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Professor Roger Morris

RogerMorris140x180Professor of Molecular Neurobiology

Contact: Pauline Stephens (PA)
Tel: +44 (0) 20 7848 6979




Laboratory of membrane pathology

We study how the neuronal trafficking of prion protein, during biosynthesis, on the cell surface and when endocytosed, affects its pathogenic misfolding into a form that kills neurons, causing Creutzfeld-Jacob disease.

Prion diseases (transmissible spongiform encephalopathies)

Although Alzheimer’s is by a considerable margin the most common of the neurodegenerative diseases caused by aberrant processing of normal cellular proteins, to study the mechanism of the development of amyloid-type neurodegeneration, the prion diseases are particularly accessible because, in addition to arising spontaneously or by mutation, they can also be transferred by infection, allowing the very early stages of the disease to be reliably studied. The critical infection event in the prion diseases is an encounter between normal cellular prion protein (that we all have at reasonably high levels in our brains) and a pathogenic form of this protein, that differs from the normal form only in how it is folded. As a result of this encounter, some of the cellular protein converts to the pathogenic form, a process that continues until the abnormal form builds up as amyloid fibrils that kill only neurons expressing the cellular prion protein.

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Our contribution

Although aspects of this conversion can be reproduced by pure molecules in the test tube, efficient production of new infectious agent, and the distinctive neurotoxicity of prions that affects only neurons expressing prion protein, is found only with living neurons. To define what it is that cells add to the conversion process, we first identified which cells express prion protein in vivo [1, 2] and showed that on the neuronal surface membrane, prion protein occupies a particular ordered lipid environment (called a ‘lipid raft’), distinct from that occupied by similarly lipid-anchored proteins on the neuronal surface [3, 4]. We have further shown that prion protein leaves these rafts to cross normal fluid membrane as it enters coated pits to be endocytosed and recycles very rapidly (within minutes) back to the neuronal surface [5]. In so doing, prion protein traverses a number of very different membrane environments [6]. We suspect it is susceptible to pathogenic conversion in only a subset of these.

Our demonstration that prion protein, and the major lipid-anchored protein of the neuronal surface (Thy-1) occupy adjacent but distinct lipid ‘rafts’ was one of the first, and the most comprehensive, demonstration that rafts were not a single membrane environment, but differed to accommodate functionally different proteins [3, 4]. The critical step in this field is to develop an isolation of ‘lipid rafts’ that allows their biochemical characterization. We have demonstrated the deficiencies of the commonly used method based on insolubility of ‘rafts’ in the detergent Triton X-100 at 4ÚC [3, 4], and progressively developed a method based on the insolubility of these membrane patches in Brij 96 at 37ÚC that allows the immunoisolation of membrane patches that increasingly have the properties expected of membrane ‘rafts’ [7-9].

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Our ongoing work

We have shown that the trafficking of both normal cellular prion protein, and of small infectious prion fibrils, is mediated by their very high affinity binding to a unique site within ligand cluster 4 of the mega-receptor, LRP1 [10, 11]. Normal Prion Protein is rapidly recycled back to the cell surface; infectious prions are moved to lysosomes that attempt to proteolyse them. Prion fibrils, however, largely resist degradation, with around 50% of the initial inoculum persisting for months within infected neurons. After 8 weeks, production of new, protease-resistant prions can be detected [11]. The immediate questions are why this single receptor traffics the two forms of prion protein so differently; in which compartment the endocytosed prion fibrils persist for months; and at what site the conversion of cellular prion into the pathogenic form occurs?

We have further refined the isolation of lipid rafts at 37ÚC by rapidly removing them from the solubilization mix using immunoaffinity methods, yielding membrane fragments of <100 nm [12]. Aki Kusumi (Kyoto) is using single molecule tracking with µs resolution to determine the size of the surface domains in which prion protein and Thy-1 are confined on the neuronal surface. This will provide an independent measure of the size of neuronal rafts, guiding our isolation of ‘raft’ membrane and testing our previous EM immunohistochemical determination of raft heterogeneity on neurons. As these different approaches converge we will be able to determine whether is really is possible to isolate membrane patches corresponding to ‘raft’ membrane on the basis of detergent insolubility.

Recycling of endocytosed prion protein to the cell surface

Cellular prion protein on the cell surface was labeled with Alexa 488 –Fab to prion protein (mouse aa 142-160) at 37ÚC. The film runs for 5mins of real time, starting from when much of the labeled surface PrPC has been endocytosed and concentrated in recycling endosomes. Note two recycling vesicles (in the lower part of the picture) returning PrPC to the cell surface, and one endocytic vesicle crossing this same region to enter the recycling endosomes. Also note the intense vesicle trafficking between the recycling endosomes.

A bifurcating axon of an adult dorsal root ganglion neuron

A bifurcating axon of an adult dorsal root ganglion neuron, growing on a laminin-coated glass slide for 3min 55s, taken with a RichardsonTM microscope with phase contrast optics optimized for contrasting cell membranes, and with optics optimized to give maximum resolution and ~1µm depth of field. Mitochondria are the highly mobile ‘worms’ (note about a dozen of them pile into the rapidly expanding growth cone at the top right). The small dark ‘blobs’ inside the axon are probably lysosomes (the large ones outside are detritis). Looking carefully, you will see, at the very limit of resolution, myriad small trafficking vesicles speeding along the axon to deliver new membrane to the growth cones. These are most readily seen by concentrating upon where the axon expands into the growth cone on the lower left, or along the horizontal stretch of axon in the middle of the picture.

3RM-image-3LRP1 is a mega-receptor: 600kDa, it is the 3rd largest transcript in the human genome, and contains 31 repeats of its ligand-binding domains (complement like repeats) that bind with high (nM) affinity a wide range of ligands which it rapidly and constitutively endocytoses via coated pits. Cellular Prion Protein (PrPC) is 30 times smaller than LRP1.PrPC is unusual in binding to LRP1 directly in the endoplasmic reticulum (the binding of other ligands is blocked by the escort chaperone Receptor Associated Protein) and by binding only once, in a site within receptor cluster IV (LRP1 is divalent for most other natural ligands, which bind to both ligand clusters II and IV). Infectious prions, provided they are small oligomers and not 1+ µm amyloid fibrils, bind competitively with PrPC to ligand cluster 4.In trafficking cellular and infectious PrP differently, LRP1 acts to separate them on the cell surface and so keep them apart, thereby providing a natural defence against prion infection.

Adult DRG neurons growing in culture
4RM-4These are optical slices through the central cell body of adult DRG neurons growing in culture, 2mins after it has been placed at 37ÚC having first had its surface PrPC labeled red with Alexa 594-Fab antibody fragment to PrPC, and its surface transferrin labeled green with Alexa 488-Transferrin, at 12-15ÚC. These surface ligands were washed away (except for Tf in A) before the temperature was raised to allow endocytosis to commence.

A shows normal conditions (except that Alexa 488-Tf has been left in the medium to delineate neuronal surface via its labeling of the Tf receptor). Essentially all surface-labelled PrPC has been endocytosed by 2mins into the perinuclear recycling compartments.

B was performed in the presence of 1 µM Receptor Associated Ligand, the natural LRP1 antagonist, which results in almost all PrPC(red) remaining on the cell surface, not endocytosed.

C was performed in the presence of an siRNA that reduced LRP1 expression by >80%; again, most of the PrPC (red) remaining on the cell surface.

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  1. Ford, M.L., L.J. Burton, H. Li, C.H. Graham, Y. Frobert, J. Grassi, S.M. Hall and R.J. Morris, A marked disparity between the expression of prion protein and its message by neurons of the central nervous system. Neuroscience, 2002. 111: p. 533-51.
  2. Ford, M.L., L.J. Burton, R.J. Morris and S.M. Hall, Selective expression of prion protein in peripheral tissues of the adult mouse. Neuroscience, 2002. 113: p. 177-192.
  3. Brügger, B., C.H. Graham, I. Leibrecht, E. Mombelli, A. Jen, F.T. Wieland and R.J. Morris, The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition. J. Biol. Chem., 2004. 279(9): p. 7530-6.
  4. Madore, N., K.L. Smith, C.H. Graham, A. Jen, K. Brady, S. Hall and R. Morris, Functionally different GPI proteins are organised in different domains on the neuronal surface. EMBO J., 1999. 18: p. 6917-26.
  5. Sunyach, C., A. Jen, J. Deng, K. Fitzgerald, Y. Frobert, M. McCaffrey and R.J. Morris, The mechanism of internalisation of GPI anchored prion protein. EMBO J, 2003. 22(14): p. 3591-3601.
  6. Morris, R.J., C.J. Parkyn and A. Jen, Traffic of prion protein between different compartments on the neuronal surface, and the propagation of prion disease. FEBS Lett, 2006. 580: p. 5565-71.
  7. Chen, X., M. Jayne Lawrence, D.J. Barlow, R.J. Morris, R.K. Heenan and P.J. Quinn, The structure of detergent-resistant membrane vesicles from rat brain cells. Biochim Biophys Acta, 2009. 1788: p. 477–483.
  8. Chen, X., A. Jen, A. Warley, M.J. Lawrence, P.J. Quinn and R.J. Morris, Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition. Biochem J, 2009. 417(2): p. 525-33.
  9. Morris, R.J., Ionic control of the metastable inner leaflet of the plasma membrane: Fusions natural and artefactual. FEBS Lett, 2009. 584(9): p. 1665-9.
  10. Parkyn, C.J., E.G. Vermeulen, R.C. Mootoosamy, C. Sunyach, C. Jacobsen, C. Oxvig, S. Moestrup, Q. Liu, G. Bu, A. Jen and R.J. Morris, LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein. J Cell Sci, 2008. 121(Pt 6): p. 773-83.
  11. Jen, A., C.J. Parkyn, R.C. Mootoosamy, M.J. Ford, A. Warley, Q. Liu, G. Bu, I.V. Baskakov, S. Moestrup, L. McGuinness, N. Emptage and R.J. Morris, Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4. J Cell Sci, 2010. 123(Pt 2): p. 246-55.
  12. Morris, R.J., A. Jen and A. Warley, Isolation of nano-meso scale Detergent Resistant Membrane that has properties expected of lipid 'rafts'. J. Neurochem., 2011. in press.

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