Fiona Pickford

The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid β accumulation in mice

Autophagy is the principal cellular pathway for degradation of long-lived proteins and organelles and regulates cell fate in response to stress. Recently, autophagy has been implicated in neurodegeneration, but whether it is detrimental or protective remains unclear. Here we report that beclin 1, a protein with a key role in autophagy, was decreased in affected brain regions of patients with Alzheimer disease (AD) early in the disease process. Heterozygous deletion of beclin 1 (Becn1) in mice decreased neuronal autophagy and resulted in neurodegeneration and disruption of lysosomes. In transgenic mice that express human amyloid precursor protein (APP), a model for AD, genetic reduction of Becn1 expression increased intraneuronal amyloid beta (Abeta) accumulation, extracellular Abeta deposition, and neurodegeneration and caused microglial changes and profound neuronal ultrastructural abnormalities. Administration of a lentiviral vector expressing beclin 1 reduced both intracellular and extracellular amyloid pathology in APP transgenic mice. We conclude that beclin 1 deficiency disrupts neuronal autophagy, modulates APP metabolism, and promotes neurodegeneration in mice and that increasing beclin 1 levels may have therapeutic potential in AD.

Aβ42 Is Essential for Parenchymal and Vascular Amyloid Deposition in Mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimers disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.

Regulation of amyloid precursor protein processing by the Beclin 1 complex.

Autophagy is an intracellular degradation pathway that functions in protein and organelle turnover in response to starvation and cellular stress. Autophagy is initiated by the formation of a complex containing Beclin 1 (BECN1) and its binding partner Phosphoinositide-3-kinase, class 3 (PIK3C3). Recently, BECN1 deficiency was shown to enhance the pathology of a mouse model of Alzheimer Disease (AD). However, the mechanism by which BECN1 or autophagy mediate these effects are unknown. Here, we report that the levels of Amyloid precursor protein (APP) and its metabolites can be reduced through autophagy activation, indicating that they are a substrate for autophagy. Furthermore, we find that knockdown of Becn1 in cell culture increases the levels of APP and its metabolites. Accumulation of APP and APP C-terminal fragments (APP-CTF) are accompanied by impaired autophagosomal clearance. Pharmacological inhibition of autophagosomal-lysosomal degradation causes a comparable accumulation of APP and APP-metabolites in autophagosomes. Becn1 reduction in cell culture leads to lower levels of its binding partner Pik3c3 and increased presence of Microtubule-associated protein 1, light chain 3 (LC3). Overexpression of Becn1, on the other hand, reduces cellular APP levels. In line with these observations, we detected less BECN1 and PIK3C3 but more LC3 protein in brains of AD patients. We conclude that BECN1 regulates APP processing and turnover. BECN1 is involved in autophagy initiation and autophagosome clearance. Accordingly, BECN1 deficiency disrupts cellular autophagy and autophagosomal-lysosomal degradation and alters APP metabolism. Together, our findings suggest that autophagy and the BECN1-PIK3C3 complex regulate APP processing and play an important role in AD pathology.

Expression of mBRI2 in mice.

Mutations in the BRI(2) gene cause the autosomal dominant neurodegenerative diseases familial British dementia (FBD) and familial Danish dementia (FDD). BRI(2) is a member of a family of type 2 integral transmembrane spanning proteins, including mBRI(2), its murine homologue. The function of BRI(2) is unknown. Northern and Western analyses and in situ hybridization were employed to determine the expression of mBRI(2) in the mouse. mBRI(2) mRNA was expressed in several tissues including the liver, heart, lung, and ubiquitously throughout the brain. mBRI(2) protein was detected at high levels in many brain regions. Murine BRI(2) expression is similar to that described in the human brain but does not fully explain the distribution of pathology seen in FBD and FDD.

Modeling familial British dementia in transgenic mice.

The chromosome 13 linked amyloidopathies familial British dementia (FBd) and familial Danish dementia (Fdd) are caused by mutations in the c‐terminus of the BRI2 gene. In both diseases, novel peptides are deposited in amyloid plaques in the brain. several laboratories have attempted to model these diseases in BrI2 transgenic mice with limited success. While high expression levels of BRI protein were achieved in transgenic lines, no aBri‐amyloidosis was observed in aged mice. this review discusses the strategies chosen and problems experienced with the development of FBd/Fdd models and suggests novel approaches to model the diseases in murine models.

Sequence conservation between mouse and human synphilin-1

Synphilin-1 has been shown to interact with alpha-synuclein, which in turn is associated with Parkinsons disease. However, the function of synphilin-1 is unknown. We have cloned mouse synphilin in an attempt to describe conserved and therefore likely functional domains. The deduced amino acid sequence of the protein shows extensive homology with its human counterpart, with greatest similarities in those regions that contain ankyrin-like motifs and the coiled-coil domain. Expression of mouse synphilin-1 across tissues is similar to its human counterpart and not limited to brain. The results show that the synphilin-1 sequence and expression patterns are conserved across species.

O4-05-02

Background: Autophagy is involved in the intracellular turnover of proteins and cell organelles and has a key role in regulating cell fate in response to stress. However, it is controversial whether autophagy exerts a pathological or protective role in neurodegenerative diseases, and it is unknown whether defects in the autophagy pathway contribute to neurodegeneration. Objectives: To determine the role of the autophagy protein Beclin 1 in Alzheimer’s Disease. Methods: Beclin 1 downregulation in affected regions of AD brains was identified by microarray analysis, and confirmed by immunoblotting. Beclin 1 deficient transgenic mice (Beclin 1 ) were crossed with mice expressing human amyloid precursor protein (APP) and analyzed for neurodegeneration and amyloid deposition. Beclin 1 expression was knocked down by siRNA in B103/APP neuroblastoma cells and APP metabolism studied. The affect of pharmacological induction of autophagy on APP metabolism in the same cell line was also studied. Results: In a search for genes associated with Alzheimer’s disease (AD) we discovered that expression of Beclin 1, a protein essential for autophagy, is reduced by 50-70% in affected entorhinal and frontal cortex of AD human brains. Heterozygous deletion of the beclin 1 gene decreased neuronal autophagy and promoted neuronal degeneration in normal mice. In transgenic mice that express human APP, a model for AD, genetic reduction of beclin 1 expression resulted in increased accumulation of APP fragments and -amyloid (A ), increased neurodegeneration and increased microgliosis. In addition, reducing Beclin 1 expression in B103/APP neuroblastoma cells by siRNA increased cellular APP, APP-CTF and secreted A levels. In contrast, inducing autophagy in B103/APP cells decreased cellular APP and APP-CTF levels. Conclusions: Beclin 1 deficiency disrupts neuronal autophagy and promotes neurodegeneration and AD-like disease. This work was funded by the John Douglas French Alzheimer’s Foundation and the National Institute of Aging.