Intracellular interactions of the BRICHOS domain with amyloid

4.2.1 ProSP-C BRICHOS general anti-amyloid properties

In Paper II, the effect of a BRICHOS domain on artificial β-sheet proteins (β17, β23) designed to form amyloid-like fibrils (West, et al., 1999) were studied. These designed β-sheet proteins have earlier been used to study cytotoxicity associated with amyloid-like aggregation in the cytosol of a human cell line and were then shown to cause cytotoxicity according to their predicted β-sheet propensity, β23>β17 (Olzscha, et al., 2011). Co-expression of proSP-C BRICHOS and proSP-C (residues 1-58) in trans, in the secretory pathway of HEK293 cells, stabilizes the otherwise aggregation prone proSP-C (1-58) from degradation (Johansson, et al., 2009a). It has been proposed that a β-hairpin structure, an early motif in amyloid aggregation, is a target motif for BRICHOS domains (Knight, et al., 2013). The model β-proteins most likely form this motif, and we therefore redesigned them

Face A

for co-expression in trans with the proSP-C BRICHOS domain in the secretory pathway of HEK293 cells, in order to study the anti-amyloid activity of proSP-C BRICHOS.

Co-expression of proSP-C BRICHOS leads to an increase of soluble β-protein, without affecting α-helical control protein levels. BRICHOS could not be detected when expressed with β23, and therefore focus was centered on β17. β17 accumulated in inclusion body-like structures and BRICHOS co-expression reduced their size. Co-localization of BRICHOS and β17 in inclusion body-like structures were detected by immunofluorescence, and a complex could be detected between β17 and BRICHOS with both immunoprecipitation and PLA.

Proteasome involvement in β17 degradation was investigated with MG132 (a proteasome inhibitor) treatment and WB, and it was found that β17 was partly degraded by the proteasome, but co-expression of BRICHOS reduced this degradation pathway. Cells expressing β17 without BRICHOS, contained ubiquitinated protein and formed aggresome-like structures, as detected by the ProteoStat assay. The ProteoStat dye (Shen, et al., 2011) cannot distinguish between inclusionbodies formed in a microtubuledependent or -independent manner, making it hard to distinguish between aggresomes or other types of protein aggregates, therefore non-transfected cells treated with proteasome inhibitor were used as control. The MG132 treated cells formed abundant structures that were stained with the ProteoStat dye, as well as with ubiquitin-antibodies. This control does not prove that the detected structures are aggresomes but suggest that the aggregated protein structures detected in Paper II, accumulate as an effect of a stressed cell and indicate proteasome involvement.

Expression of β17 alone gave rise to formation of aggresomes-like structures and

ubiquitinated protein, suggesting that the ubiquitin-proteasomal system (UPS) is affected.

Proteasome activity was further studied by analyzing the chymotrypsin-like activity in transfected cells and β17 was found to decrease proteasome activity compared with non-transfected controls, whereas BRICHOS co-expression reduced the inhibitory effects of β17 on proteasome activity.

The results in Paper II suggest that β17 expression in the secretory pathway of HEK293 cells leads to the formation of inclusions in the ER, and to some β17 being retrotranslocated to the cytosol were it is targeted for proteasome degradation by ubiquitination. The formation of ubiquitinated inclusions and structures stained with ProteoStat, together with inhibitory effects on proteasome activity suggest that the proteasome is having problems degrading β17.

With BRICHOS co-expression, a model is suggested were β17’s accumulation into inclusions is reduced along with reduced need for retrotranslocation to the cytosol, ubiquitination, and proteasome degradation (see Figure 10).

Figure 10. Model of β17 and BRICHOS. Left: β17 protein expressed in the secretory pathway aggregates and forms inclusions in the ER. β17 is retrotranslocated to the cytosol and ubiquitinated (Ub) for degradation by the proteasome. Some β17 is degraded but the proteasome is blocked with misfolded β17, and its activity decreases.

Proteasomal inhibition leads to accumulation of aggresomes, as detected using the Proteastat dye. Right:

BRICHOS decreases the aggregation of β17, resulting in less retrotranslocated β17. BRICHOS binds to β17, which leads to smaller inclusions that contain both BRICHOS and β17. When less β17 is targeted to the proteasome, the proteasomal inhibition is reduced and no aggresomes are formed. Used with permission, this figure was originally published in Dolfe et al, 2016 (Dolfe, et al., 2016).

4.2.2 Interaction between Bri2 and Bri3 BRICHOS with Aβ in neurons

In Paper III, Bri2 and Bri3 BRICHOS were studied in relation to Aβ and AD. Bri2 and Bri3 have been shown to bind AβPP and decrease Aβ secretion in transgenic cell lines, and therefore we decided to study endogenous Bri2 and Bri3 interactions with AβPP and Aβ in primary neurons from WT mice, using PLA (see section 3.1). Their interaction with AβPP could be verified, and interactions with Aβ40 were also detected. Moreover Bri2 BRICHOS, without its linker region, hence after proteolytic release of the BRICHOS domain, interacted with endogenous Aβ42 in WT primary neurons. Both isolated recombinant Bri2 and Bri3

BRICHOS domains, inhibit in vitro Aβ42 fibril formation. Therefore we wanted to determine if Bri3 as well as Bri2 BRICHOS interacted specifically with Aβ in cells. It was found that Bri3 has abundant interactions with Aβ42, however in contrast to Bri2, Bri3 BRICHOS was not found shed from neurons, and it could not be made certain if the BRICHOS domain or/and other parts of Bri3 are responsible for the interaction with Aβ. Considering that Bri3 BRICHOS inhibits Aβ42 fibril formation, similar to Bri2 BRICHOS, it seems plausible that the BRICHOS domain is responsible for binding Aβ. Mouse brain tissue, transgenic for the arctic mutation, tgAβPParc (Ronnback, et al., 2012) were used to verify the interaction with AβPP and showed abundant signals in the CA1 region. A complementary control in AβPP knockout mice were performed and no interaction could be detected. Unspecific signal is a potential problem in this study and the results are dependent on the specificity of the

antibodies. One factor that cannot be ruled out is that although Aβ and the BRICHOS domain are in close proximity to one another, <40 nm (see section 3.1), they could both bind a third protein and not actually each other. At least the control experiments, especially from the AβPP knockout mice show that the detected interaction is directly dependent on AβPP expression and not due to unspecific antibody binding. Another aspect, which is not investigated in Paper III, is whether Bri2 and Bri3 BRICHOS interact with monomeric or oligomeric Aβ. Previous data support that proSP-C BRICHOS bind fibrillar and not

monomeric Aβ42 (Cohen, et al., 2015). However these experiments have not been performed for Bri2 and Bri3 BRICHOS. Moreover, experiments show that Bri2 BRICHOS inhibits both elongation and secondary nucleation during Aβ fibril formation, whilst proSP-C BRICHOS inhibits only secondary nucleation (Arosio, et al., 2016), suggesting that Bri2/Bri3 BRICHOS potentially inhibit Aβ fibril formation by blocking different pathways than proSP-C

BRICHOS. Studies in transgenic cells indicate that the binding of Bri2 and Bri3 to AβPP is mediated by the linker region and not the BRICHOS domain, these studies also suggest that the linker region is responsible for effects on AβPP processing (see section 1.9-1.10.1). It is therefore interesting to note that expressing a Bri2 construct from residue 90-236, including part of the linker region (residues ∼76-129) and the BRICHOS domain (residues ∼130-230) in vivo in a mouse model of AβPP/PS1 overexpression, had no effects on AβPP processing, whilst reducing Aβ aggregation and subsequent neuropathological effects (Paper IV). The results in Paper III and IV together support that the BRICHOS domain in itself, without the full linker region, is important for binding to Aβ, and reducing its aggregation.

Figure 11. Model of AβPP and Bri2/Bri3 interactions. AβPP is produced in the ER and then transported through the trans-golgi network (TGN) to the plasma membrane (PM) and/or the endosomal pathway. At the plasma membrane AβPP is cleaved by α-secretase and/or internalized. AβPP is cleaved by BACE1 and γ-secretase mainly in the endosomal pathway, but also in the ER and TGN, hence, Aβ can be generated in both the secretory and endosomal pathway (van der Kant and Goldstein, 2015). Bri2 is generated in the ER and similarly to AβPP it could be transported through the TGN to the PM or endosomal pathway. At the PM it can be cleaved by ADAM10 and/or internalized. Bri2 still containing its linker region, interacts with AβPP (Paper III) and possibly other AβPP processing products, although this was not investigated in Paper III. Processed Bri2 BRICHOS interacts with Aβ (Paper III). Bri2 BRICHOS could come into contact with lumenal Aβ, either in the TGN or the endosomal pathway. Extracellular Aβ and Bri2 BRICHOS can be found deposited in plaques (Paper IV). Bri3 is biosynthesized in the ER and similarly to AβPP and Bri2 it could be transported to the PM or the endosomal pathway. Bri3 BRICHOS is not shed (Paper III), but could become internalized. Bri3 interacts with AβPP (Paper III), and possibly with other processing products. Bri3 interacts with Aβ (Paper III) and similarly to Bri2 it could come into contact with lumenal Aβ either in the TGN or in the endosomal pathway. Bri3 is found co-localized with plaques in AD brain (Paper III) and is possibly released with Aβ by exosomes. The AβPP processing products sAβPPα, C83 and p3, are left out of the illustration.

4.3 BRICHOS EFFECTS ON IN VIVO Aβ AGGREGATION (PAPER III AND IV)