synapses, and severing dendrites. In fact, upon autopsy, paralysed patients can have 20% fewer synapses compared with ambulatory patients. There is also a significant increase in apoptotic neurons in chronic active and inactive lesions, and it is also possible, although not proven, that neurons might sever their axons using mechanisms related to apoptosis. Certainly, in transected axons, the process of degeneration appears to be an active process, and not simply the consequence of being disconnected from the cell body. Thus permanent loss of function might be the result of several different mechanisms. Potential avenues for therapy: axonal protection
Several of the potential routes of axonal damage implicate a role for inflammation, and this observation supports a role for anti-inflammatory therapies in MS. Indeed, the administration of steroids during relapses, or more recently of maintenance on immunomodulatory therapies such as recombinant interferonβ, are now well established in MS to affect relapses and, in some cases, progression. However, it is worth considering that some components of the inflammatory response are probably beneficial, and the blanket dampening of inflammation might well prove to be too blunt a therapy. It is even possible that therapies designed to promote autoimmune responses might be beneficial for axonal survival and regeneration5, although it is probably imprudent to
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explore this possibility in patients without more fundamental research. One novel potential therapy arises from the observation that axonal impulse activity in conjunction with nitric oxide exposure can prove lethal to axons. Building on research in anoxic axons, preliminary findings have revealed that partial Na+ channel blockade can provide axonal protection, indicating the need for further research in other MS models. Demyelinated axons are common in MS, and an important question is whether it is necessary to repair the axons by remyelination, or provide trophic support for them, if they are to be protected from degeneration over the long term. Certainly remyelination is known to have the advantage of restoring secure conduction to axons, and advances in the transplantation of myelinating cells encourage a view that the repair of lesions is an achievable goal. Intravenous administration of cells in order to seed multiple lesions, and the use of cells, including stem cells, engineered to express desired molecules are possible strategies that might assist repair by remyelination. The provision of trophic support for axons is also a realistic goal but appropriate routes of administration need to be established. The meeting was exceptionally well organized, and the topic held the interest of the participants so tightly that even the seductive attractions of the neighbouring Bourbon Street were insufficient to distract the attendees from the meeting hall.
References 1 Kornek, B. et al. (2000) Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am. J. Pathol. 157, 267–276 2 McDonald, W. (2000) Relapse, remission, and progression in multiple sclerosis. New Engl. J. Med. 343, 1486–1487 3 Pitt, D. et al. (2000) Glutamate excitotoxicity in a model of multiple sclerosis. Nat. Med. 6, 67–70 4 Waxman, S. et al. (2000) Sodium channels and their genes: dynamic expression in the normal nervous system, dysregulation in disease states. Brain Res. 886, 5–14 5 Schwartz, M. et al. (1999) Innate and adaptive immune responses can be beneficial for CNS repair. Trends Neurosci. 22, 295–299 The conference was supported by the National Multiple Sclerosis Society, Multiple Sclerosis International Federation, Acorda Therapeutics, Bayer AG, Berlex Laboratories, Biogen, Immunex Corporation, Serono Laboratories, Inc. and Teva Marion Partners.
Peter Rieckmann Clinical Research Unit for Multiple Sclerosis and Neuroimmunology, Dept of Neurology, Julius-Maximilians University, JosefSchneider-Str. 11, D-97080 Würzburg, Germany. Kenneth J. Smith* Dept Neuroimmunology, Neuroinflammation Research Group, Guy’s, King’s and St. Thomas’ School of Medicine, Guy’s Campus, King’s College, London, UK SE1 9RT. *e-mail: [email protected]
Functional recovery after spinal cord injury: basic science meets clinic Jan M. Schwab, Christian A. Leppert, Karl-Heinz Kaps and Philippe P. Monnier Functional Recovery after Spinal Cord Injury. Held at Ascona, Monte Verità, Switzerland; 1–5 April, 2001. Organizers: Volker Dietz, Martin E. Schwab and Karim Fouad.
The Ascona meeting combining basic science with aspects of clinical, rehabilitative medicine was held in the picturesque environment of the northern Lago Maggiore. The inability of the adult mammalian CNS to regenerate after injury has confounded clinicians and scientists alike for centuries. Regenerative axon sprouting is limited in the adult http://tins.trends.com
mammalian CNS by intrinsic neuronal and local micro-environmental factors, such as axon growth inhibitors embedded in mature myelin or in the developing scar. By contrast, in the immature CNS and the PNS neurons will readily extend axons and successfully regenerate after injury. Several inhibitory molecules of the adult myelin and scar were identified and cloned. In addition, re-expressed guidance molecules might also contribute to the inability of axon regrowth in the adult CNS (Refs 1,2) adding further complexity to our current understanding of spinal cord injury. Potential new targets to
improve regenerative processes were identified, but none have reached clinical application so far. Because of space restraints, this report is not comprehensive, instead focusing on the most exciting findings presented at the meeting. Growth inhibition Inhibition by myelin
The role of a well-characterized myelinassociated inhibitor, the recently cloned Nogo-A (Ref. 3), was illustrated by Martin Schwab (Brain Research Institute, University of Zurich, Switzerland) and his
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Key conference outcomes • Nogo-A blocking might promote prevailing plasticity. • Need for the identification of the most effective, less harmful cellular substrate (olfactory ensheathing cells, Schwann cells, serotinergic cells) for cell transplantation strategy. • Therapeutic immunization: are pro-regenerative effects a result of cellular or antibody-mediated effects? • Insufficient comparability of clinical endpoint measurements to distinguish intrinsic regenerative potential from effects of therapeutic intervention.
group. The Nogo gene encodes three protein products (Nogo-A, B and C), which represent the fourth subgroup of the reticulon family. The transmembrane protein Nogo-A is a component of CNS myelin and is recognized by the monoclonal antibody IN-1, which promotes axonal regeneration and functional recovery of the injured rat spinal cord. Nogo-A is expressed by oligodendrocytes and is predominantly localized to the endoplasmic reticulum, but is also found on the cell surface. By using antibodies against different regions of
Nogo-A, Schwab presented further evidence that the Nogo-A specific domain is located at the outer surface of oligodendrocytes. In comparison with at least two additional inhibitory regions, this domain appears to be the main contributor to the inhibitory effect of Nogo-A. In the endoplasmic reticulum, however, Nogo-A can adopt different topologies and the biological function in this region is unresolved. In another elegant study, O. Rainetau (Brain Research Institute, University of Zurich, Switzerland) showed that blocking of Nogo-A achieved sprouting of unlesioned bridging fibers. After bilateral pyramidotomy targeting the corticospinal tract, in combination with application of the IN-1 antibody, strong collateral sprouting by intact rubrospinal axons projecting to the cervical gray matter of the spinal cord could be observed. This was accompanied by a nearly complete functional recovery in the grasping test. Furthermore, K. Fouad (Brain Research Institute, University of Zurich, Switzerland) showed that Nogo-Ablocking effects require significant numbers of spared axons. His results suggested a profound plasticity-mediated effect as a result of branching of intact spared axonal fibers. Inhibition by the gliotic scar
Fig. 1. Regeneration of corticospinal tract axons after ‘therapeutic’ vaccination (CNS myelin homogenate emulgated with incomplete Freund’s adjuvant, IFA) signified by large numbers of long fiber tracts three weeks following spinal cord injury6. (a) Dark-field micrograph of two adjacent longitudinal tissue sections from immunized mice in which most of the wheatgerm agglutinin–conjugated horseradish peroxydase (WGA-HRP) traced fibers in the tract course through the area of the lesion (asterisk). Caudal is located to the right side of the lesion. (b) A bright-field image of one of the sections from which the montage in (a) was made. Note the lesion at the center (asterisk), which is seen as the area of high cellularity. By contrast, the dorsal columns away from the lesion towards the left and right margins of the micrograph, have markedly fewer cells and a paler appearance. With kind permission of Sam David (Montreal, Canada). Scale bar, 200 µm. http://tins.trends.com
Spinal cord injury results in the formation of scar tissue, to which reactive astrocytes, meningeal cells, oligodendrocyte precursor cells and microglia/macrophages contribute4,5. Among others, James Fawcett (Centre for Brain Repair, Cambridge University, UK) has identified proteoglycans expressed in the developing scar after CNS injury that are inhibitory to axon regeneration. These proteoglycans, particulary NG2, neurocan, versican and phosphacan are made by oligodendrocyte precursors that are recruited to CNS injuries. Fawcett showed that removal of chondroitin sulfate from glial scar tissue by chondroitinase ABC, or inhibition of
glial cell proliferation by arabinoside and ethidium bromide treatments promoted axonal elongation beyond the lesion site. Hans-Werner Mueller (Dept Neurology, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany) showed that deposition of basement membrane, which contains collagen type IV in addition to numerous other proteins (e.g. glycoproteins and proteoglycans), is a strong inhibitory environment for axon regeneration. To diminish scar formation and the resulting basement membrane biosynthesis, his team investigated specific pharmacological modulators. A reduction of the lesion size and an increase in numbers of regenerating corticospinal axons were obtained with a protocol including, (1) multiple immediate postlesion injections of the iron chelator 2-2′-bipyridine-5,5′-dicarboxylic acid (BPY-DCA), a potent inhibitor of prolyl 4-hydrolase, which is a key enzyme of collagen synthesis, (2) continuous local release of BPY-DCA, and (3) selective inhibition of fibroblast extracellular matrix (ECM)/collagen production and proliferation by 8-Br-cAMP. Modulation of the immune response
Although there is long-standing evidence that unspecific immunostimulation by LPS (lipopolysaccharide) mimicking bacterial infections can be beneficial for the neuroregenerative outcome, in recent years inflammation has been attributed to wide aspects of secondary injury phenomena, such as lipid peroxidation, cell membrane damage, free radical formation and edema formation. Therapeutic vaccination
Sam David (Centre for Research in Neuroscience, the Montreal General Hospital Research Institute and McGill University, Quebec, Canada) continued earlier work identifying inflammatory cells that alter the non-permissive nature of adult CNS. In his recent work a postinjury therapeutic vaccine approach was used in which an animal’s own immune system is stimulated to produce polyclonal antibodies that block myelin-associated inhibitors6. Adult immunized mice (spinal cord homogenate which is rich in myelin and some inhibitory proteoglycans emulgated in incomplete Freund’s adjuvant, IFA) showed regeneration of large numbers of corticospinal tract (CST) axons after dorsal hemisection (Fig. 1).
Similarly, antisera from myelin immunized mice were able to block myelinderived inhibitors and to promote neurite growth on myelin in vitro. Immunizationinduced antibodies might block already identified myelin-bound inhibitory molecules such as Nogo-A or MAG (myelin associated glycoprotein), and probably several other, as yet, unidentified proteins1,2. However, to date, it is still unknown whether pro-regenerative effects are antibody-mediated alone or as a result of cellular effects resulting from alteration of lesional immune cell populations and their activation status. Knockouts
Mice are distinct in their pathophysiological response after SCI, for example, there is almost absent cavity formation resulting in a reduced secondary damage pathophysiology. However, modeling spinal cord injury in mice takes advantage of investigating knockout animals (Brad T. Stokes, Dept of Physiology and Cell Biology, Ohio State University, College of Medicine and Public Health, Columbus, Ohio, USA). In order to
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examine these features, standardized and behavioral outcome parameters in normal mice following SCI were addressed. One answer to all questions?
In order to anticipate future clinical intervention it was noteworthy that approaches that block even a single CNS inhibitor enhance the growth state of neurons and lead to a remarkable regenerative axonal growth under experimental conditions. In conclusion, a combination of strategies will be the most promising way for a successful therapy after spinal cord injury. Although, it will increase the need (1) to clearly distinguish epi-phenomena from effector cascades and (2) to identify the interwoven mechanisms (receptor binding effects, non-effector binding effects, second messenger effects) and their cellular targets to prevent antagonistic interactions. References 1 Behar, O. et al. (2000) Putting the spinal cord together again. Neuron 26, 291–293 2 Horner, P.J. and Gage, F.H. (2000) Regenerating the damaged central nervous system. Nature 407, 963–970
3 Chen, M.S. et al. (2000) Nogo-A is a myelinassociated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403, 434–439 4 Schwab, M.E. and Bartholdi, D. (1996) Degeneration and regeneration of axons in the lesioned spinal cord. Physiol. Rev. 2, 319–369 5 Tator, C.H. (1998) Biology of neurological recovery and functional restoration after spinal cord injury. Neurosurgery 42, 696–707 6 Huang, D.W. et al. (1999) A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord. Neuron 24, 639–647
Jan M. Schwab* Institute of Brain Research, University of Tübingen, Medical School, Neuroimmunology, Calwerstr. 3, Germany. *e-mail: [email protected]
Christian A. Leppert Philippe P. Monnier Migragen AG, Max-Planck Campus, Spemannstr. 34, D-72076 Tübingen, Germany. Karl-Heinz Kaps BG Trauma Center, Dept of Spinal Cord Injury, Orthopedics and Rehabilitation Medicine, Schnarrenbergstr. 95, D-72076 Tübingen, Germany.
SNAREdinburgh: the molecular mechanisms of exocytosis and endocytosis Mike Cousin, David Apps, Mike Shipston and Bob Chow Molecular Mechanisms of Exocytosis and Endocytosis. Held at Edinburgh, UK; 25–27 March, 2001.
Junior academics drawn from leading research labs in the UK, Europe and the USA converged on Edinburgh to discuss advances in the field of vesicle recycling. In passionate and open discussions the ‘new generation’ of group leaders debated the role of SNARE complex oligomerization and munc-18 in exocytosis, the function of adapter proteins in endocytosis, and recent developments in real-time imaging of vesicle recycling. This was the third such meeting organized by the Membrane Biology Group (University of Edinburgh, UK) and, true to form, this biennial event produced a lively and highly interactive meeting that reflected the multidisciplinarity of the field. The fusion and fission of vesicles with the plasma membrane is fundamental to http://tins.trends.com
both cell and neuronal function. Thus, identification of the molecular mechanisms that underlie these processes is essential to understand how excitable cells communicate. SNARE complex formation and oligomerization
SNARE complexes are widely believed to mediate vesicle fusion with the plasma membrane, and several presentations generated vigorous debate on their mechanism of action. One area of controversy was the occurrence and function of SNARE complex oligomers. Dirk Fasshauer (Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany) presented in vitro evidence for intermediates in the assembly of SNARE complexes and questioned the recently proposed role of complexins in SNARE complex oligomerization. However, keynote speaker George Augustine (Duke
University, Durham, USA) presented evidence for a SNARE complex incorporating complexins that drove their oligomerization. Furthermore, a peptide that disrupted complexin–syntaxin interactions abolished exocytosis in the squid giant synapse, suggesting this interaction was essential for vesicle fusion. Many existing controversies in this field should be settled by the identification and purification of SNARE complexes in vivo, but this has proven to be technically difficult. Bazbek Davletov (LMB, Cambridge, UK) and Gary Lawrence (Imperial College, London, UK) reported the successful characterization of such complexes from stimulated nerve terminals and neuroendocrine cells. Davletov isolated two distinct ternary SNARE complexes (slow and fast migrating on SDS-PAGE). Interestingly, addition of complexins promoted formation of the slow-migrating form,
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