“Overall, these findings are important contributions to our understanding of the cell biology of γ-secretase, identifying partner proteins that can modulate protease activity and affect amyloid production,” wrote Michael Wolfe, Harvard Medical School, in an e-mail to ARF. “Going forward, it will be worthwhile to investigate possible roles of tetraspanin genes in the pathogenesis of AD or whether the levels of the encoded proteins are otherwise altered,” said Wolfe, who was not involved in the work (see complete text of Wolfe comment below).

To be active, the γ-secretase complex must contain four proteins, the presenilins (PS1 or PS2), nicastrin, Aph1, and Pen-2. In the new study, first author Tomoko Wakabayashi went fishing for other members in a γ-secretase preparation from cultured cells. The researchers expressed doubly affinity-tagged PS1 or PS2 in presenilin knockout mouse embryo fibroblasts, and then purified the complex over tandem affinity columns. Mass spectrometry analysis of the resulting mixture yielded 59 proteins that co-purified with either PS1 or PS2, but not with the PS homolog SPPL3. All 46 of the proteins that reproducibly co-purified with PS2 were also found with PS1, suggesting that the two presenilins associate with a similar range of proteins. Among the 46 were some familiar proteins including Tmp21 (see ARF related news story), as well as some new players. The novel finds included the tetraspanins CD81 and Upk1b, and a tetraspanin partner protein, the cell surface immunoglobulin superfamily protein EWI-2. Coimmunoprecipitation experiments confirmed the association between γ-secretase and CD81, and identified additional interactions with a third tetraspanin, CD9, and a second EWI protein, EWI-F.

The tetraspanins play a role in γ-secretase function, as indicated by RNAi knockdown and overexpression experiments. Depletion of CD81, EWI-F, or the tetraspanin-associated protein CD98hc decreased production of Aβ in HEK293 or Hela cells. Cells from CD81 or CD9 knockout mice showed an increase in C-terminal fragments of endogenous γ-secretase substrates including the amyloid precursor protein, suggesting that in the absence of the tetraspanins, either the activity of γ-secretase or its access to substrates is disrupted. Consistent with this idea, HEK293 cells treated with anti-CD9 monoclonal antibodies had their Aβ production reduced by more than half.

Cell fractionation experiments supported the idea that γ-secretase localizes to tetraspanin-enriched membrane domains (TEMs). The tetraspanins and γ-secretase proteins co-migrate in the low-density fractions of sucrose density gradients, the researchers show. Dissociation of TEMs by detergent treatment also resulted in loss of presenilin from the same fraction. Previously, γ-secretase as well as β-secretase have been localized to lipid rafts (Wahrle et al., 2002, and see ARF related news story on Hattori et al., 2006), which are detergent-insoluble, cholesterol-rich membrane microdomains similar to TEMs. However, TEMs are different from lipid rafts in their protein and lipid makeup and in their detergent sensitivity. (There are no tetraspanins in lipid rafts, for instance.) The authors propose that the association with TEMs might account for a large part of the γ-secretase activity previously attributed to lipid rafts.

The work raises many questions: Does the disruption of the tetraspanin interactions affect γ-secretase localization, stability, activity, or all of the above? Are the interactions and functional effects direct or indirect? Do tetraspanins contribute to AD pathology, and might they make plausible drug targets? Tetraspanins have been previously shown to regulate the activity of an α-secretase, ADAM10 (Xu et al., 2009), suggesting that these little proteins may hold more than one clue to Aβ production in their tangled web.—Pat McCaffrey.

Reference:
Wakabayashi T, Craessaerts K, Bammens L, Bentahir M, Borgions F, Herdewijn P, Staes A, Timmerman E, Vandekerckhove J, Rubinstein E, Boucheix C, Gevaert K, De Strooper B. Analysis of the gamma-secretase interactome and validation of its association with tetraspanin-enriched microdomains. Nat Cell Biol. 2009 Oct 18. Abstract