Brains Of APP Transgenic Mice Research Articles

Deficiency of IKKβ in neurons ameliorates Alzheimer's disease pathology in APP- and tau-transgenic mice.

In Alzheimer's disease (AD) brain, inflammatory activation regulates protein levels of amyloid-β-peptide (Aβ) and phosphorylated tau (p-tau), as well as neurodegeneration; however, the regulatory mechanisms remain unclear. We constructed APP- and tau-transgenic AD mice with deletion of IKKβ specifically in neurons, and observed that IKKβ deficiency reduced cerebral Aβ and p-tau, and modified inflammatory activation in both AD mice. However, neuronal deficiency of IKKβ decreased apoptosis and maintained synaptic proteins (e.g., PSD-95 and Munc18-1) in the brain and improved cognitive function only in APP-transgenic mice, but not in tau-transgenic mice. Additionally, IKKβ deficiency decreased BACE1 protein and activity in APP-transgenic mouse brain and cultured SH-SY5Y cells. IKKβ deficiency increased expression of PP2A catalytic subunit isoform A, an enzyme dephosphorylating cerebral p-tau, in the brain of tau-transgenic mice. Interestingly, deficiency of IKKβ in neurons enhanced autophagy as indicated by the increased ratio of LC3B-II/I in brains of both APP- and tau-transgenic mice. Thus, IKKβ deficiency in neurons ameliorates AD-associated pathology in APP- and tau-transgenic mice, perhaps by decreasing Aβ production, increasing p-tau dephosphorylation, and promoting autophagy-mediated degradation of BACE1 and p-tau aggregates in the brain. However, IKKβ deficiency differently protects neurons in APP- and tau-transgenic mice. Further studies are needed, particularly in the context of interaction between Aβ and p-tau, before IKKβ/NF-κB can be targeted for AD therapies.

Collagenous Alzheimer amyloid plaque component impacts on the compaction of amyloid-\u03b2 plaques

Massive deposition of amyloid β peptides (Aβ) as senile plaques (SP) characterizes the brain pathology of Alzheimer’s disease (AD). SPs exhibit a variety of morphologies, although little is known about the SP components that determine their morphology. Collagenous Alzheimer amyloid plaque component (CLAC) is one of the major non-Aβ proteinaceous components of SP amyloid in AD brains. Here we show that overexpression of CLAC precursor (CLAC-P) in the brains of APP transgenic mice results in a significant remodeling of amyloid pathology, i.e., reduction in diffuse-type amyloid plaques and an increase in compact plaques laden with thioflavin S-positive amyloid cores. In vivo microdialysis revealed a significant decrease in Aβ in the brain interstitial fluid of CLAC-P/APP double transgenic mice compared with APP transgenic mice. These findings implicate CLAC in the compaction of Aβ in amyloid plaques and the brain dynamics of Aβ.

A novel BACE inhibitor NB-360 shows a superior pharmacological profile and robust reduction of amyloid-β and neuroinflammation in APP transgenic mice.

BackgroundAlzheimer’s disease (AD) is the most common form of dementia, the number of affected individuals is rising, with significant impacts for healthcare systems. Current symptomatic treatments delay, but do not halt, disease progression. Genetic evidence points to aggregation and deposition of amyloid-β (Aβ) in the brain being causal for the neurodegeneration and dementia typical of AD. Approaches to target Aβ via inhibition of γ-secretase or passive antibody therapy have not yet resulted in substantial clinical benefits. Inhibition of BACE1 (β-secretase) has proven a challenging concept, but recent BACE1inhibitors can enter the brain sufficiently well to lower Aβ. However, failures with the first clinical BACE1 inhibitors have highlighted the need to generate compounds with appropriate efficacy and safety profiles, since long treatment periods are expected to be necessary in humans.ResultsTreatment with NB-360, a potent and brain penetrable BACE-1 inhibitor can completely block the progression of Aβ deposition in the brains of APP transgenic mice, a model for amyloid pathology. We furthermore show that almost complete reduction of Aβ was achieved also in rats and in dogs, suggesting that these findings are translational across species and can be extrapolated to humans. Amyloid pathology may be an initial step in a complex pathological cascade; therefore we investigated the effect of BACE-1 inhibition on neuroinflammation, a prominent downstream feature of the disease. NB-360 stopped accumulation of activated inflammatory cells in the brains of APP transgenic mice. Upon chronic treatment of APP transgenic mice, patches of grey hairs appeared.ConclusionsIn a rapidly developing field, the data on NB-360 broaden the chemical space and expand knowledge on the properties that are needed to make a BACE-1 inhibitor potent and safe enough for long-term use in patients. Due to its excellent brain penetration, reasonable oral doses of NB-360 were sufficient to completely block amyloid-β deposition in an APP transgenic mouse model. Data across species suggest similar treatment effects can possibly be achieved in humans. The reduced neuroinflammation upon amyloid reduction by NB-360 treatment supports the notion that targeting amyloid-β pathology can have beneficial downstream effects on the progression of Alzheimer’s disease.

Transport of cargo from periphery to brain by circulating monocytes

The misfolding and aggregation of the Aβ peptide – a fundamental event in the pathogenesis of Alzheimer׳s disease – can be instigated in the brains of experimental animals by the intracranial infusion of brain extracts that are rich in aggregated Aβ. Recent experiments have found that the peripheral (intraperitoneal) injection of Aβ seeds induces Aβ deposition in the brains of APP-transgenic mice, largely in the form of cerebral amyloid angiopathy. Macrophage-type cells normally are involved in pathogen neutralization and antigen presentation, but under some circumstances, circulating monocytes have been found to act as vectors for the transport of pathogenic agents such as viruses and prions. The present study assessed the ability of peripheral monocytes to transport Aβ aggregates from the peritoneal cavity to the brain. Our initial experiments showed that intravenously delivered macrophages that had previously ingested fluorescent nanobeads as tracers migrate primarily to peripheral organs such as spleen and liver, but that a small number also reach the brain parenchyma. We next injected CD45.1-expressing monocytes from donor mice intravenously into CD45.2-expressing host mice; after 24h, analysis by fluorescence-activated cell sorting (FACS) and histology confirmed that some CD45.1 monocytes enter the brain, particularly in the superficial cortex and around blood vessels. When the donor monocytes are first exposed to Aβ-rich brain extracts from human AD cases, a subset of intravenously delivered Aβ-containing cells migrate to the brain. These experiments indicate that, in mouse models, circulating monocytes are potential vectors by which exogenously delivered, aggregated Aβ travels from periphery to brain, and more generally support the hypothesis that macrophage-type cells can participate in the dissemination of proteopathic seeds.

Peptides of Presenilin-1 Bind the Amyloid Precursor Protein Ectodomain and Offer a Novel and Specific Therapeutic Approach to Reduce ß-Amyloid in Alzheimer’s Disease

β-Amyloid (Aβ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer’s disease (AD). Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP) and Presenilin (PS), as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

A potential function for neuronal exosomes: Sequestering intracerebral amyloid-β peptide

Elevated amyloid-β peptide (Aβ) in brain contributes to Alzheimer’s disease (AD) pathogenesis. We demonstrated the presence of exosome-associated Aβ in the cerebrospinal fluid (CSF) of cynomolgus monkeys and APP transgenic mice. The levels of exosome-associated Aβ notably decreased in the CSF of aging animals. We also determined that neuronal exosomes, but not glial exosomes, had abundant glycosphingolipids and could capture Aβ. Infusion of neuronal exosomes into brains of APP transgenic mice decreased Aβ and amyloid depositions, similarly to what reported previously on neuroblastoma-derived exosomes. These findings highlight the role of neuronal exosomes in Aβ clearance, and suggest that their downregulation might relate to Aβ accumulation and, ultimately, the development of AD pathology.

Aβ seeds resist inactivation by formaldehyde.

Cerebral β-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated β-amyloid (Aβ) into young, pre-depositing Aβ precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aβ itself. Here we report that the β-amyloid-inducing activity of Alzheimer's disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aβ are maintained in fixed tissues. The resistance of Aβ seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aβ aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Xanthoceraside rescues learning and memory deficits through attenuating beta-amyloid deposition and tau hyperphosphorylation in APP mice

Xanthoceraside, a triterpenoid saponin, has been shown to reverse cognitive deficits in several Alzheimer's disease (AD) animal models. However, the effects of xanthoceraside on the Aβ deposition pathology and the APP processing in AD are unclear. Here, we show that xanthoceraside at doses of 0.08 and 0.32mg/kg/d for 6 months significantly improved learning and memory impairment in APP transgenic mice assessed by the Y maze and novel object recognition tests. Immunohistochemical analyses revealed that xanthoceraside strongly attenuated β-amyloid deposition in the brains of APP transgenic mice. Western blotting revealed that xanthoceraside decreased tau phosphorylation protein levels at Ser396 and Ser404 in the hippocampus; xanthoceraside also decreased APP protein levels and GSK-3β phosphorylation. These results suggest that xanthoceraside could be a promising novel candidate for the therapy of AD.

Tenascin-C deficiency ameliorates Alzheimer's disease-related pathology in mice

Alzheimer's disease (AD) is a neurodegenerative disease characterized by deposits of amyloid β peptide (Aβ) and microglia-driven inflammatory activation. Tenascin-C (tnc) is an extracellular matrix protein that is upregulated in inflammation and induces further inflammatory responses. We hypothesized that tnc contributes to the inflammatory pathology in AD. Using real-time polymerase chain reaction, we observed that tnc gene transcription was upregulated in cultured microglia after Aβ challenge and in the brain of an AD mouse model that overexpresses mutated amyloid precursor protein (APP) in neural cells. By cross-breeding APP-transgenic mice and tenascin–C-deficient mice, we demonstrated using real-time polymerase chain reaction, Western blot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry that tnc deficiency reduces pro- but enhances anti-inflammatory activation in the mutated APP-transgenic mouse brain, associated with a reduced cerebral Aβ load and higher levels of the postsynaptic density protein 95. Thus, our study indicates that functional inhibition of tnc exerts beneficial effects on AD pathogenesis, suggesting a potential for tnc as a new therapeutic target in AD.

Aβ43 Is the Earliest-Depositing Aβ Species in APP Transgenic Mouse Brain and Is Converted to Aβ41 by Two Active Domains of ACE

Amyloid-β protein (Aβ) varies in length at its carboxyl terminus. The longer Aβ species, Aβ43 and Aβ42, are highly amyloidogenic and deposit more frequently than Aβ40 in the brain of Alzheimer disease (AD) patients. However, the characterization of Aβ43 deposition in the brain and the relationship between Aβ43 and Aβ42 or Aβ40 remain unclear. We provide evidence that Aβ43 deposition appears earlier than Aβ42 and Aβ40 deposition in the brain of mutant amyloid precursor protein transgenic (APPtg) mice, suggesting that Aβ43 is the earliest-depositing species. In addition, we found increased Aβ43 levels and Aβ43/Aβ42 ratios in the serum of AD patients, suggesting their use as diagnostic blood biomarkers for AD. We further show that angiotensin-converting enzyme (ACE) converts Aβ43 to Aβ41. Notably, this Aβ43-to-Aβ41 converting activity requires two active domains of ACE. Inhibition of ACE activity significantly enhanced Aβ43 deposition in APPtg mouse brain. Our results suggest that Aβ43 is the earliest-depositing species in brain parenchyma and that Aβ43 may trigger later Aβ42 and Aβ40 deposition or may be converted to Aβ42 and Aβ40 plaques. Activities of both ACE domains may be important for reducing Aβ43 levels in serum and reducing brain Aβ43 deposition.

Bivalent Ligand Containing Curcumin and Cholesterol as a Fluorescence Probe for Aβ Plaques in Alzheimer’s Disease

A recently developed bivalent ligand BMAOI 14 (7) has been evaluated for its capability to label and detect aggregated β-amyloid (Aβ) peptide as a fluorescent probe. This probe contains curcumin as the Aβ recognition moiety and cholesterol as an anchorage to the neuronal cell membrane/lipid rafts. The results demonstrate that 7 binds to the monomers, oligomers as well as fibrils of Aβ42 with low micromolar to submicromolar binding affinities. This chemical probe also has many of the required optical properties for use in imaging and can rapidly cross the blood-brain barrier (BBB) in vivo. Furthermore, 7 specifically binds to Aβ plaques in both AD human patients and APP transgenic mouse brain tissues. Collectively, these results suggest that 7 is a strong candidate as an Aβ-imaging agent and encourage further optimization of 7 as a new lead to develop the next generation of Aβ-imaging probes.

The distribution profile and oxidation states of biometals in APP transgenic mouse brain: dyshomeostasis with age and as a function of the development of Alzheimer's disease

The enrichment of transition metals in the brain and the dyshomeostasis of metals are thought to be important etiological factors for elderly people in a number of neurodegenerative diseases, including Alzheimer's disease (AD). However, the understanding of how biometals dynamically dysregulate in the stages of AD development, such as the exact time-dependent and site-dependent accumulation in the brain with AD progression, is still limited. Herein, by using the APP/V717I transgenic mouse model and age-matched mice as control, we offer distinctive in situ and quantitative images of metals (Cu, Fe, Zn and Ca) in brain sections by synchrotron radiation micro beam X-ray fluorescence (SR-μXRF). The images show that Fe and Ca increased with brain aging in both AD and control (CNT) mice, and Cu, Fe, Zn and Ca appeared significantly elevated in AD mice and showed an obvious age-dependent rise. Fe, Cu and Zn were obviously specifically enriched in the cortex and hippocampus, which were also the plaque-formation sensitive brain regions. Our results demonstrate that the enrichment of transition metals with age and metals' dyshomeostasis in specific regions may contribute together to the etiology and development of AD in elderly people. The XANES measurements of Cu and Fe show evidence that Cu may have redox properties in the AD brain.

Chronic Intranasal Treatment with an Anti-Aβ30-42 scFv Antibody Ameliorates Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice.

Deposition of lactoferrin in fibrillar-type senile plaques in the brains of transgenic mouse models of Alzheimer's disease

We and others have previously reported that lactoferrin (LF), which acts as both an iron-binding protein and an inflammatory modulator, is strongly up-regulated in the brains of patients with Alzheimer's disease (AD). We have also studied the expression and localization of LF mRNA in the brain cortices of patients with AD. In this study, we investigated immunohistochemically the localization of LF in the brains of APP-transgenic mice, representing a model of AD. No LF immunoreactivity was detected in the brains of the wild-type mice. In the transgenic AD mice, LF deposition was detected in the brains. Double-immunofluorescence staining with antibodies directed against the amyloid-β peptide (Aβ) and LF localized the LF depositions to amyloid deposits (senile plaques) and regions of amyloid angiopathy. Senile plaque formation precedes LF deposition in AD. In the transgenic mice aged <18 months, most of senile plaques were negative for LF. LF deposits appeared weakly at about 18 months of age in these mice. Both the intensity and number of LF-positive depositions in the transgenic mice increased with age. Double-staining for LF and thioflavin-S revealed that LF accumulated in thioflavin-S-positive, fibrillar-type senile plaques. The up-regulation of LF in the brains of both AD patients and the transgenic mouse model of AD provides evidence of an important role for LF in AD-affected brain tissues.

Potent inhibition of ACE leads to Alzheimer-like pathology in APP transgenic mouse brain

Potent inhibition of ACE leads to Alzheimer-like pathology in APP transgenic mouse brain

Alzheimer-like pathology induced by potent ACE inhibition in APP transgenic mouse brain

Alzheimer-like pathology induced by potent ACE inhibition in APP transgenic mouse brain

P2-312: Human antibodies in intravenous immunoglobulin alter the assembly of amyloidogenic peptides into soluble oligomers

these 2 peptides following daily i.p. injections (50 mg/kg/day) significantly reduced fibrillar beta-amyloid protein load in brains of APP transgenic mice (containing the Swedish and London mutations) by 38-63% (as assessed by image analysis quantitation of Thioflavin S fluorescence and Congo red staining). One of these peptides, DP-74 also caused a marked and significant improvement (by 52%) in hippocampus-dependent memory determined by Morris water maze testing. Methods: In the present study, DP-74 was radiolabelled with tritium and the blood-brain-barrier (BBB) permeability and kinetics were determined. Results: Using capillary depletion analysis following 10 minutes of an i.v. injection of 3H-DP-74, the results demonstrated the apparent uptake was composed mainly of brain parenchymal permeation (with minimal capillary entrapment) indicating that the peptide crossed the BBB and entered the brain parenchyma. Stability studies indicated that 1 hour following i.v. administration intact 3H-DP-74 (analyzed by HPLC and mass spectroscopy) was completely stable in the circulation, and represented 45% of the radioactivity recovered in the brain homogenate. Following intranasal administration, 3H-DP-74 was identified in olfactory bulb, hypothalamus, cortex, subcortical regions, brainstem, cerebellum and spinal cord in a time and dose-dependent matter suggesting that intranasal delivery might be a viable option for administration using this peptide. Conclusions: Together, these results indicated the identification of a novel disease-modifying small peptide (DP-74) that crosses the BBB, and has the ability to reduce brain amyloid load and improve memory in APP transgenic animals. These studies suggest that small novel peptides show promise for the development of disease-modifying therapeutics for the prevention and treatment of AD and related disorders. Funded by ProteoTech and a SBIR Phase II Award (AG017787).

S5-03-03: Induction of cerebral beta-amyloidosis in transgenic mice

We have previously shown (Meyer-Lühmann et al., Science 313: 1781–84, 2006) that intracerebral injections of dilute, amyloid-beta (Abeta)-containing human or APP transgenic mouse brain extract can induce cerebral beta-amyloidosis and associated pathology in APP transgenic mice in a time- and concentration-dependent manner. After injecting extracts from APP/PS1 transgenic mice into APP hosts and vice versa, our results suggest the occurrence of polymorphic Abeta species, reminiscent of prion strains. We use dilute brain extracts as described in Meyer-Lühmann et al., Science 313: 1781–84, 2006. Results from ongoing experiments will be presented. The nature of the seeding agent and of the induced amyloid will be further characterized and peripheral routes of extract applications studied.

Cystatin C modulates cerebral β-amyloidosis

The CST3 Thr25 allele of CST3, which encodes cystatin C, leads to reduced cystatin C secretion and conveys susceptibility to Alzheimer's disease. Here we show that overexpression of human cystatin C in brains of APP-transgenic mice reduces cerebral amyloid-beta deposition and that cystatin C binds amyloid-beta and inhibits its fibril formation. Our results suggest that cystatin C concentrations modulate cerebral amyloidosis risk and provide an opportunity for genetic risk assessment and therapeutic interventions.

Practical Issues in Stem Cell Therapy for Alzheimers Disease

We have demonstrated that aged animals show significant improvements in cognitive function and neurogenesis after brain transplantation of human neural stem cells or of human adult mesenchymal stem cells that have been dedifferentiated by transfection of the embryonic stem cell gene. We have also demonstrated that peripheral administration of a pyrimidine derivative increased cognition, endogenous brain stem cell proliferation and neurogenesis. These results indicate a bright future for stem cell therapies in Alzheimer's disease (AD). Before this is realized, however, we need to consider the affect of AD pathology on stem cell biology to establish an effective stem cell therapy for this disease. Although amyloid-beta (Abeta) deposition is a hallmark of AD, an absence of a phenotype in the beta-amyloid precursor protein (APP) knockout mouse, might lead one to underestimate the potential physiological functions of APP and suggest that it is unessential or can be compensated for. We have found, however, that APP is needed for differentiation of neural stem cells (NSCs) in vitro, and that NSCs transplanted into a APP-knockout mouse did not migrate or differentiate -- indicating that APP plays an important role in differentiation or migration process of NSCs in the brain. Then again, treatment with high a concentration of APP or its over-expression increased glial differentiation of NSCs. Human NSCs transplanted into APP-transgenic mouse brain exhibited less neurogenesis and active gliosis around the plaque like formations. Treatment of such animals with the compound, (+)-phenserine, that is known to reduce APP protein levels, increased neurogenesis and suppressed gliosis. These results suggest APP levels can regulate NSC biology in the adult brain, that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases, and that a combination of stem cell therapy and regulation of APP levels could provide a treatment strategy for these disorders.