RIPK1

The innate immune system is intrinsically fascinating, dealing with invaders long before antibodies or cytotoxic cells are on the scene.  It is even more fascinating to a chemist because it works in part by forming amyloid inside the cell.  And you thought amyloid was bad.

The system becomes even more fascinating because blocking one part of it (RIPK1) may be a way to treat a variety of neurologic diseases (ALS, MS,Alzheimer’s, Parkinsonism) whose treatment could be improved to put it mildly.

One way to deal with an invader which has made it inside the cell, is for the cell to purposely die.  More and more it appears that many forms of cell death are elaborately programmed (like taking down a stage set).

Necroptosis is one such, distinct from the better known and studied apoptosis.   It is programmed and occurs when a cytokine such as tumor necrosis factor binds to its receptor, or when an invader binds to members of the innate immune system (TLR3, TLR4).

The system is insanely complicated.  Here is a taste from a superb review — unfortunately probably behind a paywall — https://www.pnas.org/content/116/20/9714 — PNAS vol. 116 pp. 9714 – 9722 ’19.

“RIPK1 is a multidomain protein comprising an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The intermediate domain of RIPK1 contains an RHIM [receptor interacting protein (rip) homotypic interaction motif] domain which is important for interacting with other RHIM-containing proteins such as RIPK3, TRIF, and ZBP1. The C-terminal DD mediates its recruitment by interacting with other DD-containing proteins, such as TNFR1 and FADD, and its homodimerization to promote the activation of the N-terminal kinase domain. In the case of TNF-α signaling, ligand-induced TNFR1 trimerization leads to the assembly of a large receptor-bound signaling complex, termed Complex I, which includes multiple adaptors (TRADD, TRAF2, and RIPK1), and E3 ubiquitin ligases (cIAP1/2, LUBAC complex).”

Got that?  Here’s a bit more

“RIPK1 is regulated by multiple posttranslational modifications, but one of the most critical regulatory mechanisms is via ubiquitination. The E3 ubiquitin ligases cIAP1/2 are recruited into Complex I with the help of TRAF2 to mediate RIPK1 K63 ubiquitination. K63 ubiquitination of RIPK1 by cIAP1/2 promotes the recruitment and activation of TAK1 kinase through the polyubiquitin binding adaptors TAB2/TAB3. K63 ubiquitination also facilitates the recruitment of the LUBAC complex, which in turn performs M1- type ubiquitination of RIPK1 and TNFR1. M1 ubiquitination of Complex I is important for the recruitment of the trimeric IκB kinase complex (IKK) through a polyubuiquitin-binding adaptor subunit IKKγ/NEMO . The activation of RIPK1 is inhibited by direct phosphorylation by TAK1, IKKα/β, MK2, and TBK1. cIAP1 was also found to mediate K48 ubiquitination of RIPK1, inhibiting its catalytic activity and promoting degradation.”

So why should you plow through all this?  Because inhibiting RIPK1 reduces oxygen/glucose deprivation induced cell death in neurons, and reduced infarct size in experimental middle cerebral artery occlusion.

RIPK1 is elevated in MS brain, and inhibition of it helps an animal model (EAE).  Mutations in optineurin, and TBK1 leading to familial ALS promote the onset of RIPK1 necroptosis

Inflammation is seen in a variety of neurologic diseases (Alzheimer’s, MS) and RIPK1 is elevated in them.

Inhibitors of RIPK1 are available and do get into the brain.  As of now two RIPK1 inhibitors have made it through phase I human safety trials.

So it’s time to try RIPK1 inhibitors in these diseases.  It is an entirely new approach to them.  Even if it works only in one disease it would be worth it.

Now a dose of cynicism.  Diseased cells have to die one way or another.  RIPK1 may help this along, but it tells us nothing about what caused RIPK1 to become activated.  It may be a biomarker of a diseased cell.  The animal models are suggestive (as they always are) but few of them have panned out when applied to man.

 

Is a rational treatment for Multiple Sclerosis in our future?

Two very recent papers taken together point the way to a rational treatment of multiple sclerosis (and probably all autoimmune disease). The short story:
Paper #1 found a way to find the antigen or antigens patients with MS are reacting to
Paper #2 found a way to selectively impair the response to an inciting antigen without clobbering the whole immune system

Some history: Some evening in 1966 or 1967 a fellow neurology resident and I were sitting on the ward having dealt with the complications of high doses corticosteroids for a case of optic neuritis (often the first sign of MS). I said, some day they’ll look at what we’re doing the way we look at docs of 200 years ago using leeches (and bloodletting). As a kid, I remember my parents driving into Philly. Shortly after getting over the Ben Franklin bridge we’d pass a pharmacy offering leeches on its sign.

It was obvious even back then that MS in some way was an attack by the immune system on the brain. Finding the particular antigen the system was reacting to would lead us to the cause and hopefully less simplistic treatment than clobbering the immune system. We didn’t know all the proteins we had or even how many, so people would look for antibodies to a variety causes (which they’d arrived at by reasoning, not data). Increased antibody titers to a variety of viruses were found, but that led nowhere. No one ever isolated a virus from MS brain, although sightings on electron microscopy were eagerly reported. Eventually it became obvious that the immune system was on high alert with increased antibodies to lots of things.

This leads to paper #1 [ Proc. Natl. Acad. Sci. vol. 113 pp. 2188 – 2193 ’16 ] To make a long story short they used something called the Human Protein Atlas Program to find what proteins the antibodies in MS patients were reacting to. So rather than having a theory about what MS patients might be reacting to and testing it, they looked at all proteins and watched. It’s the difference between being a Greek philosopher reasoning things out from first principles and collecting data. Only when the technology is available can you stop a priori theorizing and just look. Don’t be too hard on the earlier researchers, they didn’t have the tools.

The found that MS patients were reacting to a protein called anoctamin2, which actually showed increased expression near and inside the demyelinating plaques of MS.

For the gory details keep reading, otherwise skip to **** where I’ll discuss paper #2

Gory details — The Human Protein Atlas produces human protein fragments, selected on the basis of their low similarity to other proteins in the proteome. [ Science vol. 347 1260419 (23 Jan) ’15 ] The atlas hopes to find out where and how much of each our proteins is at the tissue and cellular level. It is based on antibody based profiling on tissue microarrays (of proteins?). This based on transcript expression (RNA-Seq), and immunohistochemistry (24,028 antibodies coresponding to 16,975 protein coding genes). 44 tissues were studied. The antibodies produced more than 13 million tissue based mmunohistochemistry images. They also report subproteomes (secreted proteins n = 3,171, and membrane bound proteins n = 5,570). Interstingly there was an overall concurrence between mRNA and protein levels for a given gene product across various tissues.

The PNAS paper profiled 2,169 plasma samples from MS cases and population based controls (with neurologic disease) using bead arrays built with 384 human protein fragments seleted from an initial screening with 11,520 antigens. There was increased reactivity to anoctamin2 (aka TMEM16B) in MS vs. controls (by how much?). This was corroborated in independent assay with alternative protein constructs and by epitope mapping with peptides covering the identified region of anoctamin 2.

ImmunoFLuorescence in human MS brain tissue showed increased anoctamin2 expression as small cellular aggregates near and inside MS lesions. The controls had other neurologic disease. There was a 5.3 fold change in fluorescence intensity in the MS group. The antibodies are directed against the amino terminal region.

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Paper #2 — [ Nature vol. 530 pp. 422 – 423, 434 – 440 ’16 ] basically found a way to knock out the immune system’s response to a single antigen — not all of them. The point is that just an antigen by itself isn’t enough to turn the immune system on. A costimulatory molecule must also be present on the antigen presenting cell. If it isn’t there the immune system is actually turned off by forming regulatory T cells (which even though they are part of the immune system they actually turn it off).

One can form models of human autoimmune disease in mice. Two such are EAE (Experimental Allergic Encephalomyelitis) formed by giving the animal myelin basic protein (a constituent of myelin which is attacked in MS), and rheumatoid arthritis (formed by giving collagen to the animals). What is so great about this paper is that MHC II carrying peptides from collagen suppress disease in a mouse model of rheumatoid arthritis, but NOT in mice with EAE. MHC-II carrying CNS antigen peptides control EAE but not collagen induced arthritis.. In addition neither treatment impaired the immune response to infection — something that almost always happens when you clobber the immune system.

Well it’s a long way from the lab to the bedside, but imagine finding what the immune system is reacting to and stopping it (without stopping the immune system). That’s what these two papers portend. Exciting times.