I’d like to add to Dr. Shen’s background text my perspective on the specific aspect of distinguishing between soluble and membrane-tethered TNF. The safety issues around TNF inhibition and the recent black box warning on etanercept can be better understood in the context of the difference between soluble and membrane-tethered TNF, especially since the newest generation of biologics (i.e., the DN-TNFs) are selective for soluble TNF while sparing its membrane-tethered form. My comment first describes how soluble versus membrane-tethered TNF link up with their receptors normally, then summarizes what’s known about soluble versus membrane-tethered TNF in Alzheimer and Parkinson disease, as well as multiple sclerosis where a phase 1 trial failed over this issue. I finally address differences in the prospects of these two forms of TNF as therapeutic targets and, to that end, briefly mention some unpublished data from our lab in AD mouse models.

Physiologic Pairing of TNF Ligands and Receptors: We Need to Distinguish Between Soluble and Membrane-tethered TNF
TNF belongs...  Read more

I’d like to add to Dr. Shen’s background text my perspective on the specific aspect of distinguishing between soluble and membrane-tethered TNF. The safety issues around TNF inhibition and the recent black box warning on etanercept can be better understood in the context of the difference between soluble and membrane-tethered TNF, especially since the newest generation of biologics (i.e., the DN-TNFs) are selective for soluble TNF while sparing its membrane-tethered form. My comment first describes how soluble versus membrane-tethered TNF link up with their receptors normally, then summarizes what’s known about soluble versus membrane-tethered TNF in Alzheimer and Parkinson disease, as well as multiple sclerosis where a phase 1 trial failed over this issue. I finally address differences in the prospects of these two forms of TNF as therapeutic targets and, to that end, briefly mention some unpublished data from our lab in AD mouse models.

Physiologic Pairing of TNF Ligands and Receptors: We Need to Distinguish Between Soluble and Membrane-tethered TNF
TNF belongs to a superfamily of ligands with pivotal roles in immune system function [1, 2]; many of these ligands have been implicated in the etiology of several acquired and genetic diseases [3, 4]. TNF is synthesized as a monomeric 26 kD type-2 transmembrane protein that assembles into trimers and is cleaved by TACE metalloprotease to a soluble circulating form [5, 6]. Both forms of TNF are biologically active and can be synthesized in the brain by microglia, astrocytes and subsets of neurons [7-9]. TNF receptors, commonly referred to as type 1 receptor (R1, p55, Tnfrsf1a) and type 2 receptor (R2, p75, Tnfrsf1b) bind the two forms of TNF with different affinities and are constitutively expressed on neurons and glia in the CNS [10]. R1 is activated equally well by soluble and membrane-tethered TNF; whereas R2 is preferentially activated by membrane-tethered TNF and to a lesser extent by soluble TNF [11, 12]. TNFR1 and R2 have a cysteine-rich extracellular domain with 28 percent shared homology and have completely distinct transmembrane and cytoplasmic domains with no homology between them [13]. The death domain (DD) present in the cytoplasmic tail of R1 recruits TNFR1-associating death domain (TRADD) protein and at least 20 different proteins including FADD, TRAF2 and receptor interacting protein kinase 1 (RIP) to form a cascade leading to activation of sphingomyelinase-ceramide, caspases, NFκB transcription, ASK1/c-Jun kinase (JNK), and p38 MAPK pathways. Depending on cellular context, R1 and R2 can activate many of the same downstream pathways and act synergistically or in opposing fashion [11, 14] but less is known regarding integration of signaling pathways through R2. In the nervous system, the kinetics and upstream mediators for activation of the NFκB pathway by R1 and R2 have been shown to be different and to result in opposing effects on cortical neuron survival [15] and hippocampal neurons [16, 17]. The RIP adaptor protein, which associates with both TNFRs, may be a molecular switch that allows R2 signaling to alternate between anti-apoptotic NFκB activation and death induction through caspase mechanisms [18, 19].

TNF-immunoreactive neurons have been reported in the hypothalamus, in the caudal raphe nuclei, in the bed nucleus of the stria terminalis, and along the ventral pontine and medullary surface. They are also found in areas involved in autonomic and neuroendocrine regulation, such as the hypothalamus, amygdala, parabrachial nucleus, dorsal vagal complex, nucleus ambiguous, and the thoracic sympathetic preganglionic cell column [20]. TNF receptor expression has been detected in brainstem, cortex, cerebellum, thalamus and basal ganglia [21]. TNFR1 is expressed in many cell types, whereas TNFR2 is expressed less broadly and primarily by cells of the immune system including microglia [22]. However, R2 expression has also been reported in cardiac myocytes, endothelial cells [5], dopaminergic [23], cortical [15], and hippocampal [16, 24] neurons. CNS functions which have been ascribed to TNF include activation of microglia and astrocytes [25, 26], regulation of endothelial cell permeability at the blood–brain barrier [27], generation of the febrile response [28], enhancement of slow-wave sleep [29] and synaptic strength [30, 31]. TNF exerts potent actions on glutamatergic synaptic transmission and modulates synaptic plasticity [32]. TNF and its downstream targets appear to regulate hippocampal neuron development as mice doubly deficient in R1 and R2 (double knockout; dko) have decreased arborization of the apical dendrites of the CA1 and CA3 regions and accelerated dentate gyrus development [33]. Membrane-tethered TNF is critical for lymphoid organ development [34] but its role in cellular responses related to brain function is less well understood. It appears to be important for proliferation of oligodendrocytes and nerve remyelination [35] and proliferation of hippocampal neuroblasts after stroke [16].

Inflammatory signals activated by TNF are primarily mediated through soluble TNF binding to R1 [34, 36, 37]; although some have proposed that under ischemic conditions R2 can contribute to inflammatory responses [38]. In cells that co-express R1 and R2, the functional outcome of a TNF stimulus in vivo (neurotoxic versus neuroprotective) may be determined by the R1/R2 expression ratio [4]; if this is true in the brain, it may be possible to design new pharmacological and/or gene therapy-based approaches to preferentially upregulate R2 activity and/or expression to achieve neuroprotection in brain regions where TNF normally exerts neurotoxic effects.

Much of our understanding of TNF and its receptors in the CNS has come from detailed evaluation of the phenotypes of a number of genetic mouse models developed in the last 15 years and their phenotypes after exposure to various toxins or pathogens (reviewed in [39, 40]. Consistent with a role of TNF in modulating synaptic plasticity, hippocampal brain slices from TNFR-deficient mice do not display long-term depression induced by low-frequency stimulation of Schaffer collateral axons [41]. Whole animal studies in which TNF knockouts were compared to normal animals indicated TNF-deficient animals performed better in spatial memory and learning tasks (Morris Water Maze) [33]. Conversely, two mouse lines overexpressing hTNF show significant impairment in spatial learning [42]. One obvious caveat in interpreting these studies is that the “substrate” of learning and memory is not the same in knockout and wild-type mice since TNF deficiency affects hippocampal development. At pathophysiological levels, TNF has been shown to have inhibitory effects via the p38 mitogen activated kinase pathway on hippocampal long-term potentiation (LTP) [43, 44]. Elevated levels of TNF, also through a p38 MAPK-dependent pathway, may further impair LTP through upregulation of RGS7 (a regulator of G-protein signaling) expression [45].

In addition to the reported modulatory effects of TNF on LTP and LTD, work by Malenka and colleagues revealed an important role for glial-derived TNF in modulating homeostatic activity-dependent regulation of synaptic connectivity [31, 45]. This previously unrecognized neuronal function for constitutively released TNF suggests that traditional immune signaling molecules have been adapted to serve different roles in nervous system function. It also implies that changes in the levels of immune molecules in particular regions of the brain during disease may lead to unexpected dysfunction, not only because of their direct neural actions but perhaps also due to disruption of their normal adaptive roles in the brain. Lastly, studies using TNF over-expressing mice demonstrated an indirect role of TNF in influencing survival of basal forebrain cholinergic neurons via direct regulation of the levels of nerve growth factor (NGF) [46, 47], a key survival factor for this and other neuronal populations. Genetic ablation of TNF or TNF receptors in rodent models of PD, which show neurotoxin-induced loss of dopaminergic neurons, has yielded variable results [41, 48-53]. However, because the lack of TNF signaling during development results in arrested dendritic cell development [36] and stunted microglial responsiveness in adult animals [52], it is difficult to implicate TNF directly in neurodegeneration based on these studies.

Alzheimer Disease. Interest in identifying polymorphisms in the TNF or TNF receptor genes linked to AD was largely fueled by the presence of this cytokine at amyloidogenic plaques and by results of genome-wide screening of families affected with late-onset AD. While a few individual studies find associations between polymorphisms in the TNF gene or the TNFR2 receptor gene with late-onset AD in families with no individuals possessing the APOE ε4 allele [54], others find no significant associations of three polymorphisms in the TNFR1 gene to AD [55]. Meta-analyses of genetic association studies will be required to assess overall the genetic effect of genetic susceptibility loci and other cytokine genes on AD risk [56, 57]. Since polymorphisms in cytokine genes have already been linked to peripheral inflammatory disorders, such as juvenile rheumatoid arthritis, myasthenia gravis, and periodontitis, associations between cytokine gene polymorphisms and several chronic degenerative diseases may eventually be demonstrated [58]. Dysregulated levels of TNF and other cytokines have been reported in AD patients and mouse models of AD, raising the possibility that they have disease-modifying effects and could be targeted in therapies. Higher serum TNF and TNF/IL-1β ratio have been detected in patients with severe AD compared to mild-moderate AD [59], whereas other studies have found no significant differences between studied groups [60]. Further investigation is warranted to validate these findings and assess their functional significance.

In mouse models of AD-like pathology, elevated TNF and MCP-1 transcript levels were reported in entorhinal cortex of 3-month old triple transgenic (3 × Tg) AD mice developed in LaFerla’s lab [61] coincident with accumulation of intraneuronal Ab in these brain regions [62]. Since these mice carry three transgenes encoding mutant proteins linked to familial Alzheimer disease (FAD), these findings suggest that a sensitized genetic background may trigger an early chronic neuroinflammatory response that may involve (but not be limited to) TNF-dependent JNK activation leading to increased γ-secretase activity and enhanced progression of AD-like plaque and tau pathology [61]. Indeed, chronic exposure to systemic lipopolysaccharide (LPS) was shown to hasten pathology in these mice [63]. In the Tg2576 mouse model of AD-like amyloid pathology, elevated TNF levels are detectable around amyloid plaques [64, 65] and exposure to systemic LPS worsens their pathology [66]. Shen and colleagues recently published that inactivation of TNF signaling in the APP23 transgenic mouse model of AD significantly decreased amyloid burden [67] and suggested that anti-TNF biologics may prevent or delay AD-like pathology. Our laboratory has tested the role of soluble TNF in contributing to early (pre-plaque) amyloid pathophysiology in 3xTgAD mice [manuscript under review at J Neuroscience]. Briefly, chronic infusion of soluble TNF-selective dominant negative TNF protein (XENP345), or a single injection of a lentivirus encoding DN-TNF, blocked accumulation of amyloid-associated pathology induced by chronic peripheral inflammation. Similarly, genetic inactivation of soluble TNF signaling in 3xTgAD mice prevented the appearance of AD-like pathology. Taken together, these findings strongly suggest that TNF may be an important modulator of early AD-associated pathology via multiple cellular mechanisms, including modulation of microglial phenotypes and regulation of the various proteases that process APP through TNF-dependent molecular signaling pathways.

Parkinson Disease. The levels of several cytokines including TNF are significantly increased in the substantia nigra pars compacta (SNpc) of PD patients compared to normal controls [68], particularly in the area of maximal destruction where the vulnerable melanin-containing dopamine-producing neurons reside. The genes for various cytokines, chemokines and acute phase proteins have been surveyed and individual reports demonstrate that certain single nucleotide polymorphisms in the TNF promoter that drive transcriptional activity are over-represented in a cohort of early onset Parkinson’s disease patients [69]. However, these findings have not been replicated or reported in other populations. Once such studies become available, meta-analyses of multiple such association studies will be needed to assess the overall genetic effect of TNF gene polymorphisms.

In experimental models of PD, significantly elevated levels of TNF mRNA and soluble TNF protein can be detected in the rodent midbrain substantia nigra within hours of in vivo administration of two neurotoxins widely used to model parkinsonism in rodents, 6-hydroxydopamine (6-OHDA) [70] and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [49, 51, 71]. Consistent with a role of soluble TNF in contributing to dopaminergic neuron death in chronic parkinsonism, circulating levels of soluble TNF in plasma were shown to remain elevated in MPTP-treated non-human primates 1 year after administration of the neurotoxin [72]. In addition, mice deficient in TNF or both TNF receptors have been reported to have altered dopamine metabolism and reduced survival of dopaminergic terminals [51] or reduced sensitivity to MPTP-induced neurotoxicity [49, 71]. Additional evidence that inflammation, and possibly TNF, is involved in nigral DA neuron degeneration comes from studies involving two recently developed endotoxin models of PD. In the first model, chronic low dose lipopolysaccharide (LPS) infusion into SNpc of rats results in delayed, selective and progressive loss of nigral DA neurons [73]. In the second model, exposure of pregnant rats to LPS and thus, in utero exposure of embryos to the endotoxin, caused a loss of DA neurons in postnatal brains [74]. Our laboratory demonstrated that neutralization of soluble TNF via chronic infusion of dominant negative TNF (DN-TNF) inhibitor protein into SNpc of adult rats protected nigral DA neurons from LPS and 6-OHDA induced degeneration [75]. In brief, given that TNF receptors are expressed in nigrostriatal dopamine neurons [76, 77] and these neurons are selectively vulnerable to TNF-induced toxicity [23, 78, 79], these early genetic studies and the more recent chronic inflammation models of PD strongly implicate soluble TNF-dependent mechanisms and/or one or more of its downstream targets in neurotoxin- and endotoxin-induced loss of nigral DA neurons. Together, this work suggests that high levels of soluble TNF in the midbrain may increase susceptibility for PD in humans.

Multiple Sclerosis. The role of TNF in the autoimmune dysregulation characteristic of multiple sclerosis has been extensively investigated. TNF and its receptors are upregulated in active MS lesions, and TNF levels in the CSF of MS patients correlate with disease severity [80-83]. Experimental rodent models of MS provided strong evidence that TNF is important in the MS disease process. In particular, TNF blockade was shown to prevent or treat the development of experimental autoimmune encephalomyelitis (EAE) in rodents [84, 85]. A role for TNF in the induction phase of EAE via modulation of leukocyte traffic into the CNS parenchyma [84, 86] and a role for TNFR1 in demyelination were demonstrated [87] using TNF genetic models. However, an important role for membrane-tethered TNF and its preferred receptor TNFR2 in oligodendrocyte precursor proliferation and remyelination was also demonstrated, using TNF genetic models in the cuprizone toxin model of MS [35]. In fact, these data offered a mechanistic explanation for the unfortunate failure of lenercept, an Fc-fused p55/TNFR1, in phase I clinical trials with MS patients whose symptoms worsened between bouts of relapsing-remitting episodes due to the lack of TNF-mediated remyelination [88]. In theory, selective targeting of soluble TNF/TNFR1 signaling would be of therapeutic benefit in these patients given what we now know about the need to spare membrane-tethered TNF-mediated signaling to preserve the myelination process.

TNF as a Pharmacological Target: Selective Versus Non-selective TNF Inhibitors
A wealth of pre-clinical studies support the link between inflammation and oxidative stress in the underlying pathophysiology of neurodegenerative disease (rev by [89, 90]. General anti-oxidants [73, 91-94] as well as anti-inflammatory agents [95-101] are being intensely investigated for their ability to protect dopamine neurons in experimental models of PD and other models of neurodegeneration including Huntington’s and AD. Consistent with a role of inflammation in the pathophysiology of PD, a prospective study of hospital workers found that daily use of non-steroidal anti-inflammatory drugs (NSAIDs) for a period greater than 2 years lowered their risk of developing PD by 46 percent [102], strongly suggesting that neuroinflammation contributes to dopamine neuron loss and development of PD in humans. Although these findings were confirmed and extended in another study by the same group of investigators [103], other recent studies reported lack of a protective effect ([104]).

The selective targeting of soluble TNF to treat neurodegenerative conditions is attractive, and various options may be available for consideration in clinical trials. Many molecules have been shown to inhibit TNF synthesis or bioactivity with varying specificity. Endogenous inhibitors are particularly important for limiting TNF production. They include soluble TNF decoy receptors, glucocorticoids, prostaglandins, and cAMP (reviewed in [105]). In addition, “anti-inflammatory” responses initiated by IL-10 and IL-4, as well as vagus nerve-mediated central cholinergic activation through muscarinic receptors, counteract TNF action in the periphery independent of muscarinic receptors present on macrophages [106, 107]. Engineered Fc-fused versions of soluble TNF receptors like lenercept (Fc-TNFR1) and etanercept (Fc-TNFR2) were subsequently developed and modified further by PEGylation to increase their half-life in the circulation. Anti-TNF antibodies have been humanized (e.g. infliximab, adalimumab). Many of these TNF antagonists have been used successfully in patients with inflammatory diseases like rheumatoid arthritis and Crohn’s (reviewed in [108]), and their success in the clinic underscores the deleterious effects of TNF overproduction in the periphery. However, the use of drugs like Enbrel (etanercept) and Remicade (infliximab) has been associated with an increased number of infections [109] and demyelinating disease [35, 110] due to their ability to block not only soluble TNF but also membrane-tethered TNF function, which is critical in host defense, innate immunity, and nerve myelination.

Since then, the newest generation of specific TNF inhibitors was designed by engineering a native agonistic protein (monomeric TNF) into a dominant negative one (DN-TNF) that does not bind TNFRs but still exchanges with native soluble TNF to form heterotrimers with attenuated activity. Systemic administration of DN-TNFs reduced TNF-induced hepatotoxicity and collagen-induced arthritis [111], and intranigral administration of DN-TNFs attenuated loss of midbrain dopaminergic neurons in rat models of Parkinson’s [75]. A recent study demonstrated that the membrane-tethered TNF-sparing effects of soluble TNF-selective DN-TNFs attenuate experimental arthritis without suppressing innate immunity to infection [112]. This strongly suggests that inactivation of soluble TNF may be a safer second-generation anti-TNF therapy for systemic administration. Given the demonstrated protective role of membrane-tethered TNF in the hippocampus, selective inhibition of soluble TNF to attenuate TNF-dependent neuroinflammatory processes that contribute to neurodegeneration is likely to be the more prudent approach for CNS applications. However, since none of the currently FDA-approved selective TNF inhibitors cross the blood–brain barrier, further modifications or development of alternative delivery modes will be needed if they are to be used to treat CNS diseases.

The effectiveness of anti-TNF biologics has prompted investigations to identify small molecules that might be administered orally to act as TNF inhibitors. Several classes of drugs, including inhibitors of Nuclear Factor-kappa B(NFκB) activation [113, 114], TNF-α-converting enzyme (TACE) metalloprotease [115], p38 MAPK kinase inhibitors [116], and thalidomide analogs which inhibit TNF synthesis [117], have been reported to act in this manner and merit investigation in pre-clinical animal models of neurodegeneration.

Clearly, additional research will be needed to firmly establish whether anti-inflammatory therapy could delay or prevent onset of AD or PD, and to identify the neuroinflammatory mechanism that contributes to neuronal death in order to selectively target those players in a region-specific manner. In multiple epidemiological investigations, a significant risk reduction has been observed in long-term as opposed to short-term users of traditional NSAIDs (reviewed in [118]). More recently, early promising findings with off-label use of the TNF inhibitor etanercept include the report that chronic extrathecal administration of this biologic was able to improve cognitive performance in patients with mild-to-severe AD [119]. These findings will need to be confirmed and investigated further in randomized, placebo-controlled clinical trials. On the other side of the spectrum, data from studies with TNF antibodies in animal models of stroke suggest that use of drugs that target TNF pathways to treat stroke or traumatic brain injury may have deleterious effects on hippocampal repair and neurogenesis. Therefore, the most rational approach will be to direct the inflammatory machinery via selective regional targeting of inflammatory mediators with neurotoxic effects rather than suppressing microglia activation in general [57, 99, 120]. This is true especially in light of clear and unequivocal data that indicate that certain inflammatory responses in the brain are beneficial and necessary for neural repair after injury.

Financial Disclosure: Dr. Tansey is a former employee of Xencor, Inc., a privately held biotech company in Monrovia, California, that is developing DN-TNFs for therapeutic applications. She has no significant financial holdings in the company, is not a consultant for the company, and does not receive financial support from the company for herself or her research.

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View all comments by Malu G. Tansey

A new case report mentions three cases of psychosis in patients treated with etanercept (McGregor et al., 2008). If there is a causal relationship, this would suggest another reason for caution, but also perhaps offer clues to mechanisms that can be exploited for safer therapies. There is a patchwork of evidence that inflammatory processes may play a role in schizophrenia, for example, as a mediator of prenatal insults (e.g., maternal infection or malnutrition) that might predispose to the disorder (e.g., Brown, 2006; Meyer et al., 2008). A specific role for TNF-α has been investigated in both gene expression and association studies, but there is only sparse evidence that variation in the protein or gene alters schizophrenia risk (see   Read more

A new case report mentions three cases of psychosis in patients treated with etanercept (McGregor et al., 2008). If there is a causal relationship, this would suggest another reason for caution, but also perhaps offer clues to mechanisms that can be exploited for safer therapies. There is a patchwork of evidence that inflammatory processes may play a role in schizophrenia, for example, as a mediator of prenatal insults (e.g., maternal infection or malnutrition) that might predispose to the disorder (e.g., Brown, 2006; Meyer et al., 2008). A specific role for TNF-α has been investigated in both gene expression and association studies, but there is only sparse evidence that variation in the protein or gene alters schizophrenia risk (see SchizophreniaGene entry). The most replicated result in this vein, as McGregor and colleagues note, is the large number of studies finding a lower incidence of schizophrenia among people with rheumatoid arthritis (see meta-analysis by Oken and Schulzer, 1999).

View all comments by Hakon Heimer