Hervé Maurin

Increasing Brain Protein O-GlcNAc-ylation Mitigates Breathing Defects and Mortality of Tau.P301L Mice

The microtubule associated protein tau causes primary and secondary tauopathies by unknown molecular mechanisms. Post-translational O-GlcNAc-ylation of brain proteins was demonstrated here to be beneficial for Tau.P301L mice by pharmacological inhibition of O-GlcNAc-ase. Chronic treatment of ageing Tau.P301L mice mitigated their loss in body-weight and improved their motor deficits, while the survival was 3-fold higher at the pre-fixed study endpoint at age 9.5 months. Moreover, O-GlcNAc-ase inhibition significantly improved the breathing parameters of Tau.P301L mice, which underpinned pharmacologically the close correlation of mortality and upper-airway defects. O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of O-GlcNAc-ase. Conversely, biochemical evidence for protein Tau.P301L to become O-GlcNAc-ylated was not obtained, nor was its phosphorylation consistently or markedly affected. We conclude that increasing O-GlcNAc-ylation of brain proteins improved the clinical condition and prolonged the survival of ageing Tau.P301L mice, but not by direct biochemical action on protein tau. The pharmacological effect is proposed to be located downstream in the pathological cascade initiated by protein Tau.P301L, opening novel venues for our understanding, and eventually treating the neurodegeneration mediated by protein tau.

Early Improved and Late Defective Cognition Is Reflected by Dendritic Spines in Tau.P301L Mice

Cognitive demise correlates with progressive brain tauopathy in dementing patients. Improved cognition of young Tau.P301L mice contrasts with dysfunction later in life and remains unexplained (Boekhoorn et al., 2006). To unravel early mechanisms, we composed a correlative time line of clinical symptoms, cognitive defects, and biochemical and pathological traits, including comprehensive analysis of dendritic spines in specified regions of the cortex and hippocampus of young and adult Tau.P301L mice. Remarkably, young Tau.P301L mice have not more, but more mature spines than wild-type mice, revealing the anatomical substrate for their improved cognition and LTP. Spine maturation remained high in the hippocampus of adult Tau.P301L mice. However, spines regressed in length paralleling impaired cognition and increased Tau phosphorylation (Terwel et al., 2005). Conversely, spine maturation was unaffected in adult Tau.4R mice, while spine density was increased and length reduced similar to Tau.P301L mice. To explain how protein Tau promoted spinogenesis, we analyzed hippocampal synaptosomes and dendritic spines for mouse and human Tau. While synaptosomes were positive for both mouse and human Tau, weak variable reaction in spines was observed only after fixation according to Bouin. Mouse Tau was absent from spines in wild-type mice, dissociating the pathological actions of Tau in transgenic mice by relocalization into dendrites and spines from the physiological actions of protein Tau in axons only. We conclude that mutant protein Tau modulates cognition and morphology of spines similarly and in both directions, with pathology later in life coinciding with increased phosphorylation and relocalization of Tau from axons to soma and processes.

Neurological characterization of mice deficient in GSK3α highlight pleiotropic physiological functions in cognition and pathological activity as Tau kinase

BackgroundGSK3β is involved in a wide range of physiological functions, and is presumed to act in the pathogenesis of neurological diseases, from bipolar disorder to Alzheimer’s disease (AD). In contrast, the GSK3α isozyme remained largely ignored with respect to both aspects.ResultsWe generated and characterized two mouse strains with neuron-specific or with total GSK3α deficiency. Behavioral and electrophysiological analysis demonstrated the physiological importance of neuronal GSK3α, with GSK3β not compensating for impaired cognition and reduced LTP. Interestingly, the passive inhibitory avoidance task proved to modulate the phosphorylation status of both GSK3 isozymes in wild-type mice, further implying both to function in cognition. Moreover, GSK3α contributed to the neuronal architecture of the hippocampal CA1 sub-region that is most vulnerable in AD. Consequently, practically all parameters and characteristics indicated that both GSK3 isoforms were regulated independently, but that they acted on the same physiological functions in learning and memory, in mobility and in behavior.ConclusionsGSK3α proved to be regulated independently from GSK3β, and to exert non-redundant physiological neurological functions in general behavior and in cognition. Moreover, GSK3α contributes to the pathological phosphorylation of protein Tau.

Positive and negative early life experiences differentially modulate long term survival and amyloid protein levels in a mouse model of Alzheimer’s disease

Stress has been implicated as a risk factor for the severity and progression of sporadic Alzheimers disease (AD). Early life experiences determine stress responsivity in later life, and modulate age-dependent cognitive decline. Therefore, we examined whether early life experiences influence AD outcome in a bigenic mouse model which progressively develops combined tau and amyloid pathology (biAT mice). Mice were subjected to either early life stress (ELS) or to ‘positive’ early handling (EH) postnatally (from day 2 to 9). In biAT mice, ELS significantly compromised long term survival, in contrast to EH which increased life expectancy. In 4 month old mice, ELS-reared biAT mice displayed increased hippocampal Aβ levels, while these levels were reduced in EH-reared biAT mice. No effects of ELS or EH were observed on the brain levels of APP, protein tau, or PSD-95. Dendritic morphology was moderately affected after ELS and EH in the amygdala and medial prefrontal cortex, while object recognition memory and open field performance were not affected. We conclude that despite the strong transgenic background, early life experiences significantly modulate the life expectancy of biAT mice. Parallel changes in hippocampal Aβ levels were evident, without affecting cognition of young adult biAT mice.

Early Structural and Functional Defects in Synapses and Myelinated Axons in Stratum Lacunosum Moleculare in Two Preclinical Models for Tauopathy

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer’s disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3β with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer’s disease.

Tauopathy Differentially Affects Cell Adhesion Molecules in Mouse Brain: Early Down-Regulation of Nectin-3 in Stratum Lacunosum Moleculare

Cell adhesion molecules are important structural substrates, required for synaptic plasticity and synaptogenesis. CAMs differ widely in their expression throughout different brain regions and their specific structural and functional roles in the brain remain to be elucidated. Here, we investigated selected cell adhesion molecules for alterations in expression levels and neuronal localization in validated mouse models for Alzheimers disease that mimic the age-related progression of amyloid accumulation and tauopathy. Among the cell adhesion molecules analyzed, Nectin-3 expression was affected most and specifically in all mouse models with tauopathy. In particular was Nectin-3 depleted from the specific region of the hippocampus, known as the stratum lacunosum and moleculare, in mice that express wild-type or mutant human protein Tau, either chronically or sub-acutely. Tauopathy progresses from the entorhinal cortex to the hippocampus by unknown mechanisms that could involve transport by the myelinated axons of the temporoammonic and perforant pathways. The decreased expression of Nectin-3 in the stratum lacunosum moleculare is an early marker of impaired transport, and eventual synaptic problems, caused by beginning tauopathy.

Phosphorylation of protein Tau by GSK3β prolongs survival of bigenic Tau.P301L × GSK3β mice by delaying brainstem tauopathy

Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010). In individual mice, the clinical phenotype evolves progressively and rapidly (3-6weeks) from clasping, over general motor impairment to severe reduction in body-weight into the terminal phase that announces imminent death (<3days). Surprisingly, co-expression of GSK3β with Tau.P301L significantly prolonged survival of bigenic biGT mice (Terwel et al., 2008), which we here assign to delayed development of brainstem tauopathy. Eventually, brainstem tauopathy became as prominent in old biGT mice in the specified brainstem nuclei as in the parental Tau.P301L mice, resulting in similar clinical deterioration and terminal phase preceding death, although at later age. Biochemically, in both genotypes the pathway to neurofibrillary tangles and neuropil threads was similar: phosphorylation of protein Tau and formation of soluble oligomers and insoluble aggregates, ending in the typical tangles and threads of tauopathy. The extra GSK3β activity led to expected increased phosphorylation of protein Tau, particularly at residues S262 and S396, which we must conclude to delay the aggregation of protein Tau in the brainstem of aging biGT mice. The unexpected, paradoxical alleviation of the brainstem problems in biGT mice allowed them to grow older and thereby develop more severe tauopathy in forebrain than Tau.P301L mice, which succumb at younger age.

Transcriptional upregulation of myelin components in spontaneous myelin basic protein-deficient mice

Myelin is essential for efficient signal transduction in the nervous system comprising of multiple proteins. The intricacies of the regulation of the formation of myelin, and its components, are not fully understood. Here, we describe the characterization of a novel myelin basic protein (Mbp) mutant mouse, mbp(jive), which spontaneously occurred in our mouse colony. These mice displayed the onset of a shaking gait before 3 weeks of age and seizure onset before 2 months of age. Due to a progressive increase of seizure intensity, mbp(jive) mice experienced premature lethality at around 3 months of age. Mbp mRNA transcript or protein was undetectable and, accordingly, genetic analysis demonstrated a homozygous loss of exons 3 to 6 of Mbp. Peripheral nerve conductance was mostly unimpaired. Additionally, we observed grave structural changes in white matter predominant structures were detected by T1, T2 and diffusion weighted magnetic resonance imaging. We additionally observed that Mbp-deficiency results in an upregulation of Qkl, Mag and Cnp, suggestive of a regulatory feedback mechanism whereby compensatory increases in Qkl have downstream effects on Mag and Cnp. Further research will clarify the role and specifications of this myelin feedback loop, as well as determine its potential role in therapeutic strategies for demyelinating disorders.

Epileptogenicity and precocious mortality of bigenic biat mice that express amyloid and tau

Background:Across the three clinical profiles caused by frontotemporal lobar degeneration (FTLD) (behavioural variant (bvFTD), semantic dementia (SD) and progressive non-fluent aphasia (PNFA)), pathology may be characterized at a molecular level by either tau or TDP-43 proteinopathies. In sporadic cases, correlations between profile and proteinopathy are weak, though among patients with SD around 90% have TDP-43 positive pathology in post mortem series. There is emerging evidence that inflammation underpins TDP-43 proteinopathy: in one study, antibodies against progranulin were found in up to 43% of patients with systemic autoimmune conditions, compared with <1% of controls. Another study found a history of autoimmunity in significantly more cases of definite (familial) or highly probable (SD) TDP-43 pathology than controls. Might autoimmune disease and TDP-43 proteinopathy therefore be linked by the presence of antibodies affecting the activity of proand anti-inflammatory cytokines such as PGRN and TNFa? If so, we may expect to see evidence of FTLD-TDP in patients with autoimmunity. Since TDP43 correlates strongly with an SD phenotype, subclinical pathology may be marked by subtle semantic deficits. Methods: Participants with rheumatoid arthritis (RA) or psoriatic arthritis (PsA) and a group of matched controls with osteoarthritis (OA) completed three tests of semantic memory: Graded Naming Test (GNT), Pyramids and Palm Trees (PPT) and verbal fluency, a cognitive screening questionnaire and the MMSE. Plasma samples were obtained to measure levels of PGRN and TNFa. Results: We present the semantic memory performance in individuals with and without an autoimmune condition. The putative inflammatory mechanism is examined based on correlations between aggregated semantic test performance and levels of PGRN and TNFa. Conclusions: The differential prevalence of subclinical semantic deficits in patients with a common autoimmune disease and a similar condition without an autoimmune mechanism contributes to the debate concerning a possible autoimmune, inflammatory mechanism underpinning FTLD-TDP.

Time-line of pathology and defects in biAT mice with combined amyloid-tau pathology

Background:Alzheimer’s disease (AD) post-mortem pathology consists of amyloid plaques, neurofibrillary tangles and neuropil treads, but their relation to early clinical and functional defects remains not precisely defined in humans. In mouse models, the effects of APP and Tau expression in and on neurons can be studied in spatial and temporal resolution, unattainable in human brain. Thereby the mouse models render important insights into AD, notwithstanding the necessary and evident caveats inherent to modelbuilding. The relation between the different pathologies and the functional defects are assessed, and eventually therapeutically addressed by us in mouse models that present either amyloid or tau pathology, or both combined, all in the same genetic background. Methods: We defined the comprehensive time-line of cognitive defects and pathological traits of bigenic APP.V717IxTau.P301L (biAT) mice, relative to their parental single transgenic strains suffering amyloid or tau pathology separately. In addition, biAT mice are comparatively analyzed with bigenic GSK3ßxTau.P301L mice (biGT) expressing the identical Tau.P301L transgene in combination with the GSK3ß[S9A] transgene (Spittaels et al, 2000; Terwel et al, 2008). All genotypes are in the same FvB/N genetic background. While the late pathology in the bigenic models is now well analyzed and understood (Muyllaert et al, 2006; Terwel et al, 2008) the early defects and their inter-relations remain open for experimental analysis. Results: The bigenic biAT mice present with increased phosphorylation of protein tau already at young age (2-4 months) when neurons became burdened with intracellular amyloid (icAß). Both beginning pathologies co-exist in the same neurons in cortex and hippocampus. Remarkably, dendritic spine density is increased at 4-6 months while LTP and cognition are impaired in biAT mice, similar to the parental APP.V717I mice (Moechars et al, 1999). At this age, in both APP.V717I and biAT mice the GSK3 isozymes are activated as indicated by increased tyrosine auto-phosphorylation (pY279/pY216-GSK3a/b). The activation of the GSK3 kinases coincides with the augmented phosphorylation of protein Tau, resulting in aggravated tauopathy in cortex and hippocampus of biAT mice compared to Tau.P301L mice. GSK3 is thereby confirmed as major link from amyloid to tau pathology (Muyllaert et al, 2007; Terwel et al, 2008). Observations on large cohorts (65 M/74 F) in our expanded colony of biAT mice, revealed important mortality in the age-window of 3-6 months in males and females alike, with less than 50% of biAT mice surviving over 6 months. In general, female biAT mice survive markedly longer (some up to 20-22 months) than males that do not become older than 13-14 months. This is in sharp contrast with the parental Tau.P301L mice that die without exception before age 12 months (mean 9.4 months, no gender difference). The analogy with the prolonged survival of the biGT mice is thereby corroborated (Terwel et al, 2008), underlining the hypothesis that GSK3 increases phosphorylation of protein tau and promotes its aggregation. This then is concluded to be beneficial at least for the respiratory control centers in the brain-stem (Dutschmann et al, 2010), an aspect of interest that we are analyzing in depth. A remarkable point is the early and progressive motor problems of the biGT mice that are much more severe than in the biAT mice or parental Tau.P301L mice. They necessitates feeding of biGTmice on the cage-floor from early age onwards, which nevertheless allows them to survive longer than Tau.P301L and biAT mice, making the case of GSK3 as controlling protein tau phosphorylation even stronger. Conclusions: Our continued studies of young and old, single and bigenic amyloid and tau transgenic mice define important analogies and major differences in functional, biochemical and pathological aspects of their AD-related phenotypes. The activation of GSK3 kinases by amyloid and the resulting phosphorylation of protein Tau is a most early event in the biAT model. The elucidation of the mechanism by which Ab increases tyrosine auto-phosphorylation of GSK3a/b is a fundamental biochemical and cell-biological challenge with far-reaching physiological and pathological implications. P3-130 TREHALOSE ENHANCES THE DEGRADATION OF TAU THROUGH THE AUTOPHAGY PATHWAY Ulrike Krueger*, Yipeng Wang, Xiaoyu Li, Eva Mandelkow, 1 Max-Planck Institute, Hamburg, Germany.