Introduction

Recent experience with an anti-TNFα drug has made scientists suspect that inhibiting this proinflammatory cytokine may hold promise as a protective treatment for diseases that feature neuroinflammation, such as Alzheimer's. Yet it is unclear how best to exploit this target to develop a safe and effective medicine. AD researcher Yong Shen, TNFα expert Malu Tansey (read .pdf of Tansey comment), and synaptic biologist Robert Malenka, as well as Cindy Lemere, Tsuneya Ikezu, and other leaders in the field, held a live discussion of how the field can advance the existing TNFα literature toward that goal. This Webinar/live discussion was held Thursday, June 12, from noon to 1:30 p.m. (EST).

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Transcript:

Participants: Yong Shen, Sun Health Research Institute; Rob Malenka, Stanford University; Malu Tansey, U.T. Southwestern Medical School; Cynthia Lemere, Brigham and Women's Hospital and Harvard Medical School; David Szymkowski, Xencor; Jiqing Xu, University of Nebraska Medical Center; Tsuneya Ikezu, University of Nebraska Medical Center; Gregory Brewer, Southern Illinois University Medical Center; Felicia McColl, Peter Jones, Bill Watson, Prasad Gabbita, P2D; Boobalan Pachaiyappan, University of Illinois at Chicago; Fran Shaller, Gabrielle Strobel, Alzheimer Research Forum.

Note: Transcript has been edited for clarity and accuracy.

Gabrielle Strobel
Welcome back, everyone! As people continue to come, I'd like to start off by giving our panelists, including Rob Malenka, Cindy Lemere, and Tsunea Ikezu, a first round of questions/comments to each other and to guests they see in attendance—particularly from industry if they want. Then I'll open the discussion to everyone.

Rob Malenka
Very nice presentations; I learned a lot.

Malu Tansey
Rob, in your studies, you're using the TNF knockout and a soluble R1 to block the TNF actions. Do you have any thoughts on whether this is glial-derived soluble or tmTNF juxtracrine signaling?

Rob Malenka
All of our results are consistent with it being soluble TNF. Most convincing evidence for this is that we could take media from cultured neurons that were manipulated pharmacologically and then apply this to normal cultures and see clear effects that were blocked by soluble R1. Caveat to all this work is that it was done in cultured neurons, not in vivo.

Malu Tansey
That is indeed consistent with solTNF, and it would be great to see if you can extend that with DN-TNF.

Rob Malenka
I agree about trying DN-TNF. But that will be up to Dave Stellwagen, my ex-postdoc who now has his own lab.

Malu Tansey
He should be able to obtain these directly from Xencor as they are giving them out to academic collaborators.

Gabrielle Strobel
Rob, would you be interested in working with any AD mouse models? It might be interesting to compare TNFα synaptic effects in normally aging mice to aging mice that overexpress mutant human APP or mutant tau. No?

Rob Malenka
Gabrielle, that would be interesting, but my lab is pretty busy doing other projects at the moment. Of course, if you want to give me $$$$$—I can be convinced.

Cynthia Lemere
Thanks for the excellent presentations! Malu, I may have missed this, but are the DN-TNF (soluble TNF inhibitors) able to cross the blood-brain barrier (BBB)? Do the other TNF inhibitors such as infliximab or etanercept?

Malu Tansey
Cindy, to my knowledge, none of the anti-TNF biologics would be expected to cross the BBB to any significant extent because they are very large proteins. However, we have not administered these systemically in our models of neurodegeneration and checked brain penetrance. On the other hand, under conditions in which the permeability of the BBB is compromised I would not be surprised if they did cross.

Gabrielle Strobel
To Malu and our industry guests, or anyone who knows, are any biologics that are dominant-negative proteins approved as drugs or in late clinical trials? I mean generally, for indications beyond AD/PD/MS/ALS. Or would small-molecule inhibitors hitting soluble TNF selectively have to be developed?

Malu Tansey
Gabrielle, Xencor has an open IND for trials in rheumatoid arthritis. I've got no knowledge of any small molecule that would selectively neutralize solTNF. For additional questions specific to DN-TNF trials, please direct them to Dave Szymkowski who has joined the chatroom.

Gabrielle Strobel
We have a contingent of scientists from Wyeth, Merck, Genentech. Are any of you able to share what programs are ongoing in this area? And Dave, what about DN-TNF trials?

David Szymkowski
Gabrielle, our primary focus with DN-TNF biologics has been in RA. As Malu, said, we're at the IND stage. However, with Dr. Tansey and other collaborators, we are actively exploring the effects of soluble TNF inhibition in various in vivo models of neurodegeneration.

Yong Shen
Rob, TNFα was found to promote synaptic transmission during the developmental stage; however, in an AD case, while TNFα is increased, the synapses are lost. Is this due to aging versus development?

Rob Malenka
Yong, as your question implies, I do think it is important to note that all of the work on the synaptic effects of TNF was done in very young neurons (i.e., cultured neurons) or during early postnatal development. Whether TNF has the same effects in adult or aged brain is an open question. It also may be that TNF has the same effect in adult/aged brain and the increase in AMPA receptors contributes to the eventual synapse loss or dysfunction.

Jiqing Xu
I have a question to Yong. You show TNFR1 action is detrimental to neurons and can cause neuron loss. Do you have any evidence to explain why neurons from certain anatomical sources are selectively more vulnerable?

Yong Shen
Jiqing, yes. In AD, for example, R1 is increased in the cortex; in PD cases R1 is increased in the substantia nigra.

Jiqing Xu
…specifically elevated in cortex in AD?

Malu Tansey
Jiqing and Yong, our working hypothesis is that the vulnerability to death induced by TNF comes from the ratio of R1/R2 receptors in the cell, and we are actively testing that now in models of PD.

Gabrielle Strobel
Hi, Tsuneya! Feel free to type in your questions.

Tsuneya Ikezu
Hello! I enjoyed the talk of Yong and Malu very much. I have a few points that I would like to share.

Gregory Brewer
Yong, we have two recent papers that show major effects of TNFR1 and R2 with age in old rat neurons in our culture model. The effect is dramatically evident from exposure to β amyloid and TNF together.

Yong Shen
Greg, that’s a very interesting finding.

Tsuneya Ikezu
Regarding the alternative approach for modulating the TNF signaling, there are a couple of papers regarding a new type of NFκB inhibitor, which blocks its translocation: dehydroxymethylepoxyquinomicin (Kubota et al., 2007). Lovastatin was also shown to enhance neuroprotection via upregulation of TNFR2 (Dolga et al., 2008). These may fit as alternative compounds.

Yong Shen
Greg, do R1 and R2 have similar/different effects with regard to TNFα and Aβ?

Tsuneya Ikezu
Another point is that in terms of Aβ clearance, TNF not only suppresses uptake but also suppresses Aβ degradation in microglia and macrophages. Since this degradation pathway is mediated through multiple steps of endocytosis, sorting, unfolding, and site specific/non-specific processing of Aβ, there are many to be addressed in this mechanism, and some of them may be a therapeutic target as well.

Felicia McColl
As a caregiver who has seen the amazing effects of perispinal etanercept in my mom, and in others at the clinic and through forum discussion, I would like to know the reason this hasn't been researched further in a clinical trial? How do clinical trials get started? Isn't it right that Dr. Tobinick did a preliminary study, and even though placebos weren't used, the data showed improvement in all the patients, which should have been enough to justify a clinical trial. Isn't this how many trials get started? Don't researchers many times stumble across something and do a small trial to get pharma companies interested? Why do they keep saying Dr. Tobinick did it wrong?

Gabrielle Strobel
Felicia, Dr. Tobinick was invited to attend but declined. It would have been interesting to hear from him where things stand with regard to conducting a controlled, blinded trial on intrathecal etanercept. Does anyone know the latest?

Rob Malenka
Sorry, but I have to go to another meeting. Thanks for including me. I did learn a lot.

Peter Jones
Gabrielle, regarding Dr Tobinick, he has progressed.

Yong Shen
Gabrielle, there was a new study by Dr Tobinick released yesterday, but we do not know the details yet.

Peter Jones
It’s “Perispinal Etanercept Produces Rapid Improvement in Primary Progressive Aphasia: Identification of a Novel, Rapidly Reversible TNF-Mediated Pathophysiologic Mechanism,” posted in Medscape, his usual source, 06/10/2008.

Gabrielle Strobel
Were there any controls in this report on PPA?

Felicia McColl
No controls. He didn't look for this patient; she came to him for treatment as far as I know. It worked, so he wrote a publication.

Bill Watson
Over what time frame should changes in TNFα in the brain be expected after any treatment, through any portal, with etanercept? I ask because Dr. Tobinick reported seeing gross cognitive changes in AD patients 20 minutes after “perispinal” injection. It is not clear what effect placebo had in his experimental situation.

Malu Tansey
Bill W., your question is a good one. Because Rob has left, I'd say that it is likely that the rapid effects of etanercept (if they are indeed real, and if it's somehow affecting TNF levels directly or indirectly) were mediated through the gliotransmitter role of TNF and not an inflammatory component. This would be interesting and may be very different than the mechanisms that contribute to plaque deposition/removal.

Prasad
Malu, which model is the best suited AD animal model for examining small-molecule therapeutics?

Malu Tansey
Prasad, it depends on the question you want to ask: if you want to explore synaptic effects, you don't need a plaque-making mouse, but if you want to explore the TNF regulation of plaque removal and deposition, you will need a plaque-making mouse and one which also displays neurodegeneration. Bob Vassar at Northwestern has a 5xTgAD which makes lots of plaques and also shows neuronal loss (Oakley et al., 2006).

Prasad
Malu, does Bob Vassar's model show elevated TNFα in brain region...similar to the LaFerla model?

Malu Tansey
Prasad, I'm not certain but I'm willing to bet it does.... Most of them do if there is a microglial component.

Prasad
Malu, I will follow up on Vassar's model. I enjoyed this session and learnt a lot. Regards. Catch up later. Thanks.

Gregory Brewer
Tsuneya, our paper in press with Exp. Neurology with Jigisha Patel shows that NFκB translocation is even higher in old neurons than middle-age after stimulation with Aβ + TNF (dose-dependent on TNF).

Peter Jones
Here’s Tobinick’s Medscape abstract. Perispinal Etanercept Produces Rapid Improvement in Primary Progressive Aphasia: Identification of a Novel, Rapidly Reversible TNF-Mediated Pathophysiologic. Primary progressive aphasia (PPA) is an uncommon form of progressive dementia for which there exists no established treatment. The underlying pathology may be that of either frontotemporal dementia or Alzheimer's disease. Increasing evidence suggests that excess tumor necrosis factor (TNF) may play a central role in Alzheimer's disease. Additionally, excess TNF has been documented in patients with frontotemporal dementia. Excess TNF may therefore represent a therapeutic target in PPA. Etanercept, an anti-TNF fusion protein, binds to TNF, thereby reducing its biologic effect. Emerging evidence suggests that perispinal administration of etanercept may have therapeutic efficacy for Alzheimer's disease. This evidence, in combination, supports a rationale for the use of perispinal etanercept for the treatment of PPA. This report documents rapid improvement in verbal abilities, beginning within 20 minutes of perispinal etanercept, in a patient with severe PPA. With repeated weekly dosing, sustained improvement at 1 month is documented, with a more than 10-point improvement in the patient's abilities to perform activities of daily living as measured by a standardized instrument, the Alzheimer's Disease Cooperative Study-Activities of Daily Living inventory. Rapid clinical improvement in PPA following perispinal etanercept administration may be related to TNF's role as a gliotransmitter and modulator of synaptic communication in the brain. These results may provide insight into the basic pathophysiologic mechanisms underlying PPA and related forms of dementia and suggest the existence of novel, rapidly reversible, TNF-mediated pathophysiologic mechanisms in both PPA and Alzheimer's disease. Further study of this therapeutic method is indicated. Introduction Primary progressive aphasia (PPA) is an uncommon form of progressive dementia without established treatment. One third of these patients have underlying Alzheimer's disease pathology, and two thirds have pathology characteristic of frontotemporal dementia.[1] These patients characteristically present with progressive difficulty with language as the most prominent initial manifestation of the disease, which advances in an unrelenting fashion until all language abilities are lost.[2] No effective treatment has been established.[1,2] Basic science and genetic, epidemiologic, and clinical evidence suggest that excess tumor necrosis factor-α (TNF-α) may play a central role in the pathogenesis of Alzheimer's disease [3-23]. In addition, excess TNF has been documented in the cerebrospinal fluid of patients with frontotemporal dementia.[24] Excess TNF may, therefore, represent a therapeutic target in PPA. Etanercept, a recombinant dimeric anti-TNF fusion protein, binds to TNF and blocks its interaction with cell-surface TNF receptors, thereby reducing the biologic effect of excess TNF. Emerging evidence suggests that perispinal administration of etanercept may have therapeutic efficacy in Alzheimer's disease [25-29]. This evidence, in combination, supports a rationale for the use of perispinal etanercept for the treatment of PPA.

Felicia McColl
Thanks, Peter. The publication was to give researchers a clue, and to let you know what is happening, so that someone with the $$$ will do a clinical trial.

Tsuneya Ikezu
Dr. Tansey, normally a DN molecule has to be in excess amount against wt TNF to block homodimerization. How much molecular ratio (in other words, how much DN-TNF) is necessary for the inhibitory effect?

David Szymkowski
Tsuneya, good question. DN works (as the name implies) by a dominant-negative mechanism. It's a mass action exchange effect. In other words, equimolar or slight excess DN-TNF eliminates the majority of native soluble TNF. The Science (Steed et al., 2003) and J. Immunology (Zalevsky et al., 2007) papers give the details.

Malu Tansey
Tsuneya, the stoichiometry tells us that a twofold excess should work, but you can drive it with larger amounts. The 2003 Science paper demonstrates this in detail.

Tsuneya Ikezu
Thank you, Malu. I have to leave for another appointment. Thank you for inviting me to the discussion panel.

Yong Shen
Malu, are there any similar/different effects of TNFα on different types of neurons? For example, dopamine neurons versus cortical neurons?

Malu Tansey
Yong, there is definitely a difference in sensitivity of DA neurons versus cortical. DA neurons are much more sensitive, but if you couple it with Aβ you can really affect them adversely, consistent with your data....

Bill Watson
Malu, what results have implicated the gliotransmitter role of TNFα in AD?

Malu Tansey
Bill W., your question about what data implicate TNF as a gliotransmitter in AD: I would say some of the LTP data in brain slices and clinically, the Tobinick data, but it's not clear how the etanercept is getting to the sites in the brain....

Gregory Brewer
Bill, we show neuroprotective effects of low-dose TNF for middle-age neurons against Aβ toxicity (Patel and Brewer, 2008).

Bill Watson
Gregory, in vivo?

Gregory Brewer
Yes, in culture...but old neurons are killed by doses that are protective in middle-age neurons, and it is TNF receptor dependent.

Gabrielle Strobel
Yong, in your talk you made a case for a number of potential targets besides TNFα —FADD, TRADD, NFκB, Apaf-1? Do you know if there is any drug discovery around those?

Yong Shen
Gabrielle, yes. For example, several companies are working on NFκB as a target, but there are several side effects on toxicity.

Boobalan Pachaiyappan
I've a general question for either Dr. Yong Shen or Dr. Malu. I have a feeling that TNFα1 inhibitors can make a huge impact on late-AD patients and BACE1 inhibitor on early-AD patients. How do you see this? Also, can one envision the synergistic effect by combining these inhibitors?

Yong Shen
Boobalan, we need a chemist to create the idea of a combination of a BACE inhibitor and a TNFα inhibitor.

Boobalan Pachaiyappan
Dr. Shen, in theory, if a combination inhibitor can be made, how do you envision the outcome?

Yong Shen
Boobalan, Aβ may be decreased and TNFα-induced inflammation may be diminished.

Prasad
Malu/Yong, has anyone looked at the effects of TNFα inhibition on α- or β-secretase activity in vivo?

Malu Tansey
Boobalan, I like your idea, too, but again, we must be very cautious not to affect TNF signaling in areas of the brain where it is protective against neurotoxicity or excitotoxicity. Lindvall's work with TNF antibodies after stroke in rat models says that TNF is important for hippocampal neuroblast survival. So my feeling is that targeted delivery may be in order and regulated delivery even better...but we gotta start somewhere.

Gregory Brewer
Malu, our 2001 paper with John Viel showed that microglia from old rat brains made much more TNF in response to Aβ than microglia from young brains (Viel et al., 2001).

Malu Tansey
Greg, that is very interesting and consistent with some of the data emerging from our lab that the microglia may become dysfunctional as one ages.... We should not be neuron-centric but also consider that the neighborhood matters (as Don Cleveland likes to say!).

Gregory Brewer
Malu, I agree. We need a TNF modulator, not a blocker. A little inflammation is part of the protective stress response. We need something like Namenda that only acts when there is excess TNF.

Peter Jones
Is it appropriate to use etanercept on AD patients, as it does not cross the BBB yet lowers the immune system?

Yong Shen
Peter, the concern I have is the way etanercept is given; it may cause injury to patients, especially with continuous use.

Gregory Brewer
Peter, our conclusion in our J. Neurosci. Res. paper urges caution with etanercept because of blocking the remaining protective effect of TNFα.

Peter Jones
Do you have an opinion on CSVS as a way past the BBB? Here is another quote from Tobinick: “The cerebrospinal venous system (CSVS), a term coined by me, plays a central role in the vascular circulation of the brain, the spinal cord, and the nerve roots. Detailed knowledge of the CSVS is therefore of consequence when considering therapeutic intervention for diseases involving these structures. The seminal work regarding the anatomy and physiology of the CSVS was done by Oscar Batson, a pioneering professor at the University of Pennsylvania, in the 1940s. Although ‘Batson's Plexus’ has achieved widespread recognition, much of his highly important work never achieved the scientific dissemination it deserved. Perhaps the use of highly potent and selective biologic therapeutics capable of transport via the CSVS (as described in my pending patent applications) will help illuminate the brilliance of Dr. Batson's early discoveries.” From: The Cerebrospinal Venous System: Anatomy, Physiology, and Clinical Implications. By Edward Tobinick. Source MedGenMed 2006; 8(1):53. Abstract: There is substantial anatomical and functional continuity between the veins, venous sinuses, and venous plexuses of the brain and the spine. The term "cerebrospinal venous system" (CSVS) is proposed to emphasize this continuity, which is further enhanced by the general lack of venous valves in this network. The first of the two main divisions of this system, the intracranial veins, includes the cortical veins, the dural sinuses, the cavernous sinuses, and the ophthalmic veins. The second main division, the vertebral venous system (VVS), includes the vertebral venous plexuses which course along the entire length of the spine. The intracranial veins richly anastomose with the VVS in the suboccipital region. Caudally, the CSVS freely communicates with the sacral and pelvic veins and the prostatic venous plexus. The CSVS constitutes a unique, large-capacity, valveless venous network in which flow is bidirectional. The CSVS plays important roles in the regulation of intracranial pressure with changes in posture, and in venous outflow from the brain. In addition, the CSVS provides a direct vascular route for the spread of tumor, infection, or emboli among its different components in either direction.

Malu Tansey
Peter, I wouldn't be surprised if etanercept is getting in because the BBB is probably leaky in AD patients (and in other neurodegenerative diseases like PD and others with chronic neuroinflammation)...but it would be nice to show this by labeling it.

Peter Jones
Does any scientist here support Dr. Tobinick?

Malu Tansey
Peter, I think the finding, if true, would be incredibly interesting. It needs to be confirmed because it has potential. If one is worried about the immune system, some of the solTNF selective inhibitors should be tried. He should contact Xencor if he is interested in more selective and hence possibly safer drugs. To understand the mechanism, perhaps we need to do more studies in animal models.

Gabrielle Strobel
Malu, is the data available now sufficient to recommend follow-up with a controlled, blinded human trial at this point?

Malu Tansey
Gabrielle, I'm not a neurologist nor a clinician so I can't advise but I'd like to see one done. More importantly, we could do the animal experiments to better understand the mechanism. We're considering it but as Rob mentioned, $$$ would be needed.

Gabrielle Strobel
The Alzforum received a comment to this discussion pointing out a recent paper that reports acute psychosis in three people receiving etanercept for RA (comment on Live Discussion page). To the scientists/clinicians here, is this a surprise? Or have psychiatric side effects been known before for the existing antiTNF biologics?

Yong Shen
Gabrielle, this is not a surprise because TNFα does involve some schizophrenia-type syndrome.

Cynthia Lemere
Yong and Malu, is TNF upregulated during sprouting and regeneration?

Yong Shen
Cynthia, that’s a good question. I have not seen the data yet, but I assume, during the regeneration, there might also have to be an inflammation response, so I wouldn't be surprised if TNFα is changed.

Malu Tansey
Cindy, do you mean centrally or peripherally (regeneration)?

Cynthia Lemere
Malu, I'm thinking more of sprouting. One can see sprouting of neurons following bungarotoxin treatment in goldfish optic tectum in brain, for example. I think some APP tg mice have been shown to have sprouting as well. May be a last-ditch effort by the neuron to survive.

Malu Tansey
Cindy, your question is a very good one on sprouting and regeneration. I'm not aware that anyone has looked directly, but in terms of hippocampal sprouting, it will be a good thing to have around (Olle Lindvall’s data).

Gabrielle Strobel
Cindy, has not Carl Cotman published fairly extensively about sprouting, as well? This is an old literature, but I think it's generally thought to happen in AD brain like you say, no?

Cynthia Lemere
Yes, I believe so. It would be interesting to know if these TNF inhibitors have any effect on sprouting in vivo.

Malu Tansey
Cindy, if you or anyone else is interesting in looking, we may be able to provide some reagents.

Yong Shen
Greg, the TNFα you used to treat neurons—is this soluble TNFα?

Gregory Brewer
Yong, yes—10-1000 ng/ml soluble TNFα. You all might be interested in our findings in cultured neurons, young and old, with and without anti-TNFR1 or R2 antibodies: the best correlation for neuron survival was not the nuclear NFκB, but the cytoplasmic ratio of its downstream action on Bcl-2/Bax ratio.

Malu Tansey
Greg, I'm not surprised, as TNF signaling to apoptotic pathways may be stronger than the opposing NFκB counterarm depending on strength of activation and duration of R1 versus R2 during any given stimulus, plus the signaling proteins intracellularly that modulate those responses.

Prasad
Malu, has anyone looked at the effects of TNFα inhibition on α- or β-secretase activity in vivo?

Malu Tansey
Prasad, in our paper coming out soon (we hope), we looked at inhibition of solTNF with lenti-DN-TNF. We did not see significant effects on BACE mRNA or protein, but a negative result is difficult to interpret when you're grinding up brains...limit of detectability is poor.

Peter Jones
I am stating here, after much research I challenge Dr. Tobinick in his claims with the use of etanercept for Alzheimer’s and all the other diseases he has claimed in the past. The entire works are fiction. The AD caregiver community is vulnerable to this because of the dearth of therapies in AD. There may be some truth to inflammation as a causation in the pathology in AD, but to come out with this magic potion. It is time that the AD research community regrouped. Dr. Tobinick is a dermatologist. Any person from neuroscience willing to challenge Dr. Tobinick?

Fran Shaller
Peter Jones, have you met with Dr. Tobinick or have you witnessed the post-injection reaction of any of his patients?

Cynthia Lemere
Peter, scientists and clinicians have an obligation to perform rigorous, scientifically sound clinical trials before announcing to the world that they have found the types of effects Dr. Tobinick described. Even preclinical trials must be rigorous and scientifically sound. Until this is done, the results may mislead patients and their families into believing in something that has not been validated and may put them at risk.

Peter Jones
I am sorry to intrude in your discussion, but Dr. Tobinick has entered into your domain and is messing with your research and AD patients.

Gabrielle Strobel
Peter, to reply to your call on scientists to refute Dr. Tobinick. Very few in the AD scientific community have embraced Dr. Tobinick's methods or claims, or his successive tapping of one disease market after another, now apparently moving on to some tauopathies where the treatment situation is even more dire than in AD. But there is genuine and broad-based interest in the concept of developing drugs against TNFα—doing so in the right way. No one likes to publicly attack a person, but I think you will see, are already seeing, research effort going in the direction of rigorously studied TNFα drugs that are approvable.

Malu Tansey
Peter J., discoveries about mechanism and potential for therapeutic intervention will have to take place by doing the necessary preclinical studies, so we should focus on taking his observations and rigorously testing the hypothesis with mechanistic studies.

Cynthia Lemere
Malu, I completely agree.

Peter Jones
Dr. Tobinick should be discounted completely; he is delaying the advances in this area. He has nothing to offer.

Fran Shaller
If he has "nothing to offer," then my eyes are lying to me.

Gabrielle Strobel
Let's move on. Does anyone have further questions or ideas regarding Yong's and Malu's research?

Peter Jones
I apologise for my interference. Please, scientists, Google Tobinick and see what comes up. You will all advance with real science.

Cynthia Lemere
Sorry, I have to go now. Thanks everyone!

Gabrielle Strobel
Bye, Cindy; thanks for coming!

Yong Shen
Bye, Cynthia; thanks for participating.

Malu Tansey
Cindy, talk later! To members of industry, educate me: what would it take in terms of preclinical evidence to have your company move forward with an approved biologic for a CNS application?

Gabrielle Strobel
Hi Steve, are you still there? Perhaps you could take Malu's question?

Malu Tansey
Gabrielle, the reason I ask is because I'm curious whether they would have to unequivocally know the mechanism of action (gliotransmitter versus pro-inflammatory cytokine driving neurotoxicity) before moving forward? If so, we and others need to do much more work, and we should get going.

Gabrielle Strobel
Our scheduled time has passed, but all who are still interested are most welcome to stay and chat. The room will stay open. But before people start dropping out, I'd like to invite Yong and Malu to make a wish: which study/trial would you like to see done next if funding was no issue? What would be most informative next? That can be your concluding statement.

Yong Shen
We would like to try clinical studies by using a protein-based and small-molecule-based TNFα inhibitor and see whether cognition in AD patients improves. We would also like to test approved drugs in MCI patients to see if we can slow the conversion from MCI to AD.

Malu Tansey
Gabrielle, if you mean preclinical study, I'd like to see clear-cut effects of centrally or peripherally administered TNF inhibitors (solTNF selective versus non-selective) in rodent models where neuropathology can be examined histologically and behavior outcomes can be measured objectively. In patients, we may need to do much more work and perhaps use the solTNF selective inhibitors in MS as proof of concept that we can modulate the course of an inflammatory condition without affecting immunity or synaptic plasticity (cognition).

Gabrielle Strobel
Malu, it’s unfortunate, though no surprise, that the industry scientists cannot speak here. But that so many came, from at least five companies, suggests that they are actively interested.

Malu Tansey
Gabrielle, absolutely, no problem. I understand and appreciate their interest and their hard work in this area. For all these neurodegenerative diseases with chronic inflammation, I'd like to see early diagnosis so we can run neuroprotective trials!

Gabrielle Strobel
Amen to early diagnosis! Go biomarkers! We'll host a Webinar/town hall meeting on early diagnosis next month. Yong, by approved, do you mean etanercept, infliximab etc.?

Yong Shen
Gabrielle, yes, or experimental BACE inhibitor. We just need to figure out how to give it to the patient. Realistically, small molecules may be easier to get approval for.

Malu Tansey
Yong, my concern with small molecules is that they are going to go everywhere in the brain and would be expected to inhibit TNF signaling where it may have protective roles…like hippocampus.

Yong Shen
Malu, it’s true, but we want to see in the disease state which one has the more important role. Maybe at a young stage a BACE inhibitor is not appropriate but at an older age is sensible.

Gabrielle Strobel
Malu, as a practical matter, how would you target a biologic to a subarea of the brain?

Malu Tansey
It may be possible to do targeted delivery via minipump or via gene transfer of DN-TNF.... Then all you have to do is get through the regulatory hurdles of viral vectors :-).

Fran Shaller
Malu, if AD starts in the hippocampus, is it possible that the TNF signaling is malfunctioning at that level?

Malu Tansey
Fran, it is possible that TNF signaling in the brain antagonizes trophic factor pathways (insulin-like growth factor-1, or IGF-1, perhaps) that get the brain into an energy crisis. Some have suggested that AD is type 3 diabetes. As you know, TNF is involved in the insulin resistance in diabetes. This is speculative but worth looking into.... To those doing AD research, I'm curious to know whether anyone is looking at the effects of TNF signaling on GLUT transporters in cortical neurons? Yong?

Yong Shen
Malu, yes.

Malu Tansey
Yong, good, because I have a feeling that TNF is playing very different roles at various stages in the neuronal dysfunction and pathology, which may be very different. Synaptic, APP processing, and glucose utilization effects may precede all the late events that involve plaque deposition where TNF may be playing other roles in regulation of microglia activation, etc. And, of course, TNF may be required if one is to do immunotherapy, as many have shown that microglia must step up to the plate to remove plaque.... It is very complex!

Yong Shen
Malu, indeed. Let's continue to keep in touch and we will chat more about this later.

Gabrielle Strobel
I have to leave, but that does not mean you do. Yong and Malu, thank you both tremendously for your texts, presentations, and the significant preparation going into today. Thank you, audience, for your interest. The Alzforum will stay on this topic.

Malu Tansey
Yong, sounds like a great idea. Will do. Unless others have additional questions for us, we should also get back to pushing back the frontiers of science or at least get back to work.... To all, if anyone has questions, please feel free to e-mail us: malu.tansey@utsouthwestern.edu.

Yong Shen
I also welcome questions: yong.shen@sunhealth.org.

Background

Background Text
By Yong Shen, Sun Health Research Institute, Sun City, Arizona

While current Alzheimer disease treatments remain woefully inadequate, a diversity of ideas toward novel therapies deserves rigorous exploration in the academic and pharmaceutical research communities. Tumor necrosis factor inhibition is one such potential target area. This discussion will focus on what we know about this complex and two-faced molecule, which precise targets have the most scientific support, and how research should proceed from here on out. One recent report has raised hope among patients and caregivers, while causing concern about the appropriate way to explore the risks and benefits of TNFα inhibition. We suggest the following questions for discussion:

• Which targets other than TNFα itself should be explored (receptors, upstream and downstream players, regulators)?
• What is the status of drug discovery on those?
• Do existing experimental drugs give promising effects?
• Which existing approved drugs besides etanercept are options for clinical study?
• Are they, in fact, being explored for potential effects in AD?
• What do we know about etanercept’s side effects in aged patients with neurologic disease?
• Do we know how patients on perispinal etanercept fare over the long term, past the six-week injection schedule?
• Is the evidence strong enough to warrant federally funded, or ADCS trials?

What Is TNFα? Overview of Main Properties
Produced mainly by cells of the immune system, tumor necrosis factor α (TNFα) operates in various parts of the body as a key cytokine in inflammation and immune processes (1). In the peripheral immune system, activated macrophages and monocytes release TNFα. In the brain, TNFα is expressed by neurons and glia, and promotes inflammatory responses by recruiting microglia or astrocytes to lesion sites, leading to glial cell activation. After release, TNFα binds specific receptors (TNFR1 and II) to elicit biological effects by mechanisms not fully understood.

The two receptor subtypes, TNFR1 and TNFR2, differ considerably in amino acid sequence, with just 24 percent homology in the extracellular region and about 10 percent homology in the intracellular domain. The receptors also differ functionally. TNFR1, but not TNFR2, contains an intracellular “death domain” (DD) that activates NF-κB signaling pathways leading to apoptosis (2). On the other hand, knockdown studies have shown that TNFR2 plays a trophic or protective role in neuronal survival (3).

TNFα in Normal and Diseased Brain
Released by activated microglia or astrocytes, TNFα can be trophic or toxic, depending on stage of development, target cell, and receptor subtype. For example, TNFα protects fetal and postnatal neurons after glucose deprivation (4), and adult cortical neurons after traumatic brain injury (5). In rats, administration of exogenous TNFα results in region-specific attenuation of excitotoxic damage in the brain. Given that TNFα is rapidly produced in glial cells and neurons after excitotoxic insults, the findings suggest that endogenous TNFα may modulate responses to excitotoxic brain injury.

TNFα can also have detrimental effects. For example, it potently influences aspects of synaptic transmission and plasticity, such as long-term potentiation and synaptic scaling in learning and memory (6-8). Studies in TNFα-deficient mice suggest that TNFα may disrupt synaptic plasticity by modulating synaptic vesicle proteins (9). The same doses of TNFα that aid development of fetal rat hippocampal neurons can kill human cortical neurons and oligodendrocytes. Indeed, TNFα can become destructive, and even toxic, during aging, injury, and in some disease states.

Most chronic neurodegenerative diseases are accompanied by a cytokine-mediated inflammatory response termed neuroinflammation. Though most cytokines, including TNFα, are expressed at very low levels in the healthy brain, disease-related neuroinflammation can be detected years before neurons die in significant numbers. Microglial cells and astrocytes are activated in diseased brains, and TNFα is secreted by microglia (10).

TNFα Receptor Signaling and Neuron Death
Several lines of evidence suggest that TNFα and its receptors may enlighten our understanding of neural protection and therapy. First, TNFα expression in the brain is high during development, low during adulthood, and high in people with Alzheimer and Parkinson diseases, multiple sclerosis, and stroke. Our group has identified a direct link between death receptor activation and signal cascade-mediated neuron death in brains with neurodegenerative disorders.

The TNF receptor superfamily contains several members with homologous death domains (DD). The DD is critical in initiating signaling pathways after ligand binding. In the absence of ligand, TNFR1 (DD-containing) receptors remain inactive. Under pathological conditions in which glial cells in the brain are activated and TNFα is secreted, TNFR1 gets activated. Other studies, including our work with knockout strains (2), support this notion and expand on these findings. Morphologically, neurons from TNFR2-/- mice are much more vulnerable to TNFα than are neurons from TNFR1+/- or wild-type animals. This observation is supported by the results from lactate dehydrogenase (LDH) release, an indicator of cytotoxicity. However, at lower doses of TNFα, LDH release from TNFR1-/- hippocampal neurons does not differ significantly from that of wild-type mice. This suggests that neuronal death induced by TNFα through TNFR1 involves an apoptotic process. Typically, neurons can survive the harmful effects of TNFα by activating TNFR2, which overrides death signals delivered through TNFR1. However, in a disease state, TNFR1 is robustly activated by TNFα secreted by surrounding glial cells, and the resulting apoptotic signal may be too strong for the neurons to override.

Using virally infected primary neurons that overexpress TNFR1 or neurons from TNFR1 knockout mice, we have shown that Aβ peptide induces neuronal apoptosis through TNFR1 (2). Neuronal death was mediated via alteration of apoptotic protease-activating factor (Apaf-1) expression that in turn induced activation of NF-κB. Aβ-induced neuronal apoptosis was reduced with lower Apaf-1 expression, and little NF-κB activation occurred in neurons with mutated Apaf-1 or a deletion of TNFR1, compared with wild-type neurons. Our studies suggest a novel neuronal response to Aβ, which occurs through a TNFR signaling cascade and a caspase-dependent death pathway.

Our in vivo studies on target-depleted TNFR in mice show that TNFα has little effect on hippocampal neurons from TNFR1-/- knockout mice, whereas neurons from TNFR2-/- mice are typically vulnerable to TNFα even at low doses (2). Moreover, TNFα induces little NF-κB translocation in TNFR1-/- neurons, whereas the NF-κB subunit p65 is still translocated from the cytoplasm into the nucleus in neurons from wild-type and TNFR2+/- mice. Furthermore, p38 mitogen-activated protein (MAP) kinase activity is upregulated in both wild-type and TNFR1-/- neurons; in contrast, no alteration of p38 MAP kinase was found in neurons from TNFR2-/- mice.

Results from TNFR overexpression studies further support the above findings. NT2 neuronal-like cells transiently transfected with TNFR1 are very sensitive to TNFα, whereas TNFα is not toxic and even seems to be trophic to cells that overexpress TNFR2. Radioligand-binding experiments demonstrate that TNFα binds TNFR1 with high affinity, whereas TNFR2 shows lower binding affinity to TNFα in NT2 transfected cells. Subsequent neuronal death or survival may ultimately depend on a particular subtype of TNF receptor that is predominately expressed in brain neurons during neural development or during neurological disease.

TNF Receptor Signaling, APP Processing, and Aβ
In addition to mediating neuron survival and death, TNFR signaling is involved in APP processing, which affects Aβ production. Several groups have identified a binding site for the transcription factor NF-κB, a component of the TNFR1 signaling pathway, in the β-secretase (BACE1) promoter (11,12). Interestingly, we have shown that TNFα treatment results in increased BACE1 activity in vitro and that the TNFR1 cascade is required for Aβ production in vivo (13; see also ARF related news story). We have reported that deletion of the TNFR1 gene in APP23 transgenic mice (APP23/TNFR1-/-) inhibits Aβ generation and diminishes Aβ plaque formation in the brain. Genetic deletion of TNFR1 leads to reduced BACE1 expression and activity via the NF-κB pathway, and deletion of TNFR1 in APP23 transgenic mice prevents learning and memory deficits. These findings suggest that TNFR1 not only contributes to neurodegeneration but also is involved in APP processing and Aβ plaque formation. Thus, TNFR1 is a novel therapeutic target for AD.

TNFα and Genetics
Proinflammatory cytokines, such as TNFα, and acute-phase proteins play an important role in AD neurodegeneration. Common polymorphisms associated with TNFα upregulation have been shown to be associated with AD. TNFα is upregulated in AD patients, and TNFα genetic variation could contribute to the risk of developing AD or influence the age of disease onset. Data from family-based association tests have revealed an association between AD and TNFα haplotype, as well as a significant increase in mean age of disease onset among AD patients carrying the haplotype associated with TNFα upregulation. The lowest age at onset was observed in patients carrying the 308A TNFα+/ApoE4+ genotype. This suggests that TNFα is a disease modifier gene in patients genetically predisposed to AD, bringing to light the importance of genetic variation in proinflammatory components in the progression of AD. Inheritance of the rs1799724-T allele appears to synergistically increase the risk of AD in ApoE4 carriers and is associated with altered CSF Aβ42 levels. Further investigation is warranted to assess the significance of these findings (14-17; see also TNF Alzgene page).

As mentioned, TNFα binds two receptors, TNFR1 and TNFR2, which activate distinct transduction pathways in the immune system and brain. Interestingly, the genes for TNFR2 and TNFR1 are found in regions of chromosome 1p and chromosome 12p that are linked to late-onset AD. A TNFR2 exon 6 polymorphism has been found to be significantly associated with late-onset AD in families with no ApoE4 homozygotes (16). Given these and other results that strengthen the case for the involvement of TNFα in AD pathogenesis, anti-TNFα therapy for AD deserves serious exploration.

Anti-TNFα Therapy
Thus far, most anti-TNFα agents in clinical trials have been used to treat heart failure and rheumatoid arthritis.

Infliximab (Remicade), a human-murine chimeric IgG1-k monoclonal antibody that binds and neutralizes TNFα, has FDA approval for rheumatoid arthritis and other autoimmune diseases. In addition, it has been shown to improve left ventricular function and to limit heart failure in transgenic mice that overexpress TNFα. However, in the ATTACH (Anti TNFα Therapy Against Chronic Heart failure) trial, TNFα antagonism with infliximab did not improve heart failure, and in fact adversely affected the clinical status of patients with moderate to severe heart failure (18-20).

Etanercept (Enbrel) is a recombinant, soluble human TNF receptor that binds and neutralizes circulating TNFα. Similarly to infliximab, etanercept is widely used to treat rheumatoid arthritis, psoriasis, and ankylosing spondylitis. Despite encouraging results in small pilot trials, etanercept used on patients with moderate to severe heart failure in three large multicenter trials—RENAISSANCE (Randomized Etanercept North American Strategy to Study AntagoNism of CytokinEs), RECOVER (Research into Etanercept CytOkine antagonism in VEntriculaR dysfunction), and RENEWAL (Randomized EtaNercEpt Worldwide evALuation)—did not demonstrate clinical benefit and furthermore indicated that etanercept in these patients may have adversely affected the course of disease (21,22). Nevertheless, it may be worth testing whether etanercept has a therapeutic effect in AD patients.

Pentoxifylline is a xanthine-derived agent that has been shown to inhibit the production of TNFα. In a single-center, placebo-controlled investigation, pentoxifylline has been shown to improve left ventricular performance in idiopathic dilated cardiomyopathy. In this small trial of 28 patients, improvement in left ventricular ejection fraction appeared to be greater in people treated with pentoxifylline than in untreated patients. The advantages of pentoxifylline over infliximab and etanercept include easier administration and lower cost. Large clinical trials are needed to critically evaluate the use of pentoxifylline in AD patients (23).

Among the three compounds described above, pentoxifylline may hold the most promise for treating AD because it directly modulates TNFα mRNA overexpression and TNFα production rather than simply neutralizing its effect.

Future Directions
Another agent that warrants consideration as a possible treatment for patients with dementia is Lysofylline, a lysophosphatidic acid acyl transferase (LPAAT) inhibitor that has been shown to attenuate lipopolysaccharide-induced TNFα synthesis (24). Lipopolysaccharide (LPS) binds to a variety of serum proteins—most notably, LPS-binding protein (LBP)—that influence the macrophage-mediated proinflammatory response.

Inhibition of p38 MAP kinase may also be a useful therapeutic avenue for ameliorating neuronal dysfunction in AD. Given the role of p38 MAP kinase in TNFα production during neurodegeneration in patients with AD or PD with dementia, it is logical to assume that the p38 MAP kinase inhibitors FR-167653, SB-239068, SC-409, and RWJ-67657 may prevent TNFα synthesis and thereby affect AD progression. Moreover, NF-κB is a key transcription factor that regulates the inflammatory process and has been shown to enhance TNFα synthesis in the brains of AD patients. We have demonstrated that deletion of TNFR1 prevents the translocation of NF-κB into the nucleus and thus prevents transcription of the TNFα gene (2).

Other plausible compounds for treating AD include pyrrolidine dithiocarbamates (PDTC), which antagonize DNA binding by NF-κB and thereby prevent TNFα transcription (25,26). A possible post-translational therapeutic target in AD is TACE (TNFα converting enzyme), which is required for processing pro-TNFα into its mature form. Metalloproteinase inhibitors and aprotinin are reported to decrease TNFα processing by inhibiting TACE non-selectively. Selective inhibitors of TACE like DPH-067517 and GM-6001 may also be potential candidates for treatment of neurodegenerative diseases.

Celacade^™ is a new immunomodulation therapy being developed by Ontario, Canada-based Vasogen Inc., to target chronic inflammation by activating the immune system’s physiological anti-inflammatory response. During treatment, patients are subjected to venipuncture, where approximately 10 cc of venous blood is taken and processed in Vasogen’s V.C 7001 medical device. There the blood is exposed to stress by heating at 108 degrees F (42.2 degrees C) followed by zapping with ultraviolet light and mixing with ozone gas. Oxidative stress is known to induce apoptosis, during which phosphatidylserine molecules move to the surface of apoptotic cells. The exposed molecules interact with specific receptors on the surface of antigen-presenting cells of the immune system, including macrophages and dendritic cells, which leads to production of anti-inflammatory cytokines such as IL-10. IL-10 downregulates inflammatory cytokines, e.g., TNFα, in AD patients. Celacade^™ is undergoing clinical trials in various U.S. cardiac centers.

In summary, agents that modulate TNFα production—such as inhibitors of LPAAT, p38 MAPK, NF-κB, and TACE or TNF receptor antagonists—may be potential future candidates for treatment of AD and other neurodegenerative disorders.

References:
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7. Stellwagen D, Beattie EC, Seo JY, Malenka RC. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha. J Neurosci. 2005 Mar 23;25(12):3219-28. Erratum in: J Neurosci. 2005 Jun 1;25(22):1 p following 5454. Abstract

8. Stellwagen D, Malenka RC. Synaptic scaling mediated by glial TNF-alpha. Nature. 2006 Apr 20;440(7087):1054-9. Epub 2006 Mar 19. Abstract

9. Zhao C, Ling Z, Newman MB, Bhatia A, Carvey PM. TNF-alpha knockout and minocycline treatment attenuates blood-brain barrier leakage in MPTP-treated mice. Neurobiol Dis. 2007 Apr;26(1):36-46. Abstract

10. Lue LF, Rydel R, Brigham EF, Yang LB, Hampel H, Murphy GM Jr, Brachova L, Yan SD, Walker DG, Shen Y, Rogers J. Inflammatory repertoire of Alzheimer's disease and nondemented elderly microglia in vitro. Glia. 2001 Jul;35(1):72-9. Abstract

11. Christensen MA, Zhou W, Qing H, Lehman A, Philipsen S, Song W. Transcriptional regulation of BACE1, the beta-amyloid precursor protein beta-secretase, by Sp1. Mol Cell Biol. 2004 Jan;24(2):865-74. Abstract

12. Li Y, Zhou W, Tong Y, He G, Song W. Control of APP processing and Abeta generation level by BACE1 enzymatic activity and transcription. FASEB J. 2006 Feb;20(2):285-92. Abstract

13. He P, Zhong Z, Lindholm K, Berning L, Lee W, Lemere C, Staufenbiel M, Li R, and Shen Y. (2007). Deletion of tumor necrosis factor death receptor inhibits amyloid β generation and prevents learning and memory deficits in Alzheimer’s mice. J Cell Biol. 178(5): 829-41. Abstract

14. Ramos EM, Lin MT, Larson EB, Maezawa I, Tseng LH, Edwards KL, Schellenberg GD, Hansen JA, Kukull WA, Jin LW. Tumor necrosis factor alpha and interleukin 10 promoter region polymorphisms and risk of late-onset Alzheimer disease. Arch Neurol. 2006 Aug;63(8):1165-9. Abstract

15. Culpan D, MacGowan SH, Ford JM, Nicoll JA, Griffin WS, Dewar D, Cairns NJ, Hughes A, Kehoe PG, Wilcock GK. Tumour necrosis factor-alpha gene polymorphisms and Alzheimer's disease. Neurosci Lett. 2003 Oct 16;350(1):61-5. Abstract

16. Perry RT, Collins JS, Harrell LE, Acton RT, Go RC. Investigation of association of 13 polymorphisms in eight genes in southeastern African American Alzheimer disease patients as compared to age-matched controls. Am J Med Genet. 2001 May 8;105(4):332-42. Abstract

17. Bertram L, McQueen MB, Mullin K, Blacker D, Tanzi RE. (2007). Systematic meta-analyses of Alzheimer disease genetic association studies: the AlzGene database. Nat Genet. 2007, 39(1):17-23. Abstract

18. Strand V, Singh JA. Improved health-related quality of life with effective disease-modifying antirheumatic drugs: evidence from randomized controlled trials. Am J Manag Care. 2008 Apr;14(4):234-54. Abstract

19. Klotz U, Teml A, Schwab M. Clinical pharmacokinetics and use of infliximab. Clin Pharmacokinet. 2007;46(8):645-60. Abstract

20. Adachi T, Toishi T, Takashima E, Hara H. Infliximab neutralizes the suppressive effect of TNF-alpha on expression of extracellular-superoxide dismutase in vitro. Biol Pharm Bull. 2006 Oct ;29 (10):2095-8. Abstract

21. Dhillon S, Lyseng-Williamson KA, Scott LJ. Etanercept: a review of its use in the management of rheumatoid arthritis. Drugs. 2007;67(8):1211-41. Abstract

22. Mann DL, McMurray JJ, Packer M, Swedberg K, Borer JS, Colucci WS, Djian J, Drexler H, Feldman A, Kober L, Krum H, Liu P, Nieminen M, Tavazzi L, van Veldhuisen DJ, Waldenstrom A, Warren M, Westheim A, Zannad F, Fleming T. Targeted anticytokine therapy in patients with chronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL). Circulation. 2004 Apr 6;109(13):1594-602. Epub 2004 Mar 15. Abstract

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Comments

1. • Malu G. Tansey
     The University of Florida College of Medicine
   • Posted: 04 Jun 2008

   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. See also: Aggarwal, B.B., Samanta, A., Feldmann, M., TNFa, in Cytokine Reference, M.L. J. J. a. F. Oppenheim, Editor. 2000, Academic Press. p. 414-434. Aggarwal, B.B., Samanta, A., and Feldmann, M., TNF Receptors, in Cytokine Reference, J.J.a.F. Oppenheim, M., Editor. 2000, Academic Press: London. p. pp. 1620-1632. Tansey, M.G.a.W.-C., T., Cytokines in CNS Inflammation and Disease, in Central Nervous System Diseases and Inflammation, T.E.C. Lane, M.; Bergmann, C.; Wyss-Coray, T. , Editor. 2008, Springer: New York. p. 59-106. Corti, A. and P. Ghezzi, Tumor Necrosis Factor: Methods and Protocols. First ed. Methods in Molecular Medicine, ed. J.M. Walker. Vol. 1. 2004, Totowa, New Jersey: Humana Press. 279.

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   View all comments by Malu G. Tansey
2. • Hakon Heimer
     Schizophrenia Research Forum
   • Posted: 11 Jun 2008

   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).

   References:

   McGregor L, Saunders SA, Hunter JA, Murphy E. Acute psychosis in three patients receiving anti-tumour necrosis factor-alpha therapy. Rheumatology (Oxford). 2008 Aug;47(8):1254-5. PubMed.

   Brown AS. Prenatal infection as a risk factor for schizophrenia. Schizophr Bull. 2006 Apr;32(2):200-2. PubMed.

   Meyer U, Feldon J, Yee BK. A review of the fetal brain cytokine imbalance hypothesis of schizophrenia. Schizophr Bull. 2009 Sep;35(5):959-72. Epub 2008 Apr 11 PubMed.

   Oken RJ, Schulzer M. At issue: schizophrenia and rheumatoid arthritis: the negative association revisited. Schizophr Bull. 1999;25(4):625-38. PubMed.

   View all comments by Hakon Heimer

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References

News Citations

1. Breakthrough or False Hope? Etanercept Case Report Draws Scrutiny 21 Jan 2008
2. How Does Aβ Do Harm? New Clues on Insulin Signaling, Spines, Inflammation 14 Sep 2007

Webinar Citations

1. Targeting Tumor Necrosis Factor—A Therapeutic Strategy for AD 29 May 2008

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Other Citations

1. Yong Shen

External Citations

1. TNF Alzgene page
2. Medscape

Further Reading