Glucosepane Crosslinks and Undoing Age-Related Tissue Damage

Yale University

Principal Investigator: David Spiegel
Research Team: Prof. Jason Crawford, Nam Kim, Venkata Sabbasani, Matthew Streeter

The long-lived collagen proteins that give structure to our arteries and other tissues are continuously exposed to blood sugar and other highly reactive molecules necessary for life. Occasionally, these sugar molecules will bind to tissue collagen by sheer chemistry, and if not quickly reversed these initial links will in turn bind adjacent strands of collagen, reducing the range of motion of the tissue like the legs of runners in a three-legged race. As a result, these tissues slowly stiffen with age, leading to rising systolic blood pressure, kidney damage, and increased risk of stroke and other damage to the brain.

It is currently thought that the single most common of these Advanced Glycation End-products (AGE) crosslinks is a molecule called glucosepane. A rejuvenation biotechnology that could cleave glucosepane crosslinks would allow bound arterial proteins to move freely again, maintaining and restoring the elasticity of the vessels and preventing the terrible effects of their age-related stiffening. SRF has provided funding to the Yale GlycoSENS group for several years now, in order to develop tools necessary for enabling the development of glucosepane-cleaving drugs.

Research Highlights:

The Yale group’s first major milestone – the first complete synthesis of glucosepane itself – was a sufficient tour de force to earn publication in the prestigious journal Science. In 2018, they were able to scale up this pilot-level method to produce glucosepane in quantities useful for industrial production, and also to synthesize three conformational variants (diastereomers) of glucosepane that may occur in vivo. They are now working on two more such variants. They have also used their synthetic glucosepane to develop glucosepane-targeting antibodies capable of labeling glucosepane in aging tissues, which they are now working up into a monoclonal antibody for mass production that will be compatible with human metabolism and will allow researchers to track the effects of potential glucosepane-cleaving drugs.

Finally, and most excitingly, they have now identified a lead candidate glucosepane-cleaving biocatalyst, and completed the evaluation of seven significant variants and their AGE-breaking mechanism. Today, work continues on synthesizing pentosinane (another common AGE crosslink) and additionally on the AGE-related compounds iso-imidazole and 2-aminoimidazole.

Maximally Modifiable Mouse

Applied StemCell, Inc.

Principal Investigator: Dr. Ruby Yanru Chen-Tsai

The CRISPR/Cas9 gene editing system has the ability to make precisely-targeted changes in the genetic sequence – a clear strength of the platform – but is limited in its lack of an obvious delivery mechanism. It’s reasonably easy to use CRISPR/Cas9 to modify individual cells, but there is no known (or clearly-foreseeable) way to deliver the system to human tissues in vivo while still retaining strong precision and without the risk of either silencing or mutating introduced or non-targeted genes.

Moreover, CRISPR/Cas9 is really only able to make relatively small changes to an existing gene. That’s great for correcting small but catastrophic mutations in existing genes, or rendering genes with a toxic gain-of-function inoperable — but it’s not much use for delivering the new genes that will be necessary to take advantage of it for delivery of rejuvenation biotechnologies.

The Maximally-Modifiable Mouse project aims to overcome this problem by allowing us to make use of a powerful gene insertion system (the integrase) used by phages — viruses that target bacteria as their hosts. The mycobacteriophage Bxb1 catalyzes precisely-targeted, one-way insertion of even very large genes into the host genome. Unfortunately, mammals lack the genetic “docking sites” that this integrase targets. To enable the development of models of diseases of aging and the rapid testing and eventual human delivery of rejuvenation biotechnologies, SENS Research Foundation has been funding Stanford gene therapy spinoff Applied StemCell (ASC) to create a line of Maximally-Modifiable Mice (MMM). The MMM will have two of the needed docking sites engineered directly into their genomes, which will then be ready for the insertion of new therapeutic transgenes at any time during the lifespan.

Research Highlights:

Currently, ASC is testing the ability of the system to deliver and integrate convenient test genes into the mice’s cells in vivo. They will then test the expression and functional protein production of those genes. We are especially excited by the potential to use this technology to both develop better models in which to test the allotopically-expressed mitochondrial genes that our in-house Mito team has been testing in cells, and to deliver those genes and actually test them in such mice.

Remediation of Aberrant Intracellular Tau

Buck Institute for Research on Aging

Principal Investigator: Dr. Julie Andersen
Research Team: Cyrene Arputhasamy, Manish Chamoli, Anand Rane

Aging brains accumulate aggregates composed of aberrant forms of the protein tau, both inside and outside of neurons. These aggregates are an important driver of “normal” age-related cognitive decline, as well as neurodegenerative diseases of aging like Parkinson’s (PD) and Alzheimer’s (AD) diseases. A number of rejuvenation biotechnologies targeting aberrant tau outside of cells are currently in clinical trials, with the idea that capturing these “seeds” of tau aggregates will interrupt its “infectious” cell-to-cell transmission. But to prevent the problem entirely – and eventually reverse it – requires new strategies to target aberrant tau inside of brain neurons.

Dr. Andersen’s team is being funded by SRF to test the idea that this tau accumulation may result from age-related dysfunction of the cellular “recycling centers” (lysosomes) due to the buildup of other kinds of intracellular aggregates, such as beta-amyloid, the other major damaged protein characteristic of the AD brain. If this is indeed the case, then the most effective remediation method for aberrant tau could entail using rejuvenation biotechnology to target these primary aggregates, thus allowing the cell to clear out its own burden of aberrant tau once lysosomal function is restored.

Neurons of patients with AD and other neurodegenerative aging diseases are often full of autophagosomes (APGs), the vesicles that form around targets for autophagy and in which they are dragged to the lysosome for degradation. This buildup is thought to result from a failure of lysosomal function, as the already-overburdened organelle refuses to take up any more cargo.

Research Highlights:

The Andersen lab has developed lines of human and rat neuronal cells that produce APGs with molecular tags that allow them to track the production and disappearance of APGs in neurons. They can use these tags to screen for compounds that increase the successful trafficking of APGs and their cargo to the lysosome. Compounds that pass this preliminary test will then be evaluated in neurons treated with small, soluble beta-amyloid aggregates, to see if these compounds will prevent or reverse the formation of insoluble aggregates of both beta-amyloid and tau.

Target Prioritization of Tissue Crosslinking

The Babraham Institute

Principal Investigator: Jonathan Clark
Research TeamMelanie Stammers

As discussed in the project summary for “Glucosepane Crosslinks and Undoing Age-Related Tissue Damage”, adventitious crosslinking of collagen (and elastin) contributes to the slow stiffening of our arteries and other tissues with age. Some of these crosslinks are the kind of chemistry that can happen spontaneously (like AGE crosslinking), but others are the unintended consequences of metabolic processes that modify collagen — either as “collateral damage,” or to help us get through short-term problems at the cost of contributing to the long-term burden of crosslinking damage that eventually compromises function. Amidst all of this, it’s not obvious that the sheer number of crosslinks of a given kind is a good measure of how high a priority it is for rejuvenation biotechnology: some crosslinks may have a disproportionate effect on tissue elasticity depending on where they occur in the protein strand, how tightly they bind, and how much they interfere with the body’s ability break down and renew the tissue.

Recognizing the importance of prioritizing our targets, SRF is funding a systematic study of this question in the tissues of “normally”-aging, nondiabetic mice at the Babraham Institute in Cambridge. The mice have been administered labeled building blocks for protein, which are then incorporated into extracellular matrix proteins, whose turnover can then be studied. The study has required the development and validation of new experimental methods and assays, which were published in a Royal Society of Chemistry journal in 2018.

Research Highlights:

An early and surprising finding is that crosslinks that one might think permanent in tissue are continuously being broken apart and re-forming under the stress and strain of normal activity: it is the balance between these reversible crosslinks and the truly irreversible ones that gives rise to many of the changing mechanical properties of aging collagen. They have also confirmed the expectation that the crosslink profile in each tissue is distinct from others (which is only partially explained by the tissue-specific mixture of elastin and collagen), and that both the mixture of proteins and the pattern of protein-specific crosslinks changes with age. Importantly, some of the crosslinks that have been reported by others to accumulate in aging tissues were not detected. They are also complementing chemical analysis of the tissues with functional tests of the effect of these crosslinks on tissue mechanical function. Drilling down into these issues will be critical to identifying the next targets as glucosepane crosslink-breakers enter into animal testing.

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