Amyloid-β Precursor Protein Modulates the Sorting of Testican-1 and Contributes to Its Accumulation in Brain Tissue and Cerebrospinal Fluid from Patients with Alzheimer Disease

Abstract

The mechanisms leading to amyloid-β (Aβ) accumulation in sporadic Alzheimer disease (AD) are unknown but both increased production or impaired clearance likely contribute to aggregation. To understand the potential roles of the extracellular matrix proteoglycan Testican-1 in the pathophysiology of AD, we used samples from AD patients and controls and an in vitro approach. Protein expression analysis showed increased levels of Testican-1 in frontal and temporal cortex of AD patients; histological analysis showed that Testican-1 accumulates and co-aggregates with Aβ plaques in the frontal, temporal and entorhinal cortices of AD patients. Proteomic analysis identified 10 fragments of Testican-1 in cerebrospinal fluid (CSF) from AD patients. HEK293T cells expressing human wild type or mutant Aβ precursor protein (APP) were transfected with Testican-1. The co-expression of both proteins modified the sorting of Testican-1 into the endocytic pathway leading to its transient accumulation in Golgi, which seemed to affect APP processing, as indicated by reduced Aβ40 and Aβ42 levels in APP mutant cells. In conclusion, patient data reflect a clearance impairment that may favor Aβ accumulation in AD brains and our in vitro model supports the notion that the interaction between APP and Testican-1 may be a key step in the production and aggregation of Aβ species.

Keywords: APP Swedish mutation; Alzheimer disease; Early endosomes; Neuritic plaques; Testican-1; β-Amyloid.

FIGURE 1

Protein levels and histological characterization of Testican-1 in human brain tissue. (A) Western blots of Testican-1 in frontal cortex and temporal cortex homogenates from healthy individuals and sporadic AD patients. (B) Dot-plot graphs of Testican-1 in frontal cortex and temporal cortex homogenates from healthy individuals and sporadic AD patients (FC: p = 0.0280, TC: p = 0.0027, RU = Relative units). Testican-1 immunostaining showing Testican-1 immunoreactivity in cortical neurons in a control case (C), neurons with granulovacuolar degeneration in an AD case (D), ghost tangles (E), and small cell processes within diffuse amyloid plaques (F–H). (I) Immunofluorescence staining of an Aβ plaque showing colocalization of Aβ and Testican-1. (J) Number of Testican-1- and Aβ-positive deposits per specimen in TMAs of AD patients and controls using immunohistochemical staining. Results are means of an average of 6.9 tissue samples per region per patient. The black lines within the boxes represent medians; the boxes encompass 25th and 75th percentiles of the distribution; the whisker indicates the range of values. Results were analyzed using a Student t-test; ***p < 0.001, **p < 0.005, *p < 0.05. (K) Immunohistochemical staining of frontal cortex sequential sections of an AD case for amyloid beta (6E10), Testican-1, hyperphosphorylated Tau (AT8), and microglial markers (CD68 and HLA-DR). Only some Testican-1 deposits showed spatial correlation with amyloid plaques (black arrowheads). Testican-1 deposits were not associated with Tau deposits or microglial staining. Scale bars = 50 µm.

FIGURE 2

Subcellular distribution of Testican-1 and APP in HEK293T cells. Untransfected HEK cells and cells expressing APPwt and APPsw were cotransfected with Mock or Testican-1. The distribution of APP (red) and Testican-1 (green) was analyzed 24 hours after transfection using confocal microscopy. Colocalization plots are shown at the bottom. Scale bar = 10 μm.

FIGURE 3

Subcellular localization of Testican-1 and APP in the endocytic pathway of HEK293T cells. Untransfected HEK cells and cells expressing APPwt and APPsw were cotransfected with Mock or Testican-1. (A) Representative pictures of a double staining showing the localization of Testican-1 or APP (green) and Rab4 (red). Testican-1 and APP colocalize with Rab4 in APPsw cells showing a puncta pattern. (B) APP and Testican-1 (green) localization with the late endosome marker Rab9 (red) was analyzed 24 hours after transfection using confocal microscopy. Neither the shape, distribution nor colocalization of Rab9 is modified in APPwt and APPsw cell lines after Testican-1 transfection. Scale bars = 10 μm.

FIGURE 4

Sorting of Testican-1 into the TGN of HEK293T cells. Untransfected HEK cells or cells expressing APPwt and APPsw were cotransfected with Mock or Testican-1 and analyzed 24 hours after transfection using confocal microscopy. (A) Testican-1 (green) localization in the TGN labeled by Adaptin-γ (Red) was evaluated 24 hours after transfection. Testican-1 colocalizes with Adaptin-γ in all the cell lines analyzed. (B) Testican-1 (Green) localization with the Golgi marker GM130 (Red) was analyzed 24 hours after transfection. Testican-1 was located in the Golgi apparatus and transport vesicles of the APPsw cells. Scale bar = 10 μm.

FIGURE 5

Protein levels of Aβ, APP, and enzymes involved in the processing of APP and degradation of Aβ in HEK293T cells. (A) Levels of Aβ40, Aβ42, and Aβ42/Aβ40 ratio in supernatant from HEK293T cells transfected with Testican-1. After 24 hours, the levels of Aβ40 and Aβ42 present in the supernatant were measured by ELISA. A significant decrease in the concentration of Aβ40 (p < 0.0001) and Aβ42 (p = 0.0006) was observed in the HEK cells expressing APPsw. Results were analyzed using a t-test; p < 0.05 was considered significant. Data are expressed as mean ± SEM. (B) Representative Western blots and histograms APP full-length from HEK293T cells (WT, APPwt, and APPsw) transfected with empty vector (Mock) or Testican-1. Decreased levels of APP were visible for APPsw cells (p = 0.05, RU = Relative units). (C) Representative Western blots of BACE1, ADAM10, PS1, cathepsin-L, and IDE in HEK293T cells (WT, APPwt, and APPsw) transfected with empty vector (Mock) or Testican-1.