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Parkinson's disease (PD) is a heterogeneous multi-systemic disorder unique to humans characterized by motor and non-motor symptoms. Preclinical experimental models of PD present limitations and inconsistent neurochemical, histological, and behavioral readouts. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD is the most common in vivo screening platform for novel drug therapies; nonetheless, behavioral endpoints yielded amongst laboratories are often discordant and inconclusive. In this study, we characterized neurochemically, histologically, and behaviorally three different MPTP mouse models of PD to identify translational traits reminiscent of PD symptomatology. MPTP was intraperitoneally (i.p.) administered in three different regimens: (i) acute-four injections of 20 mg/kg of MPTP every 2 h; (ii) sub-acute-one daily injection of 30 mg/kg of MPTP for 5 consecutive days; and (iii) chronic-one daily injection of 4 mg/kg of MPTP for 28 consecutive days. A series of behavioral tests were conducted to assess motor and non-motor behavioral changes including anxiety, endurance, gait, motor deficits, cognitive impairment, circadian rhythm and food consumption. Impairments in balance and gait were confirmed in the chronic and acute models, respectively, with the latter showing significant correlation with lesion size. The sub-acute model, by contrast, presented with generalized hyperactivity. Both, motor and non-motor changes were identified in the acute and sub-acute regime where habituation to a novel environment was significantly reduced. Moreover, we report increased water and food intake across all three models. Overall, the acute model displayed the most severe lesion size, while across the three models striatal dopamine content (DA) did not correlate with the behavioral performance. The present study demonstrates that detection of behavioral changes following MPTP exposure is challenging and does not correlate with the dopaminergic lesion extent.

Tau protein aggregations are important contributors to the etiology of Alzheimer’s disease (AD). Hydromethylthionine (HMT) is a potent inhibitor of tau aggregation in vitro and in vivo and is being developed as a possible anti-dementia medication. HMT was also shown to affect the cholinergic system and to interact with mitochondria. Here, we used tau-transgenic (L1 and L66) and wild-type NMRI mice that were treated with HMT, rivastigmine and memantine and with combinations thereof, for 2–4 weeks. We measured HMT concentrations in both brain homogenates and isolated mitochondria and concentrations of glucose, lactate and pyruvate in brain by microdialysis. In isolated brain mitochondria, we recorded oxygen consumption of mitochondrial complexes by respirometry. While rivastigmine and memantine lowered mitochondrial respiration, HMT did not affect respiration in wild-type animals and increased respiration in tau-transgenic L1 mice. Glucose and lactate levels were not affected by HMT administration. The presence of HMT in isolated mitochondria was established. In summary, traditional anti-dementia drugs impair mitochondrial function while HMT has no adverse effects on mitochondrial respiration in tau-transgenic mice. These results support the further development of HMT as an anti-dementia drug.

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.

Purkinje cells are the only output cell of the cerebellar cortex. Their spatiotemporal activity is controlled by molecular layer interneurons (MLIs) through GABAA receptor-mediated inhibition. Recently, it has been reported that the cerebellar cortex is required for consolidation of conditioned fear responses during fear memory formation. Although the relevance of MLIs during fear memory formation is currently not known, it has been shown that synapses made between MLIs and Purkinje cells exhibit long term plasticity following fear conditioning. The present study examined the role of cerebellar MLIs in the formation of fear memory using a genetically-altered mouse line (PC-∆γ2) in which GABAA receptor-mediated signaling at MLI to Purkinje cell synapses was functionally removed. We found that neither acquisition nor recall of fear memories to tone and context were altered after removal of MLI-mediated inhibition.

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.

Mental disorders represent a significant public health burden worldwide due to their high prevalence, chronically disabling nature, and substantial impact on quality of life. Despite growing knowledge of the pathological mechanisms that underlie the development of these disorders, a high percentage of patients do not respond to first-line clinical treatments; thus, there is a strong need for alternative therapeutic approaches. During the past half-century, after the identification of the endocannabinoid system and its role in multiple physiological processes, both natural and synthetic cannabinoids have attracted considerable interest as putative medications in pathological conditions such as, but not exclusive to, mental disorders. Here, we provide a summary of cannabinoid effects in support of possible therapeutic applications for major depression, bipolar disorder, anxiety, posttraumatic stress disorder, and schizophrenia. Considering this evidence, highlighted benefits and risks of cannabinoid use in the management of these illnesses require further experimental study. .Los trastornos mentales representan una carga importante para la salud pública en todo el mundo debido a su alta prevalencia, su naturaleza crónica con discapacidad y su impacto significativo en la calidad de vida. A pesar del creciente conocimiento de los mecanismos patológicos que subyacen al desarrollo de estos trastornos, un alto porcentaje de pacientes no responde a los tratamientos clínicos de primera línea; por lo que existe una gran necesidad de enfoques terapéuticos alternativos. Durante los últimos cincuenta años, después de la identificación del sistema endocannabinoide y su papel en múltiples procesos fisiológicos, tanto los cannabinoides naturales como los sintéticos han concentrado un alto interés como posibles fármacos para trastornos mentales y otras patologías. En este artículo se resumen los efectos de los cannabinoides en apoyo de posibles aplicaciones terapéuticas para la depresión mayor, el trastorno bipolar, la ansiedad, el trastorno por estrés postraumático y la esquizofrenia. Teniendo en cuenta esta evidencia, el destacar tanto los riesgos como los beneficios del empleo de los cannabinoides en el tratamiento de estas enfermedades requiere de más trabajo experimental.La prévalence élevée des troubles mentaux, leur chronicité et leur impact important sur la qualité de vie pèsent significativement sur la santé publique mondiale. La connaissance des mécanismes pathologiques qui sous-tendent ces troubles a progressé mais, un pourcentage élevé de patients ne répondant pas aux traitements de première ligne, d’autres approches thérapeutiques sont indispensables. L’étude du système endocannabinoïde et la mise en évidence de son rôle dans de multiples processus physiologiques au cours des cinquante dernières années, ont fortement attiré l’attention sur les cannabinoïdes naturels et synthétiques en tant que médicaments potentiels dans certaines pathologies comme, entre autres, les troubles mentaux. Nous résumons dans cet article les effets cannabinoϊdes qui pourraient s’inscrire dans le traitement de la dépression majeure, des troubles bipolaires, de l’anxiété, de l’état de stress post-traumatique et de la schizophrénie. Ces arguments nous incitent à explorer davantage les avantages et les risques des cannabinoïdes dans la prise en charge de ces maladies.

The prevention of tau protein aggregations is a therapeutic goal for the treatment of Alzheimer's disease (AD), and hydromethylthionine (HMT) (also known as leucomethylthioninium-mesylate [LMTM]), is a potent inhibitor of tau aggregation in vitro and in vivo. In two Phase 3 clinical trials in AD, HMT had greater pharmacological activity on clinical endpoints in patients not receiving approved symptomatic treatments for AD (acetylcholinesterase (AChE) inhibitors and/or memantine) despite different mechanisms of action. To investigate this drug interaction in an animal model, we used tau-transgenic L1 and wild-type NMRI mice treated with rivastigmine or memantine prior to adding HMT, and measured changes in hippocampal acetylcholine (ACh) by microdialysis. HMT given alone doubled hippocampal ACh levels in both mouse lines and increased stimulated ACh release induced by exploration of the open field or by infusion of scopolamine. Rivastigmine increased ACh release in both mouse lines, whereas memantine was more active in tau-transgenic L1 mice. Importantly, our study revealed a negative interaction between HMT and symptomatic AD drugs: the HMT effect was completely eliminated in mice that had been pre-treated with either rivastigmine or memantine. Rivastigmine was found to inhibit AChE, whereas HMT and memantine had no effects on AChE or on choline acetyltransferase (ChAT). The interactions observed in this study demonstrate that HMT enhances cholinergic activity in mouse brain by a mechanism of action unrelated to AChE inhibition. Our findings establish that the drug interaction that was first observed clinically has a neuropharmacological basis and is not restricted to animals with tau aggregation pathology. Given the importance of the cholinergic system for memory function, the potential for commonly used AD drugs to interfere with the treatment effects of disease-modifying drugs needs to be taken into account in the design of clinical trials.

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.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Numerous analyses conducted to date have clearly identified measures that need to be taken to improve research rigor. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e., performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

Aim The β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) has been identified as the central initiator of amyloid β (Aβ) generation in the brain, the key hallmark of Alzheimer's disease (AD). However, recent studies provided evidence that BACE1 also plays a crucial role in metabolic regulation, and we have shown that neuronal human BACE1 knock-in mice (PLB4) display type 2 diabetes mellitus (T2DM)-like symptoms alongside AD-like impairments. Hence, we here investigated if targeted BACE1 inhibition using LY2886721, an active site BACE1 inhibitor, would improve glucose homeostasis, insulin sensitivity and motor performance in PLB4 mice. Materials and methods LY2886721 was administered as a dietary supplement (0.02% wt/wt) for six consecutive weeks. Physiological, metabolic and motor assessments were performed during the last two weeks of treatment, followed by molecular tissue analyses post-mortem. Results LY2886721 treatment improved glucose homeostasis and hepatic gluconeogenesis in diabetic PLB4 mice, as determined by improvements in basal glucose and glucose/pyruvate tolerance tests. Furthermore, LY2886721 improved hepatic insulin sensitivity, as indicated by enhanced basal hyperphosphorylation of insulin receptors. In PLB4 brains, we detected altered basal conditions of APP expression and processing, with beneficial effects on APP processing achieved by LY2886721 treatment. No improvements in motor coordination were found. Conclusions Our data provide support for a role of BACE1 as a regulator of systemic glucose homeostasis and suggest BACE1 inhibitors for the treatment of T2DM-associated pathologies, especially in cases where diabetes is comorbid to AD.

BackgroundThe prevalence of Alzheimer’s Disease (AD) is greater in women compared to men, but the reasons for this remain unknown. This sex difference has been widely neglected in experimental studies using transgenic mouse models of AD.ObjectiveHere, we studied behaviour and molecular pathology of 5-month-old 5XFAD mice, which express mutated human amyloid precursor protein and presenilin-1 on a C57BL/6J background, vs. their wild-type littermate controls, to compared both sex- and genotype-dependent differences.MethodsA novel behavioural paradigm was utilised (OF-NO-SI), comprising activity measures (Open Field, OF) arena, followed by Novel Object exploration (NO) and Social Interaction (SI) of a sex-matched conspecific. Each segment consisted of two repeated trials to assess between-trial habituation. Subsequently, brain pathology (amyloid load, stress response and inflammation markers, synaptic integrity, trophic support) was assessed using qPCR and Western blotting.ResultsFemale 5XFAD mice had higher levels of human APP and beta-amyloid (Aβ) and heightened inflammation vs males. These markers correlated with hyperactivity observed in both sexes, yet only female 5XFAD mice presented with deficits in object and social exploration. Male animals had higher expression of stress markers and neurotrophic factors irrespective of genotype, this correlated with cognitive performance.ConclusionThe impact of sex on AD-relevant phenotypes is in line with human data and emphasises the necessity of appropriate study design and reporting. Differential molecular profiles observed in male vs. female mice offer insights into possible protective mechanisms, and hence treatment strategies.

Over the last two decades, awareness of the negative repercussions of flaws in the planning, conduct and reporting of preclinical research involving experimental animals has been growing. Several initiatives have set out to increase transparency and internal validity of preclinical studies, mostly publishing expert consensus and experience. While many of the points raised in these various guidelines are identical or similar, they differ in detail and rigour. Most of them focus on reporting, only few of them cover the planning and conduct of studies. The aim of this systematic review is to identify existing experimental design, conduct, analysis and reporting guidelines relating to preclinical animal research. A systematic search in PubMed, Embase and Web of Science retrieved 13 863 unique results. After screening these on title and abstract, 613 papers entered the full-text assessment stage, from which 60 papers were retained. From these, we extracted unique 58 recommendations on the planning, conduct and reporting of preclinical animal studies. Sample size calculations, adequate statistical methods, concealed and randomised allocation of animals to treatment, blinded outcome assessment and recording of animal flow through the experiment were recommended in more than half of the publications. While we consider these recommendations to be valuable, there is a striking lack of experimental evidence on their importance and relative effect on experiments and effect sizes.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Numerous analyses conducted to date have clearly identified measures that need to be taken to improve research rigor. Supported through the European Union’s Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e., performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system. Further, EQIPD is preparing training support and assessment services for those research units that successfully implement the quality system and would like to seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

The primeval function of the mammalian hippocampus (HPC) remains uncertain. Implicated in learning and memory, spatial navigation, and neuropsychological disorders, evolutionary theory suggests that the HPC evolved from a primeval chemosensory epithelium. Deficits in sensing of internal body status ('interoception') in patients with HPC lesions argue that internal sensing may be conserved in higher vertebrates. We studied the expression patterns in mouse brain of 250 endocrine receptors that respond to blood-borne ligands. Key findings are (i) the proportions and levels of endocrine receptor expression in the HPC are significantly higher than in all other comparable brain regions. (ii) Surprisingly, the distribution of endocrine receptor expression within mouse HPC was found to be highly structured: receptors signaling 'challenge' are segregated in dentate gyrus (DG), whereas those signaling 'sufficiency' are principally found in cornu ammonis (CA) regions. Selective expression of endocrine receptors in the HPC argues that interoception remains a core feature of hippocampal function. Further, we report that ligands of DG receptors predominantly inhibit both synaptic potentiation and neurogenesis, whereas CA receptor ligands conversely promote both synaptic potentiation and neurogenesis. These findings suggest that the hippocampus acts as an integrator of body status, extending its role in context-dependent memory encoding from 'where' and 'when' to 'how I feel'. Implications for anxiety and depression are discussed.

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., We present here two tau transgenic models; L1 expressing a truncated tau fragment and L66 full-length, mutant human tau. A cholinergic deficit and region-specific inflammation in L1 mice confirms an Alzheimer's disease-like phenotype, whilst Line 66 revealed no measurable change in cholinergic expression, a phenotypic trait of frontotemporal lobar degeneration., Graphical Abstract Graphical Abstract

Background Hydromethylthionine is a potent inhibitor of pathological aggregation of tau and TDP-43 proteins. Objective To compare hydromethylthionine treatment effects at two doses and to determine how drug exposure is related to treatment response in bvFTD. Methods We undertook a 52-week Phase III study in 220 bvFTD patients randomized to compare hydromethylthionine at 200 mg/day and 8 mg/day (intended as a control). The principal outcomes were change on the Addenbrookes Cognitive Examination - Revised (ACE-R), the Functional Activities Questionnaire (FAQ), and whole brain volume. Secondary outcomes included Modified Clinical Global Impression of Change (Modified-CGIC). A population pharmacokinetic exposure-response analysis was undertaken in 175 of the patients with available blood samples and outcome data using a discriminatory plasma assay for the parent drug. Results There were no significant differences between the two doses as randomized. There were steep concentration-response relationships for plasma levels in the range 0.3-0.6 ng/ml at the 8 mg/day dose on clinical and MRI outcomes. There were significant exposure-dependent differences at 8 mg/day for FAQ, Modified-CGIC, and whole brain atrophy comparing patients with plasma levels greater than 0.346 ng/ml with having minimal drug exposure. The exposure-response is biphasic with worse outcomes at the high concentrations produced by 200 mg/day. Conclusions Hydromethylthionine has a similar concentration-response profile for effects on clinical decline and brain atrophy at the 8 mg/day dose in bvFTD as recently reported in AD. Treatment responses in bvFTD are predicted to be maximal at doses in the range 20-60 mg/day. A confirmatory placebo-controlled trial is now planned.

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.

Electroencephalography (EEG) records fast-changing neuronal signalling and communication and thus can offer a deep understanding of cognitive processes. However, traditional data analyses which employ the Fast-Fourier Transform (FFT) have been of limited use as they do not allow time- and frequency-resolved tracking of brain activity and detection of directional connectivity. Here, we applied advanced qEEG tools using autoregressive (AR) modelling, alongside traditional approaches, to murine data sets from common research scenarios: (a) the effect of age on resting EEG; (b) drug actions on non-rapid eye movement (NREM) sleep EEG (pharmaco-EEG); and (c) dynamic EEG profiles during correct vs incorrect spontaneous alternation responses in the Y-maze. AR analyses of short data strips reliably detected age- and drug-induced spectral EEG changes, while renormalized partial directed coherence (rPDC) reported direction- and time-resolved connectivity dynamics in mice. Our approach allows for the first time inference of behaviour- and stage-dependent data in a time- and frequency-resolved manner, and offers insights into brain networks that underlie working memory processing beyond what can be achieved with traditional methods.

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.

Understanding the contribution of transmitter systems in behavioural pharmacology has a long tradition. Multiple techniques such as transmitter-specific lesions, and also localized administration of pharmacological toxins including agonists and antagonists of selected receptors have been applied. More recently, modern genetic tools have permitted cell-type selective interferences, for example by expression of light-sensitive channels followed by optogenetic stimulation in behaviourally meaningful settings or by engineered channels termed DREADDS that respond to peripherally administered drugs. We here took a similar approach and employed a Cre recombinase-dependent viral delivery system (adeno-associated virus) to express tetanus toxin light chain (TeLc) and thus, block neural transmission specifically in parvalbumin-positive (PV +) neurons of the limbic and infralimbic prefrontal circuitry. PV-TeLc cohorts presented with normal circadian activity as recorded in PhenoTyper home cages, but a reproducible increase in anxiety was extracted in both the open field and light–dark box. Interestingly, working memory assessed in a spontaneous alternation Y-maze task was impaired in PV-TeLc mice. We also recorded local field potentials from a separate cohort and found no global changes in brain activity, but found a behaviourally relevant lack of modulation in the gamma spectral band. These anomalies are reminiscent of endophenotypes of schizophrenia and appear to be critically dependent on GABAergic signalling through PV neurones. At the same time, these observations validate the use of viral vector delivery and its expression in Cre-lines as a useful tool for understanding the role of selective components of the brain in behaviour and the underpinning physiology.

Background: LMTM is being developed as a treatment for AD based on inhibition of tau aggregation. Objectives: To examine the efficacy of LMTM as monotherapy in non-randomized cohort analyses as modified primary outcomes in an 18-month Phase III trial in mild AD. Methods: Mild AD patients (n = 800) were randomly assigned to 100 mg twice a day or 4 mg twice a day. Prior to unblinding, the Statistical Analysis Plan was revised to compare the 100 mg twice a day as monotherapy subgroup (n = 79) versus 4 mg twice a day as randomized (n = 396), and 4 mg twice a day as monotherapy (n = 76) versus 4 mg twice a day as add-on therapy (n = 297), with strong control of family-wise type I error. Results: The revised analyses were statistically significant at the required threshold of p

The primeval function of the mammalian hippocampus (HPC) remains uncertain. Implicated in learning and memory, spatial navigation, and neuropsychological disorders, evolutionary theory suggests that the HPC evolved from a primeval chemosensory epithelium. Internal sensing deficits in patients with HPC lesions argue that internal sensing may be conserved in higher vertebrates. We studied the expression of 250 endocrine receptors in mouse brain. Key findings are (i) the proportions and levels of endocrine receptor expression in the HPC are significantly higher than in all other comparable brain regions. (ii) Surprisingly, the distribution of endocrine receptor expression within mouse HPC was found to be highly structured: receptors signaling ‘challenge’ are segregated in dentate gyrus (DG), whereas those signaling ‘sufficiency’ are principally found in cornu ammonis (CA) regions. Selective expression of endocrine receptors in the HPC argues that internal sensing remains a core feature of hippocampal function. Further, we report that ligands of DG receptors predominantly inhibit both synaptic potentiation and neurogenesis, whereas CA receptor ligands conversely promote both synaptic potentiation and neurogenesis. These findings suggest that the hippocampus acts as an integrator of body status, extending its role in context-dependent memory encoding from ‘where’ and ‘when’ to ‘how I feel’. Implications for anxiety and depression are discussed.

Background Although hydromethylthionine is a potent tau aggregation inhibitor, no difference was found in either of two Phase III trials in mild to moderate Alzheimer's disease (AD) comparing doses in the range 150-250 mg/day with 8 mg/day intended as a control. Objective To determine how drug exposure is related to treatment response. Methods A sensitive plasma assay for the drug was used in a population pharmacokinetic analysis of samples from 1,162 of the 1,686 patients who participated in either of the Phase III trials with available samples and efficacy outcome data. Results There are steep concentration-response relationships for steady state plasma levels in the range 0.3-0.8 ng/ml at the 8 mg/day dose. Using a threshold based on the lower limit of quantitation of the assay on Day 1, there are highly significant differences in cognitive decline and brain atrophy in patients with above threshold plasma levels, both for monotherapy and add-on therapy, but with effect sizes reduced by half as add-on. Plasma concentrations in the range 4-21 ng/ml produced by the high doses are not associated with any additional benefit. Conclusions Hydromethylthionine has pharmacological activity on brain structure and function at the 8 mg/day dose as monotherapy or as add-on to symptomatic treatments. This combined with a plateau at higher doses is consistent with the lack of dose-response seen in the Phase III trials. Treatment benefit is predicted to be maximal at 16 mg/day as monotherapy. A placebo-controlled trial in mild/moderate AD is now ongoing to confirm efficacy at this dose.

Aims β-Secretase 1 (BACE1) is a key enzyme in Alzheimer’s disease pathogenesis that catalyses the amyloidogenic cleavage of amyloid precursor protein (APP). Recently, global Bace1 deletion was shown to protect against diet-induced obesity and diabetes, suggesting that BACE1 is a potential regulator of glucose homeostasis. Here, we investigated whether increased neuronal BACE1 is sufficient to alter systemic glucose metabolism, using a neuron-specific human BACE1 knockin mouse model (PLB4). Methods Glucose homeostasis and adiposity were determined by glucose tolerance tests and EchoMRI, lipid species were measured by quantitative lipidomics, and biochemical and molecular alterations were assessed by western blotting, quantitative PCR and ELISAs. Glucose uptake in the brain and upper body was measured via 18FDG-PET imaging. Results Physiological and molecular analyses demonstrated that centrally expressed human BACE1 induced systemic glucose intolerance in mice from 4 months of age onward, alongside a fatty liver phenotype and impaired hepatic glycogen storage. This diabetic phenotype was associated with hypothalamic pathology, i.e. deregulation of the melanocortin system, and advanced endoplasmic reticulum (ER) stress indicated by elevated central C/EBP homologous protein (CHOP) signalling and hyperphosphorylation of its regulator eukaryotic translation initiation factor 2α (eIF2α). In vivo 18FDG-PET imaging further confirmed brain glucose hypometabolism in these mice; this corresponded with altered neuronal insulin-related signalling, enhanced protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4) levels, along with upregulation of the ribosomal protein and lipid translation machinery. Increased forebrain and plasma lipid accumulation (i.e. ceramides, triacylglycerols, phospholipids) was identified via lipidomics analysis. Conclusions/interpretation Our data reveal that neuronal BACE1 is a key regulator of metabolic homeostasis and provide a potential mechanism for the high prevalence of metabolic disturbance in Alzheimer’s disease. Electronic supplementary material The online version of this article (doi:10.1007/s00125-016-3960-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Gene mutations within amyloid precursor protein (APP or AβPP) and/or presenilin 1 (PS1) genes are determinants of familial Alzheimer's disease (fAD) and remain fundamental for experimental models. Here, we generated a neuronal knock-in mouse (PLB2APP) with mutated human APPSwe/Lon and investigated histopathology and behavioral phenotypes. Additionally, PLB2APP mice were cross-bred with a presenilin (PS1A246E) line to assess the impact of this gene combination. Immunohistochemistry determined amyloid-β (Aβ) pathology, astrogliosis (via GFAP labelling), and neuronal densities in hippocampal and cortical brain regions. One-year old PLB2APP mice showed higher levels of intracellular Aβ in CA1, dentate gyrus, and cortical regions compared to PLBWT controls. Co-expression of PS1 reduced hippocampal but elevated cortical Aβ build-up. Amyloid plaques were sparse in aged PLB2APP mice, and co-expression of PS1 promoted plaque formation. Heightened GFAP expression followed the region-specific pattern of Aβ in PLB2APP and PLB2APP/PS1 mice. Behaviorally, habituation to a novel environment was delayed in 6-month-old PLB2APP mice, and overall home-cage activity was reduced in both lines at 6 and 12 months, particularly during the dark phase. Spatial learning in the water maze was impaired in PLB2APP mice independent of PS1 expression and associated with reduced spatial navigation strategies. Memory retrieval was compromised in PLB2APP mice only. Our data demonstrate that low expression of APP is sufficient to drive histopathological and cognitive changes in mice without overexpression or excessive plaque deposition. AD-like phenotypes were altered by co-expression of PS1, including a shift from hippocampal to cortical Aβ pathology, alongside reduced deficits in spatial learning.

Background Rodent electroencephalography (EEG) in preclinical research is frequently conducted in behaving animals. EEG analysis is complicated by a number of confounds, particularly 1. The close relationship between EEG power and movement speed must be controlled for prior to further analysis. 2. The difficulty inherent in identifying EEG epochs associated with a particular behaviour. New Method We utilized infra-red event stamping to accurately synchronize EEG recorded superficially above the hippocampus and prefrontal cortex with motion tracking data in a transgenic Alzheimer’s disease (AD) mouse model (PLB1APP) and wild-type controls (PLBWT) performing a Y-maze spontaneous alternation task. Video tracking synchronized epochs capturing specific behaviours were automatically identified and extracted prior to auto-regressive spectral analysis. Results Despite comparable behavioural performance, PLB1APP mice demonstrated region and behavioural context specific deficits in regulation of Gamma power: In contrast to controls, hippocampal Gamma response to speed as well as prefrontal activity associated with correct vs. incorrect alternations was absent in PLB1APP mice. Regulation of hippocampal Gamma power in response to direction of movement did not differ. Comparison with existing Methods This method allows for the first time to detect behaviour-specific differences in EEG response to speed that can be quantified and actively controlled for. Analysis across multiple parameters engaging different brain regions can now be used for detailed EEG profiling of brain-region specific functions. Conclusion Combining infra-red event-stamping and auto-regressive modelling enables rapid, automated and sensitive phenotyping of AD mouse models. Subtle alterations in brain signalling can be detected prior to overt behavioural impairments.

Objective Within the last years, there has been growing awareness of the negative repercussions of unstandardized planning, conduct and reporting of preclinical and biomedical research. Several initiatives have set the aim of increasing validity and reliability in reporting of studies and publications, and publishers have formed similar groups. Additionally, several groups of experts across the biomedical spectrum have published experience and opinion-based guidelines and guidance on potential standardized reporting. While all these guidelines cover reporting of experiments, an important step prior to this should be rigours planning and conduction of studies. The aim of this systematic review is to identify and harmonize existing experimental design, conduct and analysis guidelines relating to internal validity and reproducibility of preclinical animal research. The review will also identify literature describing risks of bias pertaining to the design, conduct and analysis of preclinical biomedical research. Search strategy PubMed, Embase and Web of Science will be searched systematically to identify guidelines published in English language in peer-reviewed journals before January 2018 (box 1). All articles or systematic reviews in English language that describe or review guidelines on the internal validity and reproducibility of animal studies will be included. Google search for guidelines published on the websites of major funders and professional organisations can be found in (Box 2). Screening and annotation Unique references will be screened in two phases: screening for eligibility based on title and abstract, followed by screening for definitive inclusion based on full text. Screening will be performed in SyRF (http://syrf.org.uk). Each reference will be randomly presented to two independent reviewers. Disagreements between reviewers will be resolved by additional screening of the reference by a third, senior researcher. Data management and reporting All data, including extracted text and guidelines, will be stored in the SyRF platform. Elements of the included guidelines will be identified using a standardized extraction form. Reporting will follow the PRISMA guidelines as far as applicable.