Clearing Wastes Inside Cells
Like any household, cells produce a lot of garbage. Sooner or later, all the proteins and other constituents of our cells sustain damage from biochemical accidents that occur as side-effects of metabolism — and even some undamaged proteins will effectively become mere cellular clutter by simply outliving their usefulness. To avoid choking on their own garbage and also wasting valuable raw materials, cells are equipped with a variety of in-house recycling systems, which allow them to not only get rid of unwanted structures, but to break them down and reuse their building blocks to build fresh new cellular components.
A key part of this system is the lysosome, a versatile cellular recycling center whose high acidity and powerful enzymes allow it to safely break mangled molecules down into their molecular building blocks in a safe compartment separated from the rest of the cell. However, sometimes materials sent to the lysosome are so torturously tangled-up that not even its high acidity and potent hydrolytic enzymes can tear them apart. And if something can’t be broken down in the lysosome, there’s nowhere else for it to go: the stubborn waste just builds up inside the chamber, interfering with its function as its enzymes become diluted.
In some cases, the accumulating waste will cause the lysosome to swell to the point that its sheer size may interfere with cellular function by crowding together other components of the cell, creating an obstacle that impedes the efficient shuttling of organelles and materials around it. In other cases, materials inside or adjacent to the lysosome will damage the lysosomal membrane, triggering an inflammatory cascade. In more severe cases, the lysosome can be damaged to the point that it will rupture, spewing out not only the accumulated waste but also the toxic chemicals (acid and enzymes) that enable it to tear into the molecules that are sent to it. Releasing this destructive brew can damage or even destroy the cell.
Lysosomal membrane damage can trigger an inflammatory cascade inside the cell. IL-1β is an inflammatory cytokine. Credit: J Mol Med (Berl) 92(4):307-19.
Meanwhile, other kinds of damaged molecules aren’t effectively targeted to the lysosome in the first place, and aren’t effectively broken down by other cellular recycling systems (such as the proteasome). These wastes just build up in the main body of the cell, like hazardous waste stored on-site at a chemical plant in more and more haphazard places as the original storage shed fills up.
Exactly what kinds of waste accumulate within a given kind of cell — and where a given kind of waste builds up within it — depends on the metabolic processes that a given cell type is responsible for, just as a chemical plant that makes nonstick coatings produces different kinds of waste than does a paper mill. But whatever the underlying waste, the rising burden of toxic material and the increasing dysfunction of the cell’s recycling machinery progressively impair the function of the cell as a whole. This is an especially important problem for cells that have to last for a lifetime, such as heart muscle cells and nerve cells in the brain and elsewhere. And as more and more cells in a tissue become dysfunctional over time, the tissue’s specialized function is impaired, resulting in age-related disease centered on that tissue.
Consider what happens to macrophages, for example — immune cells that (amongst other things) protect our arteries from oxidized LDL particles when they become entrapped in the artery wall. Macrophages shield our arteries from these toxic particles by surrounding and swallowing them (their name means “big eater”) and then sending the particles to their lysosomes, where their cholesterol is extracted and removed from the cell via HDL particles in the blood — at least, at first.
The big problem is that macrophages are unable to regulate the number of oxidized LDL particles they take up: they just keep gobbling them up as they encounter them — until they suddenly can’t take any more. By that point, their lysosomes are engorged with either the sheer quantity of cholesterol from the unregulated uptake of oxidized LDL particles, or from the direct toxicity of oxidized cholesterol byproducts and other materials they can’t break down. As their lysosomes fail, arterial macrophages become dysfunctional and immobilized in the artery wall, and ultimately die outright. As sick and dying macrophages in the artery wall accumulate over time, the injury swells and festers, becomes inflamed and unstable, and eventually bursts, its contents spewing out and triggering clots that result in heart attacks and strokes.
Macrophages gorge on oxidized LDL cholesterol, which converts them to foam cells and initiates and exacerbates atherosclerotic plaque. Credit: N Engl J Med 337(6):408-16.
The buildup of these “foam cells” in the artery wall is the root cause of atherosclerosis, the disease of fatty, inflamed plaques in our arteries that is the number one killer in developed countries and is responsible for at least 40% of deaths worldwide. And there’s some evidence that the same problem of lysosomal storage of oxidized cholesterol products also plagues the resident macrophages in the liver and drives the deadly inflammation in fatty liver disease.
Similarly, the inability of other kinds of cells to break down their specific cellular waste products drives or contributes to diseases of aging involving the loss of function of those cell types. Prominent amongst these are neurodegenerative aging diseases, such as Alzheimer’s (AD) and Parkinson’s (PD) diseases. While not as well-known as the beta-amyloid aggregates that accumulate in plaques outside the cell, abnormal forms of the protein tau accumulate inside brain neurons as one part of aging — especially in people who develop clinical Alzheimer’s. Although there’s good evidence that beta-amyloid ultimately gets much of the overall degenerative process rolling, aberrant tau is more directly tied to cognitive dysfunction and neurodegeneration in AD. Additionally, while the insoluble beta-amyloid plaques that form outside of aging neurons are the most standout feature of the AD subset of degenerative brain aging, beta-amyloid also accumulates inside aging neurons in the form of smaller, soluble aggregates called oligomers, and it’s widely thought that these oligomers may be more of a problem than the plaques themselves.
There’s a similar set of problems in PD and related neurodegenerative aging diseases, which are collectively called “synucleinopathies” because they share in common aggregates comprised of yet another malformed protein called alpha-synuclein. When most people (including most doctors) think of PD, they think first of the motor symptoms of the disease: the shaking of the head and hands, slowness of motion, and bouts of freezing up. These symptoms are driven primarily by the loss of specialized neurons that inhibit the inappropriate firing of other neurons that control motion. Alpha-synuclein aggregates are instead tied with the less-well-known but also debilitating “nonmotor” symptoms of PD and other synucleinopathies, such as REM sleep disorder (in which sufferers’ brains fail to suppress their bodies’ movements during dreams); constipation and incontinence; loss of taste and smell; and cognitive and erectile dysfunction.
The course of motor and nonmotor symptoms in Parkinson’s disease. Credit: J Neurol Neurosurg Psychiatry 91(8):795-808.
Another important disease of aging that is substantially driven by intracellular aggregates is age-related macular degeneration (ARMD), which is the main cause of blindness in people over the age of 65. Here, the affected cells are retinal pigmented epithelial (RPE) cells, which are macrophage-like waste-clearing cells that “eat” the damaged membranes of photoreceptors —the cells that convert light coming into the eye into an electrical signal that the brain can turn into sensory images. The photoreceptors’ light-sensing function, however, depends on a chemical flip-flop between two forms of vitamin A — a flip-flop whose more reactive side can generate a range of aggregated molecules including A2E. As RPE gobble up and digest dying photoreceptor debris, they eventually become poisoned by A2E and its cousins, and they become dysfunctional, die, and decompose.
The progressive death of RPE cells leads to the creeping vision loss of ARMD for two distinct reasons. First, without functional RPE to maintain their membranes, rising numbers of the light-sensing cells in the eye become dysfunctional. Second, the dead RPE and their burden of waste products get deposited as drusen in the space between the light-sensing cells and the vascular layer of the eye, disrupting a sensitive part of the eye’s anatomy. In the advanced stages of “dry” ARMD — which is the form of ARMD that afflicts 80-90% of the people suffering from the disease, and which unlike the less common “dry” ARMD has no treatment — this critical region is progressively converted into a toxic graveyard of waste materials and dead cells. Blindness creeps progressively outward from the central focus area of the visual field until all sight is lost.
Lipofuscin is the longest-known but in many ways least-understood intracellular aggregate in human degenerative aging. This hodgepodge of damaged organelles, proteins, lipids, a small amount of carbohydrates, and traces of metals accumulates in critical long-lived cell types like brain neurons and heart and skeletal muscle cells.
But despite having known about it for more than a hundred years, scientists still don’t know very much about how lipofuscin is formed, or exactly from what, or what its molecular composition is. Lipofuscin fluoresces across a wide range of the spectrum, which suggests its composition is heterogenous — and those constituents are heavily crosslinked within and amongst each other, which makes it impossible to identify individual constituents, even when using fairly high-tech methods like mass spectroscopy. It’s generally agreed that incompletely-degraded mitochondria are one important constituent of lipofuscin — but this impression may mostly arise from the fact that scientists’ tools can readily pick up their molecular signature, while everything else in lipofuscin degenerates into an inchoate goo.
Whatever its origin, lipofuscin seems to grow once formed, probably because its surface is reactive and thus snags onto everything with which it comes in contact, like the Blob from the campy 1950s horror flick. One extreme example: German scientists found that lipofuscin occupies as much as 70% of the cellular volume of centenarians’ motor neurons!
It gets worse. Much of what scientists believe about lipofuscin is not based on studies of actual lipofuscin, but of artificial “lipofuscin” preparations produced by various lab techniques, often in easily-cultured cells — which are not the kinds of cells in which true lipofuscin accumulates! This is because lipofuscin is a paradoxically precious kind of waste: despite being a major constituent of the cells of people reaching the limits of current lifespans, it’s still only a tiny amount of material in absolute terms. Moreover, lipofuscin is only present in relatively large amounts in a few cell types, and only in the cells of people who reach what are today’s standards long-lived, which makes the material scarce. Plus, it’s technically difficult to get lipofuscin out of cells without somehow chemically altering or contaminating it with other cellular materials.
We don’t really even know what lipofuscin doing to our cells, though it seems hard to doubt that it’s up to no good. In experimental systems, lipofuscin causes cell death and impairs heart muscle cell contraction, and lipofuscin has been implicated in neurodegenerative aging. Remarkably, it has been reported that a high burden of lipofuscin in a transplanted heart is associated with rapid-onset coronary artery disease in the organ after transplant — a phenomenon called cardiac allograft vasculopathy that is responsible for a large share of the failures of transplanted hearts, and for 10-15% of post-transplant deaths.
So that’s a survey of some of the important forms of intracellular aggregate that form in the aging body and the kinds of age-related disease and debility to which they contribute. The question is, what can we do about all of this cellular garbage?
A Debunk on Cell Junk
First a note on a couple of things that won’t work. In recent years, the ages-long religious and health practice of fasting has been given a scientific facelift in the prolongevist biohacker community, based on the idea that fasting slows or even reverses the accumulation of intracellular aggregates by ramping up autophagy. And seeing a marketing opportunity, vendors of dietary supplements have jumped on the autophagy bandwagon too.
Fasting does have a demonstrable effect on autophagy, which is more than can be said for many dietary supplements that have been promoted to do so — and unlike supplements, most dietary regimens aren’t directly tied to a product for sale. But while fasting is quite effective in upregulating autophagy in young adults, the aging process progressively weakens its effects on the autophagy machinery in both mice and humans. (Aging also blunts the ability of fasting to activate ketosis). So ironically, the aging body becomes resistant to the effects of fasting at exactly the time of life when people are most likely to start looking for a way to intervene in their own aging process, and when they are most in need of tools to clear garbage out of their cells.
Moreover, a full 24 hours of complete (water-only) fasting in humans only boosts autophagy in one subset of circulating white blood cells (neutrophils). To nudge the full range of white blood cells into high levels of autophagy seems to take several days of complete fasting, at which point autophagy is also upregulated in muscle. Granted those timelines, the widely-promoted idea that the less rigorous practice of daily time-restricted feeding could give us a daily anti-aging kickstart via autophagy with no net reduction of energy intake seems unlikely. (By contrast, people on long-term Calorie restriction enter into a state of elevated autophagy every morning after undergoing the unremarkable overnight “fast” of a night’s sleep).
Another source of misunderstanding comes from the fact that even a scientific study that finds that some supplement or fasting program increases “autophagy” generally implies a lot less than many people think it does. The standard methods of testing for an effect on autophagy only show whether some intervention mobilizes the machinery that drags junk to the lysosome: they don’t actually show that the lysosome takes up the waste that’s delivered to it, let alone that it successfully degrades such wastes if it does accept them. And by definition, the kinds of waste that accumulate in our cells with age and then sicken and kill us are exactly the ones that our cells do a poor job of clearing out themselves, either because our cells lack any machinery to degrade such wastes in the first place, or because the inevitable residuum of partially-degraded damaged material becomes increasingly resistant to that machinery with the accumulation of further damage over time. As autophagy scientist Tim Sargeant put it: “A therapy could, for instance, help the cell to package more trash. But if your trash compactor isn’t working properly, you’re just going to end up with a room full of trash bags.”
Additionally, while some interventions that have substantial anti-aging effects in lab animals (such as rigorous Calorie restriction and the drug rapamycin) are well-known to upregulate the complete autophagy process, it’s important to remember that these interventions do a lot of other things in the aging body as well. We still don’t really know how much of these interventions’ anti-aging benefits are the result of autophagy versus these interventions’ myriad other effects. Rapamycin, for instance, inhibits protein synthesis significantly more strongly than it takes the brakes off of autophagy, and alternative drugs that ramp up autophagy but have less effect on protein synthesis than rapamycin have not been tested for anti-aging effects.
Another pretender to the LysoSENS space is the drug centrophenoxine, which has long been touted as able to somehow remove lipofuscin from the cell. This was reported in both cells and lab rodents in the 1970s, and the drug was promptly popularized for lipofuscin removal by books and public figures in the early healthy longevity movement. This notion continues to be perpetuated to this day, despite the fact that later scientists found that centrophenoxine has no effect on lipofuscin in the cerebellum, hippocampus, or frontal cortex of the brain of rodents, nor in the only nonhuman primate study in which it was tested.
When scientists specializing in lipofuscin revisited the question at the turn of the current century, they found that while centrophenoxine did slow the accumulation of lipofuscin over time in rat and human cells cultured in a dish (and note that this happened when the cells were continuously bathed in the stuff, which doesn’t model what cells would actually be exposed to when a person takes centrophenoxine pills once or twice a day), centrophenoxine fails to eliminate pre-existing lipofuscin as the earlier studies had claimed. There was also no sign that centrophenoxine exerts effects on the autophagy machinery or on the lysosome, as you would expect a lipofuscin-buster to do: there was no change in the formation of autophagic vacuoles that would carry it to the lysosome, and no evidence that lysosomes were newly engaged in degrading their contents.
Centrophenoxine has no effect on the cell’s disposal of autophagic “trash bags” through the lysosome. Credit: J Anti-Aging Med 2(3):265-273.
Why the difference? There’s no way to be sure what was happening in the earlier positive studies, but the investigators pointed to several limitations to the lab techniques used by the 1970s scientists as possible sources of experimental artifact — limitations to which their own study using modern techniques was not vulnerable.
Enough of these feeble or illusory solutions. What would it take to really deal with the stubborn, toxic waste products that accumulate in our cells with age?
The Rejuvenation Biotechnology Solution
The lysosome’s central function is to break down cellular wastes, yet many kinds of intracellular aggregate accumulate in our cells with age. For those wastes that reach the lysosome but aren’t degraded once they get there — or aren’t degraded completely, and slowly become more snarled and stubborn as they persist and interact with other, different kinds of waste — the most direct solution is conceptually straightforward. Essentially, we must fortify the lysosome with new enzymes designed to degrade those exact wastes.
Fortunately, we know of at least one source of existing enzymes that are already used by other organisms to break these materials down: enzymes present in the soil microbes that help to decompose dead bodies. If such enzymes did not exist, the planet would be ankle-deep in the undegraded lysosomal wastes left over from the half-decomposed bodies of humans and other animals that have died over the course of 600 million years of animal life on this planet. So a key strategy identified by Dr. de Grey in one of his earliest published papers is to identify the enzymes that these organisms use to digest lysosomal wastes and tweak them at the molecular level so they can be delivered to and function within the human lysosome, where they’re needed.
There’s already a good proof-of-concept for this approach: the use of “enzyme replacement therapy” (ERT) to treat lysosomal storage disorders (LSDs) such as Gaucher and Fabry diseases. LSDs are rare congenital disorders in which patients are born with missing or defective genes that encode some element of the cell’s machinery for handling cellular waste. Some of these are genes for an enzyme that degrades particular wastes in our lysosomes; others are genes for proteins involved in trafficking such wastes into or out of the lysosome. Either way, the result is that LSD patients’ lysosomes are unable to break down and recycle specific kinds of wastes that most people’s lysosomes handle with high efficiency, and the waste product instead accumulates in the lysosome or in other parts of the affected cell types. Over time, victims fall prey to a range of disabilities corresponding to dysfunction of the cells in which those wastes are produced and accumulate — just as happens in aging, except that many of these diseases lead to neurological dysfunction and death in childhood, or in the first few decades of life.
Today, many LSDs can be successfully treated with ERT, in which doctors give patients engineered versions of the missing or defective enzymes that have been modified to allow them to survive in circulation, be taken up by the cell, and be transported into the lysosome. Scientists continue to work to make these treatments more convenient and effective by using gene therapy to let the affected cells produce the enzyme themselves.
Enzyme replacement therapy (ERT) removes aggregates and restores cell function in Fabry disease. Credit: Fabry Institute
Instead of replacing enzymes that most people are born with in people who genetically lack them, many potential LysoSENS rejuvenation biotechnologies are forms of ERT that deliver entirely new enzymes to target cells. Such enzymes would be engineered to break down wastes that no one’s lysosomes are currently able to process, and that therefore accumulate in our cells with age and cause dysfunction and disease.
For example, enzymes capable of breaking down the toxic cholesterol byproducts responsible for foam cell formation would give superpowers to macrophages. Such enzymes would rehabilitate existing foam cells, allowing them to mobilize safely out of atherosclerotic lesions, and healthy enzyme-enhanced macrophages would be able to remove toxic cholesterol products from the artery wall as well as dead and dying foam cells without harm to themselves. Nascent atherosclerotic plaques would not progress, and existing ones would regress.
Another engineered enzyme would remove A2E and related compounds from the RPE cells in the eye, which would arrest and reverse RPE cell loss; macular degeneration would be prevented, and its early stages reversed.
A different approach is needed to clear out aggregates that accumulate inside aging cells but outside of the lysosome. Examples include aberrant forms of the proteins tau and alpha-synuclein, which accumulate inside neurons and are drivers of key aspects of Alzheimer’s and Parkinson’s as well as other neurological disorders of aging, including the so-called “normal” cognitive and motor decline of aging that doesn’t reach the “disease” threshold in a given person.
It’s not always clear why these aggregates aren’t sent to the lysosome and degraded. Some kinds of aggregate may only build up in the main body of the cell once the lysosome begins to fail with age due to the accumulation of those aggregates that are sent to the lysosome but not successfully degraded. Such lysosomes are known to become dysfunctional and to stop accepting delivery of new materials for degradation, which in this case might include the precursors of the aggregates that tend to accumulate with age outside of the neurons, reminiscent of the kind of logistical snarls that have bedeviled supply chains as the industrialized world has emerged from the pandemic. In cases where this is the core problem, it should clear up once we purge aging lysosomes of these other, primary kinds of waste, freeing them up to once again accept aggregates that currently sit outside the lysosome like so many container ships adrift in the waters surrounding the Port of Long Beach.
However, some of the intracellular aggregates that accumulate in the main body of the cell instead of in the lysosome them may not be targeted to the lysosome in the first place, because our cells have never evolved machinery to drag them there. Such cases would require novel strategies for their more direct removal, which is a tricky business because it would have to be done without the protective membrane and otherwise-toxic concentrations of acid and powerful enzymes that make the lysosome so effective at degrading damaged molecules. As we will discuss below, a strategy to potentially handle such cases has only recently emerged.
Where We Are Now
No LysoSENS therapy has yet become available for human use, or even gotten as far as human trials (with some partial exceptions — more below). But a number have gotten well into animal testing and some could reach clinical trials in the next few years.
LYSOCLEAR to Degrade A2E
To date, the candidate LysoSENS therapy that came closest to becoming a licensed therapeutic for humans was LYSOCLEAR. SRF-funded research found that enzymes capable of breaking down peroxides were also effective in destroying A2E in vitro. Kelsey Moody and his colleagues at Ichor Life Sciences selected the best of these (the common enzyme manganese peroxidase, or MnP) and engineered it to allow it to pass harmlessly through the circulation, penetrate cells, and reach the lysosome.
Dubbed LYSOCLEAR, this LysoSENS candidate showed great promise. When tested in RPE cells that were pre-loaded with A2E, the engineered enzyme broke down A2E and several related compounds with limited toxic effects. Ichor scientists then tested it in mice with a genetic form of macular degeneration by injecting it directly into their eyes; this may sound like a risky procedure, but patients with the less common “wet” form of macular degeneration receive several injections of antibodies that block blood vessel growth by the same route each year without incident. Remarkably, just six weekly doses of LYSOCLEAR eliminated nearly a third of the A2E and similar stubborn wastes in the mice’s eyes.
Unfortunately, the LYSOCLEAR program stalled out over two problems. First, although the treatments did not damage the light-sensing neurons or the architecture of the retina, something about the injections still caused the treated animals’ eyesight to worsen more than in the control group, despite the removal of A2E. The reasons for this were not clear, but may have been related to the escape of the engineered enzyme in its active form out of the eyes and into the circulation, where it may have caused an immune reaction or been directly toxic to the surrounding tissue.
It’s possible that whatever the problem was, it might have been solved by simply testing LYSOCLEAR in a larger animal than the mouse, whose eyes are so small that the injections themselves might have caused enough damage to the eye’s barriers to let the enzyme escape. It’s also possible that such testing would have allowed the Ichor scientists to more confidently nail down the cause of the toxicity and address it.
But when Ichor tried manufacturing LYSOCLEAR at the quality standards required for human drug testing, they found that the branching of the sugars that allow the enzyme to enter cells and be directed to the lysosome was highly inconsistent — so inconsistent, in fact, that it wouldn’t be acceptable for use as a human therapy. Even when they tried an entirely new biomanufacturing technique to produce the enzyme, they still couldn’t get the branching pattern consistent. So despite its promise, these “Chemistry, Manufacturing, and Controls” (CMC) problems spelled the end for the original LYSOCLEAR enzyme. Ichor has moved on to looking at alternative enzymes that might do the essential task (safely breaking down A2E) but be free of this limitation.
Repair Biotechnologies’ Cholesterol-Degrading Platform
Another important startup with a promising LysoSENS candidate therapy is Repair Biotechnologies, founded by prolongevist advocate Reason of Fight Aging! to turn promising basic research into a working rejuvenation biotechnology. Dr. Richard Honkanen, a professor at the University of South Alabama, was struck by the discovery that Mycobacteria —the pathogen responsible for tuberculosis — can persist for decades in the challenging environment inside human macrophages by living off of energy and carbon they derived from breaking down the macrophages’ pools of cholesterol.
Unlike Mycobacteria, human cells aren’t able to actually break down excess cholesterol: instead, the lysosomes of healthy macrophages and other cells either re-use it as a raw material or send any excess to the liver, where can it be eliminated from the body via the bile. But it’s exactly the inability of foam cells to process an overwhelming burden of cholesterol that gives them their “foamy” appearance and drives them to become the basis for atherosclerotic plaque.
Thinking about this, it occurred to Dr. Honkanen that if the enzymes that allow these pathogens to degrade macrophage cholesterol could be identified, such enzymes could be engineered into human macrophages to allow them to clear out excess cholesterol instead of being sickened by it. With some extra engineering, the enzymes could be held in check unless cholesterol levels rose to excessive levels, preventing them from unintentionally depleting the macrophage or the body of cholesterol needed for essential cellular structures.
Engineered enzymes could degrade cholesterol products, rehabilitating macrophages and allowing them to exit the plaque. Modified by Anne Corwin from N Engl J Med 360(11):1144-6.
Dr. Honkanen won an NIH Transformative Research Award to pursue a LysoSENS strategy for the rescue of foam cells based on this hypothesis, and with his graduate students Brandon D’Arcy and Mark Swingle he was able to identify the key Mycobacteria enzyme, engineer a version of it into human liver cells and macrophages, and demonstrate that the enzyme enabled these cells to degrade cholesterol into harmless breakdown products. After the technology was spun out to Repair Biotechnologies, a mouse model of atherosclerosis was allowed to develop severely advanced plaques and then given this “Cholesterol-Degrading Platform” (CDP) as a gene therapy. Remarkably, CDP very rapidly eliminated half of the pre-existing atherosclerotic plaque in the root of the mice’s aortas!
In the APOE-knockout mouse model of atherosclerosis, Repair Biotechnologies’ CDP rapidly cut the pre-existing atherosclerotic plaque at the aortic arch by 48%. Credit: Repair Biotechnologies.
Meanwhile, for the last decade the biotech industry and some Big Pharmas have invested a lot of capital on developing true, direct “damage-repair” therapies to clear some of the same malformed proteins that occur as intracellular aggregates, but targeting them outside the cell. This isn’t as off base as it sounds: both aberrant tau and alpha-synuclein aggregates do accumulate outside of neurons as well as inside, and it’s generally accepted that the extracellular presence of these aggregates is also harmful. Plus, capturing and removing these aggregates in the synapses where neurons communicate with one another would slow down or arrest the weed-like cell-to-cell spread of these aggregates across the aging brain.
The other reason that industry players have taken this approach instead of targeting these aggregates inside of neurons is that there’s an established way to do it: use active vaccines and monoclonal antibodies to bind the extracellular aggregate and drag it out of the brain — a now relatively mature AmyloSENS approach, albeit one that has not yet led to breakthrough therapies. By contrast, it’s been hard to come up with good ways to target aggregates like these that accumulate inside of neurons but outside of the lysosome. Junk that reaches the lysosome can be safely targeted for destruction with enzymes without worrying about breaking down essential proteins in the rest of the cell. Plus, such therapeutic enzymes can take advantage of the lysosome’s high acidity and its extensive complement of protein-degrading enzymes to finish the job once one or a few key bonds in the abnormal protein are cleaved. By contrast, there’s just a lot more opportunity to cause problems when you send a wrecking crew after aggregates squatting in the middle of the cytosol, where the cell’s organelles and other functional structures are busy carrying on the business of life.
SENS Research Foundation Research
As we mentioned early on, the most widely-applicable strategy for removing stubborn wastes accumulating inside cells is the one put forward by Dr. de Grey in 2002: fortifying the lysosome with engineered versions of enzymes discovered in the environment that are able to cleave the key vulnerable bonds in a given aggregate. Enthusiasm for this original ERT-like LysoSENS approach mounted following the National Institute on Aging’s sponsorship of the fourth SENS Roundtable, a 2004 meeting focused on this intervention attended by high-caliber scientists who contributed to the discussion and signed on to the resulting detailed proposal for the development of the therapeutic platform.
Before SRF was founded as a separate, research-oriented organization from the prize-oriented Methuselah Foundation, some of the first scientific projects that SRF funded as part of MF took this classical ERT-like LysoSENS approach. The early work on 7KC and A2E by John Schloendorn and others at Arizona State University identified a variety of fungal peroxidases and other enzymes that eventually led to LYOSOCLEAR. Later, SRF funded focused work on 7KC in Pedro Alvarez’s lab at Rice University that led to the discovery of an enzyme that detoxified it in fibroblasts.
More recently, we invested in lipofuscin research at Rice University. Although the cell culture system the researchers used was very artificial, it was one of the first studies looking at the effects of cyclodextrin on age-related intracellular aggregates. This work informed later research conducted by Dr. Matthew O’Connor and others at SRF’s Research Center on novel cyclodextrin formulations as an alternative LysoSENS approach for atherosclerosis. This work has advanced rapidly in the last couple of years, and has been spun out into the biotech startup Cyclarity Therapeutics (formerly Underdog Pharmaceuticals) to get it ready for human clinical trials.
Instead of breaking down 7KC, Cyclarity’s new molecules highly selectively drag 7KC from the cell by capturing it inside their barrel-like structures (perhaps by removing it from the affected cell’s cellular or lysosomal membranes — Cyclarity scientists are still working to uncover the exact mechanism of action). Because of its high toxicity and potential to interfere with lysosomal function, pulling 7KC from foam cells might be enough to restore normal cholesterol processing, returning them to health. Based on prior work using a more common form of cyclodextrin to treat children with a cholesterol lysosomal storage disorder, the Cyclarity team expects that the novel cyclodextrins would then carry the 7KC safely to the liver for excretion with the bile.
And at the Buck Institute, SRF funded Dr. Julie Andersen to develop a system for tracking the complete autophagy process in cells. As we discussed above, “autophagy” strictly speaking is only the process of packaging up wastes in order to drag them to the lysosome for recycling. Researchers have often been fooled by this: seeing the machinery of autophagy activated in cells, they have jumped to the conclusion that the waste will also be taken up by the lysosome and that the lysosome will then proceed to break it down.
In reality, a large jump in levels of autophagic proteins can often be evidence of the opposite: a traffic jam in autophagy resulting from a choke point at one step or another in the complete autophagy process. With SRF funding, Dr. Anderson developed a dual-labeling system to get a better handle on each step in the process in neurons, tracking the production of the vesicles that form to surround wastes, the fusion of these vesicles with lysosomes to deposit those wastes, and the acidification and functionality of the lysosomes after engulfing such cargo.
Currently, SRF is funding several new projects in the LysoSENS space. In our Research Center, Dr. Amit Sharma and colleagues are working on a novel strategy identified by Dr. de Grey to break down aggregates that accumulate inside the cell but outside the lysosome — a tough problem that needs to be overcome for such critical damaged molecules as aberrant tau and alpha-synuclein aggregates, for which no one had previously identified a strong damage-repair strategy.
Meanwhile, we are funding work in Dr. Tilman Grune’s lab at the German research institute DiFE to identify enzymes capable of breaking down lipofuscin. Dr. Grune is using the classic LysoSENS strategy of discovering microbes that can survive on lipofuscin as their sole energy source, and then isolating and characterizing the enzymes that allow such microbes to do it. Dr. Grune is able to do this research and get results on which we can reliably build therapies because of two key developments: one of innovation, and one of access.
First, while previous scientists have traditionally been stuck working on various kinds of artificial “lipofuscin” preparations, Dr. Grune has developed a novel extraction method that finally allows him to get real lipofuscin directly out of cells with no artifacts or contaminants. Second, he has secured access to large enough amounts of human and horse heart tissue to yield sufficient quantities of authentic lipofuscin to carry out the requisite research.
This ability to work with real human lipofuscin means that for the first time, any enzymes that Dr. Grune identifies as capable of breaking down this material will have credible clinical potential, whereas the fact that an enzyme can degrade an artificial “lipofuscin” preparation might mean nothing at all about its ability to break down actual lipofuscin in cells. Once identified, such enzymes might be engineered to do the same thing in human cells, clearing them of this stubborn waste and rejuvenating the aging cell’s function.
Between new research projects in critical areas of rejuvenation biotechnology and the emergence of biotech startups that have seized the fruits of previous LysoSENS research and run with it toward the clinic, the pipeline for this strand of SENS rejuvenation biotechnologies is flowing smoothly toward working longevity therapeutics for cellular, tissue, and human health.