Scientists believe that of the different γ complexes that exist in vivo, some are tuned for Notch, some for APP, some to yet other substrates. This tuning probably happens during the assembly phase, implying that there must be regulation at this level. Christoph Kaether, formerly of Haass’ group and now at the Leibniz Institute for Age Research in Jena, Germany, studies where in the cell the complex is put together, and what other proteins control this process. Prior work in this area has established that the complex fully assembles in the ER, where Aph-1 and nicastrin first get together, stabilize presenilin, and finally are joined by Pen-2. The complete complex then leaves the ER, moving through the TGN and up to the cell membrane, where it later becomes endocytosed and degraded. A recent study answered, at least in cultured cells, if not yet for neurons in vivo, the longstanding debate about where in the cell γ-secretase is active. It showed that the complex does not begin to be active until it has reached the cell membrane. Cleavage then occurs there, in endosomes, and also in lysosomes, Kaether said (Kaether et al., 2006). This finding ties into an emerging line of thought about dysregulation of endocytosis and autophagy in early AD.

Assembly in the ER is a characteristic the γ-secretase complex has in common with channels, Kaether said. They, too, are made up of subunits that each have an ER retention signal on them, and this signal becomes masked once the subunits have clicked together and are ready to “move up” to the plasma membrane. Surface proteins such as the acetylcholine receptor and B and T cell receptors also fall into this class.

At the Eibsee, Kaether outlined new work detailing retention/retrieval signals in the complex components, as well as additional proteins, which together govern this process. Kaether found two such signals, one in PS1’s C-terminus and one in Pen-2’s first transmembrane domain. A trafficking protein recognizing these signals might be Rer1, the human homolog of a small yeast protein known to retain unassembled proteins in the ER (Sato et al., 2003). Kaether and colleagues found that human Rer1 resides in ER and Golgi, where it binds to the retention signal in Pen-2. Rer1 limits the rate of γ-secretase assembly. The scientists propose that Rer1 cycles between the ER and the Golgi, where it traps unassembled Pen-2 that has escaped from the ER and returns it. Completion of the complex masks the Pen-2 retention signal, and the complex can slip past Rer1’s surveillance in the Golgi. (For more on APP trafficking, see Walter and Lichtenthaler talks.)

Like the γ-secretase components, APP percolates from the ER through the TGN and to the plasma membrane, where it later undergoes endocytosis and degradation in lysosomes. Researchers think that, at least in experimental cell models, APP is subject to α cleavage while in the secretory half of this life cycle and to β and γ cleavage while at the cell membrane and in endosomes, but they do not know how membrane lipids govern this segregation. Cholesterol is most often invoked, but at the Eibsee conference, Jochen Walter of the University of Bonn presented his work on glycosphingolipids. Conventional wisdom has viewed these membrane components as merely structural, but that is changing. Cells make different varieties of these complex molecules by taking ceramide and icing it with various sugars in the ER/Golgi compartments. Their transport through the secretory pathway closely tracks that of APP. Glycosphingolipids further intrigue scientists because neurons contain lots of them, preferentially in detergent-resistant membranes (DRMs, also called lipid rafts), and their levels change in AD and in other neurodegenerative diseases.

In earlier work, Walter inhibited the addition of glucose to ceramide, that is, the first step in glycosphingolipid synthesis. In its absence, cellular expression of APP stayed the same but secretion of both APP and Aβ dropped. Pulse-chase experiments showed that the reason lay in defective maturation, stability, and transport to the cell surface of APP (Tamboli et al., 2005). Since then, RNAi suppression of the enzyme catalyzing the second step in glycosphingolipid biosynthesis, as well as other experiments, has produced similar results in support of the hypothesis that these lipids aid APP maturation and trafficking, Walter said. This may well affect APP processing, as well, Walter noted. Experiments with particular sphingolipids, such as purified gangliosides, suggested that they can promote Aβ generation.

A second line of research in Walter’s lab explores the intraneuronal trafficking of BACE. (Besides Walter’s research, BACE drew surprisingly little attention at a conference otherwise heavily weighted toward APP processing.) During his time in Christian Haass’ lab, Walter had linked phosphorylation to BACE trafficking. Like presenilin, BACE becomes reinternalized into endosomes at the cell membrane. However, rather than being subsequently degraded, non-phosphorylated BACE molecules recycle directly up to the cell surface again, whereas a phosphorylated form of BACE1 journeys backwards through the TGN (Walter et al., 2001; Wahle et al., 2005). This phosphorylation-dependent retrograde transport of BACE1 to the TGN is mediated by sorting proteins that go by a mouthful of a name, that is, Golgi-localized, γ ear-containing, ADP ribosylation factor-binding, or GGA proteins, for short (Bonifacino, 2004). To read up on the emerging area of BACE recycling, see also He et al., 2003; ; and Scott Small’s recent study on retromer proteins.

Can this BACE trafficking change APP processing? There is no answer yet to this question, but Walter believes that GGA1 might hold a clue because these proteins are implicated in two-way transport of cargo proteins through the secretory pathway. Walter’s ongoing work focuses on GGA1, a neuronal variant whose levels he found to be decreased in brain lysate of AD patients. Reducing GGA1 function experimentally caused BACE1 to pile up in the endosomal compartment, Walter noted, where other studies have begun noticing it, as well. APP is a minor one among BACE1’s many substrates, yet scientists believe this cleavage occurs at least in part in the endosomal compartment. Adding to the intrigue around GGA proteins is the finding that they affect trafficking of SorLa, another sorting protein suspected of influencing APP processing (Andersen et al., 2005).

That lipids affect APP processing is quite widely accepted, yet the opposite—that Aβ affects lipids—is a newer, more provocative idea. Tobias Hartmann reviewed a recent discovery and added new twists for further discussion. Last November, Hartmann’s group proposed a set of interlocking feedback loops connecting γ-secretase activity and membrane lipid production, (see news summary and schematic drawing). In brief, Hartmann suggests that not only do cellular lipids such as cholesterol and sphingomyelin regulate γ-secretase activity, but also Aβ itself in turn regulates levels of those lipids. This is specific in that the products of γ-secretase substrates other than APP do not. The gist of the proposed regulatory pathways is that Aβ42 performs the normal, physiologic function of spurring the activity of the degradative enzyme sphingomyelinase, in essence lowering sphingomyelin levels. The ensuing decrease in sphingomyelin would then lift a block on γ-secretase function. For its part, Aβ40 at physiological concentration is reported to keep cholesterol levels low by blocking synthesis at the same HMG-CoA reductase step that statin drugs inhibit. This second cycle closes, as decreasing cholesterol concentrations would end this lipid’s stimulatory effect on γ-secretase activity.

This work has created a buzz in the field, and at the Eibsee, Hartmann added further twists. One concerns the question of how this interdependent system would react to FAD mutations. Hartmann noted that a growing body of data finds a strong correlation between the Aβ42/40 ratio and when disease begins. Specifically, mean age of onset tends to be lower in people who had less CSF Aβ40 and more Aβ42 for a set of FAD mutants Hartmann’s group examined. This change in the ratio would effectively uncouple a formerly integrated regulation of lipid levels: Aβ40 normally blocks HMG-CoA reductase and FAD mutations tend to weaken that inhibition, leaving the cells with more cholesterol. Aβ42 normally activates SMase and many FAD mutations enhance it, leaving the cell with less sphingomyelin and more of its metabolite ceramide, which has been linked to neuronal apoptosis. This data appear to fit with Kumar-Singh et al. (2005), and Wang et al. (2006), who suggest that Aβ40 might have a protective function that falls away in Aβ40-lowering FAD mutations (see recent Alzforum Discussion).

Exactly how might Aβ control lipid levels? Hartmann said that Aβ42 acts directly on sphingomyelinase, but how Aβ40 might inhibit HMG-CoA reductase remains a mystery. The peptide does not act on the gene’s promoter. Cells keep Aβ40 away from the enzyme’s site of residence in the endoplasmic reticulum, so it can’t be a direct action but likely requires a messenger in between. Additional challenges in this line of investigation include human testing, for example, by measuring sphingomyelinase activity and cholesterol levels in people with FAD mutations, and assessing the effect of related peptides, such as Aβ38, on cellular lipids.—Gabrielle Strobel.

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References

News Citations

  1. An Intricate Dance: α-Secretase and Its Partners
  2. A Better GRIP on the Aβ-Lipid Connection

Webinar Citations

  1. Gain or Loss of Function—Time to Shake up Assumptions on γ-Secretase in Alzheimer Disease?

Paper Citations

  1. . Amyloid Precursor Protein and Notch Intracellular Domains are Generated after Transport of their Precursors to the Cell Surface. Traffic Online Early. 2006 Feb;
  2. . Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes. Mol Biol Cell. 2003 Sep;14(9):3605-16. PubMed.
  3. . Inhibition of glycosphingolipid biosynthesis reduces secretion of the beta-amyloid precursor protein and amyloid beta-peptide. J Biol Chem. 2005 Jul 29;280(30):28110-7. PubMed.
  4. . Phosphorylation regulates intracellular trafficking of beta-secretase. J Biol Chem. 2001 May 4;276(18):14634-41. PubMed.
  5. . GGA proteins regulate retrograde transport of BACE1 from endosomes to the trans-Golgi network. Mol Cell Neurosci. 2005 Jul;29(3):453-61. PubMed.
  6. . The GGA proteins: adaptors on the move. Nat Rev Mol Cell Biol. 2004 Jan;5(1):23-32. PubMed.
  7. . Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins. Biochemistry. 2003 Oct 28;42(42):12174-80. PubMed.
  8. . Demonstration of BACE (beta-secretase) phosphorylation and its interaction with GGA1 in cells by fluorescence-lifetime imaging microscopy. J Cell Sci. 2004 Oct 15;117(Pt 22):5437-45. PubMed.
  9. . Model-guided microarray implicates the retromer complex in Alzheimer's disease. Ann Neurol. 2005 Dec;58(6):909-19. PubMed.
  10. . Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein. Proc Natl Acad Sci U S A. 2005 Sep 20;102(38):13461-6. PubMed.

Other Citations

  1. Walter

Further Reading