Karen Khan

Immunization with amyloid-β attenuates Alzheimer-disease-like pathology in the PDAPP mouse

Amyloid-β peptide (Aβ) seems to have a central role in the neuropathology of Alzheimers disease (AD). Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes,. Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Aβ42), which is the predominant form found in the amyloid plaques of Alzheimers disease,. The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimers disease in an age- and brain-region-dependent manner,. In the present study, transgenic animals were immunized with Aβ42, either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-β deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of β-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-β may be effective in preventing and treating Alzheimers disease.

Peripherally administered antibodies against amyloid β-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease

One hallmark of Alzheimer disease is the accumulation of amyloid β-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid β precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid β-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid β-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid β-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood–brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Epitope and isotype specificities of antibodies to β-amyloid peptide for protection against Alzheimer's disease-like neuropathology

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimers disease. In an age-dependent fashion, the mice develop plaques containing β-amyloid peptide (Aβ) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the Aβ peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against Aβ. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different Aβ epitopes and monoclonal antibodies with different isotypes were examined for efficacy both ex vivo and in vivo. The studies showed that: (i) of the purified or elicited antibodies tested, only antibodies against the N-terminal regions of Aβ were able to invoke plaque clearance; (ii) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble Aβ was not necessarily correlated with efficacy; (iii) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Aβ itself; and (v) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimers disease.

β-Amyloid Immunotherapy Prevents Synaptic Degeneration in a Mouse Model of Alzheimer's Disease

Alzheimers disease neuropathology is characterized by key features that include the deposition of the amyloid β peptide (Aβ) into plaques, the formation of neurofibrillary tangles, and the loss of neurons and synapses in specific brain regions. The loss of synapses, and particularly the associated presynaptic vesicle protein synaptophysin in the hippocampus and association cortices, has been widely reported to be one of the most robust correlates of Alzheimers disease-associated cognitive decline. The β-amyloid hypothesis supports the idea that Aβ is the cause of these pathologies. However, the hypothesis is still controversial, in part because the direct role of Aβ in synaptic degeneration awaits confirmation. In this study, we show that Aβ reduction by active or passive Aβ immunization protects against the progressive loss of synaptophysin in the hippocampal molecular layer and frontal neocortex of a transgenic mouse model of Alzheimers disease. These results, substantiated by quantitative electron microscopic analysis of synaptic densities, strongly support a direct causative role of Aβ in the synaptic degeneration seen in Alzheimers disease and strengthen the potential of Aβ immunotherapy as a treatment approach for this disease.

Lack of alzheimer pathology after β-amyloid protein injections in rat brain

Abstract In order to establish a direct relationship between β-amyloid protein (βAP) and in vivo neurotoxicity, we made intraparenchymal injections and Alzet pump infusions of βAP into the hippocampus and cortex of adult rats. We tested a number of synthetic βAP peptides (βAP 1–40, 1–38, and 25–35) and peptide controls (scrambled and reversed 1–40, and scrambled and reversed 25–35) over a wide range of concentrations and in a variety of vehicles. The rats were sacrificed from 2–35 days following the implant, and the brains examined by standard immunohistochemical and histological methods used to evaluate the pathologies associated with Alzheimers disease. We report the lack of Alzheimer related pathology and no significant morphological differences between the βAP peptide and the peptide and vehicle control injections. These observations indicate that the simple intraparenchymal injection of βAP in the rat brain is not an appropriate model of Alzheimer-related neurotoxicity.

Morphological characterization of thioflavin-s-positive amyloid plaques in transgenic Alzheimer mice and effect of passive Aβ immunotherapy on their clearance

Transgenic mice mimicking certain features of Alzheimers disease (AD)-pathology, namely amyloid plaques and neurofibrillary tangles, have been developed in an effort to better understand the mechanism leading to the formation of these characteristic cerebral lesions. More recently, these animal models have been widely used to investigate emergent therapies aimed at the reduction of the cerebral amyloid load. Several studies have shown that immunotherapy targeting the amyloid peptide (Abeta) is efficacious at clearing the amyloid plaques or preventing their formation, and at reducing the memory/behavior impairment observed in these animals. In AD, different types of plaques likely have different pathogenic significance, and further characterization of plaque pathology in the PDAPP transgenic mice would enhance the evaluation of potential therapeutics. In the present study, a morphological classification of amyloid plaques present in the brains of PDAPP mice was established by using Thioflavin-S staining. Neuritic dystrophy associated with amyloid plaques was also investigated. Finally, the efficacy of passive immunization with anti-Abeta antibodies on the clearance of Thio-S positive amyloid plaques was studied. Our results show that distinct morphological types of plaques are differentially cleared depending upon the isotype of the antibody.

Immunotherapy Reduces Vascular Amyloid-β in PDAPP Mice

In addition to parenchymal amyloid-β (Aβ) plaques, Alzheimers disease (AD) is characterized by Aβ in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in the majority of patients. Recent studies investigating vascular Aβ (VAβ) in amyloid precursor protein transgenic mice have suggested that passive immunization with anti-Aβ antibodies may clear parenchymal amyloid but increase VAβ and the incidence of microhemorrhage. However, the influences of antibody specificity and exposure levels on VAβ and microhemorrhage rates have not been well established, nor has any clear causal relationship been identified. This report examines the effects of chronic, passive immunization on VAβ and microhemorrhage in PDAPP mice by comparing antibodies with different Aβ epitopes (3D6, Aβ1–5; 266, Aβ16–23) and performing a 3D6 dose–response study. VAβ and microhemorrhage were assessed using concomitant Aβ immunohistochemistry and hemosiderin detection. 3D6 prevented or cleared VAβ in a dose-dependent manner, whereas 266 was without effect. Essentially complete absence of VAβ was observed at the highest 3D6 dose, whereas altered morphology suggestive of ongoing clearance was seen at lower doses. The incidence of microhemorrhage was increased in the high-dose 3D6 group and limited to focal, perivascular sites. These colocalized with Aβ deposits having altered morphology and apparent clearance in the lower-dose 3D6 group. Our results suggest that passive immunization can reduce VAβ levels, and modulating antibody dose can significantly mitigate the incidence of microhemorrhage while still preventing or reducing VAβ. These observations raise the possibility that Aβ immunotherapy can potentially slow or halt the course of CAA development in AD that is implicated in vascular dysfunction.

Appearance of Sodium Dodecyl Sulfate-Stable Amyloid β-Protein (Aβ) Dimer in the Cortex During Aging

We previously noted that some aged human cortical specimens containing very low or negligible levels of amyloid β-protein (Aβ) by enzyme immunoassay (EIA) provided prominent signals at 6∼8 kd on the Western blot, probably representing sodium dodecyl sulfate (SDS)-stable Aβ dimer. Re-examination of the specificity of the EIA revealed that BAN50- and BNT77-based EIA, most commonly used for the quantitation of Aβ, capture SDS-dissociable Aβ but not SDS-stable Aβ dimer. Thus, all cortical specimens in which the levels of Aβ were below the detection limits of EIA were subjected to Western blot analysis. A fraction of such specimens contained SDS-stable dimer at 6∼8 kd, but not SDS-dissociable Aβ monomer at ∼4 kd, as judged from the blot. This Aβ dimer is unlikely to be generated after death, because (i) specimens with very short postmortem delay contained the Aβ dimer, and (ii) until 12 hours postmortem, such SDS-stable Aβ dimer is detected only faintly in PDAPP transgenic mice. The presence of Aβ dimer in the cortex may characterize the accumulation of Aβ in the human brain, which takes much longer than that in PDAPP transgenic mice.

Prevention and Reduction of AD‐type Pathology in PDAPP Mice Immunized with Aβ1–42

Abstract: In AD certain brain structures contain a pathological density of Aβ protein deposited into plaques. The effect of genetic mutations found in early onset AD patients was an overproduction of Aβ42, strongly suggesting that overproduction of Aβ42 is associated with AD. We hypothesized that an immunological response to Aβ42 might alter its turnover and metabolism. Young PDAPP transgenic mice were immunized with Aβ1–42, which essentially prevented amyloid deposition; astrocytosis was dramatically reduced and there was reduction in Aβ‐induced inflammatory response as well. Aβ1–42 immunization also appeared to arrest the progression of amyloidosis in older PDAPP mice. Aβ immunization appears to increase clearance of amyloid plaques, and may therefore be a novel and effective approach for the treatment of AD.

Accumulation of Amyloid β-Protein in the Low-Density Membrane Domain Accurately Reflects the Extent of β-Amyloid Deposition in the Brain

To learn more about the process of amyloid β-protein (Aβ) deposition in the brain, human prefrontal cortices were fractionated by sucrose density gradient centrifugation, and the Aβ content in each fraction was quantified by a two-site enzyme-linked immunosorbent assay. The fractionation protocol revealed two pools of insoluble Aβ. One corresponded to a low-density membrane domain; the other was primarily composed of extracellular Aβ deposits in those cases in which Aβ accumulated to significant levels. Aβ42 levels in the low-density membrane domain were proportional to the extent of total Aβ42 accumulation, which is known to correlate well with overall amyloid burden. In PDAPP mice that form senile plaques and accumulate Aβ in a similar manner to aging humans, Aβ42 accumulation in the low-density membrane domain also increased as Aβ deposition progressed with aging. These observations indicate that the Aβ42 associated with low-density membrane domains is tightly coupled with the process of extracellular Aβ deposition.