMP-2, SNTX-1), but rivastigmine also interfered with the HMTM-dependent reduction in tau accumulation. These data extend our previous findings with L1 mice and provide evidence for a synaptic mechanism of interference between symptomatic and disease-modifying dementia therapies and an explanation for similar drug interactions observed in clinical trials.

Alpha-Synuclein (alpha-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-10 H -phenothiazine- 3,7-diamine dihydrochloride (LETC), a protein aggregation inhibitor, on alpha-Syn aggregation. In both cellular models and transgenic mice, alpha-Syn aggregation was achieved by the overexpression of full-length human alpha-Syn fused with a signal sequence peptide. alpha-Syn accumulated in transfected DH60.21 neuroblastoma cells and alpha-Syn aggregation was inhibited by LETC with an EC 50 of 0.066 +/- 0.047 mu M. Full-length human alpha-Syn overexpressing Line 62 (L62) mice accumulated neuronal alpha-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 alpha-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 alpha-Syn aggregation.

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.

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.

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.

Objective: As a distinct cellular signaling model, a receptor crosstalk between G protein-coupled receptors (GPCR) and epidermal growth factor receptor (EGFR) has been demonstrated in various in vitro studies. In addition, recent in vitro studies had focused on the signaling pathways of endothelin-1 (ET-1) in the pathophysiology of cancer. Accordingly, a growing interest in the analysis of the receptor crosstalk between ET-1 receptors and EGFR and functional consequences of EGFR activation of proliferative diseases evoked us to the analysis of this phenomenon in vivo. Materials and Methods: We performed a comparative study between ET-1 transgenic mice and control mice during the late pregnancy (n=7), early lactation (n=6) the mid of lactation (n=10) and involution day 14 (n=7) periods. Hematoxylin and eosin (HE)stained parallel sections from mammary glands were microscopically examined. The key signal proteins (ETAR, ETBR, ERK1/2, pEGFR) in transactivation of EGFR were analyzed employing Western blot techniques. Genes (amphiregulin, TGFa, EGF, HB-EGF, ADAM 17) known to play an important role in these activities were analyzed using real-time PCR (RT-PCR) techniques. Results: ET-1 transgenic mice exhibited hyperproliferative lesions (lactational hyperplasia) during the middle of the lactation period. Our RT-PCR analyses showed a prominent up regulation of amphiregulin and ADAM 17 in ET-1 transgenic mice. Moreover, we found higher EGFR and ERKs activations in the transgenic mammary glands. Conclusion: This study highlights a causative effect of upregulated ET-1 gene expression on the induction of proliferative lesions via EGFR transactivation in mammary glands. Further, ET-1 overexpression induced an upregulation of amphiregulin and ADAM17 expressions in the transgenic mammary glands. These results suggests that the enhanced ET-1 gene expression and its receptors might have a crucial role in proliferative diseases maintaining EGFR activation.

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.

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.

Amaç: Endotelin (ET-1), kardiyovasküler sistemde güçlü bir vazokonstrüktör rolü olmasının yanı sıra birçok fizyolojik olayda kendini göstermektedir. ET-1 ekspresyonunun özellikle gebelik ve emzirme döneminde ortaya çıkması nedeniyle hem emzirme hem de yeni doğan için önemli bir işlevi olduğu düşünülmektedir. Bu çalışmada ET-1 transgenik fare modeli kullanarak meme bezi üstünde ET-1'in etkilerinin incelenmesi amaçlanmıştır. Gereç ve Yöntem: Meme dokusunun farklı fizyolojik durumlarında (ergenlik, gebelik, laktasyon ve involusyon) ET-1 in etkisini incelemek üzere karşılaştırmalı bir çalışma gerçekleştirildi. Meme dokularından paralel kesitler hazırlanıp hematoksilen-eozin (HE) boyama ile mikroskobik inceleme yapıldı. Meme bezi gelişimi ve metabolizmasında önemli görevleri olan sinyal proteinlerinin (STAT5, AKT, STAT3) analizlerinde Western blot tekniği kullanıldı. Metabolik faaliyetlerde anahtar rolü olduğu bilinen genler (WAP, beta kazein, GLUT-1, SRBF1 Claudin 8, IL-6, LIF) gercek zamanlı polimeraz zincir reaksiyonu (RTPCR) tekniği ile analiz edildi. Bulgular: ET-1 transgenik farelerde sırasıyla sekresyonel hasar, laktasyonel yetmezlik ve erken involusyon gibi patolojik bulgular tespit edildi. Transgenik grupta STAT5’in aktivasyonunun bu durumdan etkilenmediği ancak AKT protein seviyesinde düşüş olduğu saptandı. İnvolusyonda görülen STAT3 aktivasyonu transgenik farelerde laktasyonun 3. gününde tespit edildi. Ayrıca, RT-PCR analizleri ile transgenik farelerde yüksek ET-1 ifadesine bağlı olarak WAP'ın baskılandığını LIF protein ifadesinin belirgin bir şekilde arttığı tespit edildi. Sonuç: Sadece gebelik ve laktasyon döneminde ifade edilen ET1'in laktasyon ve yeni doğan için önemli bir işlevi olduğu düşünülmektedir. Yüksek ET-1 ifadesi meme fizyolojisinde önemli olduğu bilinen genlerin sentezini doğrudan etkileyerek laktasyonel yetersizliğe sebep olmuştur. Bu çalışmada ET-1 regülasyonunun meme bezi gelişiminde ve süt sekresyonu fonksiyonunda kritik bir rol oynadığı gosterilmiştir.

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.

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-1β 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.

The ability to carry out a comprehensive molecular genetic analysis of an organism becomes more difficult and more limited as the complexity of that organism increases, and more effort is required to analyse a given developmental process, mutation or disease. Progress in the genetic analysis of mammalian development can never be as rapid as with Caenorhabditis and Drosophila. The remarkably large and varied collection of known mouse mutations has been obtained principally by relying upon serendipity to expose visible variation in behaviour and or morphology. The ability to introduce foreign genes into the mammalian germline has proved to be one of the most powerful tools in mammalian developmental genetics. The spatio-temporal regulation of gene expression during development and differentiation, and the subsequent coordinated regulation of cellular activities in response to physiological stimuli, are orchestrated primarily at the level of transcription.

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 a-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.

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.

α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson’s disease (PD). We have tested whether N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate) (leuco-methylthioninium bis(hydromethanesulfonate); LMTM), a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn) fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 μM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62) transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK)-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.

12 Targeting oncogenic gene transcription Dr. Ralph Neumüller Boehringer Ingelheim, Vienna Aberrant transcriptional regulation can drive neoplastic transformation and sustain oncogenic growth. In the course of neoplastic transformation cancer cells can become addicted to transcription from large regulators elements (called superenhancers) that maintain cancer cell identity. Interference with oncogenic gene transcription has thus been proposed as a promising therapeutic strategy. I will discuss approaches undertaken at Boehringer Ingelheim Oncology to inhibit oncogenic gene transcription by targeting super-enhancers, transcriptional elongation and chromatin remodeling complexes.

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.

α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease, with yet no disease modifying therapy available. We have generated transgenic models in which the expression and aggregation of human α-Syn is induced and have tested whether LMTM, a tau aggregation inhibitor, affects α-Syn aggregation and behavioural defects in mice. Two independent lines of mice (L58 and L62) accumulated neuronal human α-Syn to different extents. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following daily oral treatment with LMTM, evident in both transgenic lines, and a rescue of movement and anxiety-related behavioural defects in L62. LMTM may provide a potential disease modification therapy in synucleinopathies.

Alpha-Synuclein (α-Syn) accumulation is considered a major risk factor for the development of synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. We have generated mice overexpressing full-length human α-Syn fused to a membrane-targeting signal sequence under the control of the mouse Thy1-promotor. Three separate lines (L56, L58 and L62) with similar gene expression levels, but considerably heightened protein accumulation in L58 and L62, were established. In L62, there was widespread labelling of α-Syn immunoreactivity in brain including spinal cord, basal forebrain, cortex and striatum. Interestingly, there was no detectable α-Syn expression in dopaminergic neurones of the substantia nigra, but strong human α-Syn reactivity in glutamatergic synapses. The human α-Syn accumulated during aging and formed PK-resistant, thioflavin-binding aggregates. Mice displayed early onset bradykinesia and age progressive motor deficits. Functional alterations within the striatum were confirmed: L62 showed normal basal dopamine levels, but impaired dopamine release (upon amphetamine challenge) in the dorsal striatum measured by in vivo brain dialysis at 9 months of age. This impairment was coincident with a reduced response to amphetamine in the activity test. L62 further displayed greater sensitivity to low doses of the dopamine receptor 1 (D1) agonist SKF81297 but reacted normally to the D2 agonist quinpirole in the open field. Since accumulation of α-Syn aggregates in neurones and synapses and alterations in the dopaminergic tone are characteristics of PD, phenotypes reported for L62 present a good opportunity to further our understanding of motor dysfunction in PD and Lewy body dementia.

We investigated sex differences in cardiac protein patterns of intact and castrated mice using proteomics and 1D and 2D immunoblotting. To exclude differences concerning developmental aspects gonadectomy was conducted in mature mice at the age of three months. The main sex-related regulation in the protein pattern of the myocardium occurred for proteins involved in metabolic processes whereas only few proteins involved in other pathways underwent a regulation. Many regulated proteins (2/3) displayed a characteristic V form, which means that these proteins are up- or down-regulated in sexually mature compared to young mice and are back-regulated after castration, emphasizing a direct regulation by gonadal hormones. Several other spots (1/3) showed the same male/female regulation or a drastic increase in male/female spot intensity ratio after castration, suggesting either a regulation independent of sex hormones or a removal of an inhibiting feedback mechanism by gonadectomy. Technically, we found that it cannot be expected that a single spot contains only one protein species and that one protein is present in only one spot. We thus propose for proteomic investigations to identify/quantify all spots of a 2-DE pattern to obtain information about protein speciation and its potential importance for function and pathology. Biological significance: Sex related differences in cardiovascular disease, including risk factors, disease manifestation and outcomes, are far from being well understood, and improved biological understanding of these differences in the healthy myocardium is of great importance. We investigated sex related changes of myocardial protein pattern in intact and castrated mice at different ages and found metabolic proteins to be highly regulated, some of which independently from gonadal hormones.