Transgenic expression of ß1 antibody in brain neurons impairs age-dependent amyloid deposition in APP23 mice.

Heterologous expression of the functional amyloid beta (Aß) antibody ß1 in the central nervous system was engineered to maximize antibody exposure in the brain and assess the effects on Aß production and accumulation in these conditions. A single open reading frame encoding the heavy and light chains of ß1 linked by the mouth and foot virus peptide 2A was expressed in brain neurons of transgenic mice. Two of the resulting BIN66 transgenic lines were crossed with APP23 mice, which develop severe central amyloidosis. Brain concentrations at steady-state 5 times greater than those found after peripheral ß1 administration were obtained. Similar brain and plasma ß1 concentrations indicated robust antibody efflux from the brain. In preplaque mice, ß1 formed a complex with Aß that caused a modest Aß increase in brain and plasma. At 11 months of age, ß1 expression reduced amyloid by 97% compared with age-matched APP23 mice. Interference of ß1 with ß-secretase cleavage of amyloid precursor protein was relatively small. Our data suggest that severely impaired amyloid formation was primarily mediated by a complex of ß1 with soluble Aß, which might have prevented Aß aggregation or favored transport out of the brain.

High LRRK2 levels fail to induce or exacerbate neuronal alpha-synucleinopathy in mouse brain.

The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson's disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy.

Transgenic expression of intraneuronal Aß42 but not Aß40 leads to cellular Aß lesions, degeneration, and functional impairment without typical Alzheimer's disease pathology.

An early role of amyloid-ß peptide (Aß) aggregation in Alzheimer's disease pathogenesis is well established. However, the contribution of intracellular or extracellular forms of Aß to the neurodegenerative process is a subject of considerable debate. We here describe transgenic mice expressing Aß1-40 (APP47) and Aß1-42 (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 Aß concentration. The reduced solubility and increased aggregation of Aß1-42 may impair its degradation. APP48 mice develop intracellular Aß 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 Aß1-40 lesions are found in APP47 mice. Neither APP47 nor APP48 nor the bigenic mice develop extracellular amyloid plaques. While intracellular membrane expression of Aß1-42 in APP48 mice does not lead to the AD-typical lesions, Aß aggregates develop within cells accompanied by considerable neurodegeneration.

Neuropathology in mice expressing mouse alpha-synuclein.

α-Synuclein (αSN) in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD) and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN), which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occurred in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are strikingly similar to those evoked by expression of human wildtype or mutant forms.

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.

The second-generation active Aß immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects.

Immunization against amyloid-ß (Aß) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimer's 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 Aß1-6 coupled to the virus-like particle Qß. Immunization with this vaccine did not activate Aß-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aß antibody titers of different IgG subclasses mainly recognizing the Aß3-6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aß42 and Aß40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aß, which atypically was nonfibrillar. The efficacy of Aß immunotherapy depended on the Aß 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 Alzheimer's 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 Aß monomers and oligomers and blocked Aß toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aß aggregation and its downstream detrimental effects.

Rapid cerebral amyloid binding by Aß antibodies infused into ß-amyloid precursor protein transgenic mice.

BACKGROUND: Passive immunization for the treatment of Alzheimer's disease (AD) was rapidly translated into clinical trials. However, basic mechanisms of AD immunotherapy remain only partially understood. METHODS: We analyzed the dynamic changes of amyloid-ß (Aß) levels in plasma, brain, and cerebrospinal fluid (CSF) as well as cerebral amyloid binding by Aß antibody after a single ß1-antibody infusion into APP(Swedish) and APP(wildtype) transgenic mice at preplaque and plaque-bearing age. RESULTS: Following intravenous Aß antibody treatment, plasma Aß increased rapidly, reaching significantly higher levels in preplaque compared with plaque-bearing mice, whereas cerebral and CSF Aß remained unchanged. Strikingly, Aß antibodies exhibited strong cerebral amyloid plaque binding rapidly after intravenous administration in a subset of animals with more severe vascular amyloid. CONCLUSIONS: Rapid plasma Aß increase after Aß antibody infusion results primarily from stabilization of Aß. Nevertheless, the smaller plasma Aß increase in plaque-bearing mice might be of diagnostic use. Importantly, intravenously administered antibodies can rapidly bind to cerebral plaques, potentially facilitated by vascular-amyloid-mediated damage of the blood-brain barrier.

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

BACKGROUND: A causal role of the complement system in Alzheimer's 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. METHODS: APP23 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. RESULTS: High 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. CONCLUSION: Early 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.

Dynamics of Abeta turnover and deposition in different beta-amyloid precursor protein transgenic mouse models following gamma-secretase inhibition.

Human beta-amyloid precursor protein (APP) transgenic mice are commonly used to test potential therapeutics for Alzheimer's disease. We have characterized the dynamics of beta-amyloid (Abeta) generation and deposition following gamma-secretase inhibition with compound LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(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 Abeta pool in brain with rapid turnover (half-lives for Abeta40 and Abeta42 were 0.7 and 1.7 h) and a much more stable, less soluble pool. Abeta in cerebrospinal fluid (CSF) reflected the changes in the soluble brain Abeta pool, whereas plasma Abeta turned over more rapidly. In brain, APP C-terminal fragments (CTF) accumulated differentially. The half-lives for gamma-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 gamma-secretase inhibition regardless of the familial Alzheimer's disease mutations in APP. Amyloid deposition started with Abeta42, whereas Abeta38 and Abeta40 continued to turn over. Chronic gamma-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. gamma-Secretase inhibition led to a redistribution of intracellular Abeta and an elevation of CTFs in neuronal fibers. In CSF, Abeta showed a similar turnover as in preplaque animals demonstrating its suitability as marker of newly generated, soluble Abeta in plaque-bearing brain. This study supports the use of APP transgenic mice as translational models to characterize Abeta-lowering therapeutics.

Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis.

The E693Q mutation in the amyloid beta precursor protein (APP) leads to cerebral amyloid angiopathy (CAA), with recurrent cerebral hemorrhagic strokes and dementia. In contrast to Alzheimer disease (AD), the brains of those affected by hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) show few parenchymal amyloid plaques. We found that neuronal overexpression of human E693Q APP in mice (APPDutch mice) caused extensive CAA, smooth muscle cell degeneration, hemorrhages and neuroinflammation. In contrast, overexpression of human wild-type APP (APPwt mice) resulted in predominantly parenchymal amyloidosis, similar to that seen in AD. In APPDutch mice and HCHWA-D human brain, the ratio of the amyloid-beta40 peptide (Abeta40) to Abeta42 was significantly higher than that seen in APPwt mice or AD human brain. Genetically shifting the ratio of AbetaDutch40/AbetaDutch42 toward AbetaDutch42 by crossing APPDutch mice with transgenic mice producing mutated presenilin-1 redistributed the amyloid pathology from the vasculature to the parenchyma. The understanding that different Abeta species can drive amyloid pathology in different cerebral compartments has implications for current anti-amyloid therapeutic strategies. This HCHWA-D mouse model is the first to develop robust CAA in the absence of parenchymal amyloid, highlighting the key role of neuronally produced Abeta to vascular amyloid pathology and emphasizing the differing roles of Abeta40 and Abeta42 in vascular and parenchymal amyloid pathology.

Neuropeptide alterations in the hippocampal formation and cortex of transgenic mice overexpressing beta-amyloid precursor protein (APP) with the Swedish double mutation (APP23).

The role of neuropeptides and the significance of peptidergic mechanisms in neurodegenerative diseases are still unclear. In the periphery, nerve injury results in dramatic changes in the expression of neuropeptides. An important question regards to what extent similar changes occur, and similar mechanisms operate, after lesions and/or degeneration in the brain. The purpose of this work is, therefore, to study neuropeptides with regard to their presence and distribution in the APP23 mouse (HuAPP(751) K670M/N671L under the murine Thy-1 promoter), a model for Alzheimer's disease, or cerebral amyloidosis, using the immunohistochemical technique. In addition, tyrosine hydroxylase and acetylcholinesterase were analyzed. This study shows marked neuropeptide changes in the hippocampal formation and the ventral cortex, whereas the dorsolateral neocortex was less affected. There was a considerable variation with regard to peptide expression among animals of the same age which was related to the variation in Abeta deposition. Dystrophic and varicose fibers containing galanin, neuropeptide Y, enkephalin, and especially cholecystokinin were commonly seen in close proximity to amyloid plaques. In addition, generalized changes were observed, such as increases of enkephalin and neuropeptide Y in stratum lacunosum moleculare and of neuropeptide Y, enkephalin, and dynorphin in mossy fibers. In contrast, cholecystokinin was decreased in mossy fibers. Comparatively small differences were observed between wild-type and transgenic mice with regard to tyrosine hydroxylase (noradrenergic but also dopaminergic fibers) and acetylcholine esterase (mainly cholinergic fibers). The increase of neuropeptides in dystrophic fibers in this model may represent a response to nerve injury caused by the amyloid accumulation and may reflect attempts to counteract degeneration by initiating protective and/or regenerative processes.

Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid.

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartyl protease and is responsible for the beta-secretase cleavage of APP producing different endoproteolytic fragments referred to as the carboxy-terminal C99, C89 and the soluble ectodomain sAPPbeta. Here we describe two transgenic mouse lines expressing human BACE in the brain. Overexpression of BACE augments the amyloidogenic processing of APP as demonstrated by decreased levels of full-length APP and increased levels of C99 and C89 in vivo. In mice expressing huBACE in addition to human APP wild-type or carrying the Swedish mutation, the induction of APP processing characterized by elevated C99, C89 and sAPPbeta, results in increased brain levels of beta-amyloid peptides Abeta40 and Abeta42 at steady-state.