Amyloid and intracellular accumulation of BRI2

Familial British dementia (FBD) and familial Danish dementia (FDD) are caused by mutations in the BRI2 gene. These diseases are characterized clinically by progressive dementia and ataxia and neuropathologically by amyloid deposits and neurofibrillary tangles. Herein, we investigate BRI2 protein accumulation in FBD, FDD, Alzheimer disease and Gerstmann-Sträussler-Scheinker disease. In FBD and FDD, we observed reduced processing of the mutant BRI2 pro-protein, which was found accumulating intracellularly in the Golgi of neurons and glial cells. In addition, we observed an accumulation of a mature form of BRI2 protein in dystrophic neurites, surrounding amyloid cores. Accumulation of BRI2 was also observed in dystrophic neurites of Alzheimer disease and Gerstmann-Sträussler-Scheinker disease cases. Although it remains to be determined whether intracellular accumulation of BRI2 may lead to cell damage in these degenerative diseases, our study provides new insights into the role of mutant BRI2 in the pathogenesis of FBD and FDD and implicates BRI2 as a potential indicator of neuritic damage in diseases characterized by cerebral amyloid deposition

Increased Tau Phosphorylation and Tau Truncation, and Decreased Synaptophysin Levels in Mutant BRI₂/Tau Transgenic Mice

<div><p>Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the <em>BRI₂</em> gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant <em>BRI₂</em> (Tg-FDD) with mice expressing human 4-repeat mutant <em>Tau-P301S</em> (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI₂ can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new <em>in vivo</em> insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration.</p> </div

BRI₂ expression and processing in transgenic mice.

<p>Schematic diagram of the Danish amyloid precursor protein (ADanPP) (A). BRI₂ is a type-II single trans-membrane (TM) domain protein. Processing of ADanPP by pro-protein convertases (PCs) generates the 34 amino acid peptide (ADan) and a mature form of BRI₂ (m-BRI₂). Processing by ADAM10 in the ectodomain of BRI₂ releases the BRICHOS domain and an N-terminal fragment (NTF). The NTF is also the subject of additional proteolysis by SPPL2, releasing an intracellular domain (ICD) and a C-terminal peptide fragment (BRI2 C-peptide) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056426#pone.0056426-Garringer1" target="_blank">[18]</a>. Disulfide bonded loops in the BRICHOS domain and in the carboxy-terminus of BRI₂ (amino acids 5 and 22 of the ADan peptide) are indicated. The figure shows the localization of the Abs used. Western blot analysis of PNS from neocortex of Tg-FDD-Tau (2x, n = 6) mice, Tg-FDD (FDD, n-6) mice, and wild-type (WT, n = 6) control mice using the BRI₂-amino-terminal antibody 14307 (B). The ectodomain processing of the FDD mutant form of BRI₂ by PCs seems to be compromised in FDD. In Tg-FDD mice, two bands can be observed corresponding to full-length ADanPP and m-BRI₂, while in WT mice most of the detectable BRI₂ protein can be seen as m-BRI₂. Samples were run in triplicates. Representative samples are shown. The densitometric values of the bands representing ADanPP and m-BRI₂ immunoreactivity were normalized to the values of the corresponding actin band using ImageJ software. No significant differences were observed between Tg-FDD-Tau and Tg-FDD mice (independent <i>t</i> test).</p

Biochemical analysis of synaptophysin levels in transgenic mice.

<p>Representative immunoblots and densitometry analysis showing synaptophysin and PSD-95 levels in PNS from wild-type (WT, n = 6), Tg-FDD (FDD, n = 6), Tg-Tau (Tau, n = 6), and Tg-FDD-Tau (2x, n = 6) mice. Samples were run in triplicates. No significant changes were observed at 3 months of age in synaptophysin levels between Tg-FDD-Tau and WT mice (A). A significant decrease in synaptophysin levels in Tg-FDD-Tau mice is observed at 6 months of age (B). At 9 months of age, synaptophysin levels are also decreased in single transgenic mice, particularly in Tg-Tau mice (C). A short and an extended (long) exposure of the film are shown. β-actin was used to normalize protein loading. Optical densities of the individual bands were quantified using NIH ImageJ. Statistical analyses were performed with GraphPad Prism 5.04. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001, two tail t test).</p

Comparison between single transgenic mice, double transgenic mice, and littermate controls.

<p>Representative photographs of 12 months old wild-type (A), Tg-Tau (B), Tg-FDD-Tau (C), and Tg-FDD (D) showing clasping of the hindlimb and bilaterally pulling of the hind paws toward the abdomen when suspended by the tail. Performance of wild-type (WT, n = 7) and Tg-FDD-Tau (2x-Tg, n = 9) animals on an accelerating rotating rod apparatus at 6 months of age (E). No significant differences in performance were observed between Tg-FDD-Tau mice compared with age-matched WT animals. No differences in daily performances were observed in females (f) (F) and males (m) (G) WT and Tg-FDD-Tau animals.</p

Amyloid deposition and BRI₂ accumulation in transgenic mice.

<p>Amyloid deposition is seen throughout all cortical layers (A), the hippocampal formation (B), and in leptomeningeal vessels of the cerebellum (C) of Tg-FDD-Tau mice. Antibodies against ADan immunolabeled cortical blood vessels (D) and amyloid deposits in the hippocampus (E). Amyloid plaques in Tg-FDD mice are surrounded by globular dystrophic neurites (DNs) labeled by the BRI₂-amino-terminal Ab 14307 in the neocortex (F) and hippocampus (G, H). The Ab also labeled intracellular deposits and swollen neurites. Arrows indicate the presence of ADan amyloid plaques. Sections were from a 12 month old (A–E) Tg-FDD-Tau mice and a 21 month old Tg-FDD mouse (F–H). Thioflavine S (A–C). Immunohistochemistry using Abs 1699/1700 (D, E) and Ab 14307 (F–H). Scale bars: A–C, F, 100 µm; G, 50 µm; H, 25 µm.</p

Biochemical analysis of tau in transgenic mice.

<p>Representative immunoblots of tris-soluble and sarkosyl-insoluble fractions from 9 month old Tg-FDD-Tau (2x) and Tg-Tau (Tau) (four to five mice per group were analyzed). Samples were run on SDS-PAGE and immunoblotted with anti-tau Abs d29, AT8, AT100 and Tau-C3. β-actin was used to normalize protein loading.</p

Tau deposition and inflammatory changes in double transgenic mice.

<p>Reactive astrocytes in the hippocampus (A) and neocortex (B), and keratan sulfate-positive-activated microglia in the hippocampus (C). The phosphorylation-dependent anti-tau Ab AT8 immunolabeled tau deposits in the hippocampus (D, E), and the neocortex (F). Sections were from 6 (F), 10 (B), and 12 month old (A, C, D, E) Tg-FDD-Tau mice. Immunohistochemistry using anti-GFAP (A, B), anti-keratan sulfate (C), and Ab AT8 (D–F). Scale bars: A–F, 50 µm.</p