Functional interaction between amyloid-β precursor protein and peripherin neurofilaments: a shared pathway leading to Alzheimer's disease and amyotrophic lateral sclerosis?

Abstract

Background and objective: The pathology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting motor neurons, comprises aberrant accumulations of neurofilaments; mutations in the peripherin subunit of neurofilaments have been identified in some forms of ALS. Recently, the amyloid-β precursor protein (APP), a key element for the pathology of Alzheimer's disease (AD), was linked to ALS. Here, we provide evidence that the generation of the N-terminal fragment of APP, sAPP, relies on peripherin neurofilaments. This finding could relate to a novel molecular mechanism dysregulated in ALS and/or AD.

Methods and results: The production and the fate of sAPP were studied with the brainstem-derived, neuronal cell line, CAD, which expresses endogenous peripherin. We show that sAPP and C-terminal fragments (CTF) are generated to a large extent in the neuronal soma. We find that sAPP, but not CTF, associates with filamentous structures that delineate the nuclear lamina, extend to the cell periphery and immunostain for peripherin. The depletion of peripherin with siRNA eliminates the filamentous immunostaining of sAPP.

Conclusion: Our results indicate that a fraction of APP is cleaved by β-secretase in the soma and that the generated sAPP becomes associated with perinuclear peripherin neurofilaments. These findings link the metabolism of APP--which is dysregulated in AD--to the organization of neurofilaments--which is abnormal in ALS--and suggest a possible crosstalk/overlap between the molecular mechanisms of these diseases.

Figure 1

Segregated distribution of APP-derived polypeptides. a Diagram showing the polypeptide fragments resulting from the cleavage of APP by β- and γ-secretase. Cleavage by β-secretase produces NTFβ (also referred to as sAPPβ) and CTFβ. Aβ and the APP intracellular domain (AICD) are obtained from CTFβ by cleavage by γ-secretase. The dotted lines mark the transmembrane domain. b Antibodies recognizing the N- (APP_N), but not C-terminal (APP_C) epitopes of APP label a filamentous, perinuclear compartment in the soma of CAD neuronal cells. Bar = 20 µm.

Figure 2

The APP N-terminal epitopes detected with antibody 22C11 (likely sAPP) colocalize with the peripherin-positive neurofilaments in a region surrounding the nucleus of CAD neuronal cells. Bar = 20 µm.

Figure 3

Silencing the expression of prph by transfection of CAD neuronal cells with peripherin siRNA diminishes the expression of peripherin protein with over 50% (immunoblot, top right; the Ponceau S stained transfers at bottom right show comparable loads in siRNA-treated and control cells) and abrogates the characteristic, perinuclear localization of both peripherin and sAPP (immunofluorescence images). Cotransfected GFP marks the siRNA treated cells (left and middle images). A nontransfected, control cell is shown for comparison (right images). For increased resolution of sAPP labeling, the top images are shown in black and white. Bars = 20 µm. Molecular weight markers are in kilodaltons.