Heterologous expression of the functional amyloid beta (Aβ) antibody β1 in the central nervous system was engineered to maximize antibody exposure in the brain and assess the effects on Aβ production and accumulation in these conditions. A single open reading frame encoding the heavy and light chains of β1 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 β1 administration were obtained. Similar brain and plasma β1 concentrations indicated robust antibody efflux from the brain. In preplaque mice, β1 formed a complex with Aβ that caused a modest Aβ increase in brain and plasma. At 11 months of age, β1 expression reduced amyloid by 97% compared with age-matched APP23 mice. Interference of β1 with β-secretase cleavage of amyloid precursor protein was relatively small. Our data suggest that severely impaired amyloid formation was primarily mediated by a complex of β1 with soluble Aβ, which might have prevented Aβ aggregation or favored transport out of the brain.

An early role of amyloid-β peptide (Aβ) aggregation in Alzheimer's 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 Aβ 1–40 (APP47) and Aβ 1–42 (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 Aβ 1–42 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 Aβ 1–40 lesions are found in APP47 mice. Neither APP47 nor APP48 nor the bigenic mice develop extracellular amyloid plaques. While intracellular membrane expression of Aβ 1–42 in APP48 mice does not lead to the AD-typical lesions, Aβ aggregates develop within cells accompanied by considerable neurodegeneration.

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.

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.

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.

Immunization against amyloid-β (Aβ) 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 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 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 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.

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 understoodMethods We analyzed the dynamic changes of amyloid beta (A beta) levels in plasma brain and cerebrospinal fluid (CSF) as well as cerebral amyloid binding by A beta antibody after a single beta 1 antibody infusion into APP(Swedish) and APP(wildtype) transgenic mice at preplaque and plaque bearing ageResults Following intravenous A beta antibody treatment plasma A beta increased rapidly reaching significantly higher levels in preplaque compared with plaque bearing mice whereas cerebral and CSF A beta remained unchanged Strikingly A beta antibodies exhibited strong cerebral amyloid plaque binding rapidly after intravenous administration in a subset of animals with more severe vascular amyloidConclusions Rapid plasma A beta increase after A beta antibody infusion results primarily from stabilization of A beta Nevertheless the smaller plasma A beta 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

In addition to parenchymal amyloid plaques most brains from AD patients contain vascular amyloid, which is considered a risk factor for microhemorrhage. Previous investigations with Aβ antibodies showed variable results regarding their effect on vascular amyloid and microhemorrhages. In this study we have investigated the active Aβ immunotherapy CAD106 for its effect on vascular Aβ and microhemorrhages under conditions resulting in different parenchymal amyloid reduction. The APP transgenic mice used, APP23 and APP24, express human APP751 with the Swedish or the Swedish plus London mutations under control of the murine Thy1-promoter approximately 7 and 3.5-fold over endogenous APP. Accordingly, APP24 mice generate less Aβ but show a higher Aβ42/40 ratio than APP23. Amyloid deposition in the parenchyma and the vasculature is lower as well. Mice of both lines were immunized with CAD106 (Aβ1-6 coupled to the virus-like particle of E. coli phage Qβ) at various ages and for various times. Brain Aβ levels, amyloid plaque load, vascular Aβ and microhemorrhages were determined using standard methods. Following immunization with CAD106, vascular amyloid deposition was not significantly changed in APP23 even if a significant parenchymal amyloid reduction was achieved. In APP24 mice vascular amyloid was increased together with very strong parenchymal plaque reduction but remained unchanged under conditions of low amyloid plaque reduction. The elevation of vascular amyloid primarily resulted from deposition of Aβ42, the main Aβ isoform reduced in the parenchyma. It was deposited in a non-fibrillar (Congo red negative) form. Microhemorrhages were found in aged APP23 mice as described but were very rare in APP24. Neither their frequency nor their severity was changed following CAD06 immunization of both mouse lines. An increase in vascular Aβ occurs in animals showing an efficient reduction of parenchymal amyloid following CAD106 immunization. This elevation is not accompanied by an increase in hemorrhages possibly because the vascular Aβ is deposited in a diffuse, non-fibrillar form.

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.

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.

A role of the complement system in Alzheimer's disease pathogenesis has been postulated. Using histology, complement components have been detected in association with amyloid deposits in AD brain which included markers for activation up to the membrane attack complex. In vitro, fibrillar Aβ was demonstrated to activate complement suggesting an induction of the system by amyloid deposits in vivo. However, histological studies of amyloid containing APP transgenic mice have not replicated the findings in human brain, which may be due to a lack of expression, activation or antibody detection of mouse complement. On the other hand, genetic modification of the complement system in mice led to complex results regarding its role in the disease process. The expression of complement components and their induction by amyloid deposition was analyzed in mouse brain to better understand the observed differences to human AD brain. We also analyzed for complement activation. APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 and BUB/BnJ mice were used at different ages. mRNA was quantified by Realtime PCR. Western blotting and histology was according to standard protocols. While high mRNA levels were detected for early (in particular C1q) and several inhibitory complement components, expression of downstream components (in particular C9) was low. No major difference was found between the mouse strains and similar expression levels were observed with human brain material. Expression of most complement components increased with age in control mice. Several of these, most notably C1q and C3, showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was also detected on the protein level using Western blotting. In line with these results histology demonstrated amyloid plaque–associated C1q and C3 as well as C3d indicating activation of C3 convertase. These data show that the mouse complement system responds to amyloid deposition with increased expression and activation. The failure to detect activation of downstream components may be due to the lack of suited antibodies or a block of complete activation e.g. by the abundant inhibitory components.

Neurofibrillary tangles and amyloid deposits are the defining hallmarks for AD. The amyloid cascade hypothesis predicts that tau pathology is secondary to amyloid aggregation. In agreement, an enhancement of tau pathology has been described in higher brain regions of transgenic mice expressing both, mutant tau and APP as compared to mice carrying the tau transgene only. To further study the interaction of the amyloid and tau pathologies we developed bigenic mouse lines and investigated the time course of tau and amyloid deposition. TAU58/2 mice expressing mutated human (0N4R P301S) under the control of the Thy–1 promoter were generated. Mice were crossbred with APP23 (Thy1–APPSWE) and APP51/16 (Thy1–APP wildtype) mice and analyzed histologically and by Western blotting. At 6 months of age, APP23xTAU58/2 mice showed an at least fivefold increase of Gallyas positive neurons in neocortex, compared to the single transgene TAU58/2 mice. Neurodegeneration was seen in brain sections stained with the phosphorylation–dependent neurofilament antibody SMI310 and ubiquitin. Numerous axonal dilatations (axonal spheroids) and degenerating neurons were visible in neocortex, midbrain, brain stem and spinal cord. Amyloid pathology was not detectable or very minor at this time point. These data indicate that an enhancement of tau deposition does not require Aβ deposition but may be induced by soluble Aβ species or by APP overexpression.

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-β40 peptide (Aβ40) to Aβ42 was significantly higher than that seen in APPwt mice or AD human brain. Genetically shifting the ratio of AβDutch40/AβDutch42 toward AβDutch42 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 Aβ 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 Aβ to vascular amyloid pathology and emphasizing the differing roles of Aβ40 and Aβ42 in vascular and parenchymal amyloid pathology.

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.

Lesions can affect the nervous system in many different ways and lead to more or less serious consequences. Our approach has over a period of 15 years been to study the effect of injury on dorsal root ganglion (DRG) neurons projecting into the sciatic nerve and to the dorsal horn of the spinal cord. The most simple approach is to completely transect the sciatic nerve and study the consequences of this injury in the parent cell bodies in the DRGs. This model may have some significance for our understanding of the mechanisms underlying pain that emerges in some patients after various types of surgical procedures, so called neuropathic pain (see 1). It has also been suggested that this model may be relevant for studying neurodegeneration (see refs. in 2). Our work on DRGs has led us to ask questions to what extent the changes we see in DRG neurons occur also in other neuronal systems. In fact, peptide regulation occurs in several systems in the brain in response to nerve injury (see 3). We have then turned our attention to established neurodegenerative diseases. In particular, we have analysed some mouse models for Alzheimer’s disease (AD), as will be described below.

Idiopathic Parkinson's Disease (PD), dementia with Lewy bodies (DLB), and a Lewy Body variant of Alzheimer's Disease (LBVAD) are characterized pathologically by proteinaceous inclusions in neurons commonly referred to as Lewy pathology inpost mortembrain tissue samples1–6. The inclusions occur in the dystrophic (Lewy) neurites that constitute an important part of the pathology of PD and DLB4–6, in neuronal perikarya (Lewy bodies), and occasionally extracellularly11.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.

The vascular endothelial growth factor (VEGF) has been shown to be upregulated in acute hypoxia. Although an increase in blood vessel number has been described in severe chronic brain hypoxia, it is unclear whether VEGF is upregulated in this condition. We therefore investigated male inbred Wistar rats, which were exposed for 9 to 13 weeks to decreasing amounts of oxygen, down to 7% O2 (15%: 15 days; 12%, 10%, respectively; 8%: 1 day, 3 weeks, respectively; 7%: 4 weeks). The expression of VEGF was studied by Northern analysis and in situ hybridization in frozen sections of cerebral cortex, hippocampus and cerebellum in six chronic hypoxic and two control rats. We found a marked upregulation of VEGF mRNA in all brain regions investigated, being strongest in cerebral cortex and cerebellum. Our results suggest a potential role of VEGF for vascular growth and vascular permeability observed in chronic cerebral hypoxia.