G. William Rebeck

Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions.

An increased prevalence of apolipoprotein E (ApoE) epsilon 4 allele exists in late onset familial Alzheimers disease. We found, in sporadic Alzheimers disease, that 62% of patients possessed an ApoE-epsilon 4 allele, compared with 20% of controls. ApoE-epsilon 4/4 patients had more senile plaques (SPs) than epsilon 3/3 patients. ApoE immunoreactivity of SPs was equivalent in both groups. Two receptors bind ApoE complexes, the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP). In normal brain, anti-LRP antibodies strongly stained neurons and lightly stained astrocytes; anti-LDL receptor antibodies stained only the neuropil and astrocytes. In Alzheimers disease, SPs and reactive astrocytes were also strongly LRP immunoreactive. Colocalization of ApoE and LRP to SPs implies that these molecules may be involved in metabolism of components of SPs.

LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted β-amyloid precursor protein and mediates its degradation

The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimers disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.

Novel amyloid precursor protein mutation in an Iowa family with dementia and severe cerebral amyloid angiopathy

Several mutations in the amyloid precursor protein (APP) gene have been found to associate with pathologic deposition of the β‐amyloid peptide (Aβ) in neuritic plaques or in the walls of cerebral vessels. We report a mutation at a novel site in APP in a three‐generation Iowa family with autosomal dominant dementia beginning in the sixth or seventh decade of life. The proband and an affected brother had progressive aphasic dementia, leukoencephalopathy, and occipital calcifications. Neuropathological examination of the proband revealed severe cerebral amyloid angiopathy, widespread neurofibrillary tangles, and unusually extensive distribution of Aβ40 in plaques. The affected brothers shared a missense mutation in APP, resulting in substitution of asparagine for aspartic acid at position 694. This site corresponds to residue 23 of Aβ, thus differing from familial Alzheimers disease mutations, which occur outside the Aβ sequence. Restriction enzyme analysis of DNA from 94 unrelated patients with sporadic cerebral amyloid angiopathy‐related hemorrhage found no other instances of this mutation. These results suggest a novel site within Aβ that may promote its deposition and toxicity.

Clinical manifestations of cerebral amyloid angiopathy–related inflammation

To explore the clinical effects of inflammation associated with vascular deposits of the amyloid β peptide (Aβ), we analyzed 42 consecutive patients with pathologically diagnosed cerebral amyloid angiopathy (CAA) for evidence of an inflammatory response. Inflammation with giant‐cell reaction surrounding amyloid‐laden vessels was identified in 7 of the 42 cases. The clinical symptoms in each of the seven were subacute cognitive decline or seizure rather than hemorrhagic stroke, the primary clinical presentation in 33 of 35 patients with noninflammatory CAA (p < 0.001). Inflammatory CAA also was associated with radiographic white matter abnormalities, significantly younger age at presentation, and a marked overrepresentation of the apolipoprotein E ε4/ε4 genotype (71% vs 4%, p < 0.001). Of the six inflammatory CAA patients with available follow‐up information, five demonstrated clinical and radiographic improvement after immunosuppressive treatment. The syndrome of CAA‐related perivascular inflammation appears to represent a subset of CAA with clinically distinct symptoms that may respond to immunosuppressive treatment. These data add to evidence that inflammation against Aβ can cause vascular dysfunction, a potential mechanism for the toxic response recently observed in clinical trials of Aβ immunization.

Amyloid precursor protein secretases as therapeutic targets for traumatic brain injury

Amyloid-β (Aβ) peptides, found in Alzheimers disease brain, accumulate rapidly after traumatic brain injury (TBI) in both humans and animals. Here we show that blocking either β- or γ-secretase, enzymes required for production of Aβ from amyloid precursor protein (APP), can ameliorate motor and cognitive deficits and reduce cell loss after experimental TBI in mice. Thus, APP secretases are promising targets for treatment of TBI.

Females exhibit more extensive amyloid, but not tau, pathology in an Alzheimer transgenic model

Epidemiological studies indicate that women have a higher risk of Alzheimers disease (AD) even after adjustment for age. Though transgenic mouse models of AD develop AD-related amyloid beta (Abeta) and/or tau pathology, gender differences have not been well documented in these models. In this study, we found that female 3xTg-AD transgenic mice expressing mutant APP, presenilin-1 and tau have significantly more aggressive Abeta pathology. We also found an increase in beta-secretase activity and a reduction of neprilysin in female mice compared to males; this suggests that a combination of increased Abeta production and decreased Abeta degradation may contribute to higher risk of AD in females. In contrast to significantly more aggressive Abeta pathology in females, gender did not affect the levels of phosphorylated tau in 3xTg-AD mice. These results point to the involvement of Abeta pathways in the higher risk of AD in women. In addition to comparison of pathology between genders at 9, 16 and 23 months of age, we examined the progression of Abeta pathology at additional age points; i.e., brain Abeta load, intraneuronal oligomeric Abeta distribution and plaque load, in male 3xTg-AD mice at 3, 6, 9, 12, 16, 20 and 23 months of age. These findings confirm progressive Abeta pathology in 3xTg-AD transgenic mice, and provide guidance for their use in therapeutic research.

Interaction of Reelin with Amyloid Precursor Protein Promotes Neurite Outgrowth

The processing of amyloid precursor protein (APP) to Aβ is an important event in the pathogenesis of Alzheimers disease, but the physiological function of APP is not well understood. Our previous work has shown that APP processing and Aβ production are regulated by the extracellular matrix protein Reelin. In the present study, we examined whether Reelin interacts with APP, and the functional consequences of that interaction in vitro. Using coimmunoprecipitation, we found that Reelin interacted with APP through the central domain of Reelin (repeats 3–6) and the E1 extracellular domain of APP. Reelin increased cell surface levels of APP and decreased endocytosis of APP in hippocampal neurons in vitro. In vivo, Reelin levels were increased in brains of APP knock-out mice and decreased in APP-overexpressing mice. RNA interference knockdown of APP decreased neurite outgrowth in vitro and prevented Reelin from increasing neurite outgrowth. Knock-out of APP or Reelin decreased dendritic arborization in cortical neurons in vivo, and APP overexpression increased dendritic arborization. APP and Reelin have previously been shown to promote neurite outgrowth through interactions with integrins. We confirmed that APP interacted with α3β1 integrin, and α3β1 integrin altered APP trafficking and processing. Addition of an α3β1 integrin antibody prevented APP and Reelin-induced neurite outgrowth. These findings demonstrate that Reelin interacts with APP, potentially having important effects on neurite development.

DAB1 and Reelin Effects on Amyloid Precursor Protein and ApoE Receptor 2 Trafficking and Processing

Numerous cytoplasmic adaptor proteins, including JIP1, FE65, and X11α, affect amyloid precursor protein (APP) processing and Aβ production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP β-C-terminal fragment and secreted Aβ. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the β-C-terminal fragment of APP and Aβ. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands.

Induction of the Cholesterol Transporter ABCA1 in Central Nervous System Cells by Liver X Receptor Agonists Increases Secreted Aβ Levels

The expression, function, and regulation of the cholesterol efflux molecule, ABCA1, has been extensively examined in peripheral tissues but only poorly studied in the brain. Brain cholesterol metabolism is of interest because several lines of evidence suggest that elevated cholesterol increases the risk of Alzheimers disease. We found a largely neuronal expression of ABCA1 in normal rat brain by in situ hybridization. ABCA1 message was dramatically up-regulated in neurons and glia in areas of damage by hippocampal AMPA lesion after 3–7 days. Immunoblot analysis demonstrated ABCA1 protein in cultured neuronal and glial cells, and expression was induced by ligands of the nuclear hormone receptors of the retinoid X receptor and liver X receptor family. ABCA1 was induced by treatment with retinoic acid and several oxysterols, including 22(R)-hydroxycholesterol and 24-hydroxycholesterol. Expression of an ABCA1-green fluorescent protein construct in neuroblastoma cells demonstrated fluorescence in perinuclear compartments and on the plasma membrane. Because the Aβ peptide is important in Alzheimers disease pathogenesis, we examined whether ABCA1 induction altered Aβ levels. Treatment of neuroblastoma cells with retinoic acid and 22(R)-hydroxycholesterol caused significant increases in secreted Aβ40 (29%) and Aβ42 (65%). Treatment with a nonsteroidal liver X receptor ligand, TO-901317, similarly increased levels of secreted Aβ40 (25%) and Aβ42 (126%). The increase in secreted Aβ levels was reduced by RNAi blocking of ABCA1 expression. These data suggest that the cholesterol efflux molecule ABCA1 may also be involved in the secretion of the membrane-associated molecule, Aβ.

Demonstration by Fluorescence Resonance Energy Transfer of Two Sites of Interaction between the Low-Density Lipoprotein Receptor-Related Protein and the Amyloid Precursor Protein: Role of the Intracellular Adapter Protein Fe65

Amyloid-β, the major constituent of senile plaques in Alzheimers disease, is derived from the amyloid precursor protein (APP) by proteolysis. Kunitz protease inhibitor (KPI) containing forms of APP (APP751/770) interact with a multifunctional endocytic receptor, the low-density lipoprotein receptor-related protein (LRP), which modulates its proteolytic processing affecting production of amyloid-β. We used fluorescence resonance energy transfer (FRET) using labeled LRP and APP in H4 cell line to examine the subcellular localization and the molecular domains involved in the APP-LRP interaction. KPI-containing forms of APP (APP770) demonstrated FRET with LRP that was sensitive to the LRP inhibitor receptor-associated protein (RAP), suggesting an interaction between the extracellular domains of APP770 and LRP. APP695 also interacts with LRP to lesser degree (as measured by extracellular domain probes), and this ectodomain interaction is not altered by RAP. By using C-terminally tagged LRP and APP, we demonstrate a second site of interaction between the C termini of both APP695 and APP770 and the C terminus of LRP, and that the interactions at these regions are not sensitive to RAP. We next examined the possibility that the C-termini APP-LRP interaction was mediated by Fe65, an adaptor protein that interacts with the cytoplasmic tails of LRP and APP. FRET studies confirmed a close proximity between the amino Fe65 phosphotyrosine binding (PTB) domain and LRP cytoplasmic domain and between the carboxyl Fe65 PTB domain and the APP cytoplasmic domain. These findings demonstrate that LRP interaction with APP occurs via both extracellular and intracellular protein interaction domains.