Contact

Prof. Dr. Volker Haucke

phone +49 30 94793 100
haucke(at)fmp-berlin.de

 

Research Areas of the Haucke Lab

Neurotransmission and presynaptic membrane turnover


Regulation of membrane homeostasis in endocytosis & the endolysosomal system by phosphoinositides and phosphoinositide metabolizing enzymes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Research Department Molecular Pharmacology and Cell Biology (Volker Haucke)

Neurotransmission and presynaptic membrane turnover

From sensory perception to learning and memory, the functioning of the nervous system is dependent upon communication between neurons. Dysfunction of neuronal signaling results in neurological and neurodegenerative disorders ranging from autism to epilepsy and Alzheimer's disease. During neurotransmission at synapses specialized 40 nm secretory organelles termed synaptic vesicles undergo calcium-regulated exocytotic fusion with the presynaptic membrane to release their neurotransmitter content into the synaptic cleft (Fig.1) PubMed link.

Figure 1: Computational 3D-model of an "average" mammalian synapse based on combined quantitative proteomic and super-resolution imaging analyses Wilhelm et al., Science 2014.

In order to sustain neurotransmitter release under conditions of stimulation at high frequency and the corresponding massive exocytotic membrane insertion fused synaptic vesicle membranes are retrieved and synaptic vesicles are reformed at the outer margin of the active zone [see Galli & Haucke, Sci STKE (2004), Jung & Haucke, Traffic (2007), Haucke et al. Nat Rev. Mol. Cell Biol. (2011), Puchkov & Haucke, Trends Cell Biol. (2013), Haucke et al., Nat Rev. Mol Cell Biol. 2011, Kononenko & Haucke, Neuron 2015 for recent reviews].

Despite the identification of a plethora of proteins involved in endocytosis, the precise pathway by which synaptic vesicles are recycled has remained a matter of debate.

Work in the laboratory thus focusses on three fundamental questions:

1. What are the molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation?

2. How is exocytosis at the active zone coupled to endocytosis and synaptic vesicle reformation and how does this relate to disease (i.e. autism, epilepsy, ADHS) (Figure 2; Haucke et al., Nat Rev. Mol Cell Biol. 2011; Kononenko & Haucke, Neuron 2015)?

3. How is presynaptic membrane homeostasis maintained under conditions of activity, in different types of neurons, and during aging? How are synaptic proteins turned over and what is the role of autophagy in this process?

We use combined optical imaging, electron microscopy, acute perturbation, and genetic approaches including mouse models to address these questions.

Figure 2: Models of synaptic vesicle endocytosis (A) In the "kiss-and-run" model, synaptic vesicles form a transient pore through which they release their neurotransmitter contents, and are then rapidly recycled (in about a second) at the site of fusion without being absorbed into the membrane. (B) Utrafast mechanism for synaptic vesicle recycling. This process takes roughly one tenth of a second (or less) and involves the formation of large endocytic pits devoid of a clathrin coat. This mechanism is distinct from either "kiss-and-run" or clathrin-mediated endocytosis. (C) Clathrin-based endocytosis. In this model, synaptic vesicles collapse fully into the plasma membrane, before being retrieved via a slow process (taking about 10 to 20 seconds) mediated by the coat protein clathrin. This occurs at sites distant from the site of fusion. Both (B) and (C) require the GTPase dynamin to pinch off newly formed endocytic vesicles from the plasma membrane (adapted from Kononenko and Haucke & Klingauf, eLife (2013).

The Haucke lab has identified stonin 2 as the first sorting adaptor specifically dedicated to the sorting of the vesicular calcium sensor synaptotagmin 1 in the mammalian brain (Diril et al., Dev. Cell 2006) and in the in C. elegans nervous system  (Jung et al., J. Cell Biol., 2007). Using combined NMR-based structural, biochemical, and cell biological approaches we have unravelled the molecular mechanism by which endocytic AP180 family proteins sort the essential v-SNARE synaptobrevin during exo-endocytic cycling of synaptic vesicle membranes (Koo et al., Proc. Natl. Acad. Sci. USA 2011). We could show that loss of AP180 in mice in vivo causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. These data together we further genetic experiments demonstrate that a large vesicular Syb2 pool maintained by AP180 is crucial to sustain efficient neurotransmission and SV reformation (Koo et al., Neuron 2015). Endocytic adaptors also play an important role in limiting the diffusional spread of newly exocytosed synaptic vesicle proteins at the neuronal cell surface (Gimber et al., Nat. Commun. 2015).

Another major focus of the lab has been the dissection of the mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. This has resulted in several major discoveries. First, we have demonstrated (together with the group of Dr. Tanja Maritzen, FMP Berlin) based on the analysis of stonin 2 and AP180 KO mice that distinctive mechanisms control the rate and fidelity of synaptic vesicle protein sorting (Kononenko et al., Proc. Natl. Acad. Sci. USA 2013; Koo et al., Neuron 2015). Second, we have shown using lentiviral strategies and conditional KO mice that clathrin and its major adaptor protein 2 (AP-2), key essential proteins in clathrin-mediated endocytosis, in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate synaptic vesicles but, surprisingly, are dispensable for membrane retrieval. Loss of AP-2 in vivo causes the accumulation of endosome-like vacuoles and a depletion of synaptic vesicles, resulting in severely impaired neurotransmission and postnatal lethality (Figure 3).



Figure 3: Electron tomograms and 3D reconstructions of presynaptic terminals from the somatosensory cortex of wild-type (A) and AP-2(µ) KO mice (B). AP-2(µ) KO synapses display a drastic reduction in SV number and an accumulation of endosomes-like vacuoles (ELVs), most of which were disconnected from the plasma membrane (PM). Adapted from Kononenko et al. Neuron (2014).

These data unravel a clathrin-independent endocytosis involving dynamin 1/3 and endophilin as a major route for presynaptic membrane turnover at synapes and together with theoretical modelling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/ AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies (Kononenko et al., Neuron 2014; Kononenko & Haucke, Neuron 2015) (Figure 4).

Figure 4: Hypothetical model for SV membrane retrieval and reformation. (Left) High activity firing induces a steep rise in presynaptic calcium levels and triggers fast membrane retrieval via endosome-like vacuoles (ELVs) upstream of clathrin coat assembly. ELVs are subsequently converted into SVs via clathrin/ AP-2 mediated budding and possibly additional pathways (i.e. via AP-3). (Right) Under conditions of low frequency activity presynaptic calcium levels remain comparably low and membrane fission is rate limiting. This allows clathrin/ AP-2 coats to assemble prior to membrane fission, resulting in SV reformation directly from the plasma membrane. From Kononenko & Haucke Neuron (2012).

Third, we have identified molecular scaffolds that act as a bridge between the machineries for exocytosis and synaptic vesicle recycling. For example, we have found that loss of the endocytic proteins AP-2 or intersectin 1 in mice causes selective defects in fast neurotransmitter release that may be related to the clearance of release sites for neurotransmitter rather than defects in presynaptic membrane retrieval (Jung, Maritzen et al., EMBO J. 2015, Sakaba et al., Proc. Natl. Acad. Sci. USA 2013). Conversely, we have identified the active zone cytomatrix protein dGIT/ GIT1 as the first component that links the active zone cytomatrix and the endocytic machinery and thereby facilitates synaptic vesicle regeneration (Podufall et al., Cell Reports 2014).
Much of this work is performed together with a number of collaborating labs in the US, Australia, Sweden, Israel, the UK, and Germany.

Selected references

  • Koo, S.Y., Kochlamazashvili, G., Rost, B., Puchkov, D., Gimber, N., Lehmann, M., Tadeus, G., Schmoranzer, J., Rosenmund, C., Haucke, V.#, Maritzen, T.# (2015) Vesicular synaptobrevin/VAMP2 levels guarded by AP180 control efficient neurotransmission. Neuron, 88, 330-344 (#co-corresponding authors)
  • Gimber, N., Tadeus, G., Maritzen, T., Schmoranzer, J., Haucke, V. (2015) Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins. Nature Communications, 6, 8392
  • Jung, S.Y., Maritzen, T., Wichmann, C., Jing, Z., Neef, A., Revelo, N.H., Al-Moyed, H., Meese, S., Wojcik, S.M., Panou, I., Bulut, H., Schu, P., Ficner, R., Reisinger, E., Rizzoli, S.O., Haucke, V.*, Moser, T.* (2015) Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing. EMBO J, [advance online] (* co-corresponding authors)
  • Lehmann, M., Gottschalk, B., Puchkov, D., Schmieder, P., Schwagerus, S., Hackenberger, C.P., Haucke, V., Schmoranzer, J. (2015) Multicolor 'caged' dSTORM resolves the ultra-structure of synaptic vesicles in the brain. Angew. Chem. 54, 13230-13235
  • Kaempf N., Kochlamazashvili G., Puchkov D., Maritzen T., Bajjalieh S.M., Kononenko N.L., Haucke V. (2015) Proc Natl Acad Sci U S A, 112, 7297-7302
  • Kononenko, N.L. and Haucke, V. (2015) Molecular Mechanisms of Presynaptic Membrane Retrieval and Synaptic Vesicle Reformation. Neuron, 85, 484-496
  • Kononenko, N.L., Puchkov, D., Classen, G.A., Walter, A., Pechstein, A., Sawade, L., Kaempf, N., Trimbuch, T., Lorenz, D., Rosenmund, C., Maritzen, M., Haucke, V. (2014) Clathrin/ AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron, 82, 981-988
  • Podufall, J,. Tian, R., Knoche, E., Puchkov, D., Walter, A.M., Rosa, S., Quentin, C., Vukoja, A., Jung, N., Lampe, A., Wichmann, C., Böhme, M., Depner, H., Zhang, Y.Q., Schmoranzer, J., Sigrist, S.J., Haucke, V. (2014) A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling. Cell Reports, 7, 1417-1425
  • Wilhelm, B.G., Mandad, S., Truckenbrodt, S., Kröhnert, K., Schäfer, C., Rammner, B., Koo, S.J., Claßen, G.A., Krauss, M., Haucke, V., Urlaub, H., Rizzoli, S. O. (2014) Composition of Synaptic Boutons Reveals the Amounts of Vesicle Trafficking Proteins. Science, 344, 1023-1028
  • Kononenko, N., Diril, M.K., Puchkov, D., Kintscher, M., Koo, S.J., Pfuhl, G, Winter, Y., Wienisch, M., Klingauf, J., Breustedt, J., Schmitz, D., Maritzen, T., Haucke, V. (2013) Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2. Proc Natl Acad Sci U S A, 110, E526-E535
  • Sakaba, T., Kononenko, N., Bacetic, J., Pechstein, A., Schmoranzer, J., Yao, L., Barth, H., Shupliakov, O., Kobler, O., Aktories, K., Haucke, V. (2013) Fast neurotransmitter release regulated by the endocytic scaffold intersectin. Proc Natl Acad Sci U S A, 110, 8266-71
  • Puchkov, D. and Haucke, V. (2013) Greasing the synaptic vesicle cycle by membrane lipids. Trends Cell Biol., 23, 493–503
  • von Kleist, L., Stahlschmidt, W., Bulut, H., Gromova, K., Puchkov, D., Robertson, M., MacGregor, K.A., Tomlin, N., Pechstein, A., Chau, N., Chircop, M., Sakoff, J., von Kries, J., Saenger, W., Kräusslich, H.-G., Shupliakov, O., Robinson, P., McCluskey, A., Haucke, V. (2011) Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell, 146, 471-484
  • Koo, S.Y., Markovic, S., Puchkov, D., Mahrenholz, C., Beceren-Braun, F., Maritzen, T., Dernedde, J., Volkmer, R., Oschkinat, O., Haucke, V. (2011) SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors CALM and AP180 at synapses. Proc. Natl. Acad. Sci. USA, 108, 13540-13545
  • Haucke, V., Neher, E., Sigrist, S.J. (2011) Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat Rev Neurosci., 12, 125-136
  • Faelber, K., Posor, Y., Gao, S., Held, M., Roske, Y., Schulze, D., Haucke, V., Noe, F., Daumke, O. (2011) Crystal structure of nucleotide-free dynamin. Nature, 477, 556-560
  • Pechstein, A., Bacetic, J., Vahedi-Faridi, A., Gromova, K., Sundborger, A., Tomlin, N., Krainer, N., Vorontsova, O., Schäfer, J.G., Owe, S.G., Cousin, M.A., Saenger, W., Shupliakov, O., Haucke, V. (2010) Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2. Proc. Natl. Acad. Sci. USA, 107, 4206-4211
  • Wieffer, M., Maritzen, T., and Haucke, V. (2009) SnapShot: Endocytic trafficking at the plasma membrane. Cell 137, 382.e1-3.
  • Diril, M. K., Wienisch, M., Jung, N., Klingauf, J., and Haucke, V. (2006) Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization. Dev. Cell 10, 233-244

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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