Summary and discussion
MAC and peripheral nerve degeneration
MAC and peripheral nerve regeneration and recovery
MAC and neuropathies
Targeting MAC formation
The complement (C) system plays a central role in innate immunity and bridges innate and adaptive immune responses. A fine balance of C activation and regulation mediates the elimination of invading pathogens and the protection of the host from excessive C deposition on healthy tissues. If this delicate balance is disrupted, the C system may cause injury and contribute to the pathogenesis of various diseases.
Previous studies showed local synthesis of C factors and regulators in the peripheral nerve 1 , but their role in the microenvironment of the nerve was still controversial. This thesis comprises several studies aiming at understanding the role of the C system in degeneration, regeneration and disease processes of the peripheral nervous system.
In chapter 1 , the general structure of the peripheral nerve, pathological changes during degeneration and regenerative processes are described. A general introduction of the C system from its discovery to our current knowledge of its role in innate immunity, inflammatory and neurodegenerative disorders is provided. The outline of this thesis is presented. Chapter 2 reviews the present knowledge on local production and physiological role of C in the peripheral nerve and highlights open questions on the role of C in peripheral nerve injury and disease, setting the stage for the work presented in this thesis. Initial studies have shown that upstream factors of the C cascade, including C3 and C5, play a key role in macrophage recruitment and activation during Wallerian degeneration of the injured nerve. However, C3 and C5 have multiple functions as generators of opsonins, chemoattractants and anchor for the assembly of the MAC. Which of these functions is a key determinant for the progression of WD was not yet known. So the question remains whether local complement activation during WD is beneficial for nerve regeneration. In chapter 3 we use a rat model deficient in the terminal C component C6 to address this question. Since these rats (C6 -/- ) have otherwise normal upstream C components we could dissect the effects of the upstream C factors from the cytolytic effect of the MAC during WD after a crush injury of the peripheral nerve. We found that the MAC is essential for rapid WD of the peripheral nerve. At 3 days post-injury pronounced WD occurred in wildtype animals whereas the axons and myelin of C6 -/- animals appeared intact. Macrophage recruitment and activation was impaired in C6 -/- rats. Seven days after injury, the distal part of the C6 -/- nerves appeared degraded. As a consequence of a delayed WD, more myelin breakdown products were present than in wildtype nerves. We concluded that MAC deposition is essential for rapid WD and efficient clearance of myelin after acute nerve trauma of the PNS. In chapter 4 a study supporting the hypothesis that C activation pays a role in myelin clearance is described. We show the effects of lack of the complement CD59a regulator of the MAC on WD in mice. Axonal degradation in CD59a deficient mice occurred earlier than wildtypes. The number of endoneurial macrophages was significantly higher in CD59a deficient mice than wildtypes at 1 day post-injury. These findings strongly implicate MAC as determinant of axonal damage during WD, confirming the results presented in chapter 3. Chapter 5 describes the result of a study which tests the therapeutical relevance of blocking complement activation during WD. To this end, the effects of systemic treatment with soluble C receptor 1 (sCR1), inhibitor of all C pathways, or with C1 inhibitor (C1INH, Cetor) which blocks the classical and lectin but not the alternative C pathways, were tested in wildtype rats in which WD of the sciatic nerve was induced by crush injury. This study shows that treatment with sCR1 blocked both systemic and local C activation. The nerve was protected from axonal and myelin breakdown at 3 days post-injury and macrophage infiltration and activation were strongly reduced. This study also shows that activation of the alternative pathway is sufficient to cause pathology since inhibition of the classical and lectin pathway by Cetor diminished but did not abolished the load of MAC deposition in the injured nerve. It blocked myelin breakdown, inhibited macrophage infiltration and prevented their activation at 3 days post-injury but, in contrast to sCR1 treatment, early signs of axonal degradation were visible in the nerve, linking MAC deposition to axonal damage. Therefore, sCR1 protects the nerve from early axon loss after injury. In chapter 6 histological and functional studies are combined to explore the role of the complement system during regeneration of the peripheral nerve. Functional recovery of damaged peripheral axons is slow and incomplete. The study described in this chapter is based on the hypothesis that interfering with the process of post-traumatic axonal degeneration may improve the subsequent regenerative process. Lack of MAC formation prevents early axonal damage and delays the clearance of myelin and axons during WD (presented in chapter 3). Damage to nervous tissue and clearance of neuronal debris are both key determinant of regeneration. On one hand, damage to neuronal membranes (i.e. axolemma and myelin) and basal lamina directly by the MAC and indirectly by macrophages (i.e. via matrix metalloproteinases) could be detrimental for regeneration by disrupting the physical guidance necessary for an axon to reach its target muscle. On the other hand, efficient clearance of neuronal debris is considered a prerequisite for successful regeneration. Thus, the effects of C activation during WD raise the question whether there is a beneficial or detrimental consequence for the subsequent regeneration of the nerve. The study presented in this chapter sets out to answer this question. To this end the effect of C inhibition on nerve regeneration after a crush injury of the sciatic nerve in C6 -/- rats and rats treated systemically for 1 week with sCR1, are investigated. Recovery of function was assessed by footflick test every week up to 5 weeks post-injury, axonal regeneration was assessed by pathological analysis of tibial nerves at 5 weeks post-injury and by retrograde tracing of sensory neurons at 1 week post-injury. Both, genetic and pharmacological inhibition of C activation resulted in faster recovery of sensory function than PBS-treated animals whereas reconstitution with purified human C6 protein re-established the wildtype phenotype. Neuropathological analysis of the tibial nerve after 5 weeks recovery showed that C6 deficiency and sCR1 treatment resulted in better regeneration as judged by, axon diameter, myelin thickness and number of regenerative clusters. The number of retrogradelly-labelled sensory neurons in C6 -/- rats was significantly higher than wildtypes. Thus, inhibition of post-traumatic C activation accelerates axonal regeneration and recovery of motor and sensory function in a rat model of sciatic nerve injury and may offer a novel therapeutic approach to promote recovery after nerve trauma. In chapter 7 a possible involvement of the C cascade in hereditary motor and sensory neuropathies (HMSN) is tested. Secondary axon loss is a major determinant of disability in HMSNs. Whether the C system is involved in this chronic disease was not known. Nerve biopsies from HSMN patients and animal model of the disease were tested for evidence of C activation. Deposits of activated C components, including MAC, were found in nerve biopsies of HMSN patients and mice. Therefore, C is activated in a certain group of HMSN patients (and mouse model of the disease) and it may play a role in the disease.
The aim of the studies presented in this thesis was to understand the role of the C system in post-traumatic degeneration, regeneration and recovery of the peripheral nerve by genetic and pharmacological inhibition of C activation. Further, we intended to determine whether the C cascade is activated in chronic diseases of the peripheral nerve, such as hereditary neuropathies.
MAC and peripheral nerve degeneration
Local synthesis of the C system in the peripheral nerve has been established 1 and a possible role in WD has been proposed 2-4 . However, whether C cleaved products alone or the entire C cascade including the MAC is needed for WD was still matter of debate 5 .
We analyzed the role of MAC in WD and found that it is necessary for rapid axonal degradation and myelin clearance (see chapter 3 ). We propose the following model ( Figure 1 ). After crush, C1q binds axonal and myelin epitopes exposed by the mechanical injury, activating the classical pathway. The damaged nerve is then targeted by C4b, C3b and C5b which act as opsonins for macrophages. In WT animals, formation of the MAC contributes to myelin and axonal damage by creating pores on the Schwann cell membrane and axolemma. The negative regulator of the MAC, CD59, expressed on the surface of myelinating Schwann cells, can - to some extent - protect the nerve from the initial degradation, since lack of CD59 exacerbates WD after injury (see chapter 4 ). A devastating event for the fate of a nerve is the uncontrolled calcium influx through MAC-derived pores 6 . This activates calpains, calcium-dependent proteases which cleave cytoskeletal proteins including neurofilament, contributing to structural disorganization of the nerve. The MAC-induced neuronal debris is also targeted by C1q creating a positive feedback loop resulting in increased opsonization and macrophage recruitment and activation, leading to rapid degeneration.
On one hand, opsonization prevents an increase in the size of the opsonized complex maintaining its solubility, thus facilitating its clearance by activated phagocytes. This is considered a prerequisite for successful regeneration 6 . On the other hand, activated macrophages release matrix metalloproteinases, enzymes able to break down extracellular matrix proteins. These are major constituents of the Schwann cell basal lamina, which is then disrupted and penetrated by macrophages to remove the degenerating myelin 7 . Disruption of the basal lamina may impair nerve regeneration.
In the C6 -/- or sCR1-treated rats the neuronal debris produced by the mechanical injury is the only target for opsonization and not sufficient for efficient recruitment and activation of macrophages (see chapter 3 and 5 ). Thus a small number of resting macrophages is found in the injured C6 -/- nerve. This results in delayed myelin clearance. Resting macrophages do not produce MMPs. This may be critical for the maintenance of the basal lamina. WD and myelin removal in the absence of C6 is carried out in a slow, opsonin-independent manner by proliferating Schwann cells. Our findings have important implications for the design of the ideal C drug and translational studies to the clinic (discussed below).
MAC and peripheral nerve regeneration and recovery
Functional recovery after nerve trauma requires both control of axonal branching and elongation by attractive and repulsive molecular cues 8 and maintenance of intact endoneurial tubes 9 to accurately reinnervate the original target.
To date, the effect of post-traumatic MAC formation on nerve regeneration was not known and the question of a beneficial or detrimental effect remained open. On one hand, the residual myelin which is not readily cleared by phagocytes may inhibit axon growth 10-12 . On the other hand, lack of MAC- and macrophage- mediated damage, also via the action of matrix metalloproteinases 13 , may rescue the endoneurial tube, thus the architecture necessary for guiding the axon to its target.
We analyzed the role of MAC in post-traumatic nerve regeneration and recovery and found that return of motor and sensory function is accelerated in C6 -/- rats and that the same effect can be obtained by inhibition of post-traumatic complement activation with sCR1 (see chapter 6 ). sCR1 inhibits C activation at the level of the C3 convertase, but ultimately inhibits the MAC. Since we observed similar effects in C6 -/- animals, MAC - and not the upstream C members - is responsible for the poor regeneration and recovery of the wildtype rats.
In the C57BL/Wlds mouse model with slow WD, regeneration is also delayed 14,15 . This seems to be partly dependent on the inhibitory effect of myelin associated glycoprotein (MAG) on axonal regrowth since a cross of MAG-deficient mice and C57BL/Wlds mice showed an increase in the number of regrowing axons. However, no functional tests were performed 8 .
Our finding that both genetic and pharmacological inhibition of the MAC improves regeneration and recovery is surprising. We suggest that this effect is due to the inhibition of destructive complement-mediated events during nerve degeneration which may interfere with the subsequent regenerative process. In addition, the possibility that the inhibitory effect of short-lasting myelin components in the degenerating nerve may control branching and promote correct reinnervation and faster recovery is not ruled out. Interestingly, a recent study demonstrated that local application of exogenous MAG for a short time (72h) after injury reduces axonal branching without affecting axonal elongation and enhances the functional recovery after rat sciatic nerve transection, presumably by reducing hyperinnervation and misdirection 16 . Thus, residual myelin proteins in the degenerating nerve, as seen in the case of C6 -/- and sCR1-treated animals in which post-traumatic myelin clearance is delayed 17 , can prevent branching and promote the accuracy of reinnervation, improving recovery (see chapter 6 ).
MAC and neuropathies
The MAC is a key determinant of axon loss in acquired neuropathies 18 . Secondary axon loss is also a major hallmark in demyelinating forms of hereditary motor and sensory neuropathies (HMSN) and causes disability. Whether the complement (C) system is involved was unknown.
We investigated whether the C system is activated in certain forms of HMSNs and found deposition of activated C factors, including MAC, in nerve biopsies of HMSN patients and animal model of the disease (see chapter 7 ). The role of complement in HMSNs is not clear. It is possible that the abnormally folded myelin (i.e. onion bulbs), determined by the primary genetic defect, exposes epitopes which are normally sequestered within the correctly folded membrane. These epitopes may be recognized by C proteins, mainly C1q, and trigger activation of the complement cascade, leading to the production of anaphylatoxins and the assembly of the MAC. MAC deposits could damage the myelin sheath and attack the neighboring unsheathed axons, leading to axonal damage. The C anaphylatoxins could recruit macrophages which damage the nerve directly or indirectly via the secretion of matrix metalloproteinases which have been shown to degrade the Schwann cell' basal lamina in a mouse model of HMSN 19 .
The finding of activated C protein deposits in HMSN biopsies offers the opportunity to test whether new specific C drugs may alleviate the demyelinating phenotype and the progressive and disabling muscle atrophy derived from distal motor axon loss.
Targeting MAC formation
We observed that inhibition of the MAC is sufficient to delay axon loss and promote post-traumatic nerve regeneration and recovery (see chapters 3, 5 and 6 ). This has a major advantage since it allows to discriminate between the “foe” and “friend” side of C activation. The upstream factors of the C cascade have key functions in modulating an inflammatory reaction, essential to protect the body against infections and to maintain tissue homeostasis. Selective inhibition of the terminal pathway will target the “foe” side of the C cascade, leaving the upstream C factors, thus the “friend” side of the C cascade, undisturbed. This novel approach will limit the side effects which would derive from inhibition of the entire C cascade. Further, it will allow systemic administration of the selective inhibitor, circumventing the challenge of finding new venues to solely target local C activation.
Selective inhibitors of the terminal C pathway are available and they are currently in the stage of preclinical development. Inhibition at the C5 stage is an attractive proposition as C3b opsonization of pathogens and solubilization of immune complexes proceed unaffected whereas C5a-mediated inflammation and tissue damage caused by the MAC are prevented. Eculizumab, the humanized antibody against C5 already in use in clinical trials for the treatment of PNH, has been successfully used to control C activation in animal models of GBS. Eculizumab could prevent MAC deposition and terminal axonal neurofilament loss in the MFS model. It could block the large increase in the frequency of MEPPs and prevent the block in synaptic transmission 20 . The 17 kDa non-glycosylated protein produced in the salivary gland of the soft tick Ornithodoros moubata has a high binding affinity and low dissociation rate with C5. In rodents it has a circulating half-life of 30 hours and the ability to completely inhibit complement hemolytic activity. Further, the in vivo therapeutic efficacy of OmCI has been tested in a rat model of myasthenia gravis, resulting in reduced C3 and C9 deposition, and cellular infiltrates at the neuromuscular junction. The treated animals were completely protected from manifesting any clinical symptom 21 .
Intriguingly, the work described in this thesis showed that blocking formation of the MAC, downstream of C5, also limits phagocytes recruitment and activation. This probably results from inhibition of MAC-derived debris, their opsonization, thus recognition by phagocytes. Antibodies which target components of the MAC, other than C5, including C6 and C8, have been generated and their efficacy shown in an in vitro bypass model 22 but their therapeutic efficacy in vivo has not been tested. The membrane negative regulator of the MAC, CD59, has also been engineered in soluble form (sCD59) 23 . Unfortunately, sCD59 is a poor inhibitor of C since it sticks to serum protein and it is efficiently cleared by filtration in the kidney 24 . To overcome these problems, two types of antibody-CD59 hybrid molecules have been created. In the first construct, the Fab arms of the antibody are attached to CD59 as a strategy to deliver the C regulatory protein to the target location. In the second construct, CD59 replaces the Fab arms while the Fc portion of the antibody is strategically used to retain the hybrid molecule in the circulation, thus increasing plasma half-life. To date these constructs have been tested and shown to function in vitro 25 . Since the regulatory activity of the hybrid molecule is lower than that of the free regulator alone 25 , the next step will be to incorporate a cleavage site, cleaved by specific enzymes expressed at the target site, between the regulator and the Fc antibody fragment. This promising approach will generate a drug with a long half-life, little or no systemic C inhibitory activity and high specificity to the desired target site.
Activation of the C system is responsible for much of the tissue damage seen in various acute conditions such as ischemia-reperfusion injury, stroke, myocardial infarction, traumatic brain and spinal cord injuries, or in the damage occurring as a result of transplantation and cardiopulmonary bypass. It has also been implicated in the pathology of various chronic conditions including multiple sclerosis, myasthenia gravis, rheumatoid arthritis, hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and chronic idiopathic polyneuropathy 26,27 . In this thesis, we showed that C activation is also a major determinant of post-traumatic peripheral nerve degeneration and regeneration and that it may play a role in certain types of HMSNs.
It seems straightforward that controlling inappropriate C activation represents a possible therapeutic strategy for these conditions. Despite promising attempts, the design of the ideal complement drug meets various challenges. The diverse nature of acute (i.e. traumatic nerve injury) and chronic clinical conditions (i.e. neuropathies) adds complexity to the challenge. Further, the optimal C target, the time and extent of inhibition need to be critically defined for each condition.
The work presented in this thesis strongly proposes the MAC as optimal target for C therapeutics ( Chapter 3 and 4 ). In acute conditions such as nerve trauma, we showed that pre-treatment with a C inhibitor up to 1 week is sufficient to delay axon loss and affect the regenerative capacity of the nerve. This has important implications for the eventual translation to the clinic, minimizing the chances of an autoimmune response against the drug, which could result from otherwise prolonged treatment, and minimizing the costs of drug production, which would result from continuous administration.
To date, experience on the use of C inhibitory drugs in man has been very limited. sCR1 (TP10, AVANT Immunotherapeutics) is the best characterized C drug and it was tested in man with acute respiratory distress syndrome and myocardial infarction 28 . Although the drug proved safe, clinical outcome was disappointing and further development for these indications has been stopped (http://www.avantimmune.com). Another C inhibitor that is now registered for use in humans is Eculizimab. This humanized antibody directed against C5, has shown promising results in clinical trials for the treatment of myocardial infarction and paroxysmal nocturnal hemoglobinuria 29,3 0 . The validity of C therapeutics in the treatment of chronic diseases remains to be tested.
The studies described in this thesis suggest that traumatic nerve injury, acute and chronic neuropathies may benefit from C therapeutics. For example, in some neurodegenerative diseases like multiple sclerosis, axonal damage has only recently emerged as a substantial determinant of pathology 31 . Delaying axonal degeneration could give a chance for more axons to survive a period of demyelination, arresting the decline from the relapsing-remitting to the progressive phase of the disease. Traumatic brain and spinal cord injuries are characterized by diffuse axon loss and complement activation 32 . Pathology secondary to the primary mechanical damage appears to be a major determinant of clinical outcome 33 . The secondary axonal damage occurs hours after the initial insult opening a window of opportunity for therapy aimed at rescuing the axons. Inhibition of complement activation could prevent spreading of secondary axon loss and also improve regeneration.
What are the mechanisms of C-dependent improved regeneration? The studies described in this thesis strongly suggest that a detrimental event during degeneration affects the regenerative capacity of the nerve. This event is dependant on the formation of the MAC. However, lack of MAC formation blocks the damage caused directly by the unspecific pores and it also inhibits the damage caused by macrophages and their toxic mediators, including matrix metalloproteinases. Dissection of these two processes, perhaps by the use of inhibitors of macrophage activation, should be addressed in future work.
Further, we speculated that the improved regeneration which results from lack of post-traumatic MAC formation may be due to either rescue of the basal lamina during degeneration or to a transient inhibitory effect of residual myelin proteins on axonal branching, minimizing hyperinnervation and misdirection thereby improving the accuracy of reinnervation. The finding that lack of MAC formation promotes the regeneration of sensory neurons is also intriguing. Gene expression profiling of both, the crush site and DRGs of degenerating and regenerating neurons is a valuable approach to elucidate the mechanisms responsible for the improved regeneration and should be pursued.
Does C activation play a role in hereditary neuropathies? Genetic crosses between murine models of HMSNs, such as the CMT1A rat 34 , and genetically modified or naturally occurring mutant strains of rodents deficient in specific C components, will be a valuable tool to determine the effects of C activation on disease severity in this chronic disease. Follow up studies will then determine the therapeutic efficacy of specific C inhibitors in the treatment of such diseases.
MAC therapeutics. The design of the optimal drug to target MAC formation still remains a challenge. The drug of the future should be safe, highly specific for MAC blockade and/or for the target tissue, a small molecule, inexpensive and have a long half-life. Engineered C regulatory proteins and the screening of libraries of natural compounds as source of potential MAC inhibitors are interesting options. In vivo studies will then confirm their therapeutic value.
Does inhibition of C activation facilitates recovery in patients with traumatic peripheral nerve injuries and peripheral neuropathies? The ultimate goal will be to determine whether treatment with C inhibitory drugs can ameliorate severity and accelerates recovery in patients with nerve trauma, acute and chronic demyelinating neuropathies. Initially, clinical trials with MAC inhibitors for patients with traumatic nerve injuries or immune-mediated neuropathies such as Guillain-Barré syndrome (GBS), will show the therapeutic efficacy of C drugs in the acute conditions, setting the premise for a promising future in the treatment of chronic neuropathies.
It is evident that the complement system plays both a protective and detrimental role in the peripheral nerve. The beneficial effects of complement in immune surveillance and possibly in regulating energy metabolism in the microenvironment of the nerve need to be balanced against the heavy weight of its damaging effects as key determinant of early axon loss and regeneration after injury and in acute PNS diseases. Future research aimed at producing specific modulation of the complement system at the terminal level of the cascade, e.g. formation of the MAC, may represent a promising approach to control the neurotoxic effect of excessive complement activation in peripheral nerve injury and disease.
Figure 1. Model of the role of MAC in WD.
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