More Than APP—γ-Secretase, Metalloproteases Control Neurotransmission

Pondering the physiological roles of γ-secretase? A study in this week’s Journal of Neuroscience offers more reason to think outside the amyloid-β box. It finds the secretase at synapses and suggests it helps control neurotransmission. γ-secretase works with metalloproteases to cleave proteins that promote synaptic activity, according to the paper, and neurotransmission drives the proteolysis, providing a “novel form of synaptic autoregulation,” write first author Sophie Restituito of New York University Langone Medical Center, and colleagues. By suggesting a function for γ-secretase at synapses, the research could explain how familial AD mutations that cause loss of γ-secretase function give rise to neurodegenerative disease marked by synaptic failure.

Though γ-secretase appears primarily in endosomes, scientists previously detected the protease at the plasma membrane (see Lah et al., 1997), and at synapses (see Georgakopoulos et al., 1999). More recent work showed that presenilin-1 (PS1), the catalytic center of γ-secretase, interacts with δ-catenin (Kosik et al., 2005), a cell adhesion regulator that senior author Edward Ziff and others have also reported at synapses (Silverman et al., 2007). This hinted to Restituito that PS1 does something important in those tiny spaces between neurons.

By immunofluorescence, her team found PS1 co-localizing with the post-synaptic marker PSD-95 in embryonic rat hippocampal neurons. PSD-95 also shows up with the PS1 substrates N-cadherin and EphRB2, as well as with three metalloproteases (aka “α-secretases”)—ADAM10, ADAM17, and MT5-MMP. α- and γ-secretase sequentially cleave a variety of transmembrane proteins including N-cadherin and EphRB2. In addition, the researchers detected γ-secretase components and substrates on Western blots of rat brain synaptosomes, and showed that the protease is enzymatically active there, cutting N-cadherin at pre- and post-synaptic areas.

Harking back to the lab’s earlier report of PS1 associating with δ-catenin, Restituito and colleagues found that the cell adhesion factor is, in fact, required to keep PS1 at the synapse. In brain extracts from δ-catenin knockout mice, PS1 was down, relative to wild-type levels, in post-synaptic sites, but not in other subcellular fractions, the researchers found.

If α- and γ-secretase reside at the synapse, could neuronal activity determine whether they snip their synaptic substrates? The literature to date is murky, suggesting synaptic stimulation can prevent (see Tanaka et al., 2000; Tai et al., 2007) or promote N-cadherin cleavage (see Marambaud et al., 2003; Uemura et al., 2006). The experiments in the current study add support for the latter—activation of cultured rat neurons with glutamate or NMDA drove up production of the C-terminal fragment (CTF1) formed when metalloproteases cut N-cadherin. When neuronal firing was dampened using the sodium channel blocker tetrodotoxin, levels of the N-cadherin fragment dropped. Src or JNK kinase inhibitors also blocked formation of this fragment, suggesting those signaling pathways operate in the NMDAR-mediated proteolysis. However, synaptic activity had little bearing on γ-secretase cleavage of CTF1, indicating that NMDA stimulation enhances N-cadherin processing by promoting the initial cut only. Just a small fraction of full-length N-cadherin gets cleaved, and what those C-terminal fragments do is not entirely clear, though accumulating evidence points toward a synaptic function. Of the little CTF1 that neurons do produce, much of it shows up in synapses. γ-secretase cleavage of CTF1 produces a smaller CTF2 fragment that represses transcription (Marambaud et al., 2003).

If synaptic activity promotes N-cadherin proteolysis, what happens if that cleavage is blocked? The researchers showed that γ-secretase or metalloprotease inhibitors enhanced synaptic transmission (measured by mini-excitatory post-synaptic currents) and increased levels of synaptic proteins needed for neurotransmission (judged by immunofluorescent staining of PSD-95, synaptotagmin, and the AMPA receptor subunit GluA2). Taken together, the data describe a “feedback loop” where synaptic activity activates proteolysis, which in turn modulates synaptic activity, Restituito told ARF.

Showing that γ-secretase and metalloproteases are “not just physically present at the synapse, but also influence other proteins known to be important for synaptic transmission—that is a key finding,” said Jane Sullivan of the University of Washington, Seattle. In a Nature Neuroscience paper published a few weeks ago, she and colleagues reported that PS1 regulates homeostatic scaling, a type of synaptic plasticity that operates in neuronal networks (ARF related news story on Pratt et al., 2011).

Kenneth Kosik of the University of California, Santa Barbara, finds the current paper novel in that it “gets at a global mechanism for the regulation of synaptic proteins. It shows that concentrations of these proteins may be linked to synaptic transmission through proteolytic activity [of γ-secretase and MMPs],” he told ARF.

Though the present study focused on N-cadherin, Restituito’s team has also looked at amyloid precursor protein (APP), the MMP/γ-secretase substrate that grabs much of the attention in AD. Preliminary data suggest that some APP may also be processed at synapses and regulated by synaptic activity, Restituito told ARF.

The relationship between Aβ and synaptic activity was examined in a study published earlier this month by John Cirrito of Washington University School of Medicine, St. Louis, Missouri (see ARF related news story on Verges et al., 2011). That work and the current paper show that NMDAR stimulation activates α-secretase. (ADAM10, ADAM17, and MT50MMP—the metalloproteases Restituito’s team detected at synapses—are α-secretases.) Cirrito found that α-secretase activation decreases Aβ production, whereas Restituito discovered it leads to N-cadherin-dependent inhibition of synaptic activity. “It would appear NMDARs may activate one pathway which then has multiple consequences,” Cirrito suggested in an e-mail to ARF (see full comment below).

Given prior reports that some PS1 FAD mutations cause loss of γ-secretase function (Marambaud et al., 2003; Georgakopoulos et al., 2006), the new data may link dysfunction in the γ-secretase/MMP proteolytic system with the synaptic abnormalities of familial AD, Anastasios Georgakopoulos, Mount Sinai School of Medicine, New York, suggested in an e-mail to ARF (see full comment below).

Ultimately, “how NMDARs, secretases, MMPs, cadherins, and Aβ function in various combinations to regulate synaptic activity will be important to parse apart, though the relationships are likely to be very complex,” Cirrito noted.—Esther Landhuis

Comments

  1. The interesting work by Restituito et al. shows that both metalloproteinase and γ-secretase activities are localized at the synapse and regulate synaptic function. These findings, combined with previous reports that PS1 FAD mutations cause loss of γ-secretase function (Marambaud et al. 2003; Georgakopoulos et al. 2006; Litterst et al., 2007), may provide a link between the dysfunction of these proteolytic systems and the synaptic abnormalities of FAD.

    The authors present evidence that components of γ-secretase localize on synaptic membranes where this enzymatic system exerts its proteolytic function, a finding consistent with previous observations of the synaptic localization of PS1 (Georgakopoulos et al., 1999). Interestingly, the experiments with δ-catenin knockout neurons suggest that this catenin mediates the association of PS1 with post-synaptic densities, a finding consistent with reports that δ/p120-catenin, a cadherin-binding protein, binds PS1 and acts as an adaptor that recruits cadherins to γ-secretase for processing (Kouchi et al., 2009). Interestingly, what δ/p120 catenin does for the γ-secretase processing of cadherins, GSAP performs for the γ-secretase processing of APP (He et al., 2010).

    The reported work presented here nicely dissects the role of the two proteolytic activities on synaptic function, showing that they differentially affect glutamatergic transmission, a finding consistent with recent reports that γ-secretase regulates glutamate release (Pratt et al., 2011). Together, these findings provide a potential mechanism of how deregulation of those proteolytic activities may impair synaptic function in AD.

    Overall, the interesting work by Restituito et al. provides evidence for the synaptic localization of both metalloproteinase and γ-secretase activities, and shows that they affect synaptic function. Combined with literature, the work suggests that malfunctions in those proteolytic systems may be involved in the synaptic impairments observed in AD.

    References:

    Marambaud P, Wen PH, Dutt A, Shioi J, Takashima A, Siman R, Robakis NK. A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell. 2003 Sep 5;114(5):635-45. PubMed.

    Georgakopoulos A, Litterst C, Ghersi E, Baki L, Xu C, Serban G, Robakis NK. Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling. EMBO J. 2006 Mar 22;25(6):1242-52. PubMed.

    Litterst C, Georgakopoulos A, Shioi J, Ghersi E, Wisniewski T, Wang R, Ludwig A, Robakis NK. Ligand binding and calcium influx induce distinct ectodomain/gamma-secretase-processing pathways of EphB2 receptor. J Biol Chem. 2007 Jun 1;282(22):16155-63. PubMed.

    Georgakopoulos A, Marambaud P, Efthimiopoulos S, Shioi J, Cui W, Li HC, Schütte M, Gordon R, Holstein GR, Martinelli G, Mehta P, Friedrich VL, Robakis NK. Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts. Mol Cell. 1999 Dec;4(6):893-902. PubMed.

    Kouchi Z, Barthet G, Serban G, Georgakopoulos A, Shioi J, Robakis NK. p120 catenin recruits cadherins to gamma-secretase and inhibits production of Abeta peptide. J Biol Chem. 2009 Jan 23;284(4):1954-61. PubMed.

    He G, Luo W, Li P, Remmers C, Netzer WJ, Hendrick J, Bettayeb K, Flajolet M, Gorelick F, Wennogle LP, Greengard P. Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease. Nature. 2010 Sep 2;467(7311):95-8. PubMed.

    Pratt KG, Zimmerman EC, Cook DG, Sullivan JM. Presenilin 1 regulates homeostatic synaptic scaling through Akt signaling. Nat Neurosci. 2011 Sep;14(9):1112-4. PubMed.

    View all comments by Anastasios Georgakopoulos

  2. Restituito and colleagues provide interesting and compelling evidence that α-secretase/matrix metalloproteases (MMPs) and γ-secretase lead to a suppression of synaptic activity. NMDA receptors increase enzymatic activity of α-secretase. α-secretase cleaves N-cadherin, which leads to further cleavage by γ-secretase. The consequence of this is destabilized synapses and suppressed synaptic activity (or at the very least, suppressed mini-excitatory post-synaptic currents). Several groups have localized various ADAM secretases and MMP proteins to synapses as well as components of the γ-secretase complex. This paper includes a very elegant localization of all of these proteins to both pre- and post-synaptic compartments using biochemistry, histology, and electron microscopy. It also includes evidence that γ-secretase activity (not just proteins) is present at synapses, though the subcellular localization of γ-secretase activity at the synapse still needs further refinement. They also include nice data that δ-cadherin tethers γ-secretase near the synapse.

    NMDARs, α-secretase, γ-secretase, and MMPs are also responsible for Aβ metabolism (aspects of Aβ generation and Aβ clearance). Oligomeric Aβ appears to reduce synaptic activity. Roberto Malinow’s group has shown that Aβ can have an autoregulatory effect on synaptic activity. Restituito and colleagues propose the NMDAR/N-cadherin/secretase pathway also has a negative autoregulatory effect on activity. Both of these autoregulatory mechanisms would involve NMDARs. There are conceivable scenarios whereby NMDARs could simultaneously decrease Aβ generation to increase synaptic activity, while increasing N-cadherin cleavage to decrease synaptic activity. How NMDARs, secretases, MMPs, cadherins, and Aβ function in various combinations to regulate synaptic activity will be important to parse apart, though the relationships are likely to be very complex (and at the moment are mind boggling to me).

    We have data recently published in that activation of NMDARs increase α-secretase activity, which reduces Aβ generation in vivo (Verges et al., 2011). Restituito et al. show NMDARs increase α-secretase, which leads to N-cadherin-dependent suppression of synaptic activity. It will be interesting to determine if these phenomena share the same initial signaling pathway through α-secretase but result in distinct consequences.

    References:

    Verges DK, Restivo JL, Goebel WD, Holtzman DM, Cirrito JR. Opposing synaptic regulation of amyloid-β metabolism by NMDA receptors in vivo. J Neurosci. 2011 Aug 3;31(31):11328-37. PubMed.

    View all comments by John Cirrito

  3. The news article states that "γ-secretase appears primarily in endosomes," but the results of Area-Gomez et al. (Area-Gomez et al., 2009) do not support this. Most of the γ-secretase activity (at least in neurons) appears to be in the mitochondrial associated membranes (aka "MAM"). In a second paper (Schon and Area-Gomez, 2010), they describe why this has not previously been observed.

    References:

    Area-Gomez E, de Groof AJ, Boldogh I, Bird TD, Gibson GE, Koehler CM, Yu WH, Duff KE, Yaffe MP, Pon LA, Schon EA. Presenilins are enriched in endoplasmic reticulum membranes associated with mitochondria. Am J Pathol. 2009 Nov;175(5):1810-6. PubMed.

    Schon EA, Area-Gomez E. Is Alzheimer's disease a disorder of mitochondria-associated membranes?. J Alzheimers Dis. 2010;20 Suppl 2:S281-92. PubMed.

    View all comments by Michael Lardelli

  4. Very interesting results from Restituito and colleagues. It is time to move forward and find out new alternatives to complement the classic amyloid cascade hypothesis of AD.

    View all comments by Breno Diniz

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References

News Citations

  1. Homeostatic Scaling—Presenilin Repertoire Reaches New Heights?
  2. Brain Activity and Aβ—The Interstitial Plot Thickens

Paper Citations

  1. Lah JJ, Heilman CJ, Nash NR, Rees HD, Yi H, Counts SE, Levey AI. Light and electron microscopic localization of presenilin-1 in primate brain. J Neurosci. 1997 Mar 15;17(6):1971-80. PubMed.
  2. Georgakopoulos A, Marambaud P, Efthimiopoulos S, Shioi J, Cui W, Li HC, Schütte M, Gordon R, Holstein GR, Martinelli G, Mehta P, Friedrich VL, Robakis NK. Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts. Mol Cell. 1999 Dec;4(6):893-902. PubMed.
  3. Kosik KS, Donahue CP, Israely I, Liu X, Ochiishi T. Delta-catenin at the synaptic-adherens junction. Trends Cell Biol. 2005 Mar;15(3):172-8. PubMed.
  4. Silverman JB, Restituito S, Lu W, Lee-Edwards L, Khatri L, Ziff EB. Synaptic anchorage of AMPA receptors by cadherins through neural plakophilin-related arm protein AMPA receptor-binding protein complexes. J Neurosci. 2007 Aug 8;27(32):8505-16. PubMed.
  5. Tanaka H, Shan W, Phillips GR, Arndt K, Bozdagi O, Shapiro L, Huntley GW, Benson DL, Colman DR. Molecular modification of N-cadherin in response to synaptic activity. Neuron. 2000 Jan;25(1):93-107. PubMed.
  6. Tai CY, Mysore SP, Chiu C, Schuman EM. Activity-regulated N-cadherin endocytosis. Neuron. 2007 Jun 7;54(5):771-85. PubMed.
  7. Marambaud P, Wen PH, Dutt A, Shioi J, Takashima A, Siman R, Robakis NK. A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell. 2003 Sep 5;114(5):635-45. PubMed.
  8. Uemura K, Kihara T, Kuzuya A, Okawa K, Nishimoto T, Bito H, Ninomiya H, Sugimoto H, Kinoshita A, Shimohama S. Activity-dependent regulation of beta-catenin via epsilon-cleavage of N-cadherin. Biochem Biophys Res Commun. 2006 Jul 7;345(3):951-8. PubMed.
  9. Pratt KG, Zimmerman EC, Cook DG, Sullivan JM. Presenilin 1 regulates homeostatic synaptic scaling through Akt signaling. Nat Neurosci. 2011 Sep;14(9):1112-4. PubMed.
  10. Verges DK, Restivo JL, Goebel WD, Holtzman DM, Cirrito JR. Opposing synaptic regulation of amyloid-β metabolism by NMDA receptors in vivo. J Neurosci. 2011 Aug 3;31(31):11328-37. PubMed.
  11. Georgakopoulos A, Litterst C, Ghersi E, Baki L, Xu C, Serban G, Robakis NK. Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling. EMBO J. 2006 Mar 22;25(6):1242-52. PubMed.

Further Reading

Papers

  1. Inoue E, Deguchi-Tawarada M, Togawa A, Matsui C, Arita K, Katahira-Tayama S, Sato T, Yamauchi E, Oda Y, Takai Y. Synaptic activity prompts gamma-secretase-mediated cleavage of EphA4 and dendritic spine formation. J Cell Biol. 2009 May 4;185(3):551-64. PubMed.
  2. Verges DK, Restivo JL, Goebel WD, Holtzman DM, Cirrito JR. Opposing synaptic regulation of amyloid-β metabolism by NMDA receptors in vivo. J Neurosci. 2011 Aug 3;31(31):11328-37. PubMed.
  3. Pratt KG, Zimmerman EC, Cook DG, Sullivan JM. Presenilin 1 regulates homeostatic synaptic scaling through Akt signaling. Nat Neurosci. 2011 Sep;14(9):1112-4. PubMed.

Primary Papers

  1. Restituito S, Khatri L, Ninan I, Mathews PM, Liu X, Weinberg RJ, Ziff EB. Synaptic autoregulation by metalloproteases and γ-secretase. J Neurosci. 2011 Aug 24;31(34):12083-93. PubMed.

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