Centre for Integrative Physiology

Prof Mike Cousin

I am working to understand the molecular mechanisms involved in the fusion, retrieval and trafficking of synaptic vesicles within the central nervous system.

Mike Cousin

Chair of Neuronal Cell Biology, Head of Pre-Clinical Research at the Muir Maxwell Epilepsy Centre

  • Hugh Robson Building
  • room 178
  • 15 George Square
Street

Edinburgh EH8 9XD

Personal profile

  • 2013-present: Head of Pre-Clinical Research at the Muir Maxwell Epilepsy Centre
  • 2010-present: Chair of Neuronal Cell Biology, Centre for Integrative Physiology, University of Edinburgh
  • 2008-2010: Reader, Centre for Integrative Physiology, University of Edinburgh
  • 2004-2008: Senior Lecturer, Centre for Integrative Physiology, University of Edinburgh
  • 2000-2004: Lecturer, Biomedical Sciences, University of Edinburgh
  • 1998-2000: Human Frontiers of Science Long-Term Fellow, Children’s Medical Research Institute, Sydney, Australia
  • 1997-1998: Royal Society Travelling Fellow, Children’s Medical Research Institute, Sydney, Australia
  • 1994-1997: Postdoctoral Research Fellow, Neurosciences Institute, University of Dundee
  • 1994: PhD Biochemistry, University of Dundee
  • 1991: BSc (Hons I) Biochemistry, University of Edinburgh

Research

Prof Mike Cousin's research briefing

Molecular mechanisms of synaptic vesicle recycling in neurones

Neurones communicate at structures called synapses, which are tiny gaps between neurones where chemical signals are passed. These chemicals are called neurotransmitters and they are packaged inside synaptic vesicles.

Synaptic vesicles (SVs) live locally at one side of the synapse called the presynapse or nerve terminal. Upon nerve stimulation synaptic vesicles fuse with the presynaptic plasma membrane and release their neurotransmitter into the synapse, stimulating the neuron on the other side of the synapse (postsynapse). After release of neurotransmitter, the vesicle membrane is retrieved by a process called endocytosis, refilled with neurotransmitter then trafficked locally within the nerve terminal to be released again.

My research investigates how presynaptic function adapts to maintain neurotransmission across the range physiological inputs. This includes recruitment of specific SV endocytosis modes and SV cargo retrieval mechanisms. To answer these questions we use a multi-disciplinary approach employing a variety of in vitro model systems which combine population and single cell fluorescent imaging of nerve terminals with functional, biochemical, cell biological and molecular biological techniques. We are increasingly making use of preclinical disease models to determine how dysfunctional SV recycling can impact on synaptic failure.

Our research spans three interlinked research themes investigating SV recycling in response to physiological stimuli and dysfunctional SV recycling in human disease.

1) Molecular mechanism of activity-dependent bulk endocytosis

1a) Activity-dependent triggering of bulk endocytosis

Activity-dependent bulk endocytosis (ADBE) forms endosomes direct from the plasma membrane and is selectively triggered during high neuronal activity. We are particularly interested in this SV retrieval mode since it will be activated during plastic changes at the synapse. We have shown that ADBE is triggered by an activity-dependent protein dephosphorylation cascade. Intense stimulation activates the protein phosphatase calcineurin to dephosphorylate the large GTPase dynamin I. This dephosphorylation event allows an association of dynamin I with the endocytosis protein syndapin. Each step in this cascade is essential for ADBE, but not any other retrieval route. We are currently investigating the essential molecular determinants of the syndapin requirement for this SV retrieval mode.

Morton A., Marland J.K. and Cousin M.A. (2015) Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis. J. Neurochem. [In Press].

Wenzel E.M., Morton A., Ebert K., Wenzel O., Kornhuber L., Cousin M.A. and Groemer T.W. (2012) Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses. PLoS One. 7: e38188.

Xue J., Graham M.E., Novelle A.E., Sue N., Gray N., McNiven M.A., Smillie K.J., Cousin M.A. and Robinson P.J. (2011) Calcineurin selectively docks with the dynamin Ixb splice variant to regulate activity-dependent bulk endocytosis J. Biol. Chem. 286: 30295-30303.

Clayton E.L., Anggono V., Smillie K.J., Chau N., Robinson P.J. and Cousin M.A. (2009) The phospho-dependent dynamin-syndapin interaction triggers activity-dependent bulk endocytosis of synaptic vesicles. J. Neurosci. 29: 7706-7717.

1b) Control of bulk endocytosis modes by extracellular signalling molecules

After stimulation terminates dynamin I is phosphorylated by both cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3 (GSK3). Inhibition of either enzyme results in arrest of ADBE. GSK3 is tightly controlled by signalling cascades that are activated by extracellular molecules, such as brain-derived neurotrophic factor (BDNF). We have recently shown that the activity-dependent release of BDNF arrests dynamin I rephosphorylation by GSK3 and ADBE, suggesting that manipulation of this signalling cascade will modulate neurotransmission during high neuronal activity.

Smillie K.J., Pawson J, Perkins E.M., Jackson M. and Cousin M.A. (2013) Control of synaptic vesicle endocytosis by an extracellular signalling molecule. Nature Commun. 4: 2394.

Smillie K.J. and Cousin M.A. (2012) Akt/PKB controls the activity-dependent bulk endocytosis of synaptic vesicles. Traffic 13: 1004-1011.

Clayton E.L., Sue N., Smillie K.J., O’Leary T., Bache N., Cheung G., Cole, A.R., Wyllie D.J, Sutherland C., Robinson P.J. and Cousin M.A. (2010) Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles. Nature Neurosci. 13: 845-851.

1c) Synaptic vesicle budding from bulk endosomes

SVs are generated from bulk endosomes during ADBE and these SVs selectively refill the reserve pool of SVs within the nerve terminal. Little is known regarding the mechanism for cargo selection during this SV reformation process. We have shown that the endosomal adaptor protein complexes AP-1 and AP-3 are both essential for this event. We have also found that fluid phase uptake of calcium into bulk endosomes are also essential for SV formation. We are currently elucidating further molecules which are essential for this event using optical and morphological assays which track SV specifically generation from bulk endosomes.

Kokotos A.C. and Cousin M.A. (2014) Synaptic vesicle generation from central nerve terminal endosomes. Traffic [EPub ahead of print].

Cheung G. and Cousin M.A. (2013) Synaptic vesicle generation from bulk endosomes requires calcium and calcineurin. J. Neurosci. 33: 3370-3379.

Cheung G. and Cousin M.A. (2012) Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes. J. Neurosci. 32: 6014-6023.

Cheung G., Jupp O.J. and Cousin M.A. (2010) Activity-dependent bulk endocytosis and clathrin-dependent endocytosis replenish specific synaptic vesicle pools in central nerve terminals. J. Neurosci. 30: 8151-8161.

2) Self-chaperoning of SV cargo during endocytosis

2a) Synaptophysin - a synaptobrevin II chaperone

Clathrin-mediated endocytosis (CME) is the dominant SV retrieval mode during mild stimulation. Accurate sorting of both the type and quantity of SV cargo during this process is critical for formation of functional SVs. We have shown that the abundant SV protein synaptophysin is essential for the efficient retrieval of the v-SNARE synaptobrevin II from the plasma membrane during CME. We hypothesise that synaptophysin i) prevents synaptobrevin II from entering futile cis-SNARE complexes at the plasma membrane and ii) presents synaptobrevin II in the correct conformation to be recognised by its monomeric adaptor protein AP180.

Gordon S.L. and Cousin M.A. (2014) The sybtraps: control of synaptobrevin traffic by synaptophysin, α-synuclein and AP180. Traffic 15: 245-254.

Gordon S.L., Leube R.E. and Cousin M.A. (2011) Synaptophysin is required for synaptobrevin retrieval during synaptic vesicle endocytosis J. Neurosci. 31: 14032-14036.

2b) SV2A - a synaptotagmin I chaperone

The ability of SV cargo to control their sorting and retrieval is not restricted to the synaptophysin - synaptobrevin II interaction. We have shown that a phosphorylation-dependent interaction between SV2A and synaptotagmin I is critical for the accurate retrieval of synaptotagmin I during CME. In a further parallel, genomic ablation of the monomeric adaptor protein for synaptotagmin I (stonin 2) results in an identical phenotype. Thus self-chaperoning of SV cargo may be a conserved mechanism to ensure their accurate and efficient retrieval during CME.

Zhang N.*, Gordon S.L.*, Fritsch M.J*, Esoof N., Campbell D.G., Gourlay R., Velupillai S., Macartney T., Peggie M., van Aalten D.M.F., Cousin M.A.* and Alessi D.R.* (2015) Phosphorylation of SV2A at Thr84 by CK1 family kinases controls the specific retrieval of synaptotagmin-1. J. Neurosci. In Press.

3) SV recycling in human synaptic disease

It is becoming increasingly apparent that small, sometimes imperceptible, perturbation SV recycling results in formation of defective SVs that have an inaccurate complement of protein cargo, culminating in altered neurotransmitter release. This has led to the realisation that many human neurodevelopmental disorders and neurodegenerative diseases have at the core a synaptic defect, an hypothesis underpinned by extensive data from patients which display a disproportionate number of mutations in key synaptic genes.

3a) SV recycling in X-linked intellectual disability

We have shown that synaptophysin mutations identified in patients with X-linked intellectual disability (XLID) fail to rescue synaptobrevin II retrieval in knockout neurones. These mutations also revealed that this defect could be separated from a defect in the kinetics of CME in synaptophysin knockout mice. Thus defective synaptobrevin II retrieval may underlie at least in part XLID displayed by these patients.

Gordon S.L. and Cousin M.A. (2013) X-linked intellectual disability-associated mutations in synaptophysin disrupt synaptobrevin II retrieval. J. Neurosci. 33: 13695-13700.

3b) SV recycling in neurodegenerative disease

We are currently investigating dysfunctional SV recycling in preclinical models of both Huntington’s Disease (collaboration Dr. K. Smillie) and Alzheimer’s Disease.

Figure 2: Imaging of synaptic vesicle turnover using FM4-64

This picture shows primary cultures of cerebellar granule neurones loaded with the vital dye FM4-64. Dye is localised to specific puncta corresponding to nerve terminals.

The green neurone that is overlaid has been transfected with a cytosolic EGFP fusion protein.

The cultures were stimulated with a train of action potentials followed by a 50 mM KCl stimulus. Note the heterogeneity of the responses of different nerve terminals.

Figure 1: The synaptic vesicle life cycle
 Imaging of synaptic vesicle turnover using FM4-64

Funding

  • The Wellcome Trust
  • Medical Research Council
  • Biotechnology and Biological Sciences Research Council (BBSRC)
  • CHDI
  • EU (Marie-Curie Initial Training Network)

Team members

  • Jamie Marland (Postdoctoral Fellow)
  • Callista Harper (Postdoctoral Fellow)
  • Robyn McAdam (Postdoctoral Fellow)
  • Jessica Nicholson-Fish (PhD student)
  • Alexandros Kokotos (PhD student)
  • Darryl Low (PhD student)
  • Katherine Bonnycastle (PhD student)
  • Rona Wilson (Technician)

Collaborations

  • Professor Dario Alessi (University of Dundee)
  • Professor Lucy Raymond (Cambridge Institute of Medical Research)
  • Professor Phil Robinson (CMRI, Sydney, Australia)
  • Prof Rory Duncan (Heriot Watt University)
  • Prof Peter Kind (University of Edinburgh)
  • Prof David Wyllie (University of Edinburgh)
  • Dr Matt Nolan (University of Edinburgh)

Selected Publications

Zhang N., Gordon S.L., Fritsch M.J, Esoof N., Campbell D.G., Gourlay R., Velupillai S., Macartney T., Peggie M., van Aalten D.M.F., Cousin M.A. and Alessi D.R. (2015) Phosphorylation of SV2A at Thr84 by CK1 family kinases controls the specific retrieval of synaptotagmin-1. J. Neurosci. 35(6):2492-507

Baker K., Gordon S.L., Grozeva D., van Kogelenberg M., Roberts N.Y., Pike M., Blair E.,  Hurles M.E., Kling Chong W., Baldeweg T., Kurian M.A., Boyd S., UK10K consortium, Cousin M.A. and Raymond F.L. (2015) A human mutation in SYT1 that perturbs synaptic vesicle recycling. J. Clin. Invest. 125: 1670-1678.

Morton A., Marland J.K. and Cousin M.A. (2015) Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis. J. Neurochem. [In Press].

Kokotos AC and Cousin MA. (2015) Synaptic vesicle generation from central nerve terminal endosomes. Traffic 16: 229-240.

Kavanagh D.M., Smyth A.M., Martin K.J., Dun A., Brown E.R., Gordon S.L., Smillie K.J., Chamberlain L.H., Wilson R.S., Yang L., Lu, W., Cousin M.A., Rickman C. and Duncan R.R. (2014) A molecular toggle sequesters monomeric syntaxin1a during trafficking and neuronal SNARE disassembly. Nature Commun. 5:5774.

Gordon S.L. and Cousin M.A. (2014) The sybtraps: control of synaptobrevin traffic by synaptophysin, α-synuclein and AP180. Traffic 15: 245-254.

Smillie KJ, Pawson J, Perkins EM, Jackson M, Cousin MA.(2013) Control of synaptic vesicle endocytosis by an extracellular signalling molecule. Nature Commun. 4:2394.

Gordon SL, Cousin MA. (2013) X-linked intellectual disability-associated mutations in synaptophysin disrupt synaptobrevin II retrieval. J Neurosci. 33(34):13695-700.

Cheung G. and Cousin M.A. (2013) Synaptic Vesicle Generation from Activity-Dependent Bulk Endosomes Requires Calcium and Calcineurin. J. Neurosci. 33: 3370-3379.

Cheung G. and Cousin M.A. (2012) Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes. J. Neurosci. 32: 6014-6023.

Smillie K.J. and Cousin M.A. (2012) Akt/PKB controls the activity-dependent bulk endocytosis of synaptic vesicles. Traffic 13: 1004-1011.

Quan A., Xue J., Wielens J., Smillie K.J., Anggono V., Parker M.W,. Cousin M.A., Graham M.E. and Robinson P.J. (2012) Phosphorylation of syndapin F-BAR domain at two helix capping motifs regulates membrane tubulation Proc. Natl Acad Sci. 109: 3760-3765.

Gordon S.L., Leube R.E. and Cousin M.A. (2011) Synaptophysin is required for synaptobrevin retrieval during synaptic vesicle endocytosis J. Neurosci. 31: 14032-14036.

Clayton E.L., Sue N., Smillie K.J., O’Leary T., Bache N., Cheung G., Cole, A.R., Wyllie D.J, Sutherland C., Robinson P.J. and Cousin M.A. (2010) Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles. Nature Neurosci. 13: 845-851.

Mike Cousin publication list (PDF)