Valeria Melis

Abnormal Cognition, Sleep, EEG and Brain Metabolism in a Novel Knock-In Alzheimer Mouse, PLB1

Late-stage neuropathological hallmarks of Alzheimers disease (AD) are β-amyloid (βA) and hyperphosphorylated tau peptides, aggregated into plaques and tangles, respectively. Corresponding phenotypes have been mimicked in existing transgenic mice, however, the translational value of aggressive over-expression has recently been questioned. As controlled gene expression may offer animal models with better predictive validity, we set out to design a transgenic mouse model that circumvents complications arising from pronuclear injection and massive over-expression, by targeted insertion of human mutated amyloid and tau transgenes, under the forebrain- and neurone-specific CaMKIIα promoter, termed PLB1Double. Crossing with an existing presenilin 1 line resulted in PLB1Triple mice. PLB1Triple mice presented with stable gene expression and age-related pathology of intra-neuronal amyloid and hyperphosphorylated tau in hippocampus and cortex from 6 months onwards. At this early stage, pre-clinical 18FDG PET/CT imaging revealed cortical hypometabolism with increased metabolic activity in basal forebrain and ventral midbrain. Quantitative EEG analyses yielded heightened delta power during wakefulness and REM sleep, and time in wakefulness was already reliably enhanced at 6 months of age. These anomalies were paralleled by impairments in long-term and short-term hippocampal plasticity and preceded cognitive deficits in recognition memory, spatial learning, and sleep fragmentation all emerging at ∼12 months. These data suggest that prodromal AD phenotypes can be successfully modelled in transgenic mice devoid of fibrillary plaque or tangle development. PLB1Triple mice progress from a mild (MCI-like) state to a more comprehensive AD-relevant phenotype, which are accessible using translational tools such as wireless EEG and microPET/CT.

Complex Disposition of Methylthioninium Redox Forms Determines Efficacy in Tau Aggregation Inhibitor Therapy for Alzheimer’s Disease

Methylthioninium (MT) is a tau aggregation inhibitor with therapeutic potential in Alzheimer’s disease (AD). MT exists in equilibrium between reduced [leucomethylthioninium (LMT)] and oxidized (MT+) forms; as a chloride salt [methylthioninium chloride (MTC), “methylene blue”], it is stabilized in its MT+ form. Although the results of a phase 2 study of MTC in 321 mild/moderate AD subjects identified a 138-mg MT/day dose as the minimum effective dose on cognitive and imaging end points, further clinical development of MT was delayed pending resolution of the unexpected lack of efficacy of the 228-mg MT/day dose. We hypothesized that the failure of dose response may depend on differences known at the time in dissolution in simulated gastric and intestinal fluids of the 100-mg MTC capsules used to deliver the 228-mg dose and reflect previously unsuspected differences in redox processing of MT at different levels in the gut. The synthesis of a novel chemical entity, LMTX (providing LMT in a stable anhydrous crystalline form), has enabled a systematic comparison of the pharmacokinetic properties of MTC and LMTX in preclinical and clinical studies. The quantity of MT released in water or gastric fluid within 60 minutes proved in retrospect to be an important determinant of clinical efficacy. A further factor was a dose-dependent limitation in the ability to absorb MT in the presence of food when delivered in the MT+ form as MTC. A model is presented to account for the complexity of MT absorption, which may have relevance for other similar redox molecules.

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.

Different pathways of molecular pathophysiology underlie cognitive and motor tauopathy phenotypes in transgenic models for Alzheimer's disease and frontotemporal lobar degeneration.

A poorly understood feature of the tauopathies is their very different clinical presentations. The frontotemporal lobar degeneration (FTLD) spectrum is dominated by motor and emotional/psychiatric abnormalities, whereas cognitive and memory deficits are prominent in the early stages of Alzheimer’s disease (AD). We report two novel mouse models overexpressing different human tau protein constructs. One is a full-length tau carrying a double mutation [P301S/G335D; line 66 (L66)] and the second is a truncated 3-repeat tau fragment which constitutes the bulk of the PHF core in AD corresponding to residues 296–390 fused with a signal sequence targeting it to the endoplasmic reticulum membrane (line 1; L1). L66 has abundant tau pathology widely distributed throughout the brain, with particularly high counts of affected neurons in hippocampus and entorhinal cortex. The pathology is neuroanatomically static and declines with age. Behaviourally, the model is devoid of a higher cognitive phenotype but presents with sensorimotor impairments and motor learning phenotypes. L1 displays a much weaker histopathological phenotype, but shows evidence of neuroanatomical spread and amplification with age that resembles the Braak staging of AD. Behaviourally, the model has minimal motor deficits but shows severe cognitive impairments affecting particularly the rodent equivalent of episodic memory which progresses with advancing age. In both models, tau aggregation can be dissociated from abnormal phosphorylation. The two models make possible the demonstration of two distinct but nevertheless convergent pathways of tau molecular pathogenesis. L1 appears to be useful for modelling the cognitive impairment of AD, whereas L66 appears to be more useful for modelling the motor features of the FTLD spectrum. Differences in clinical presentation of AD-like and FTLD syndromes are therefore likely to be inherent to the respective underlying tauopathy, and are not dependent on presence or absence of concomitant APP pathology.

Alpha-Synuclein transgenic mice, h-α-SynL62, display α-Syn aggregation and a dopaminergic phenotype reminiscent of Parkinson's disease

HIGHLIGHTSTransgenic mice overexpressing human &agr;‐Syn under the control of the mouse Thy1‐promotor.Three transgenic lines express similar levels of &agr;‐Syn mRNA but a different number of cells expressing human &agr;‐syn.L62 has greatest level of aggregated &agr;‐Syn protein throughout brain and spinal cord and most severe motor phenotype.L62 mice display dopaminergic transmission deficits and altered D1 receptor function.L62 presents a model to study motor changes associated with Parkinsons disease. ABSTRACT Alpha‐Synuclein (&agr;‐Syn) accumulation is considered a major risk factor for the development of synucleinopathies such as Parkinsons disease (PD) and dementia with Lewy bodies. We have generated mice overexpressing full‐length human &agr;‐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 &agr;‐Syn immunoreactivity in brain including spinal cord, basal forebrain, cortex and striatum. Interestingly, there was no detectable &agr;‐Syn expression in dopaminergic neurones of the substantia nigra, but strong human &agr;‐Syn reactivity in glutamatergic synapses. The human &agr;‐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 &agr;‐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.

P2-383: Reversal of spatial problem-solving cognitive deficit in a transgenic mouse line overexpressing the repeat domain of Tau by treatment with the Tau aggregation inhibitor methylthioninium chloride

ylated at several disease-relevant epitopes, leading to progressive neuronal dystrophy and formation of RIPA-insoluble tau. AD-like tau hyperphosphorylation was reduced by the tau kinase inhibitors lithium as well as SRN 003-556, but RIPA-insoluble tau accumulated regardless of reduced tau phosphorylation. Moreover, we obtained evidence that synapse pathology, assessed by two markers for synaptic vesicles, preceded axon and cell body degeneration which is in line with previous in vivo observations. Specifically SRN 003-556, a kinase inhibitor that may partially inhibit multiple tau kinases, but not lithium was able to protect hippocampal neurons from synaptic damage that was presumably caused by a toxic soluble tau fraction. Synapse protection correlated with reduced neurodegeneration observed at later stages. Conclusions: These data provide first mechanistic insights towards the functional benefits of tau kinase inhibition that have been observed in vivo. The ex vivo model system of hippocampal tau pathology described here may facilitate the identification of drug candidates, in particular kinase inhibitors, and help to identify modulators of tau toxicity in hippocampal neurons.

A Protein Aggregation Inhibitor, Leuco-Methylthioninium Bis(Hydromethanesulfonate), Decreases α-Synuclein Inclusions in a Transgenic Mouse Model of Synucleinopathy

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.

The first third-generation Alzheimer mouse, PLB1: Motor performance and anxiety assessment

Background: Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder with a devastating prognosis. As previously reported, passive immunization using monoclonal antibodies against Abeta (Aß) was successful in improving cognitive deficits in transgenic mice models of AD. The detection of antibodies against Aß in human immunoglobulin (hIVIg) offers an alternative approach of passive immunization using natural occurring polyclonal anti Aß antibodies. The present study was arranged to test if acute treatment with hIVIg has a therapeutic effect on cognition in 9-10 months old Tg2576 mice. Methods: Tg2576 mice (9-10 months of age) were treated acutely with either hIVIg (400 mg, i.p.) or vehicle 1 day before assessing spatial and non-spatial memory using the object recognition test and the novel object location task. Results: Our data revealed no significant difference between hIVIg and control Tg2576 mice in the object recognition task. Both immunized and control group showed increased interaction time with the novel object versus the sample object. However, in contrast to the novel object recognition test, our results of the novel object location task showed a significant trend towards an improvement of spatial memory in immunized Tg2576 mice, indicated by an increase in interaction time with objects moved to a novel location. Conclusions: Acute passive immunization with hIVIg can reverse spatial memory deficits in Tg2576 mice using the object location memory task, therefore showing a therapeutic effect. These findings lead to the conclusion that acute passive immunization with hIVIg might be a promising approach for the treatment of AD.