Julia Reichwald

Brain homogenates from human tauopathies induce tau inclusions in mouse brain.

Filamentous inclusions made of hyperphosphorylated tau are characteristic of numerous human neurodegenerative diseases, including Alzheimer’s disease, tangle-only dementia, Pick disease, argyrophilic grain disease (AGD), progressive supranuclear palsy, and corticobasal degeneration. In Alzheimer’s disease and AGD, it has been shown that filamentous tau appears to spread in a stereotypic manner as the disease progresses. We previously demonstrated that the injection of brain extracts from human mutant P301S tau-expressing transgenic mice into the brains of mice transgenic for wild-type human tau (line ALZ17) resulted in the assembly of wild-type human tau into filaments and the spreading of tau inclusions from the injection sites to anatomically connected brain regions. Here we injected brain extracts from humans who had died with various tauopathies into the hippocampus and cerebral cortex of ALZ17 mice. Argyrophilic tau inclusions formed in all cases and following the injection of the corresponding brain extracts, we recapitulated the hallmark lesions of AGD, PSP and CBD. Similar inclusions also formed after intracerebral injection of brain homogenates from human tauopathies into nontransgenic mice. Moreover, the induced formation of tau aggregates could be propagated between mouse brains. These findings suggest that once tau aggregates have formed in discrete brain areas, they become self-propagating and spread in a prion-like manner.

Critical role of soluble amyloid-β for early hippocampal hyperactivity in a mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by a progressive dysfunction of central neurons. Recent experimental evidence indicates that in the cortex, in addition to the silencing of a fraction of neurons, other neurons are hyperactive in amyloid-β (Aβ) plaque-enriched regions. However, it has remained unknown what comes first, neuronal silencing or hyperactivity, and what mechanisms might underlie the primary neuronal dysfunction. Here we examine the activity patterns of hippocampal CA1 neurons in a mouse model of AD in vivo using two-photon Ca2+ imaging. We found that neuronal activity in the plaque-bearing CA1 region of older mice is profoundly altered. There was a marked increase in the fractions of both silent and hyperactive neurons, as previously also found in the cortex. Remarkably, in the hippocampus of young mice, we observed a selective increase in hyperactive neurons already before the formation of plaques, suggesting that soluble species of Aβ may underlie this impairment. Indeed, we found that acute treatment with the γ-secretase inhibitor LY-411575 reduces soluble Aβ levels and rescues the neuronal dysfunction. Furthermore, we demonstrate that direct application of soluble Aβ can induce neuronal hyperactivity in wild-type mice. Thus, our study identifies hippocampal hyperactivity as a very early functional impairment in AD transgenic mice and provides direct evidence that soluble Aβ is crucial for hippocampal hyperactivity.

The Second-Generation Active Aβ Immunotherapy CAD106 Reduces Amyloid Accumulation in APP Transgenic Mice While Minimizing Potential Side Effects

Immunization against amyloid-β (Aβ) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimers disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aβ1–6 coupled to the virus-like particle Qβ. Immunization with this vaccine did not activate Aβ-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aβ antibody titers of different IgG subclasses mainly recognizing the Aβ3–6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aβ42 and Aβ40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aβ, which atypically was nonfibrillar. The efficacy of Aβ immunotherapy depended on the Aβ levels and thus differed between animal models, brain regions, and stage of amyloid deposition. Therefore, animal studies may not quantitatively predict the effect in human Alzheimers disease. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In rhesus monkeys, CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aβ monomers and oligomers and blocked Aβ toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aβ aggregation and its downstream detrimental effects.

Dynamics of Aβ Turnover and Deposition in Different β-Amyloid Precursor Protein Transgenic Mouse Models Following γ-Secretase Inhibition

Human β-amyloid precursor protein (APP) transgenic mice are commonly used to test potential therapeutics for Alzheimers disease. We have characterized the dynamics of β-amyloid (Aβ) generation and deposition following γ-secretase inhibition with compound LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-l-alaninamide]. Kinetic studies in preplaque mice distinguished a detergent-soluble Aβ pool in brain with rapid turnover (half-lives for Aβ40 and Aβ42 were 0.7 and 1.7 h) and a much more stable, less soluble pool. Aβ in cerebrospinal fluid (CSF) reflected the changes in the soluble brain Aβ pool, whereas plasma Aβ turned over more rapidly. In brain, APP C-terminal fragments (CTF) accumulated differentially. The half-lives for γ-secretase degradation were estimated as 0.4 and 0.1 h for C99 and C83, respectively. Three different APP transgenic lines responded very similarly to γ-secretase inhibition regardless of the familial Alzheimers disease mutations in APP. Amyloid deposition started with Aβ42, whereas Aβ38 and Aβ40 continued to turn over. Chronic γ-secretase inhibition lowered amyloid plaque formation to a different degree in different brain regions of the same mice. The extent was inversely related to the initial amyloid load in the region analyzed. No evidence for plaque removal below baseline was obtained. γ-Secretase inhibition led to a redistribution of intracellular Aβ and an elevation of CTFs in neuronal fibers. In CSF, Aβ showed a similar turnover as in preplaque animals demonstrating its suitability as marker of newly generated, soluble Aβ in plaque-bearing brain. This study supports the use of APP transgenic mice as translational models to characterize Aβ-lowering therapeutics.

Changes in Amyloid-β and Tau in the Cerebrospinal Fluid of Transgenic Mice Overexpressing Amyloid Precursor Protein

Changes in Aβ and Tau in the CSF of APP transgenic mice mirror the temporal sequence and magnitude of changes in these proteins in the CSF of Alzheimer’s disease patients. From Bedside Back to Bench The pathology of Alzheimer’s disease (AD) is thought to start 10 to 20 years before the onset of the first clinical symptoms in both sporadic and familial forms of the disease. Thus, disease-modifying drugs will most likely be effective when given at a preclinical stage of disease before neurodegeneration has become severe enough to induce clinical symptoms. Amyloid-β (Aβ) peptide and Tau protein, the constituents of the pathological hallmarks of AD, amyloid plaques and neurofibrillary tangles, respectively, have shown promise as markers in cerebrospinal fluid (CSF) of early, preclinical AD. Transgenic mice overexpressing human amyloid precursor protein (APP) have been used to model Aβ pathology, but CSF markers have not been investigated. To this end, Maia et al. optimized methods for collecting mouse CSF and validated sensitive assays for detecting Aβ peptides and total Tau in mouse CSF. They then used these assays to measure Aβ peptides and total Tau in the CSF of two different APP transgenic mouse models, with different ages of onset and progression of Aβ pathology. They found that the Aβ42 concentration in mouse CSF decreased as Aβ deposition started and that CSF t-Tau increased when amyloid plaques in mouse brain became prominent. Mechanistically, these results suggest that Aβ deposition in the brain may be the driving force for changes in Aβ and Tau in the CSF of AD patients. They also suggest the translational value of APP mouse models for understanding events in human disease. Altered concentrations of amyloid-β (Aβ) peptide and Tau protein in the cerebrospinal fluid (CSF) are thought to be predictive markers for Alzheimer’s disease (AD). Transgenic mice overexpressing human amyloid precursor protein (APP) have been used to model Aβ pathology, but concomitant changes in Aβ and Tau in CSF have been less well studied. We measured Aβ and Tau in the brains and CSF of two well-characterized transgenic mouse models of AD: one expressing human APP carrying the Swedish mutation (APP23) and the other expressing mutant human APP and mutant human presenilin-1 (APPPS1). Both mouse models exhibit Aβ deposition in the brain, but with different onset and progression trajectories. We found an age-related 50 to 80% decrease in Aβ42 peptide in mouse CSF and a smaller decrease in Aβ40, both inversely correlated with the brain Aβ load. Surprisingly, the same mice showed a threefold increase in total endogenous murine Tau in CSF at the stages when Aβ pathology became prominent. The results mirror the temporal sequence and magnitude of Aβ and Tau changes in the CSF of patients with sporadic and dominantly inherited AD. This observation indicates that APP transgenic mice may be useful as a translational tool for predicting changes in Aβ and Tau markers in the CSF of AD patients. These findings also suggest that APP transgenic mouse models may be useful in the search for new disease markers for AD.

Staged decline of neuronal function in vivo in an animal model of Alzheimer's disease

The accumulation of amyloid-β in the brain is an essential feature of Alzheimers disease. However, the impact of amyloid-β-accumulation on neuronal dysfunction on the single cell level in vivo is poorly understood. Here we investigate the progression of amyloid-β load in relation to neuronal dysfunction in the visual system of the APP23×PS45 mouse model of Alzheimers disease. Using in vivo two-photon calcium imaging in the visual cortex, we demonstrate that a progressive deterioration of neuronal tuning for the orientation of visual stimuli occurs in parallel with the age-dependent increase of the amyloid-β load. Importantly, we find this deterioration only in neurons that are hyperactive during spontaneous activity. This impairment of visual cortical circuit function also correlates with pronounced deficits in visual-pattern discrimination. Together, our results identify distinct stages of decline in sensory cortical performance in vivo as a function of the increased amyloid-β-load.

Expression of complement system components during aging and amyloid deposition in APP transgenic mice

BackgroundA causal role of the complement system in Alzheimers disease pathogenesis has been postulated based on the identification of different activated components up to the membrane attack complex at amyloid plaques in brain. However, histological studies of amyloid plaque bearing APP transgenic mice provided only evidence for an activation of the early parts of the complement cascade. To better understand the contribution of normal aging and amyloid deposition to the increase in complement activation we performed a detailed characterization of the expression of the major mouse complement components.MethodsAPP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 mice were used at different ages. mRNA was quantified by Realtime PCR and the age- as well as amyloid induced changes determined. The protein levels of complement C1q and C3 were analysed by Western blotting. Histology was done to test for amyloid plaque association and activation of the complement cascade.ResultsHigh mRNA levels were detected for C1q and some inhibitory complement components. The expression of most activating components starting at C3 was low. Expression of C1q, C3, C4, C5 and factor B mRNA increased with age in control C57BL/6 mice. C1q and C3 mRNA showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was confirmed on the protein level using Western blotting, whereas immunohistology indicated a recruitment of complement to amyloid plaques up to the C3 convertase.ConclusionEarly but not late components of the mouse complement system show an age-dependent increase in expression. The response to amyloid deposition is comparatively smaller. The low expression of C3 and C5 and failure to upregulate C5 and downstream components differs from human AD brain and likely contributes to the lack of full complement activation in APP transgenic mice.

Transgenic Expression of Intraneuronal Aβ42 But Not Aβ40 Leads to Cellular Aβ Lesions, Degeneration, and Functional Impairment without Typical Alzheimer's Disease Pathology

An early role of amyloid-β peptide (Aβ) aggregation in Alzheimers disease pathogenesis is well established. However, the contribution of intracellular or extracellular forms of Aβ to the neurodegenerative process is a subject of considerable debate. We here describe transgenic mice expressing Aβ1–40 (APP47) and Aβ1–42 (APP48) with a cleaved signal sequence to insert both peptides during synthesis into the endoplasmic reticulum. Although lower in transgene mRNA, APP48 mice reach a higher brain Aβ concentration. The reduced solubility and increased aggregation of Aβ1–42 may impair its degradation. APP48 mice develop intracellular Aβ lesions in dendrites and lysosomes. The hippocampal neuron number is reduced already at young age. The brain weight decreases during aging in conjunction with severe white matter atrophy. The mice show a motor impairment. Only very few Aβ1–40 lesions are found in APP47 mice. Neither APP47 nor APP48 nor the bigenic mice develop extracellular amyloid plaques. While intracellular membrane expression of Aβ1–42 in APP48 mice does not lead to the AD-typical lesions, Aβ aggregates develop within cells accompanied by considerable neurodegeneration.

Increased CSF Aβ during the very early phase of cerebral Aβ deposition in mouse models

Abnormalities in brains of Alzheimers disease (AD) patients are thought to start long before the first clinical symptoms emerge. The identification of affected individuals at this ‘preclinical AD’ stage relies on biomarkers such as decreased levels of the amyloid‐β peptide (Aβ) in the cerebrospinal fluid (CSF) and positive amyloid positron emission tomography scans. However, there is little information on the longitudinal dynamics of CSF biomarkers, especially in the earliest disease stages when therapeutic interventions are likely most effective. To this end, we have studied CSF Aβ changes in three Aβ precursor protein transgenic mouse models, focusing our analysis on the initial Aβ deposition, which differs significantly among the models studied. Remarkably, while we confirmed the CSF Aβ decrease during the extended course of brain Aβ deposition, a 20–30% increase in CSF Aβ40 and Aβ42 was found around the time of the first Aβ plaque appearance in all models. The biphasic nature of this observed biomarker changes stresses the need for longitudinal biomarker studies in the clinical setting and the search for new ‘preclinical AD’ biomarkers at even earlier disease stages, by using both mice and human samples. Ultimately, our findings may open new perspectives in identifying subjects at risk for AD significantly earlier, and in improving the stratification of patients for preventive treatment strategies.

The Swedish APP mutation alters the effect of genetically reduced BACE1 expression on the APP processing

J. Neurochem. (2011) 119, 231–239.