The complement cascade - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2008 | 107 | 1169–1187

doi: 10.1111/j.1471-4159.2008.05668.x

*Department of Medicine, University of Chicago, Chicago, Illinois, USA Department of Physical Medicine & Rehabilitation, University of California, Irvine, California, USA àDepartment of Microbiology, University of Alabama, Birmingham, Alabama, USA §Department of Neurology, Neurobiology Program, Children’s Hospital, Harvard Medical School, Boston, MA, USA ¶Department of Molecular Biology and Biochemistry, University of California, Irvine, California, USA

Abstract The complement cascade has long been recognized to play a key role in inflammatory and degenerative diseases. It is a ‘double edged’ sword as it is necessary to maintain health, yet can have adverse effects when unregulated, often exacerbating disease. The contrasting effects of complement, depending on whether in a setting of health or disease, is the price paid to achieve flexibility in scope and degree of a protective response for the host from infection and injury. Loss or even decreased efficiency of critical regulatory control mechanisms can result in aggravated inflammation and destruction of self-tissue. The role of the complement cascade is poorly understood in the nervous system and neurological disorders.

The complement system The complement system is a powerful arm of the innate immune system, immediately participating in the recognition, trafficking, and elimination of pathogens and unwanted host material (Fig. 1). Its activation pathways include: classical, alternative, lectin, and a recently identified fourth pathway involving activation of the terminal cascade (Huber-Lang et al. 2006). The system involves more than 40 proteins, with additional regulators and functional receptors for activation products being discovered as new molecular techniques and animal models become available. Complement activation leads to cleavage of several cascade proteins including the key proteins, C3 and C5. Cleavage of C3 and C5 generates C3b, which

Novel studies have demonstrated that the expression of complement proteins in brain varies in different cell types and the effects of complement activation in various disease settings appear to differ. Understanding the functioning of this cascade is essential, as it has therapeutic implications. In this review, we will attempt to provide insight into how this complex cascade functions and to identify potential strategic targets for therapeutic intervention in chronic diseases as well as acute injury in the CNS. Keywords: Alzheimer’s, complement, glaucoma, lupus, multiple sclerosis, spinal cord injury. J. Neurochem. (2008) 107, 1169–1187.

Received April 29, 2008; revised manuscript received August 5, 2008; accepted August 21, 2008. Address correspondence and reprint requests to J. J. Alexander, Department of Medicine, University of Chicago, Chicago, IL, USA. E-mail: [email protected] Abbreviations used: AD, Alzheimer’s disease; APP, amyloid precursor protein; APPQ)/), a mouse model lacking C1q; Ab, amyloid beta; BBB, blood–brain barrier; Crry, complement receptor-related protein y; DBA, strain is homozygous for Cdh23ah1 and C5 deficient; dLGN, lateral geniculate nucleus; EAE, experimental autoimmune encephalomyelitis; ECM, extracellular matrix; ICAM-1, intercellular adhesion molecule-1; IOP, intraocular pressure; IPL, inner plexiform layer; MAC, membrane attack complex; MRL/lpr, MLR/MpJ-lpr/lpr mouse with insertion of the early transposable element ETn in the Fas gene; MS, multiple sclerosis; NP, neuropsychiatric; PMNs, polymorphonuclear leukocytes; RGCs, retinal ganglion cells; SAP, serum amyloid protein; SCI, spinal cord injury; SLE, systemic lupus erythematosus; TNF, tumor necrosis factor.

2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 1169–1187

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hepatocytes. However, many other cell types constitutively synthesize complement proteins at low levels or can be induced to produce complement components under inflammatory conditions. In the CNS complement proteins are synthesized by neurons, microglia, astrocytes, and oligodendrocytes (Gasque et al. 2000; Barnum 2001).

Complement system and inflammatory signaling proteins

Fig. 1 Model of complement activation and effector functions. Activation of the complement cascade is initiated by either the classical, lectin, or alternative pathway, but all result in the effector functions of surface opsonization [C3b deposits on the pathogen (yellow) signaling engulfment by a phagocyte], leukocyte recruitment (C3a and C5a), and pathogen lysis by formation and insertion of the C5b-9 complex in membranes (MAC, membrane attack complex) [Permission obtained from Elsevier, (Bohlson et al. 2007)].

binds to immune complexes, and C5b, which initiates the assembly of the C5b-9 complex [membrane attack complex (MAC)] that can result in cellular death or activation. In addition, the anaphylactic peptides C3a and C5a are released to diffuse from the area of activation, recruiting inflammatory cells and inducing inflammation. To prevent self-injury, regulatory proteins strictly control the spontaneous and immune complex-induced activation of the complement system. However, the complement system is not just a mediator of innate immunity, but also participates in neurogenesis (Rahpeymai et al. 2006), liver regeneration (Strey et al. 2003), B-cell proliferation (Peters et al. 1988; Ambrus et al. 1990), and neuronal synaptic plasticity (Stevens et al. 2007). In addition, the complement proteins along with their primary function of participating in the complement cascade are also linked to the adaptive immune system and several signaling pathways. Complement proteins are found in all body tissues and fluids and are synthesized primarily by

Once the complement system is activated, through various molecules and pathways, the inflammatory process intensifies. Complement proteins have the potential to activate other intracellular, and ultimately intercellular, signaling pathways. For example, activation of C3 can induce tumor necrosis factor (TNF) release, which can lead in turn to activation of downstream mediators such as IL-1, IL-6, and IL-18, facilitating and amplifying inflammation (Gasque et al. 1995). Altered cytokine profiles in CNS can result in the activation of different brain cells including astrocytes, microglia, and/or neurons. The brain is ‘protected’ from the systemic environment by the presence of the blood–brain barrier (BBB). Alteration in cytokines can lead to pathology of the BBB, thereby altering ionic homeostasis and causing edema that plays a significant role in the ultimate impairment in CNS function. Changes in cytokine levels have been shown to have the capacity to alter motivated behavior and emotional reactivity (Harden et al. 2008). Furthermore, complement activation can lead to apoptosis, which can occur directly through cellular events stimulated by C5b-9 (Rus et al. 2006a), C5a (Osaka et al. 1999), and Bb (Uwai et al. 2000), or through cytokine signaling, culminating in activation of caspase 3, the effector protein that performs the downstream function of apoptosis. It remains to be discovered what determining factors and requirements are involved in tilting the balance, allowing the complement cascade to change from being a protective mechanism normally, to an aggravating mechanism during inflammation.

What we know about the role of complement in CNS diseases In the subsequent sections we describe the role of complement in CNS diseases, in humans and experimental mouse models. Animal models are indispensable for our understanding of human disease pathogenesis and the development of new therapeutic approaches. The study of genetically altered mice has been among the most important advances in our understanding of human physiology and pathophysiology. Mice allow use of alternative methodologies, such as administration of recombinant proteins, inducers or inhibitors to study brain function and behavior.


Complement and neuroinflammation | 1171

Alzheimer disease Alzheimer disease (AD) is a progressive neurological disorder, afflicting over 5 million people in the USA alone, with the first clinical presentation being a loss of cognitive ability. It is now just over 100 years since Alois Alzheimer first described the pathologic features in the brain of an individual diagnosed with dementia that now define AD. These included accumulation of protein ‘senile’ plaques comprised mainly of an approximately 4000 Da peptide designated b-amyloid (Ab), neurofibrillar tangles composed of hyperphosphorylated tau protein, and neuronal loss. It is now known that AD is a disorder of the elderly with onset of the clinical manifestations of the disease commonly between 65 and 80 years. The identification of familial forms of the disease, all of which involve genetic mutations leading to the accumulation of Ab, has led to a plethora of information about this disease in the past two decades (Tanzi and Bertram 2005). Early events involving intracellular Ab peptide accumulation (Echeverria and Cuello 2002; Osmers et al. 2006) and/or generation of oligomeric Ab peptides (Walsh et al. 2002; Kayed et al. 2003) or complexes containing oligomeric Ab (Lambert et al. 1984) occur in humans in Down syndrome and as a precursor to AD. Which components are triggering the cognitive loss in the human disease is still being debated. Complement component synthesis and activation in the AD brain Complement components have been observed in association with plaques since the early 1980s (Eikelenboom and Stam 1984). Subsequently, several laboratories provided evidence that a specific assembly state of Ab peptide (that of b-sheet fibrils) activates both the classical and alternative complement pathways in vitro (Rogers et al. 1992; Jiang et al. 1994; Watson et al. 1997; Bradt et al. 1998) via interaction with C1q in C1 and directly with C3, respectively (see Fig. 1). Products of the activation cascades are generated in human AD brain but not in elderly non-demented controls who lack b-sheet fibrillar amyloid plaques (Afagh et al. 1996; Webster et al. 1997; Stoltzner et al. 2000) (although see below). In addition, neurofibrillary tangles have been shown to be coated with C1q (Afagh et al. 1996) and to activate the complement system when added to serum (Shen et al. 2001). Complement activation by either pathway could result in the generation of the cleavage peptides C3a and C5a that are known to be chemotactic for microglia and astrocytes (Yao et al. 1990), and indeed microglia and astrocytes localize with b-sheet (fibrillar) amyloid plaques in AD. Activated microglia recruited to the plaque area can be phagocytic, but also can be further activated to secrete several proinflammatory cytokines, as well as reactive oxygen species and proteases, essentially creating a local inflammatory nidus that could accelerate neuronal dysfunction and cognitive decline seen in the human disease (Cummings et al. 1996; Tenner and

Webster 2001). In addition, decreased levels of complement regulators with age may also reduce the ability of neurons in the area of plaques to protect against lytic effects of the MAC (Shen and Meri 2003). Loeffler et al. (2004, 2008) have reported an occasional association of complement component C3b with diffuse plaques, perhaps because of transient or bystander activation. The study of Zanjani et al. (2005) demonstrated staining of amyloid plaques in all stages of AD with anti C4d, with the density of C4d immunostaining correlating with the density of silver stained plaques. Non-demented elderly controls that had plaques also had some plaques with complementassociated proteins [as had been reported earlier (Lue et al. 1996)], but these were rare relative to the prominent association with fibrillar plaques found in AD patients. While the dependence on morphology for classification of plaques as diffuse or neuritic in these studies rather than the use of thioflavine as a more specific marker of b-sheet conformation (complement activating form) of amyloid leads to less definitive conclusions, the data are consistent with the hypothesis that complement activation by fibrillar amyloid plaques may contribute to inflammation and neuronal injury, and thus cognitive loss in AD. The synthesis of C1q, the recognition component of C1, by neurons is known to be robustly induced in response to injury in the CNS [reviewed in (Tenner and Pisalyaput 2008) and below]. Furthermore, the synthesis of all complement components are up-regulated in AD brain (Walker and McGeer 1992; Blalock et al. 2004) as well as in animal model systems of amyloid injury (reviewed in (Shen and Meri 2003; Tenner and Pisalyaput 2008). One interesting hypothesis, which has been supported by experiments in peripheral phagocytes (Bensa et al. 1983), is that complement components with ‘protective’ functions are differentially synthesized early in disease, but that as ‘injury’ increases, the remainder of the complement components are induced. The appearance of activating substances (fibrillar amyloid plaques) would then provide the stimulus for activation of complement and an acceleration of local inflammation and neuronal injury. Thus, whether the roles of these components and pathways are detrimental, protective or both (depending on the stage of the disease) has been the subject of discussion and investigation – leading to the concept of ‘Yin and Yang’ as nicely discussed and illustrated for AD by Shen and Meri (2003). Investigating the contribution of complement to pathology in murine models of AD Mice designed to express a transgene for a mutant form of human amyloid precursor protein (APP) in brain have been generated and shown to display elements of age-related AD-like pathology (Ashe 2001) Thus, animal models are available for exploration of mechanisms of initiation and pathogenesis of the disease as well as to test candidate therapeutics for stopping or slowing the progression of



disease. Recent application of intravital microscopy to image one such APP mouse model demonstrated that the appearance of fibrillar amyloid plaques was rapidly followed by an influx of microglia and subsequent degeneration of neurons in the plaque area (Meyer-Luehmann et al. 2008). Several mouse models have shown that, as seen in human AD, C1q, and C3 are associated with fibrillar amyloid plaques (Matsuoka et al. 2001; Fonseca et al. 2004; Zhou et al. 2005). To begin to assess whether the activation of the classical complement cascade plays a role in progression of pathology, APP transgenic mice (Tg2576) that develop Ab plaques (Hsiao et al. 1996), inflammatory pathology and some behavioral deficiencies were crossed to mice containing a targeted deletion of the gene encoding C1q (Botto et al. 1998). These animals [APPQ)/) (a mouse model lacking C1q) mice] therefore lack the ability to activate the classical pathway via fibrillar Ab interaction with C1q. When age-dependent pathology in brains from APPQ)/) was compared to that of APPQ+/+ mice (Fonseca et al. 2004), the APPQ+/+ and APPQ)/) mice developed comparable total amyloid and fibrillar Ab in frontal cortex and hippocampus with indistinguishable kinetics (9–16 months of age). However, both astrocytic and microglial accumulation were significantly reduced (by approximately 50%) in APPQ)/) animals at 12 and 16 months of age relative to the C1q sufficient animals. In addition, the knock out transgenic mouse demonstrated 50–65% reduction in the loss of synaptophysin and MAP2 in the CA3 region, all consistent with the detrimental role of the activated complement pathway in this model. The residual pathology in the APPQ)/) mice relative to nontransgenic animals was suggested to be either the result of alternative pathway activation (Zhou et al. 2008) or direct toxicity of the amyloid itself (oligomeric or fibrillar assemblies) (Fonseca et al. 2004). Testing of inhibitors of specific pivotal points in the complement cascade for efficacy in reducing progression of pathology and behavioral dysfunction in murine models will be an essential step toward developing effective treatments to prevent or slow the progression of pathogenic events that lead to AD. Indeed, enoxaparin, a low molecular weight heparin that inhibits Ab-induced complement activation, resulted a greater than twofold decrease in the Ab deposits and total amyloid load, and decreased astrogliosis in the APP23 transgenic mouse (Bergamaschini et al. 2004), supporting a role for complement-mediated neuropathology. Nevertheless, additional studies must be presented to assess alternative mechanisms for the enoxaparin effects such as an influence on the aggregation of amyloid and/or effects on the trafficking of amyloid out of the CNS. Potential neuroprotective effects of complement in AD As mentioned above, induced neuronal synthesis of C1q is an early response to injury. C1q has been shown to be

synthesized by cells of the CNS including neurons either damaged or in disease brains (Rozovsky et al. 1994) [and reviewed by (Gasque et al. 2000)]. C1q is known to bind apoptotic cells and thus may flag either damaged neurons or neuronal blebs for rapid removal by microglia, thereby preventing the release of neurotoxic levels of intracellular components (such as glutamate). In addition, proinflammatory cytokine expression in peripheral phagocytes is down-regulated by C1q (Nauta et al. 2004; Fraser et al. 2006), suggesting another modulating role for C1q. That is, C1q could play a protective role in the early stages of disease by enhancing the clearance of cellular debris and/or altering the effects of the amyloid peptide on microglia. Most recently, we observed a direct neuroprotective effect of C1q on primary rat and mouse neurons in vitro. Addition of C1q to the media reduced neuron loss and protected neurons against toxicity induced by Ab and serum amyloid protein (Pisalyaput and Tenner 2008). This neuroprotection was independent of caspase or calpain activation, did not alter initial mitochondrial depolarization and was not a reflection of induced neuronal proliferation. It remains to be established if, and to what extent, these C1q-mediated events contribute to preventing AD or limiting the rate of progression of the disease, but further investigation of these observations may lead to the discovery of a novel neuroprotective pathway. Other observations that suggest neuroprotective effects of complement have been reported. Wyss-Coray et al. (2002) reported that over-expression of complement receptorrelated protein y (Crry), an inhibitor of C3 and C5 cleavage, enhanced pathology in their mouse model of AD, suggesting a protective role for C3 cleavage products, perhaps via enhancement of clearance of Ab. The complexity is further reinforced by the recent publication from Lemere and colleagues reporting increased elevated Ab deposition in 17 month (i.e. very old J20) APP C3)/) relative to the APP (J20) C3 sufficient mice (Maier et al. 2008). Interestingly, the C3)/) APP animals had a cytokine profile characteristic of M2 macrophages, i.e. elevated IL-4 and IL-10. Whether this is due to anti-inflammatory signaling of C1q on plaques in the absence of C5a signaling and a lack of clearance of Ab in the absence of opsonizing C3b, remains to be determined. A protective role for C5a has also been proposed (Mukherjee and Pasinetti 2001). In summary, until more is known about the multiple, sometimes counter-balancing effects of various complement components, complement activation fragments, complement receptor expression and function in the AD brain, and particularly the possible protective roles of the early components of the complement pathway in enhancing the clearance of cellular debris, regulating inflammation and/or providing direct neuroprotective effects, selective inhibition of complement activities should be the preferred therapeutic strategy.



Glaucoma Glaucoma is a neurodegenerative eye disease and the leading cause of blindness worldwide. Vision loss is caused by the progressive and irreversible loss of retinal ganglion cells (RGCs) and their axons that is often associated with elevated intraocular pressure (IOP) (John et al. 1999; Libby et al. 2005). While there is evidence to support a link between elevated IOP and the development of glaucoma, the molecular mechanisms underlying the specific death of RGCs are still poorly understood. Insight into how and where RGCs are first damaged could have important therapeutic implications. The DBA/2J (strain is homozygous for Cdh23ah1 and C5 deficient) mouse has been an extremely useful model to investigate these and other mechanistic questions, as the time course and degenerative changes in DBA/2J retinas are well characterized and closely mimic the human disease (Jakobs et al. 2005; Libby et al. 2005). Growing evidence points to a role of C1q in glaucoma. C1q levels are significantly up-regulated in the DBA/2J retinas at early stages of glaucoma (Steele et al. 2006; Stevens et al. 2007). Microarray analysis of retinal RNA from DBA/2J mice revealed that C1q is significantly up-regulated prior to significant RGC loss (Steele et al. 2006; S. M. John, personal communication). Consistent with these findings, C1q mRNA, and protein are elevated in the retina of mouse, primate and human glaucomatous eyes (Stasi et al. 2006), and multiple complement components are up-regulated in both rat and monkey retinas after acute IOP elevation (Miyahara et al. 2003; Ahmed et al. 2004). Together these data suggest that the classical complement pathway play an important early role in the development of glaucoma. The classical complement cascade mediates developmental synapse elimination Does complement up-regulation or activation contribute to the loss of RGCs in glaucoma? Insight into this question stems from recent work from the Barres laboratory which found that C1q and the classical complement cascade mediates synapse elimination in the developing visual system (Stevens et al. 2007). We used a gene profiling approach to screen candidate neuronal genes that are regulated by astrocytes. We found that C1q mRNA (for each of the C1q A, B, and complement chains) was significantly up-regulated in purified RGCs upon exposure to immature astrocytes. RTPCR and in situ hybridization on mouse retinal sections showed that C1q mRNA levels were highest in post-natal RGCs and developmentally down-regulated after the second post-natal week. Importantly, C1q was highly localized to synapses in the inner plexiform layer (IPL) of developing, but not adult mouse retinas (Stevens et al. 2007). A similar synaptic localization was observed in the developing brain, including in the lateral geniculate nucleus (dLGN), a major target of RGC axons (Stevens et al. 2007).

As one of C1qs primary roles in the immune system is to opsonize unwanted cells or debris for removal, we asked whether C1q is similarly targeting unwanted synapses for elimination in the developing brain. Indeed, C1q expression and synaptic localization (as well as the appearance of astrocytes at CNS synapses) corresponds to the period of synaptic pruning, a process essential for the precise wiring of the developing brain. Early in development, excess numbers of synapses are generated, but inappropriate synapses are later pruned away while appropriate synapses are maintained and strengthened (Katz and Shatz 1996; Sanes and Lichtman 1999; Hua and Smith 2004). Using a combination of neuroanatomical and electrophysiological techniques, we found that mice deficient in C1q or the downstream C3 have significant and sustained defects in CNS synapse elimination. Initially, dLGN neurons are innervated by multiple (10 or more) RGC axons but by adulthood each dLGN neuron normally receives strong, stable inputs from only 1-2 RGC axons (Hooks and Chen 2006). Electrophysiological recordings from acute Lateral Geniculate Nucleus (LGN) slices revealed that dLGN neurons in C1q- and C3-deficient mice remain multiinnervated. In addition, both C1q and C3 knockout (KO) mice have significant defects in the ability of RGC axons to properly segregate into eye specific territories in the dLGN, which together provide compelling evidence that the classical complement cascade mediates synapse elimination in the developing brain (Stevens et al. 2007). Implications of complement-mediated synapse elimination in neurodegenerative disease Synapse elimination is necessary for the formation of mature functional neural circuits, but what if the same mechanisms that normally eliminate inappropriate synapses during development become aberrantly reactivated to promote destructive synapse loss in the adult brain? Indeed, growing evidence suggests that one of the earliest events in neurodegenerative diseases, such as AD, is the dysfunction and loss of synapses (Selkoe 2002). Complement expression and activation are known to be significantly up-regulated in many neurodegenerative diseases, including glaucoma and AD, but it is not known whether complement is localized to synapses. Given our findings that C1q mediates synapse elimination in the developing CNS, we investigated whether C1q is reexpressed and re-localized to synapses in the adult retina at early stages of glaucoma. Using the DBA/2J mouse model we looked at the timing and localization of C1q in DBA/2J and control retinas at various stages of disease (Libby et al. 2005; Stevens et al. 2007). Neuronal C1q is normally down-regulated in the adult CNS, however we found that C1q was up-regulated and synaptically localized in the IPL of 70% of retinas from aged DBA/2J mice (Stevens et al. 2007) compared with young DBA/2J (< 2 months), or age matched control mice



(a)

(b)

(c)

Fig. 2 Proposed model for complement-mediated synapse elimination in the developing and diseased CNS. During development, immature astrocytes can promote synapse formation, elimination, and structural plasticity of synaptic circuits via a variety of secreted and contact-dependent signals. (a) In the post-natal CNS, immature astrocytes secrete a signal that up-regulates C1q in neurons. C1q and downstream C3 are then localized to developing synapses and ‘tags’ them for elimination by activated microglia, or via other

mechanisms. (b) Complement expression is down-regulated in the healthy adult brain after the appropriate synaptic connections develop into stable, mature synaptic connections. (c) In the damaged or diseased brain, reactive astrocytes (and possibly microglia) re-express C1q and complement cascade proteins, suggesting that the same mechanisms that normally eliminate inappropriate synapses during development are reactivated to trigger destructive synapse loss in the adult CNS.

(Howell et al. 2007). Importantly, C1q was localized at synapses at very early stages of the disease, before detectable synapse or RGC loss (Stevens et al. 2007). Furthermore, punctate C1q staining was more intense in the IPL from eyes with moderate (and severe) glaucoma compared with early glaucomatous eyes, and corresponded to a decrease in the density of synapses and RGCs. Together our findings indicate that C1q becomes relocalized to synapses in glaucomatous retinas before significant synapse loss and RGC death, which suggests that complement mediated synapse elimination is recapitulated and occurs as a crucial early event in neurodegenerative disease (see Fig. 2).

resemble immature astrocytes (Cahoy et al. 2008), suggesting that reactive astrocytes may re-express the signal that induces C1q expression in developing neurons in early stages of neurodegenerative disease (Stevens et al. 2007). Consistent with this notion, the appearance of reactive astrocytes corresponds to C1q up-regulation in neurons and microglia in many CNS neurodegenerative diseases, including glaucoma. Glial fibrillary acidic protein is strongly up-regulated in Mu¨ller glia and retinal astrocytes after IOP elevation (Tanihara et al. 1997; Miyahara et al. 2003), and at early stages of glaucoma in the DBA/2J mouse model (Steele et al. 2006). Identification of the soluble signal(s) that trigger C1q up-regulation in neurons will be an important next step to investigate the specific mechanisms by which astrocytes control synapse elimination in both the developing and diseased retina. How are complement ‘tagged’ synapses eliminated? One intriguing possibility is that C1q or C3b tagged synapses are actively engulfed by activated microglia, the primary phagocytic cells in the brain. Indeed, microglia express the C3 complement receptor (alphaM bintegrin, CD11b/CD18, and Mac-1) (Perry et al. 1985), which can induce phagocytosis when bound to C3b/iC3b. Furthermore, activated microglia are enriched in synaptic brain regions during the period of developmental synapse elimination (Milligan et al. 1991; Dalmau et al. 1998; Maslinska et al. 1998; Fiske and Brunjes 2000); our unpublished data), and it has been speculated for some time that microglia could participate in synaptic remodeling. As activated microglia play a prominent role in neurodegenerative diseases, this

Role of glial cells in complement-dependent synapse elimination In the developing rodent brain, immature astrocytes persist until P15, and provide instructive signals to neurons to promote the development and plasticity of synaptic circuits (Ullian et al. 2004; Allen and Barres 2005). In the adult CNS, astrocytes and microglia become ‘reactive’ following CNS injury and in neurodegenerative disease and can secrete multiple complement components, as well as an array of cytokines and inflammatory signals that could both positively and negatively regulate synaptic function in the damaged and diseased brain. Our findings suggest that a signal produced by immature astrocytes in the normal developing brain and retina triggers the expression of C1q, thus enabling a developmental window for complement-dependent synapse elimination. Gene profiling indicates that reactive astrocytes antigenically



phagocytic mechanism of synapse elimination could also mediate synapse loss in neurodegenerative disease. In the adult CNS, activated microglia are recruited and rapidly phagocytose motor neuron synapses after axotomy in a process known as ‘synaptic stripping’, but it is not known whether the ‘stripping’ of synapses is mediated by complement, or whether a similar mechanism of synapse engulfment occurs in glaucoma and other neurodegenerative diseases. Systemic lupus erythematosus Systemic lupus erythematosus (SLE) or lupus is a debilitating chronic autoimmune disease that affects as many as 1.5 million Americans, mainly women of childbearing age (Danchenko et al. 2006). Although its etiology remains an enigma, there is evidence suggesting a role for genetics (Vyse and Kotzin 1996) and infectious and non-infectious environmental factors. While human leukocyte antigen and complement genes play a crucial role, environmental factors such as medications, Epstein Barr Virus (EBV) and ultraviolet light may initiate the disease or contribute to its progression (Estes and Christian 1971). The manifestations of lupus are diverse and characterized by multiorgan involvement. The organs involved include peripheral organs such as skin and joints and internal organs such as kidneys, lungs, heart, blood vessels, and brain. CNS changes result in neuropsychiatric (NP) manifestations such as seizures, headaches, psychosis, mood disorders, and cognitive dysfunction (Brey et al. 2002). These may occur in the absence or presence of systemic disease and oftentimes precede systemic disease. Although there is increased awareness among clinicians that CNS involvement is a devastating manifestation of SLE and 30–75% of patients demonstrate psychiatric and/or neurological symptoms, the prognosis remains poor. Diagnosis and treatment of NP-SLE is delayed because of the absence of disease markers. Cyclophosphamide and prednisolone have remained the drugs of choice for the last 40 years (Baca et al. 1999; Stojanovich et al. 2003). However, their potent side effects make the need for alternate therapies urgent. Attempts to determine diagnostic laboratory markers for NP-SLE have yielded conflicting data. Although antibody titers to dsDNA and anti-Sm were identified as markers for NP-SLE in patients, no correlation was observed between the antibody levels and CNS pathology. On the contrary, serum levels of C3, and C4 complement components, were strongly associated with NP-SLE (Jongen et al. 2000). Complement system and lupus brain Support that complement can contribute to CNS pathology in human SLE includes increased concentration of central complement proteins, C3 and C4, in the CSF of SLE patients. In addition, increased serum level of the anaphylatoxins, C3a and C5a, has been shown to correlate with CNS

disease in lupus (Belmont et al. 1986; Hopkins et al. 1988). Furthermore, the expression of factor B, an important component of the alternative pathway, was increased in lupus patients. By using accurate mouse models of SLE, we have gained insight into possible mechanisms causing the pathogenesis of human disease (Alexander and Quigg 2007). Similar to humans, increased expression of complement proteins was observed in the CNS of the established mouse model, MRL/Tnfrsf6lpr/lpr mice [MRL/lpr (MLR/MpJ-lpr/lpr mouse with insertion of the early transposable element ETn in the Fas gene)] (Segurado et al. 1990; Buyon et al. 1992; Alexander et al. 2007). As mentioned earlier, the complement cascade can be both neuroprotective and neuroinflammatory depending on the setting and the proteins involved. Once activated, the proteins C5b-9, C3a, and Bb can cause apoptosis, a key event in lupus brain through activation of caspase 3 (Uwai et al. 2000; Nauta et al. 2002), while C5a protects brain cells from apoptosis by reducing the activation of caspases indicating different participating signaling pathways (Mukherjee et al. 2008). Further, it is also notable that sublytic levels of C5b-9 have been shown to be protective in brain (Soane et al. 1999). In addition, complement and complement regulators play an important role in the removal of apoptotic cells, reducing the subsequent pathology (Trouw et al. 2008). Studies using genetically altered mice and recombinant complement inhibitors have confirmed that early complement proteins C1q and C4 are protective in lupus, whereas downstream complement activation in the pathways is deleterious. Our studies provide compelling data showing that inhibition of the complement cascade by overexpression or administration of a soluble form of the potent complement regulator Crry to MRL/lpr mice, markedly reduced the disease manifestations in brain (Alexander et al. 2005b). Our subsequent studies show that apoptosis detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in lupus brain, colocalized with neurons (Alexander et al. 2007) and was alleviated by depletion of factor B suggesting an important role for the alternative pathway in lupus setting. Given that MRL/lpr mice are Fas deficient, the mechanism of apoptosis has to be through the TNF or other pathways. Circulating levels of TNFa and its receptor TNFR1 were increased and correlated with disease in SLE patients (Aringer et al. 2002). TNF induction is synergistically up-regulated by complement activation fragments (Zhang et al. 2007). The TNF pathway can regulate different inflammatory proteins, such as a-amino-3-hydroxy-5-methylisoxazole-4-propionate-GluR1, intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS), all of which are increased in patients with SLE (Brey et al. 1997). Apoptosis was reduced in complement-inhibited lupus mice through the Akt signaling pathway, with a concomitant decrease in the proapoptotic proteins, Bcl-xL/Bcl-2-Associated Death Promoter (BAD), Bclx, and phosphatase and tensin homolog (PTEN) lending



support to the growing appreciation that apoptosis and inflammation are not mutually exclusive but may be linked. Akt/protein kinase B may be a possible link involved in apoptosis and cytoskeleton-mediated processes (Fujio and Walsh 1999). The extracellular matrix proteins (ECM) form the cerebral microvascular basal lamina and along with astrocytes form the glial scar in response to CNS injury (Eng and Ghirnikar 1994). The localization of the ECM proteins in mice suggests that collagen IV, laminin, and fibronectin are integral components of the basement membrane of the microvessels, similar to that observed earlier in humans (van Horssen et al. 2005). The complement cascade regulates the expression of ECM proteins, laminin, fibronectin, and collagen IV, in lupus brain. Increase in expression of these proteins indicates possible vascular hypertrophy and remodeling or could be a prosurvival response to counteract injury and attempt to maintain the vascular integrity in the disease tissue. These studies suggest that the integrity of the BBB could be regulated by the complement cascade. Complement and the blood–brain barrier in lupus The hypothesis that the BBB dysfunction is an important requisite for disease activity in lupus brain is strengthened by recent studies (Abbott et al. 2003). Activation of the endothelial cells by circulating mediators such as complement and autoantibodies (Husebye et al. 2005) could result in a ‘leaky’ BBB. ICAM-1 present on the endothelial cells of the BBB and regulated by both TNF and complement is an important mediator in leukocyte transmigration. Complement inhibition with Crry resulted in reduced ICAM-1 expression in MRL/lpr brain with a significant paucity in neutrophil infiltration into the CNS. This is in line with studies that show reduced CNS pathology on ICAM-1 inhibition in patients and mouse models (Brey et al. 1997; Sari et al. 2002). Further, local deposition of immune complexes in blood vessel walls with subsequent complement activation (Belmont et al. 1996) could result in vasculitis, observed in a number of NP-SLE autopsy cases. Compromise of the BBB gives access to plasma complement proteins and IgG to brain parenchyma. Increased deposition of both C3 and IgG was observed in lupus brain, colocalized in and around the capillaries, choroid plexus and in the brain parenchyma. The pericapillary distribution of IgG deposits suggests that IgG present in the periphery traverses the BBB and binds to parenchymal targets in the brain. The presence of immune complexes could cause thrombosis and lead to stroke in patients. Therefore, the role of complement and cytokines in causing the vascular pathology observed in the brains of NP-SLE patients is an area of promising research. Metabolic alterations and lupus 2-[18F] fluoro-2-deoxy-D-glucose (FDG) and positron emission tomography (PET) scans and magnetic resonance spectroscopy studies reveal neurometabolic abnormalities,

thought to reflect neuronal injury or loss in NP-SLE (Handa et al. 2003). Our recent studies demonstrated increased complement-dependent brain lactate synthesis, intracellular glutamine concentration and choline accumulation in experimental lupus brain, by NMR spectroscopic studies (Alexander et al. 2005a). Disturbance in energy metabolism and increased concentrations of different metabolites such as glutamine and glutamate can cause changes in ionic strength and thereby lead to a change in water transport and content. In the CNS, membrane water transport is involved in brain volume homeostasis, CSF production, compensation for local changes in osmolarity, and the pathogenesis of brain edema. Expression of aquaporin-4, which is predominantly found in astrocytes and plays an important role in water transport, is increased in the brains of MRL/lpr mice (Alexander et al. 2003). Concomitant with complementdependent aquaporin-4 expression there was increased water content in experimental lupus brain. These studies are consistent with the imaging studies that demonstrate alterations in brain energy metabolism, brain volume and the BBB, in lupus patients (Brooks et al. 1997; Chinn et al. 1997). Alterations in cytokines, metabolism and edema and infiltration of autoantibodies or circulating cells into the CNS could all result in behavioral alterations. Complement regulation of behavioral alterations in lupus Abnormal neurological functioning similar to that seen in SLE patients is detectable in MRL/lpr mice by 8–10 weeks of age and is severe by 18 weeks of age (Sakic et al. 1992). An exciting observation made was that the behavioral alterations that occur in MRL/lpr mice are complement dependent (Alexander et al. 2007) in line with the observation that complement level correlates with flares in lupus patients. The observed behavioral changes are not completely dependent on the systemic pathology, as we and others have observed behavioral alterations in these mice at an age when they are free of clinical symptoms. These behavioral alterations could also be cytokine driven (Zalcman et al. 1998), as deposition of immune complexes could lead to changes in cytokine profiles. As the behavioral alterations in our study were attenuated but not completely eliminated by complement inhibition, other factors may also contribute to these changes. In summary, these studies demonstrate that the complement cascade plays a very crucial role in SLE (see Fig. 3) and its absence in mice, led to a better neurological outcome. Although additional questions regarding the access of proteins administered, into brain and the challenge of immune complex handling in the face of complement cascade inhibition need to be addressed. Therefore, the complement cascade is a promising potential site for therapeutic intervention in the treatment of CNS lupus, since it markedly reduces the severity of the disease in an experimental model similar to humans, without the toxic effects of the current therapy using immunosuppressants and steroids.



Fig. 3 Schematic diagram of possible complement-mediated pathogenic mechanisms in CNS lupus.

Spinal cord injury Spinal cord injury (SCI) affects over 10 000 new people each year; there are an estimated 250 000 SCI cases severe enough to require wheelchair confinement in the USA alone. Individual lifetime medical costs for SCI average $400 000 and can reach as much as $2 000 000, while the total medical cost for all SCI individuals in the USA approaches $10 billion annually. Many events are likely to play important roles in SCI pathogenesis, but inflammation has garnered increasing attention as understanding of the interactions between the nervous and immune systems has grown and potential anti-inflammatory therapeutics for CNS injury have been identified (Anderson 2002; Donnelly and Popovich 2007). The environment in the spinal cord following initial mechanical trauma is dynamic, including both primary injury and secondary degenerative processes. In the acute period, hours to days post-SCI, the BBB is disrupted, even in closed contusion or compression models. This leads to the rapid invasion of blood and serum components including thrombin, heme, circulating antibodies, chemokines/cytokines, and complement components (Anderson 2002). Additionally, constituents of the cellular immune response are rapidly recruited to the site of damage, particularly polymorphonuclear neutrophils (PMNs) followed by macrophages/ microglia (Popovich and Jones 2003) (Jones et al. 2005). Glutamate is also acutely released from CNS cells directly damaged by the initial trauma, contributing to a wave of excitotoxic, ischemic, and oxidative secondary death of both neurons and oligodendrocytes (Kigerl et al. 2006; Belegu et al. 2007). In the sub-acute period, within days to weeks post-SCI, an astroglial scar forms. This scar may protect the CNS by repairing the disrupted BBB (Faulkner et al. 2004; Sofroniew 2005), but also includes growth-inhibitory molecules such as chondroitin-sulfate proteoglycans (Davies et al. 1997; Fitch et al. 1999; Silver and Miller 2004). The physical and molecular properties of the glial scar contribute to an environment that inhibits axonal regeneration (Fawcett and Asher 1999; Chen et al. 2002; Fawcett 2006). Further, over a period of several weeks, delayed apoptosis of white matter oligodendrocytes extends rostral and caudal to the injury epicenter, contributing to axonal demyelination (Crowe et al. 1997). Myelin disruption and degradation further confound the inhibitory environment of the glial scar by contributing myelin-associated inhibitors of regeneration

to the microenvironment (Chaudhry and Filbin 2007; Gonzenbach and Schwab 2008). The role of the complement response to the multiphasic pathogenesis of SCI is likely to be complex. Based on the traditional roles of complement, several discrete functions would be predicted in the acute to sub-acute phase postSCI. These include recognition and clearance of invading pathogens via binding bacterial cell wall proteins by the classical, alternative, and lectin pathways, accompanied by activation of the terminal pathway and initiation of bacterial cell lysis. In addition, the presence of C5b-9 on both neurons and oligodendrocytes following SCI suggests that activation of the terminal pathway could contribute to direct lysis of damaged/injured CNS cells (Anderson et al. 2004, 2005). In this regard, antibody-mediated terminal pathway activation is associated with both demyelination and axonal injury (Mead et al. 2002). Conversely, C5b-9 deposition has been suggested to affect diverse aspects of cellular function, including protection of oligodendrocytes from apoptotic death (Cudrici et al. 2006a,b). Furthermore, clearance of cellular and myelin debris via recognition and opsonization of cellular constituents such as phosphatidylserine, DNA, and myelin epitopes has long been suggested as a principal role for the complement response to CNS injury (Tornqvist et al. 1996; Mead et al. 2002; Rus et al. 2005, 2006b). Accordingly, complement has been shown to mediate myelin phagocytosis by macrophages (Bru¨ck and Friede 1990), and contribute to myelin breakdown during Wallerian degeneration in peripheral nerve (Dailey et al. 1998; Liu et al. 1999; Ramaglia et al. 2007). Taken together with the inhibitory role of myelin-associated inhibitors in spontaneous regeneration and repair after SCI, it is possible that the contribution of complement to myelin debris clearance post-SCI could alter the permissiveness of the injured spinal cord for repair/regeneration. However, excessive early activation of complement could also contribute to oligodendrocyte and myelin loss, exacerbating pathology. There have been few studies in animal models or human SCI patients assessing variables such as inflammation, cell death, scar or lesion remodeling, or other events in chronic conditions. The few studies that have been completed suggest that there may be a chronic contribution of inflammatory, and possibly other processes, to continued remodeling of the injured cord as a part of ongoing



degenerative events, regenerative events, or both (Nguyen et al. 2002; Fleming et al. 2006). One significant negative issue that emerges in the chronic period post-SCI is central pain syndromes, which are a serious clinical issue in SCI. Chronic central pain syndromes have a significant inflammatory component; in this regard, recent studies have suggested that complement activation may play a role in the evolution of hypersensitivity in spinal cord models of pain (allodynia) (Twining et al. 2005; Griffin et al. 2007). Investigation of the dynamics of the host microenvironment after SCI may improve identification of successful therapeutic targets for SCI, as well as the optimal clinical timing for their application based on cell type. Evidence for complement deposition and activation after SCI Some evidence suggesting systemic complement activation after SCI has been reported in the human clinical population (Rebhun and Botvin 1980); however, complement activation in animal models of SCI or other types of traumatic CNS injury has not been broadly studied. Accordingly, we investigated complement deposition after contusion injury to the rat spinal cord. Classical (C1q and C4), alternative (factor B), and terminal (C5b-9) complement pathways were strongly activated within 1 day of SCI following either mild or severe SCI (Anderson et al. 2004, 2005). Complement protein immunoreactivity rostral and caudal to the lesion epicenter was predominantly found in cell types vulnerable to degeneration, neurons and oligodendrocytes, and was not generally observed in inflammatory or astroglial cells in spinal cord segments isolated from regions > 5 mm from the SCI epicenter. Surprisingly, immunoreactivity for complement proteins was also evident 6 weeks after injury, and complement activation was observed at least 20 mm rostral to the site of injury. Axonal staining by C1q and factor B was also observed, suggesting a potential role for the complement cascade in demyelination or axonal degeneration, consistent with previously described myelin binding by C1q and a role for the C3b receptor in myelin phagocytosis. Complement and the cellular immune response to SCI More recently, we have reported that rat neutrophils (polymorphonuclear leukocytes, PMNs), the first infiltrating immune cells observed after traumatic SCI, may promote injury by releasing toxic soluble factors that may affect neuronal survival (Nguyen et al. 2007). We found that polymorphonuclear leukocytes (PMN)-driven neurotoxicity was dependent on matrix metalloproteinases (MMP), reactive oxygen species (ROS), and TNF-a, and suggested that these factors interact with one another to mediate PMNdriven neurotoxicity. Critically, while blood–brain/spinal barrier breakdown after traumatic injury may provide initial access of circulating liver-derived complement proteins to the spinal cord, infiltrating immune cells may represent an important local source of complement after injury as well.

Thus, PMNs could hypothetically affect the pathogenesis of SCI through mechanisms associated with complement activation/deposition as well. However, there is little data in the literature regarding the ability of PMNs to synthesize complement in vitro, and essentially no data regarding the presence of complement proteins in association with PMNs in vivo. Accordingly, we recently investigated early and terminal complement component expression by rat peritoneal PMNs in vitro and the association of complement proteins with PMNs in vivo at the epicenter of a traumatic SCI (Nguyen et al. 2008). Stimulated [phorbol-12-myristate-13acetate (PMA), TNFa, interferon-c, or TNF+ interferon] or unstimulated cultured peritoneal PMNs expressed mRNAs encoding for C1q, C3, and C4, but not C5, C6, C7, or C9, by rtPCR, an observation that was confirmed by immunofluorescence. Quantitative flow cytometry demonstrated that < 30% of cultured peritoneal PMNs exhibited C1q and C3 proteins, respectively, in vitro. In contrast, over 70% of infiltrating PMNs isolated from the injured rat spinal cord by fluorescence-activated cell sorting (FACS) stained for C1q, C3, C7, and C5b-9 3 days following SCI. Confocal analysis of double immunofluorescence for infiltrating PMNs and complement antibodies in tissue sections from SCI animals suggested that some labeling reflects internalization of complement-tagged debris from the primary injury in this case. Given the extended time course of PMN infiltration in the injured spinal cord (weeks) after SCI, this inflammatory cell population could play a role in the chronic aspects of the complement response and chronic complement activation after SCI. Complement and experimental models of SCI Although there are excellent animal research models of traumatic SCI, profound variations in terminal pathway complement sufficiency have been reported between rats and mice (Ong and Mattes 1989; Ong et al. 1992), with rats exhibiting lytic complement activity comparable to humans, and mice exhibiting little or no lytic activity in response to challenge. While the reasons for variations in terminal complement sufficiency in mice are not clear, a recent study did demonstrate that at least common laboratory mouse strains retain C3 sufficiency (Osmers et al. 2006). However, strain and species differences in the ability to generate hemolytic activity could dramatically affect the validity of testing mechanistic hypotheses and potential therapies in traumatic CNS injury models, particularly models using transgenic mice. Accordingly, we investigated the relationship of mouse strain and gender to complement activity (Galvan and Anderson 2008). For this study, we selected BUB mice (BUB/Bnj-Pdebbrd1 homozygous for Cdh23ahl and Pde6brd1 and carrying a specific T cell receptor V beta mutation), previously suggested to exhibit relative sufficiency in lytic complement activity (Ong and Mattes 1989; Ong et al. 1992), C57BL6 mice, previously suggested to exhibit sufficiency in



C3 levels (Osmers et al. 2006), and severe combined immunodeficient mice, previously reported to have a C5 deficiency. As expected, uninjured control BUB mice exhibited the highest total hemolytic complement (CH50) activity and severe combined immunodeficient mice the lowest: males exhibited greater total complement activity than females. Most critically for in vivo mouse models of SCI, only male BUB mice exhibited increased complement consumption, indicating evidence of hemolytic activation, 24 h post-SCI (Galvan and Anderson, submitted). While the mixture of reagents used to assess CH50 hemolysis could contribute some degree of variation, it is unlikely that this can account for the differences across strain and gender observed. In addition to these observations, we identified specific strain and gender differences in C3 and C5 sufficiency. In particular, while BUB and C57BL/6 mice were similar in C3 sufficiency and the percentage of CH50 activation, BUB mice exhibited greater C5 sufficiency and overall magnitude of CH50 activation 24 h post-SCI. Of particular interest for SCI models, which generally employ female animals only because of bladder complications in males post-injury, male mice of all strains exhibited greater C5 and total CH50 hemolytic sufficiency, and female mice exhibited reduced consumption of C3 post-SCI. These data appear to highlight differences in C5 as a critical point of divergence, which could affect C5a-mediated cell recruitment in addition to lytic cell death. Interestingly, there is an emerging role for C5a signaling in neuroprotective mechanisms in the CNS (Mukherjee et al. 2008). In this regard, we have tested the effect of delayed (beginning after 14 dpi) inhibition of C5a after SCI, and found that the timing of inhibition can exacerbate damage and impair recovery of function (Beck et al. 2008), suggesting that strain specific deficiency in C5 could have significant and unexpected implications for results obtained from transgenic mice with variations in complement sufficiency. Testing the role of complement in SCI using hemolytic complement-sufficient knockout models Finally, a pivotal question is how animal models constitutively deficient in complement components or in which complement has been experimentally knocked out, respond to SCI. We have tested this question by backcrossing C1q)/), C3)/), and C6)/) mice onto the BUB background to congenicity, and by investigating piebald-viral Glaxo (PVG) rats, constitutively deficient in C6. In these longerterm survival experiments, both BUB C1q)/) mice and C6-deficient rats exhibited improvements in recovery of locomotor function after contusion-induced traumatic SCI as well as attenuation of histological parameters of damage (Galvan and Anderson, submitted). Experiments on BUB C3)/) mice are in progress, however, a recent study using comparing C3)/) to wildtype mice on a C57Bl/6 background described improvements in recovery after SCI

following weight drop injury in the C3)/) mice (Qiao et al. 2006). Comparison of these multiple animal models may yield additional insights into the relative role of the early versus late pathways of complement activation in mediating SCI pathophysiology, and perhaps, in mediating pro-regenerative events, after SCI. In parallel with genetic models of complement manipulation, there is emerging data using alternative approaches to manipulate complement activation and immunoregulation after SCI, including administration of a vacinia virus complement control protein, which has been shown to reduce inflammation and potentially improve recovery after SCI in rats (Reynolds et al. 2004). Further investigation of multiple models and outcomes will be essential in defining what appears likely to be a multifaceted contribution of this component of the immune response to long term deficits in, and recovery of, function after CNS injury. Multiple sclerosis Multiple sclerosis (MS) is a complex, T-cell-mediated autoimmune disease that affects over 400 000 individuals in the USA alone and several million individuals worldwide. A continually growing list of cellular and soluble immune mediators has been shown to contribute to the pathogenesis of MS or its animal model, experimental autoimmune encephalomyelitis (EAE) (Hemmer et al. 2003; Owens 2003; Sospedra and Martin 2005; McFarland and Martin 2007). Of the soluble immune mediators, the complement system has been implicated as an important contributor to the killing of oligodendrocytes and neurons (Shin and Koski 1992; Storch et al. 1998; Rus et al. 2005). If complement contributes to the pathogenesis of MS, a first question might be: how is complement activated in this disease setting? The classical pathway of complement is activated by antibody and, most individuals with MS have antibodies in CSF, (so-called oligoclonal bands) with both known and unknown specificities [reviewed in (Antel and Bar-Or 2006; Klawiter and Cross 2007)]. It is not known how critically important oligoclonalderived antibodies are to activation of complement in MS in general, or to recently described subtypes of MS (Lucchinetti et al. 2000). In EAE, at least in the C57BL/6 genetic background, antibodies are not important for the development and progression of disease and neither is the classical pathway (Hjelmstrom et al. 1998; Boos et al. 2005). This does not rule out a significant contribution for the classical pathway to the pathogenesis of MS, but suggests that it may not be essential either. Unfortunately, there are insufficient data from complement-deficient individuals with MS to assign relative importance of any of the complement activation pathways to disease development and/or progression. Although antibodies may play an undefined role in MS with respect to complement, it has been known for some time that sub-cellular components and proteins from the CNS activate complement. Myelin and myelin basic protein



activate both the classical and alternative pathways and, the peripheral nerve myelin protein P0 activates the alternative pathway. In addition, heterologous serum induces demyelination both in vivo and in vitro [reviewed in (Shin and Koski 1992; Barnum 2001)]. Care must be taken in the interpretation of many of these earlier studies since heat-inactivation of serum was frequently used to inhibit complement activation and complete inactivation of complement was not determined. The strongest evidence of the ability of myelin and myelin-derived components to activate complement through the classical pathway comes from in vitro studies. Myelin, myelin basic protein-dimers or the myelinderived proteins W1 and W2 activate the classical pathway in a Ca2+-dependent fashion. In most of these studies, complement activation occurred via the classical pathway in the presence of antibody, however myelin can activate the classical pathway in an antibody-independent fashion as well [reviewed in (Shin and Koski 1992; Barnum 2001)]. Complement activation by myelin components often leads to the formation of the MAC. As will be discussed below, the MAC is frequently found in MS lesions and CSF of MS patients, but it has no apparent value in either initial diagnosis of MS or changes in disease status (Morgan et al. 1984; Rodriguez et al. 1990). Complement as a biomarker in MS Recently a number of studies have indicated that complement may play a predominant role in the pathogenesis of the socalled type II disease pattern of MS (Lucchinetti et al. 2000; Barnett and Prineas 2004; Wingerchuk and Lucchinetti 2007). In this disease classification system, four different patterns of demyelinating lesions were identified from a small cohort of biopsies and autopsies. In the type II disease pattern, complement (based on neoC9 immunostaining) and antibody, along with T cells, are implicated as the pathological mediators of tissue destruction (Lucchinetti et al. 2000). Interestingly, the contribution of complement to demyelination has also been suggested to be location dependent in the CNS (Brink et al. 2005). In this study, deposition of complement activation products was readily detected by immunohistochemical methods in white matter lesions; however, little complement was detected in gray matter lesions or purely cortical lesions. These studies are intriguing and suggest that complement might be a useful biomarker for MS, at least in a postmortem setting. The utility of complement as a biomarker in MS is however, limited to what we understand regarding complement from in vitro studies under static conditions. In reality the true functions and impact of complement in vivo in many diseases, including MS, remain poorly understood. In addition, the inflammatory environment that provokes complement activation in MS no doubt varies among different lesions in the same patient and with respect to disease fluctuation. Therefore, depending on when and where during

the course of MS complement deposition is examined, the outcome will likely differ. Although immunohistochemical analysis is the method of choice for determining complement activation and deposition in MS, it is reasonable to suggest that deposited complement fragments are likely degraded continuously in the lesion, particularly in the active lesion. As a result, the inability to detect a given complement protein epitope might be due to proteolytic degradation of the epitope rather than complete absence of the protein. These in vivo possibilities highlight the need for better insight into the relationship between complement and the immunological life cycle of the MS lesion. Without this understanding, it is not possible to determine the role(s) of complement in any of the recently described subtypes of MS (Lucchinetti et al. 2000) or in any of the classic characterizations of MS such as relapsing-remitting, primary progressive and others. Complement in experimental autoimmune encephalomyelitis: insight for MS? Experimental autoimmune encephalomyelitis is a heavily used animal model for MS that has proven to be a valuable tool in the development of multiple therapies for MS (Ransohoff 2006; Steinman and Zamvil 2006). As a model system, EAE has proven effective in demonstrating where complement proteins and pathways contribute to the pathogenesis of this demyelinating disease and how it might be targeted therapeutically. Perhaps, the most surprising finding with regard to complement and EAE is that the terminal complement pathway leading to the formation of the MAC is not critical to the development and progression of disease [reviewed in (Barnum and Szalai 2006; Stahel and Barnum 2006)]. In studies by Weerth et al. (2003) and those from our own laboratory (R. Reiman, A. Szalai and S. R. Barnum, unpublished data) it has been shown that EAE is no different in onset or severity compared to control mice in the absence of C5. This does not preclude a role for MAC-mediated pathology in MS, but suggests that the functions of other complement proteins make greater contributions in demyelinating disease. It follows then, that if C5 deficiency does not affect EAE, deletion of the receptor for C5a would also not alter the course of EAE. This is precisely what has been observed and in fact, expression of C5a in the CNS under the control of an astrocyte-specific promoter also did not affect EAE (Reiman et al. 2002, 2005). Nevertheless, there is a wealth of data indicating that inhibition of complement activation at early stages of the pathways, particularly at the level of the C3 convertases, is effective in blocking or attenuating the course of EAE. Natural complement inhibitors including soluble complement receptor type or Crry, which disrupt C3 convertase activity, provide significant protection from disease, suggesting that C3-derived activation fragments make major contributions to disease pathology [reviewed in (Barnum and Szalai 2006; Stahel and Barnum 2006)].



CR4, respectively), two complement receptors that bind iC3b, present with remarkably attenuated EAE. Surprisingly, expression of these complement receptors on T cells plays an important role in the disease process as determined by transferred EAE studies (Bullard et al. 2005, 2007). The outcome of various complement mutant mice in EAE studies is shown in Fig. 4.

Progress in the development of therapeutics targeting the complement system

Fig. 4 Schematic of the two main complement activation pathways showing the outcome of experimental autoimmune encephalomyelitis (EAE) studies using complement mutant mice. Noted in the gray boxes are the specific complement deficient or transgenic mice used in EAE studies and the overall outcome of those studies with respect to disease phenotype. M, EAE no different than wildtype controls, ›, more severe disease than controls, fl, delayed and/or attenuated disease compared with controls. GFAP, glial fibrillary acidic protein, and astrocyte-specific protein; sCrry, mice producing sCrry in the CNS under the control of a GFAP promoter; MAC, membrane attack complex.

An important first step in determining the role of complement in EAE was to examine the contribution of the classical versus alternative pathways. In vitro studies had always suggested that the classical pathway was of greater importance in demyelinating disease most likely because the assays used serum-containing antibody and because the biochemistry of the alternative pathway, at least in early studies, remained poorly understood [reviewed in (Shin and Koski 1992)]. Unexpectedly, EAE studies using C4-deficient guinea pigs and mice demonstrated that the classical pathway is not crucial to the development or progression of disease (Morariu and Dalmasso 1978; Boos et al. 2005). Deletion of factor B (a key enzyme required for the activation of the alternative pathway) did, however, provide significant protection in the chronic phase of EAE as seen by reduced inflammation, cellular infiltration and demyelination (Nataf et al. 2000). Studies using C3-deficient mice or sCrry transgenic mice demonstrated a level of protection from disease similar to that seen in factor B-deficient mice providing additional support for the pivotal role of the alternative pathway in EAE (Davoust et al. 1999; Nataf et al. 2000). The importance of C3 derived activation fragments in EAE is also supported by studies using C3a receptordeficient mice in which EAE was attenuated in the chronic phase of disease. Furthermore, transgenic mice expressing C3a in the CNS presented with markedly worse EAE and significant mortality (Boos et al. 2004). In addition, mice deficient in CD11b or CD11c (the alpha chains of CR3 and

Currently, there are modulators of the complement system in the therapeutic pipeline and in clinical trials [reviewed by (Ricklin and Lambris 2007)], but only one complementspecific drug has been approved by the FDA, an antibody against C5 (eculizumab; Soliris, Alexion Pharmaceuticals, Cheshire, CT, USA). While this monoclonal antibody, which binds to C5 preventing its cleavage to C5a and C5b, was shown to be effective in a model of collagen-induced arthritis in a mouse model (Wang et al. 1995), it is currently marketed as a therapy for paroxysmal nocturnal hemoglobinuria, a disorder that results from the inability to prevent C5b-9 lysis of red cells, and is in pre-clinical trials for other disorders. Once it is hypothesized that specific complement-mediated events are protective or detrimental, generation of the appropriate specific drug, also may encounter a potential disadvantage for the use in the nervous system disorders because of size and thus problems crossing the BBB. As a result, more promising therapeutic possibilities currently being explored are small cyclic peptide molecules such as Compstatin (inhibitor of C3 cleavage) and C5a receptor antagonists (Ricklin and Lambris 2007). C5aR receptor antagonists may be particularly advantageous in preventing recruitment and activation of reactive glia in AD, neutrophils, and/or macrophages in SCI and lupus. While BBB issues have not yet been addressed, it also remains to be seen if the ‘leakiness’ of the BBB in certain inflammatory diseases may facilitate drug entry. Furthermore, dosing and/or tissue/cell targeting is going to be important to permit any positive effects of inhibitors to be manifest, while allowing systemic protective effects of these components to remain functional to some degree. Alternatively, as the specificity of these targets will likely be an improvement over current therapies, with careful observation infections that may escape other immune redundant protective mechanisms can be detected early and treated. Therapeutic strategies that target regulatory molecules to tissues, cells, or areas of inflammation are also under investigation. These inhibitors are generally large (such as soluble C3 receptors or bi-specific molecules such as CR2Crry which targets areas of complement activation via C3d/ dg binding CR2 and then provides an effective local concentration of an inhibitor of amplification via Crry in animal models) (Katschke et al. 2007; Atkinson et al. 2008)



(see Multiple sclerosis). Preliminary studies from one of our laboratories indicates that the complement inhibitory protein CR2-Crry, a chimeric protein composed of the complement receptor type 2 (which binds C3 activation fragments) and the extracellular portion of the Crry proteins (Atkinson et al. 2005), provides complement-mediated protection of EAE seen to date (X. Hu, S. Tomlinson, S. R. Barnum, unpublished data). The CR2-Crry protein appears to be one of the next generation in complement inhibitory proteins by virtue of its C3 targeting ability. This protein has proven successful in several other model systems including SCI and ischemic stroke (Atkinson et al. 2006; Qiao et al. 2006). The sum of all of these studies, from cobra venom factor (CVF) to CR2-Crry treatment, indicates that inhibiting complement in demyelinating disease would have therapeutic value. Although inhibition of complement in a clinical setting might be considered risky because the patient would be immunocompromised, currently used MS therapeutic reagents are also selectively immunosuppressive and the adverse effects are minimal (Fox and Ransohoff 2004; Hemmer et al. 2005; De Jager and Hafler 2007). In recent experiments in animals models, the use of gene therapy to induce these bispecific fusion proteins (Spitzer et al. 2006) showed encouraging results, and thus may permit inducible tissue specific production of complement modulators undeterred by the BBB. We would argue that complement inhibition in animal models of several neuroinflammatory diseases have shown sufficient success to merit moving towards translational studies. Success in such studies would add complement immunotherapeutics to the limited or non-existent repertoire of treatments for these diseases, a sorely needed option.

Conclusion In summary, in each of the diseases described above, detrimental effects of the activation of complement are documented. However, the use of complement deficient rodent models has provided support for beneficial roles of specific complement components in different disorders. Clearly, a more detailed understanding of the paradoxical roles of complement in infection and inflammation is required and will have significant implications for delineating the underlying mechanisms of, and developing effective treatments for, neurological pathologies. Much is yet to be learned about the interaction of the complement cascade with the different signaling mechanism(s) throughout different stages of injury and inflammation. However, the complexity and variety of functions of the complement components and the clear importance of the achievement of balance of responses to permit neuroprotection (and likely repair) but prevent neurodegeneration dictate not only a critical understanding of the various roles of these components and their activation products, but clearly require a ‘systems’ approach to collating and analyzing the data once generated (Mastellos et al. 2005).

Care must be taken in zooming in on particular complement proteins as potential therapeutic targets, since such a treatment could interfere with the protective role of the complement cascade both in the nervous system and in the periphery in terms of susceptibility to disease and/or autoimmunity. Nevertheless, novel technologies coupled with studies defining the role of complement proteins in the nervous system and utilizing animal models were documented to be appropriate, as well as carefully designed human clinical trials hold promise for the development of more effective treatments limiting the contribution of dysregulated complement activation for a variety of devastating neurologic diseases. The most efficacious treatment for most if not all of the disorders discussed above may very well consist of a combination therapy aimed at both complement components as well as other disease specific targets.

Acknowledgements The work described here performed in the author’s laboratories was supported by NS 35144 (AJT), NS 46032 (SRB), NS 43428 (AJA), and Larry L Hillblom Foundation Fellowship (BS).

References Abbott N. J., Mendonca L. L. and Dolman D. E. (2003) The blood–brain barrier in systemic lupus erythematosus. Lupus 12, 908–915. Afagh A., Cummings B. J., Cribbs D. H., Cotman C. W. and Tenner A. J. (1996) Localization and cell association of C1q in Alzheimer’s disease brain. Exp. Neurol. 138, 22–32. Ahmed F., Brown K. M., Stephan D. A., Morrison J. C., Johnson E. C. and Tomarev S. I. (2004) Microarray analysis of changes in mRNA levels in the rat retina after experimental elevation of intraocular pressure. Invest. Ophthalmol. Vis. Sci. 45, 1247–1258. Alexander J. J. and Quigg R. J. (2007) Systemic lupus erythematosus and the brain: what mice are telling us. Neurochem. Int. 50, 5–11. Alexander J. J., Bao L., Jacob A., Kraus D. M., Holers V. M. and Quigg R. J. (2003) Administration of the soluble complement inhibitor, Crry-Ig, reduces inflammation and aquaporin 4 expression in lupus cerebritis. Biochim. Biophys. Acta 1639, 169–176. Alexander J. J., Zwingmann C. and Quigg R. (2005a) MRL/lpr mice have alterations in brain metabolism as shown with [1H-13C] NMR spectroscopy. Neurochem. Int. 47, 143–151. Alexander J. J., Jacob A., Bao L., Macdonald R. L. and Quigg R. J. (2005b) Complement-dependent apoptosis and inflammatory gene changes in murine lupus cerebritis. J. Immunol. 175, 8312–8319. Alexander J. J., Jacob A., Vezina P., Sekine H., Gilkeson G. S. and Quigg R. J. (2007) Absence of functional alternative complement pathway alleviates lupus cerebritis. Eur. J. Immunol. 37, 1691–1701. Allen N. J. and Barres B. A. (2005) Signaling between glia and neurons: focus on synaptic plasticity. Curr. Opin. Neurobiol. 15, 542–548. Ambrus J. L. Jr, Peters M. G., Fauci A. S. and Brown E. J. (1990) The Ba fragment of complement factor B inhibits human B lymphocyte proliferation. J. Immunol. 144, 1549–1553. Anderson A. J. (2002) Mechanisms and pathways of inflammatory responses in CNS trauma: spinal cord injury. J. Spinal Cord Med. 25, 70–79. Anderson A. J., Robert S., Huang W., Young W. and Cotman C. W. (2004) Activation of complement pathways as a components of the



inflammatory response after contusion-induced spinal cord injury. J. Neurotrauma 21, 1831–1836. Anderson A. J., Robert S., Huang W., Young W. and Cotman C. W. (2005) Upregulation of complement inhibitors in association with vulnerable cells following acute contusion-induced spinal cord injury. J. Neurotrauma 22, 382–397. Antel J. and Bar-Or A. (2006) Roles of immunoglobulins and B cells in multiple sclerosis: from pathogenesis to treatment. J. Neuroimmunol. 180, 3–8. Aringer M., Feierl E., Steiner G., Stummvoll G. H., Hofler E., Steiner C. W., Radda I., Smole J. S. and Graninger W. B. (2002) Increased bioactive TNF in human systemic lupus erythematosus: associations with cell death. Lupus 11, 102–108. Ashe K. H. (2001) Learning and memory in transgenic mice modeling Alzheimer’s disease. Learn. Mem. 8, 301–308. Atkinson C., Song H., Lu B., Qiao F., Burns T. A., Holers V. M., Tsokos G. C. and Tomlinson S. (2005) Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection. J. Clin. Invest. 115, 2444–2453. Atkinson C., Zhu H., Qiao F., Varela J. C., Yu J., Song H., Kindy M. S. and Tomlinson S. (2006) Complement-dependent P-selectin expression and injury following ischemic stroke. J. Immunol. 177, 7266–7274. Atkinson C., Qiao F., Song H., Gilkeson G. S. and Tomlinson S. (2008) Low-dose targeted complement inhibition protects against renal disease and other manifestations of autoimmune disease in MRL/ lpr mice. J. Immunol. 180, 1231–1238. Baca V., Lavalle C., Garcia R., Catalan T., Sauceda J. M., Sanchez G., Martinez I., Ramirez M. L., Marquez L. M. and Rojas J. C. (1999) Favorable response to intravenous methylprednisolone and cyclophosphamide in children with severe neuropsychiatric lupus. J. Rheumatol. 26, 432–439. Barnett M. H. and Prineas J. W. (2004) Relapsing and remitting multiple sclerosis: pathology of the newly forming lesion. Ann. Neurol. 55, 458–468. Barnum S. R. (2001) The complement system in demyelinating disease: new insights from transgenic and complement-deficient mice, in Inflammatory Events in Neurodegeneration (Bondy S. C. and Campbell A., eds), pp. 139–156. Prominent Press, Scottsdale. Barnum S. R. and Szalai A. J. (2006) Complement and demyelinating disease: no MAC needed? Brain Res. Rev. 52, 58–68. Beck K. D., Nguyen H., Woodruff T. and Anderson A. J. (2008) Chronic complement-mediated inflammation promotes recovery after spinal cord injury. J. Neurochem. 104(S1), 105. Belegu V., Oudega M., Gary D. S. and McDonald J. W. (2007) Restoring function after spinal cord injury: promoting spontaneous regeneration with stem cells and activity-based therapies. Neurosurg. Clin. N. Am. 18, 143–168. Belmont H. M., Hopkins P., Edelson H. S., Kaplan H. B., Ludewig R., Weissmann G. and Abramson S. (1986) Complement activation during systemic lupus erythematosus. C3a and C5a anaphylatoxins circulate during exacerbations of disease. Arthritis Rheum. 29, 1085–1089. Belmont H. M., Abramson S. B. and Lie J. T. (1996) Pathology and pathogenesis of vascular injury in systemic lupus erythematosus. Interactions of inflammatory cells and activated endothelium. Arthritis Rheum. 39, 9–22. Bensa J. C., Reboul A. and Colomb M. G. (1983) Biosynthesis in vitro of complement subcomponents C1q, C1s and C1 inhibitor by resting and stimulated human monocytes. Biochem. J. 216, 385– 392. Bergamaschini L., Rossi E., Storini C., Pizzimenti S., Distaso M., Perego C., De Luigi A., Vergani C. and De Simoni M. G. (2004) Peripheral treatment with enoxaparin, a low molecular weight heparin, reduces

plaques and beta-amyloid accumulation in a mouse model of Alzheimer’s disease. J. Neurosci. 24, 4181–4186. Blalock E. M., Geddes J. W., Chen K. C., Porter N. M., Markesbery W. R. and Landfield P. W. (2004) Incipient Alzheimer’s disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc. Natl Acad. Sci. USA 101, 2173–2178. Bohlson S. S., Fraser D. A. and Tenner A. J. (2007) Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-term protective immune functions. Mol. Immunol. 44, 33–43. Boos L., Campbell I. L., Ames R., Wetsel R. A. and Barnum S. R. (2004) Deletion of the complement anaphylatoxin C3a receptor attenuates, whereas ectopic expression of C3a in the brain exacerbates, experimental autoimmune encephalomyelitis. J. Immunol. 173, 4708–4714. Boos L. A., Szalai A. J. and Barnum S. R. (2005) Murine complement C4 is not required for experimental autoimmune encephalomyelitis. Glia 49, 158–160. Botto M., Dell’Agnola C., Bygrave A. E., Thompson E. M., Cook H. T., Petry F., Loos M., Pandolfi P. P. and Walport M. J. (1998) Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat. Genet. 19, 56–59. Bradt B. M., Kolb W. P. and Cooper N. R. (1998) Complementdependent proinflammatory properties of the Alzheimer’s disease beta-peptide. J. Exp. Med. 188, 431–438. Brey R. L., Amato A. A., Kagan-Hallet K., Rhine C. B. and Stallworth C. L. (1997) Anti-intercellular adhesion molecule-1 (ICAM-1) antibody treatment prevents central and peripheral nervous system disease in autoimmune-prone mice. Lupus 6, 645–651. Brey R. L., Holliday S. L., Saklad A. R. et al. (2002) Neuropsychiatric syndromes in lupus: prevalence using standardized definitions. Neurology 58, 1214–1220. Brink B. P., Veerhuis R., Breij E. C., van der Valk P., Dijkstra C. D. and Bo L. (2005) The pathology of multiple sclerosis is locationdependent: no significant complement activation is detected in purely cortical lesions. J. Neuropathol. Exp. Neurol. 64, 147–155. Brooks W. M., Sabet A., Sibbitt W. L. Jr, Barker P. B., van Zijl P. C., Duyn J. H. and Moonen C. T. (1997) Neurochemistry of brain lesions determined by spectroscopic imaging in systemic lupus erythematosus. J. Rheumatol. 24, 2323–2329. Bru¨ck W. and Friede R. L. (1990) Anti-macrophage CR3 antibody blocks myelin phagocytosis by macrophages in vitro. Acta Neuropathol. 80, 415–418. Bullard D. C., Hu X., Schoeb T. R., Axtell R. C., Raman C. and Barnum S. R. (2005) Critical requirement of CD11b (Mac-1) on T cells and accessory cells for development of experimental autoimmune encephalomyelitis. J. Immunol. 175, 6327–6333. Bullard D. C., Hu X., Adams J. E., Schoeb T. R. and Barnum S. R. (2007) p150,95 (CD11c/CD18) expression is required for the development of experimental autoimmune encephalomyelitis. Am. J. Pathol. 170, 2001–2008. Buyon J. P., Tamerius J., Ordorica S., Young B. and Abramson S. B. (1992) Activation of the alternative complement pathway accompanies disease flares in systemic lupus erythematosus during pregnancy. Arthritis Rheum. 35, 55–61. Cahoy J. D., Emery B., Kaushal A. et al. (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278. Chaudhry N. and Filbin M. T. (2007) Myelin-associated inhibitory signaling and strategies to overcome inhibition. J. Cereb. Blood Flow Metab. 27, 1096–1107. Chen Z. J., Ughrin Y. and Levine J. M. (2002) Inhibition of axon growth by oligodendrocyte precursor cells. Mol. Cell. Neurosci. 20, 125–139.



Chinn R. J., Wilkinson I. D., Hall-Craggs M. A., Paley M. N., Shortall E., Carter S., Kendall B. E., Isenberg D. A., Newman S. P. and Harrison M. J. (1997) Magnetic resonance imaging of the brain and cerebral proton spectroscopy in patients with systemic lupus erythematosus. Arthritis Rheum. 40, 36–46. Crowe M. J., Bresnahan J. C., Shuman S. L., Masters J. N. and Beattie M. S. (1997) Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat. Med. 3, 73–76. Cudrici C., Niculescu T., Niculescu F., Shin M. L. and Rus H. (2006a) Oligodendrocyte cell death in pathogenesis of multiple sclerosis: Protection of oligodendrocytes from apoptosis by complement. J. Rehabil. Res. Dev. 43, 123–132. Cudrici C., Niculescu F., Jensen T., Zafranskaia E., Fosbrink M., Rus V., Shin M. L. and Rus H. (2006b) C5b-9 terminal complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and up-regulating FLIP. J. Immunol. 176, 3173–3180. Cummings B. J., Pike C. J., Shankle R. and Cotman C. W. (1996) Betaamyloid deposition and other measures of neuropathology predict cognitive status in Alzheimer’s disease. Neurobiol. Aging 17, 921– 933. Dailey A. T., Avellino A. M., Benthem L., Silver J. and Kliot M. (1998) Complement depletion reduces macrophage infiltration and activation during Wallerian degeneration and axonal regeneration. J. Neurosci. 18, 6713–6722. Dalmau I., Finsen B., Zimmer J., Gonzalez B. and Castellano B. (1998) Development of microglia in the postnatal rat hippocampus. Hippocampus 8, 458–474. Danchenko N., Satia J. A. and Anthony M. S. (2006) Epidemiology of systemic lupus erythematosus: a comparison of worldwide disease burden. Lupus 15, 308–318. Davies S. J., Fitch M. T., Memberg S. P., Hall A. K., Raisman G. and Silver J. (1997) Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390, 680–683. Davoust N., Nataf S., Reiman R., Holers M. V., Campbell I. L. and Barnum S. R. (1999) Central nervous system-targeted expression of the complement inhibitor sCrry prevents experimental allergic encephalomyelitis. J. Immunol. 163, 6551–6556. De Jager P. L. and Hafler D. A. (2007) New therapeutic approaches for multiple sclerosis. Annu. Rev. Med. 58, 417–432. Donnelly D. J. and Popovich P. G. (2007) Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp. Neurol. 209, 378–388. Echeverria V. and Cuello A. C. (2002) Intracellular A-beta amyloid, a sign for worse things to come? Mol. Neurobiol. 26, 299–316. Eikelenboom P. and Stam F. C. (1984) An immunohistochemical study on cerebral vascular and senile plaque amyloid in Alzheimer’s dementia. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 47, 17–25. Eng L. F. and Ghirnikar R. S. (1994) GFAP and astrogliosis. Brain Pathol. 4, 229–237. Estes D. and Christian C. L. (1971) The natural history of systemic lupus erythematosus by prospective analysis. Medicine (Baltimore) 50, 85–95. Faulkner J. R., Herrmann J. E., Woo M. J., Tansey K. E., Doan N. B. and Sofroniew M. V. (2004) Reactive astrocytes protect tissue and preserve function after spinal cord injury. J. Neurosci. 24, 2143–2155. Fawcett J. W. (2006) Overcoming inhibition in the damaged spinal cord. J. Neurotrauma 23, 371–383. Fawcett J. W. and Asher R. A. (1999) The glial scar and central nervous system repair. Brain Res. Bull. 49, 377–391. Fiske B. K. and Brunjes P. C. (2000) Microglial activation in the developing rat olfactory bulb. Neuroscience 96, 807–815. Fitch M. T., Doller C., Combs C. K., Landreth G. E. and Silver J. (1999) Cellular and molecular mechanisms of glial scarring and progressive

cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma. J. Neurosci. 19, 8182–8198. Fleming J. C., Norenberg M. D., Ramsay D. A., Dekaban G. A., Marcillo A. E., Saenz A. D., Pasquale-Styles M., Dietrich W. D. and Weaver L. C. (2006) The cellular inflammatory response in human spinal cords after injury. Brain 129, 3249–3269. Fonseca M. I., Zhou J., Botto M. and Tenner A. J. (2004) Absence of C1q leads to less neuropathology in transgenic mouse models of Alzheimer’s disease. J. Neurosci. 24, 6457–6465. Fox R. J. and Ransohoff R. M. (2004) New directions in MS therapeutics: vehicles of hope. Trends Immunol. 25, 632–636. Fraser D. A., Bohlson S. S., Jasinskiene N., Rawal N., Palmarini G., Ruiz S., Rochford R. and Tenner A. J. (2006) C1q and MBL, components of the innate immune system, influence monocyte cytokine expression. J. Leukoc. Biol. 80, 107–116. Fujio Y. and Walsh K. (1999) Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchoragedependent manner. J. Biol. Chem. 274, 16349–16354. Galvan M. and Anderson A. J. (2008) Deficiency in complement C1q improves histological and functional locomotor outcome after SCI. J. Neurosci. (in press). Gasque P., Fontaine M. and Morgan B. P. (1995) Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines. J. Immunol. 154, 4726–4733. Gasque P., Dean Y. D., McGreal E. P., VanBeek J. and Morgan B. P. (2000) Complement components of the innate immune system in health and disease in the CNS. Immunopharmacology 49, 171– 186. Gonzenbach R. R. and Schwab M. E. (2008) Disinhibition of neurite growth to repair the injured adult CNS: focusing on Nogo. Cell. Mol. Life Sci. 65, 161–176. Griffin R. S., Costigan M., Brenner G. J., Ma C. H., Scholz J., Moss A., Allchorne A. J., Stahl G. L. and Woolf C. J. (2007) Complement induction in spinal cord microglia results in anaphylatoxin C5amediated pain hypersensitivity. J. Neurosci. 27, 8699–8708. Handa R., Sahota P., Kumar M., Jagannathan N. R., Bal C. S., Gulati M., Tripathi B. M. and Wali J. P. (2003) In vivo proton magnetic resonance spectroscopy (MRS) and single photon emission computerized tomography (SPECT) in systemic lupus erythematosus (SLE). Magn. Reson. Imaging 21, 1033–1037. Harden L. M., Plessis I. D., Poole S. and Laburn H. P. (2008) Interleukin (IL)-6 and IL-1beta act synergistically within the brain to induce sickness behavior and fever in rats. Brain Behav. Immun.. 22, 838– 849. Hemmer B., Kieseier B., Cepok S. and Hartung H. P. (2003) New immunopathologic insights into multiple sclerosis. Curr. Neurol. Neurosci. Rep. 3, 246–255. Hemmer B., Stuve O., Kieseier B., Schellekens H. and Hartung H. P. (2005) Immune response to immunotherapy: the role of neutralising antibodies to interferon beta in the treatment of multiple sclerosis. Lancet Neurol. 4, 403–412. Hjelmstrom P., Juedes A. E., Fjell J. and Ruddle N. H. (1998) B-celldeficient mice develop experimental allergic encephalomyelitis with demyelination after myelin oligodendrocyte glycoprotein sensitization. J. Immunol. 161, 4480–4483. Hooks B. M. and Chen C. (2006) Distinct roles for spontaneous and visual activity in remodeling of the retinogeniculate synapse. Neuron 52, 281–291. Hopkins P., Belmont H. M., Buyon J., Philips M., Weissmann G. and Abramson S. B. (1988) Increased levels of plasma anaphylatoxins in systemic lupus erythematosus predict flares of the disease and may elicit vascular injury in lupus cerebritis. Arthritis Rheum. 31, 632–641.



van Horssen J., Bo L., Vos C. M., Virtanen I. and de Vries H. E. (2005) Basement membrane proteins in multiple sclerosis-associated inflammatory cuffs: potential role in influx and transport of leukocytes. J. Neuropathol. Exp. Neurol. 64, 722–729. Howell G. R., Libby R. T., Marchant J. K., Wilson L. A., Cosma I. M., Smith R. S., Anderson M. G. and John S. W. (2007) Absence of glaucoma in DBA/2J mice homozygous for wild-type versions of Gpnmb and Tyrp1. BMC Genet. 8, 45. Hsiao K., Chapman P., Nilsen S., Eckman C., Harigaya Y., Younkin S., Yang F. and Cole G. (1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274, 99–102. Hua J. Y. and Smith S. J. (2004) Neural activity and the dynamics of central nervous system development. Nat. Neurosci. 7, 327–332. Huber-Lang M., Sarma V., Zetoune F. S. et al. (2006) Generation of C5a in the absence of C3: a new complement activation pathway. Nat. Med. 12, 682–687. Husebye E. S., Sthoeger Z. M., Dayan M., Zinger H., Elbirt D., Levite M. and Mozes E. (2005) Autoantibodies to a NR2A peptide of the glutamate/NMDA receptor in sera of patients with systemic lupus erythematosus. Ann. Rheum. Dis. 64, 1210–1213. Jakobs T. C., Libby R. T., Ben Y., John S. W. and Masland R. H. (2005) Retinal ganglion cell degeneration is topological but not cell type specific in DBA/2J mice. J. Cell Biol. 171, 313–325. Jiang H., Burdick D., Glabe C. G., Cotman C. W. and Tenner A. J. (1994) beta-amyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain. J. Immunol. 152, 5050–5059. John S. W., Anderson M. G. and Smith R. S. (1999) Mouse genetics: a tool to help unlock the mechanisms of glaucoma. J. Glaucoma 8, 400–412. Jones T. B., McDaniel E. E. and Popovich P. G. (2005) Inflammatorymediated injury and repair in the traumatically injured spinal cord. Curr. Pharm. Des. 11, 1223–1236. Jongen P. J., Doesburg W. H., Ibrahim-Stappers J. L., Lemmens W. A., Hommes O. R. and Lamers K. J. (2000) Cerebrospinal fluid C3 and C4 indexes in immunological disorders of the central nervous system. Acta Neurol. Scand. 101, 116–121. Katschke K. J. Jr, Helmy K. Y., Steffek M. et al. (2007) A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis. J. Exp. Med. 204, 1319–1325. Katz L. C. and Shatz C. J. (1996) Synaptic activity and the construction of cortical circuits. Science 274, 1133–1138. Kayed R., Head E., Thompson J. L., McIntire T. M., Milton S. C., Cotman C. W. and Glabe C. G. (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489. Kigerl K. A., McGaughy V. M. and Popovich P. G. (2006) Comparative analysis of lesion development and intraspinal inflammation in four strains of mice following spinal contusion injury. J. Comp. Neurol. 494, 578–594. Klawiter E. C. and Cross A. H. (2007) B cells: no longer the dominant arm of multiple sclerosis. Curr. Neurol. Neurosci. Rep. 7, 231– 238. Lambert W. C., Cohen P. J., Klein K. M. and Lambert M. W. (1984) Cellular and molecular mechanisms in wound healing: selected concepts. Clin. Dermatol. 2, 17–23. Libby R. T., Anderson M. G., Pang I. H. et al. (2005) Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration. Vis. Neurosci. 22, 637–648. Liu L., Lioudyno M., Tao R., Eriksson P., Svensson M. and Aldskogius H. (1999) Hereditary absence of complement C5 in adult mice influences Wallerian degeneration, but not retrograde responses,

following injury to peripheral nerve. J. Peripher. Nerv. Syst. 4, 123–133. Loeffler D. A., Camp D. M., Schonberger M. B., Singer D. J. and LeWitt P. A. (2004) Early complement activation increases in the brain in some aged normal subjects. Neurobiol. Aging 25, 1001–1007. Loeffler D. A., Camp D. M. and Bennett D. A. (2008) Plaque complement activation and cognitive loss in Alzheimer’s disease. J. Neuroinflammation 5, 9. Lucchinetti C., Bruck W., Parisi J., Scheithauer B., Rodriguez M. and Lassmann H. (2000) Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann. Neurol. 47, 707–717. Lue L. F., Brachova L., Civin W. H. and Rogers J. (1996) Inflammation, A beta deposition, and neurofibrillary tangle formation as correlates of Alzheimer’s disease neurodegeneration. J. Neuropathol. Exp. Neurol. 55, 1083–1088. Maier M., Peng Y., Jiang L., Seabrook T. J., Carroll M. C. and Lemere C. A. (2008) Complement C3 deficiency leads to accelerated amyloid beta plaque deposition and neurodegeneration and modulation of the microglia/macrophage phenotype in amyloid precursor protein transgenic mice. J. Neurosci. 28, 6333–6341. Maslinska D., Laure-Kamionowska M. and Kaliszek A. (1998) Morphological forms and localization of microglial cells in the developing human cerebellum. Folia Neuropathol. 36, 145– 151. Mastellos D., Andronis C., Persidis A. and Lambris J. D. (2005) Novel biological networks modulated by complement. Clin. Immunol. 115, 225–235. Matsuoka Y., Picciano M., Malester B. et al. (2001) Inflammatory responses to amyloidosis in a transgenic mouse model of Alzheimer’s disease. Am. J. Pathol. 158, 1345–1354. McFarland H. F. and Martin R. (2007) Multiple sclerosis: a complicated picture of autoimmunity. Nat. Immunol. 8, 913–919. Mead R. J., Singhrao S. K., Neal J. W., Lassmann H. and Morgan B. P. (2002) The membrane attack complex of complement causes severe demyelination associated with acute axonal injury. J. Immunol. 168, 458–465. Meyer-Luehmann M., Spires-Jones T. L., Prada C., Garcia-Alloza M., de Calignon A., Rozkalne A., Koenigsknecht-Talboo J., Holtzman D. M., Bacskai B. J. and Hyman B. T. (2008) Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer’s disease. Nature 451, 720–724. Milligan C. E., Cunningham T. J. and Levitt P. (1991) Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain. J. Comp. Neurol. 314, 125–135. Miyahara T., Kikuchi T., Akimoto M., Kurokawa T., Shibuki H. and Yoshimura N. (2003) Gene microarray analysis of experimental glaucomatous retina from cynomologous monkey. Invest. Ophthalmol. Vis. Sci. 44, 4347–4356. Morariu M. A. and Dalmasso A. P. (1978) Experimental allergic encephalomyelitis in cobra venom factor-treated and C4-deficient guinea pigs. Ann. Neurol. 4, 427–430. Morgan B. P., Campbell A. K. and Compston D. A. (1984) Terminal component of complement (C9) in cerebrospinal fluid of patients with multiple sclerosis. Lancet 2, 251–254. Mukherjee P. and Pasinetti G. M. (2001) Complement anaphylatoxin C5a neuroprotects through mitogen-activated protein kinasedependent inhibition of caspase 3. J. Neurochem. 77, 43–49. Mukherjee P., Thomas S. and Pasinetti G. M. (2008) Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo. J. Neuroinflammation 5, 5.



Nataf S., Carroll S. L., Wetsel R. A., Szalai A. J. and Barnum S. R. (2000) Attenuation of experimental autoimmune demyelination in complement-deficient mice. J. Immunol. 165, 5867–5873. Nauta A. J., Daha M. R., Tijsma O., van de Water B., Tedesco F. and Roos A. (2002) The membrane attack complex of complement induces caspase activation and apoptosis. Eur. J. Immunol. 32, 783– 792. Nauta A. J., Roos A. and Daha M. R. (2004) A regulatory role for complement in innate immunity and autoimmunity. Int. Arch. Allergy Immunol. 134, 310–323. Nguyen M. D., Julien J. P. and Rivest S. (2002) Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat. Rev. Neurosci. 3, 216–227. Nguyen H. X., O’Barr T. J. and Anderson A. J. (2007) Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-alpha. J. Neurochem. 102, 900–912. Nguyen H. X., Galvan M. D. and Anderson A. J. (2008) Neutrophils express the early components of the complement system. J. Neuroinflammation 5, 26–38. Ong G. L. and Mattes M. J. (1989) Mouse strains with typical mammalian levels of complement activity. J. Immunol. Methods 125, 147–158. Ong G. L., Baker A. E. and Mattes M. J. (1992) Analysis of high complement levels in Mus hortulanus and BUB mice. J. Immunol. Methods 154, 37–45. Osaka H., Mukherjee P., Aisen P. S. and Pasinetti G. M. (1999) Complement-derived anaphylatoxin C5a protects against glutamate-mediated neurotoxicity. J. Cell. Biochem. 73, 303–311. Osmers I., Szalai A. J., Tenner A. J. and Barnum S. R. (2006) Complement in BuB/BnJ mice revisited: serum C3 levels and complement opsonic activity are not elevated. Mol. Immunol. 43, 1722–1725. Owens T. (2003) The enigma of multiple sclerosis: inflammation and neurodegeneration cause heterogeneous dysfunction and damage. Curr. Opin. Neurol. 16, 259–265. Perry V. H., Hume D. A. and Gordon S. (1985) Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain. Neuroscience 15, 313–326. Peters M. G., Ambrus J. L. Jr, Fauci A. S. and Brown E. J. (1988) The Bb fragment of complement factor B acts as a B cell growth factor. J. Exp. Med. 168, 1225–1235. Pisalyaput K. and Tenner A. J. (2008) Complement component C1q inhibits beta-amyloid- and serum amyloid P-induced neurotoxicity via caspase- and calpain-independent mechanisms. J. Neurochem. 104, 696–707. Popovich P. G. and Jones T. B. (2003) Manipulating neuroinflammatory reactions in the injured spinal cord: back to basics. Trends Pharmacol. Sci. 24, 13–17. Qiao F., Atkinson C., Song H., Pannu R., Singh I. and Tomlinson S. (2006) Complement plays an important role in spinal cord injury and represents a therapeutic target for improving recovery following trauma. Am. J. Pathol. 169, 1039–1047. Rahpeymai Y., Hietala M. A., Wilhelmsson U. et al. (2006) Complement: a novel factor in basal and ischemia-induced neurogenesis. EMBO J. 25, 1364–1374. Ramaglia V., King R. H., Nourallah M. et al. (2007) The membrane attack complex of the complement system is essential for rapid Wallerian degeneration. J. Neurosci. 27, 7663–7672. Ransohoff R. M. (2006) EAE: pitfalls outweigh virtues of screening potential treatments for multiple sclerosis. Trends Immunol. 27, 167–168. Rebhun J. and Botvin J. (1980) Complement elevation in spinal cord injury. Ann. Allergy 44, 287–288.

Reiman R., Gerard C., Campbell I. L. and Barnum S. R. (2002) Disruption of the C5a receptor gene fails to protect against experimental allergic encephalomyelitis. Eur. J. Immunol. 32, 1157–1163. Reiman R., Torres A. C., Martin B. K., Ting J. P., Campbell I. L. and Barnum S. R. (2005) Expression of C5a in the brain does not exacerbate experimental autoimmune encephalomyelitis. Neurosci. Lett. 390, 134–138. Reynolds D. N., Smith S. A., Zhang Y. P., Mengsheng Q., Lahiri D. K., Morassutti D. J., Shields C. B. and Kotwal G. J. (2004) Vaccinia virus complement control protein reduces inflammation and improves spinal cord integrity following spinal cord injury. Ann. NY Acad. Sci. 1035, 165–178. Ricklin D. and Lambris J. D. (2007) Complement-targeted therapeutics. Nat. Biotechnol. 25, 1265–1275. Rodriguez M., Wynn D. R., Kimlinger T. K. and Katzmann J. A. (1990) Terminal component of complement (C9) in the cerebrospinal fluid of patients with multiple sclerosis and neurologic controls. Neurology 40, 855–857. Rogers J., Schultz J., Brachova L., Lue L. F., Webster S., Bradt B., Cooper N. R. and Moss D. E. (1992) Complement activation and beta-amyloid-mediated neurotoxicity in Alzheimer’s disease. Res. Immunol. 143, 624–630. Rozovsky I., Morgan T. E., Willoughby D. A., Dugichi-Djordjevich M. M., Pasinetti G. M., Johnson S. A. and Finch C. E. (1994) Selective expression of clusterin (SGP-2) and complement C1qB and C4 during responses to neurotoxins in vivo and in vitro. Neuroscience 62, 741–758. Rus H., Cudrici C. and Niculescu F. (2005) C5b-9 complement complex in autoimmune demyelination and multiple sclerosis: dual role in neuroinflammation and neuroprotection. Ann. Med. 37, 97–104. Rus H., Cudrici C. and Niculescu F. (2006a) C5b-9 complement complex in autoimmune demyelination: dual role in neuroinflammation and neuroprotection. Adv. Exp. Med. Biol. 586, 139–151. Rus H., Cudrici C., David S. and Niculescu F. (2006b) The complement system in central nervous system diseases. Autoimmunity 39, 395– 402. Sakic B., Szechtman H., Keffer M., Talangbayan H., Stead R. and Denburg J. A. (1992) A behavioral profile of autoimmune lupusprone MRL mice. Brain Behav. Immun. 6, 265–285. Sanes J. R. and Lichtman J. W. (1999) Development of the vertebrate neuromuscular junction. Annu. Rev. Neurosci. 22, 389–442. Sari R. A., Taysi S., Erdem F., Yilmaz O., Keles S., Kiziltunc A., Odabas A. R. and Cetinkaya R. (2002) Correlation of serum levels of soluble intercellular adhesion molecule-1 with disease activity in systemic lupus erythematosus. Rheumatol. Int. 21, 149–152. Segurado O. G., Gomez-Reino J. and Arnaiz-Villena A. (1990) Factor B activation in systemic lupus erythematosus. Arthritis Rheum. 33, 1598–1599. Selkoe D. J. (2002) Alzheimer’s disease is a synaptic failure. Science 298, 789–791. Shen Y. and Meri S. (2003) Yin and Yang: complement activation and regulation in Alzheimer’s disease. Prog. Neurobiol. 70, 463– 472. Shen Y., Lue L., Yang L. et al. (2001) Complement activation by neurofibrillary tangles in Alzheimer’s disease. Neurosci. Lett. 305, 165–168. Shin M. L. and Koski C. L. (1992) The complement system in demyelination, in Myelin: Biology and Chemistry, 1st edn (Martenson R. E., ed.), pp. 801–831. CRC Press, Boca Raton, FL. Silver J. and Miller J. H. (2004) Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5, 146–156. Soane L., Rus H., Niculescu F. and Shin M. L. (1999) Inhibition of oligodendrocyte apoptosis by sublytic C5b-9 is associated with



enhanced synthesis of bcl-2 and mediated by inhibition of caspase3 activation. J. Immunol. 163, 6132–6138. Sofroniew M. V. (2005) Reactive astrocytes in neural repair and protection. Neuroscientist 11, 400–407. Sospedra M. and Martin R. (2005) Immunology of multiple sclerosis. Annu. Rev. Immunol. 23, 683–747. Spitzer D., Wu X., Ma X., Xu L., Ponder K. P. and Atkinson J. P. (2006) Cutting edge: treatment of complement regulatory protein deficiency by retroviral in vivo gene therapy. J. Immunol. 177, 4953– 4956. Stahel P. F. and Barnum S. R. (2006) The role of the complement system in CNS inflammatory diseases. Expert Rev. Clin. Immunol. 2, 445–456. Stasi K., Nagel D., Yang X., Wang R. F., Ren L., Podos S. M., Mittag T. and Danias J. (2006) Complement component 1Q (C1Q) upregulation in retina of murine, primate, and human glaucomatous eyes. Invest. Ophthalmol. Vis. Sci. 47, 1024–1029. Steele M. R., Inman D. M., Calkins D. J., Horner P. J. and Vetter M. L. (2006) Microarray analysis of retinal gene expression in the DBA/ 2J model of glaucoma. Invest. Ophthalmol. Vis. Sci. 47, 977–985. Steinman L. and Zamvil S. S. (2006) How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis. Ann. Neurol. 60, 12–21. Stevens B., Allen N. J., Vazquez L. E. et al. (2007) The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178. Stojanovich L., Stojanovich R., Kostich V. and Dzjolich E. (2003) Neuropsychiatric lupus favourable response to low dose i.v. cyclophosphamide and prednisolone (pilot study). Lupus 12, 3–7. Stoltzner S. E., Grenfell T. J., Mori C., Wisniewski K. E., Wisniewski T. M., Selkoe D. J. and Lemere C. A. (2000) Temporal accrual of complement proteins in amyloid plaques in Down’s syndrome with Alzheimer’s disease. Am. J. Pathol. 156, 489–499. Storch M. K., Piddlesden S., Haltia M., Iivanainen M., Morgan P. and Lassmann H. (1998) Multiple sclerosis: in situ evidence for antibody- and complement- mediated demyelination. Ann. Neurol. 43, 465–471. Strey C. W., Markiewski M., Mastellos D., Tudoran R., Spruce L. A., Greenbaum L. E. and Lambris J. D. (2003) The proinflammatory mediators C3a and C5a are essential for liver regeneration. J. Exp. Med. 198, 913–923. Tanihara H., Hangai M., Sawaguchi S., Abe H., Kageyama M., Nakazawa F., Shirasawa E. and Honda Y. (1997) Up-regulation of glial fibrillary acidic protein in the retina of primate eyes with experimental glaucoma. Arch. Ophthalmol. 115, 752–756. Tanzi R. E. and Bertram L. (2005) Twenty years of the Alzheimer’s disease amyloid hypothesis: a genetic perspective. Cell 120, 545–555. Tenner A. J. and Pisalyaput K. (2008) The complement system in the CNS: thinking again, in Central Nervous System Disease and Inflammation (Lane T. E., Carson M. J., Bergmann C. and WyssCoray T., eds), pp. 153–174. Springer, New York. Tenner A. J. and Webster S. (2001) Complement-mediated injury and inflammation in the pathogenesis of Alzheimer’s disease, in Inflammatory Events in Neurodegeneration (Bondy S. C. and Campbell A. C., eds), pp. 119–138. Prominent Press, Scottsdale. Tornqvist E., Liu L., Aldskogius H., Holst H. V. and Svensson M. (1996) Complement and clusterin in the injured nervous system. Neurobiol. Aging 17, 695–705. Trouw L. A., Blom A. M. and Gasque P. (2008) Role of complement and complement regulators in the removal of apoptotic cells. Mol. Immunol. 45, 1199–1207. Twining C. M., Sloane E. M., Schoeniger D. K., Milligan E. D., Martin D., Marsh H., Maier S. F. and Watkins L. R. (2005) Activation of the spinal cord complement cascade might contribute to mechan-

ical allodynia induced by three animal models of spinal sensitization. J. Pain 6, 174–183. Ullian E. M., Christopherson K. S. and Barres B. A. (2004) Role for glia in synaptogenesis. Glia 47, 209–216. Uwai M., Terui Y., Mishima Y. et al. (2000) A new apoptotic pathway for the complement factor B-derived fragment Bb. J. Cell. Physiol. 185, 280–292. Vyse T. J. and Kotzin B. L. (1996) Genetic basis of systemic lupus erythematosus. Curr. Opin. Immunol. 8, 843–851. Walker D. G. and McGeer P. L. (1992) Complement gene expression in human brain: comparison between normal and Alzheimer disease cases. Brain Res. Mol. Brain Res. 14, 109–116. Walsh D. M., Klyubin I., Fadeeva J. V., Cullen W. K., Anwyl R., Wolfe M. S., Rowan M. J. and Selkoe D. J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535–539. Wang Y., Rollins S. A., Madri J. A. and Matis L. A. (1995) Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease. Proc. Natl Acad. Sci. USA 92, 8955–8959. Watson M. D., Roher A. E., Kim K. S., Spiegel K. and Emmerling M. R. (1997) Complement interactions with amyloid-beta1–42: a nidus for inflammation in AD brains. Amyloid: Int. J. Exp. Clin. Invest. 4, 147–156. Webster S., Lue L. F., Brachova L., Tenner A. J., McGeer P. L., Terai K., Walker D. G., Bradt B., Cooper N. R. and Rogers J. (1997) Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer’s disease. Neurobiol. Aging 18, 415–421. Weerth S. H., Rus H., Shin M. L. and Raine C. S. (2003) Complement C5 in experimental autoimmune encephalomyelitis (EAE) facilitates remyelination and prevents gliosis. Am. J. Pathol. 163, 1069– 1080. Wingerchuk D. M. and Lucchinetti C. F. (2007) Comparative immunopathogenesis of acute disseminated encephalomyelitis, neuromyelitis optica, and multiple sclerosis. Curr. Opin. Neurol. 20, 343–350. Wyss-Coray T., Yan F., Lin A. H., Lambris J. D., Alexander J. J., Quigg R. J. and Masliah E. (2002) Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer’s mice. Proc. Natl Acad. Sci. USA 99, 10837–10842. Yao J., Harvath L., Gilbert D. L. and Colton C. A. (1990) Chemotaxis by a CNS macrophage, the microglia. J. Neurosci. Res. 27, 36–42. Zalcman S., Murray L., Dyck D. G., Greenberg A. H. and Nance D. M. (1998) Interleukin-2 and -6 induce behavioral-activating effects in mice. Brain Res. 811, 111–121. Zanjani H., Finch C. E., Kemper C., Atkinson J., McKeel D., Morris J. C. and Price J. L. (2005) Complement activation in very early Alzheimer disease. Alzheimer Dis. Assoc. Disord. 19, 55– 66. Zhang X., Kimura Y., Fang C., Zhou L., Sfyroera G., Lambris J. D., Wetsel R. A., Miwa T. and Song W. C. (2007) Regulation of Tolllike receptor-mediated inflammatory response by complement in vivo. Blood 110, 228–236. Zhou J., Fonseca M. I., Kayed R., Hernandez I., Webster S. D., Yazan O., Cribbs D. H., Glabe C. G. and Tenner A. J. (2005) Novel Abeta peptide immunogens modulate plaque pathology and inflammation in a murine model of Alzheimer’s disease. J. Neuroinflammation 2, 28. Zhou J., Fonseca M. I., Pisalyaput K. and Tenner A. J. (2008) Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer’s disease. J. Neurochem. 106, 2080– 2092.
