07 Apr 2023

Queen Silvia of Sweden opened the 17th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 28 to April 1 in Sweden's southwestern city of Gothenburg. The queen, age 79, flew in from Stockholm through a snowstorm, toured the labs of Kaj Blennow and Henrik Zetterberg at Sahlgrenska University Hospital, and then addressed the 3,000 in-person and 1,000 virtual attendees at the city’s conference center. Her Majesty spoke movingly about losing her mother and, two years ago, her brother to Alzheimer's disease. She mentioned hope, and progress having been made on risk factors, immunotherapy, and fluid biomarkers. “Yet, there is still a lot to do, and that is why you are here,” she told the audience.

Indeed there is a lot to do, and Alzforum will do its part with a series of news summaries over the next two weeks. First up: progranulin as a therapeutic target.

In people with deleterious variants of the progranulin gene, the halving of its encoded protein that results from these mutations leads to frontotemporal dementia. This comparatively straightforward haploinsufficiency mechanism casts progranulin restoration as a leading therapeutic target, and a handful of progranulin-boosting therapies have already made their way into clinical trials. At AD/PD, scientists presented preclinical data on potential therapies coming down the pike. One was Arkuda’s ARKD-104, a small molecule that boosts progranulin levels as well as the activity of its cleavage products, the granulin peptides. Another, Denali’s DNL593, consists of a progranulin protein strapped to an antibody that whisks it across the blood-brain barrier; it relieved dramatic phenotypes in mice and human cells. Finally, scientists zeroed in on the lysosomal protease legumain as a link between progranulin deficiency and TDP-43 pathology. The findings could present opportunities to squelch the damage wrought by too little progranulin.

Though the concept of progranulin replacement sounds simple, the varied lifestyle of progranulin is anything but. In its full-length form, progranulin can be secreted, but also trafficked to the lysosomes, where proteases rapidly dice it up into multiple different granulin peptides. Considered the functional forms of progranulin, these peptides take part in processing various lipids within lysosomes, and play a critical part maintaining these organelles' acidification and function. What's more, progranulin rarely enters the lysosome alone. When outside the cell, it often buddies up with prosaposin, another protein that needs to gain access to the lysosome before being processed into its active forms, the saposin peptides. When bound together, the two full-length proteins can take advantage of different transport pathways to cooperatively traffic to the lysosome.

Multiple strategies attempt to boost progranulin levels. The furthest along is AL001, aka latozinemab. This antibody blocks the sortilin receptor, thought to internalize progranulin and whisk it to lysosomes for degradation (Aug 2021 conference news). It is in Phase 3, and had no presentations at AD/PD. Also underway are earlier-stage trials, for gene replacement using progranulin-expressing adeno associated viruses, including Prevail’s PR006 and Passage Bio’s PBFT02.  

At AD/PD, scientists presented preclinical findings about the next drugs to enter the pipeline. Arkuda Pharmaceuticals, a small company in Watertown, Massachusetts, took a small-molecule approach to up progranulin levels. Raymond Hurst presented preclinical findings on the company’s current lead, ARKD-104, a brain-penetrant compound that stokes progranulin activity. In addition to raising the concentration of secreted progranulin, the compound also ratcheted up levels of granulins and saposins within the lysosome.

In GRN knockout mice and in iPSC-derived neurons from people with FTD-GRN, these elevated progranulin cleavage products appeared to be doing their job. Hurst reported a rise in bis(monoacylglycero)phosphates (BMPs), a type of lipid that is known to be abnormally low in both progranulin mutation carriers and in mouse models of progranulin deficiency. BMPs are required for the catabolism of glycosphingolipids within the lysosome, so BMP deficiency is thought to promote lysosomal overflow (Boland et al., 2022). 

The treatment also restored flagging activity of acid lysosomal hydrolases such as GBA1. The exact mechanism of ARKD-104 remains unknown, but experiments thus far suggest that it enhances progranulin trafficking through the vesicular system, leading to its secretion, Hurst said.

In cynomolgus monkeys that received the drug orally, Hurst’s team measured a near tripling of the progranulin concentration in the cerebrospinal fluid within eight hours of the first dose. Levels held steady with daily dosing, but fell quickly back to baseline after dosing stopped. Hurst predicts that AKRD-104 could similarly normalize progranulin in humans. Arkuda plans to start a Phase 1a trial in healthy volunteers in early 2024.

Some in the audience asked whether such a bump in progranulin might be toxic, noting that elevated progranulin has been tied to cancer and inflammation. Hurst said that the goal of the treatment is to restore progranulin to physiological levels in people who are deficient. If anything, Hurst said, preclinical work suggests that restoring progranulin levels should reduce inflammation.

Others asked about off-target effects. Could the drug interfere with expression of other proteins? Hurst said that other than a small number of other lysosomal proteins that rose along with progranulin itself, no other proteins have been found to budge in response to the drug so far.

How does this small-molecule approach measure up to other ongoing strategies to boost progranulin? Hurst told Alzforum that, relative to biological drugs such as antibodies or gene therapy vectors, ease of use is the obvious benefit of ARKD-104. This is particularly true in the case of progranulin deficiency, which would need to be continually corrected throughout life. Hurst noted that while treating people with FTD-GRN is the first and foremost goal for ARKD-104, it is conceivable that using small molecules to slightly elevate progranulin could benefit people with other neurodegenerative conditions in which flagging endolysosomal dysfunction plays a part.

Another way to boost progranulin in the brain is to strap it to a shuttle. Denali’s DNL593 makes use of so-called “brain shuttle” technology. Also called a protein transfer vehicle, it comprises a progranulin protein fused to an antibody fragment that binds the transferrin receptor. This facilitates passage of the pair across the blood-brain barrier via transcytosis (Logan et al., 2021).

This drug is in Phase 1/2 and had no presentations at AD/PD; however, Christian Haass of Ludwig Maximilians University in Munich presented preclinical data exploring a related approach. Haass' team infected mice in the liver with an adeno-associated virus expressing the genes encoding this progranulin protein transport vehicle. The scientists treated mice deficient in both progranulin and TMEM106b, a gene involved in lysosomal function that modifies progranulin disease risk (Sep 2020 news on Werner et al., 2020).

Without treatment, these mice display drastic motor deficits. They quickly fall off a spinning rod, and can't get back up after being pushed over. Treatment with the progranulin shuttle not only gave them staying power on the rod, but also made them resistant to being pushed over in the first place, Haass said. The brain-shuttle-expressing virus also corrected a barrage of other disease phenotypes. It reduced soaring CSF NfL, calmed inflamed microglia, boosted autophagy, and lessened the buildup of ubiquitinated proteins. Importantly, it also reduced the accumulation of insoluble phosphorylated TDP-43 in neurons.

Haass’ group, in collaboration with Dominik Paquet and Anja Capell’s groups at LMU, also tried out the approach in human cells. Graduate students Marvin Reich and Sophie Robinson generated iPSC-derived neurons and microglia, with or without expression of progranulin and TMEM106b. Surprisingly, they found that when cultured alone, double-knockout neurons showed no signs of TDP-43 pathology. However, when either wild-type or double-knockout neurons were co-cultured with double-knockout microglia, the neurons not only developed TDP-43 aggregates, they also died. Adding Denali’s brain shuttle construct to the culture medium restored progranulin expression in the microglia, quelled their lysosomal dysfunction, and saved the neurons. Haass said the findings imply a role for neuron-microglial cross talk in the development of neuronal TDP-43 pathology and neurodegeneration caused by progranulin deficiency.

Legumain: The Missing Link?
How are progranulin-deficient microglia triggering TDP-43 aggregation in neurons? Anja Capell, also at LMU, presented data pointing to one possible explanation. She reported that in microglia lacking progranulin, the lysosomal protease legumain became much more active. Using a series of biochemical experiments, Capell unraveled a striking chain of events.She found that progranulin somehow slows the cleavage of pro-legumain into its active form. Therefore, without progranulin around, legumain activity shoots up. Legumain, it turns out, also contributes to the cleavage of progranulin into granulins, and in the activation of cathepsin proteases. What’s more, progranulin-deficient microglia churn out pro-legumain, which is taken up by nearby neurons. Once inside, legumain becomes activated, and then cleaves—none other than TDP-43. This, in turn, sets off pathological accumulation of TDP-43 in the neurons.

Using a co-culture of iPSC-derived microglia and neurons similar to those that Haass used, Capell showed that legumain released from progranulin-deficient microglia played a pivotal role in instigating TDP-43 pathology in co-cultured neurons.

In postmortem brain samples from people with FTD-GRN, Capell detected an excess of activated legumain, suggesting this pathway is active in disease, even in real-life circumstances milder than a full, experimental progranulin deletion.

That latter finding is important. One audience member noted that, in contrast to hyperactivated microglia seen in progranulin knockout mice, microglia with but one defunct copy of progranulin remain relatively "calm," hence progranulin knockouts do not truly model FTD-GRN. Capell readily agreed. She is continuing her work with human cells missing one copy of functional progranulin.

The detailed biochemical mechanisms linking progranulin, legumain, and TDP-43 remain to be ironed out. Even so, scientists at AD/PD suspect that this pathway likely will present even more opportunities for therapeutic targeting.—Jessica Shugart

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References

Therapeutics Citations

1. Latozinemab
2. PBFT02
3. DNL593

News Citations

1. AL001 Boosts Progranulin. Does it Slow Frontotemporal Dementia? 20 Aug 2021
2. Nixing TMEM106b Fans the Flames of Progranulin Deficiency 25 Sep 2020

Paper Citations

1. Boland S, Swarup S, Ambaw YA, Malia PC, Richards RC, Fischer AW, Singh S, Aggarwal G, Spina S, Nana AL, Grinberg LT, Seeley WW, Surma MA, Klose C, Paulo JA, Nguyen AD, Harper JW, Walther TC, Farese RV Jr. Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis. Nat Commun. 2022 Oct 7;13(1):5924. PubMed.
2. Logan T, Simon MJ, Rana A, Cherf GM, Srivastava A, Davis SS, Low RL, Chiu CL, Fang M, Huang F, Bhalla A, Llapashtica C, Prorok R, Pizzo ME, Calvert ME, Sun EW, Hsiao-Nakamoto J, Rajendra Y, Lexa KW, Srivastava DB, van Lengerich B, Wang J, Robles-Colmenares Y, Kim DJ, Duque J, Lenser M, Earr TK, Nguyen H, Chau R, Tsogtbaatar B, Ravi R, Skuja LL, Solanoy H, Rosen HJ, Boeve BF, Boxer AL, Heuer HW, Dennis MS, Kariolis MS, Monroe KM, Przybyla L, Sanchez PE, Meisner R, Diaz D, Henne KR, Watts RJ, Henry AG, Gunasekaran K, Astarita G, Suh JH, Lewcock JW, DeVos SL, Di Paolo G. Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic. Cell. 2021 Sep 2;184(18):4651-4668.e25. Epub 2021 Aug 26 PubMed.
3. Werner G, Damme M, Schludi M, Gnörich J, Wind K, Fellerer K, Wefers B, Wurst W, Edbauer D, Brendel M, Haass C, Capell A. Loss of TMEM106B potentiates lysosomal and FTLD-like pathology in progranulin-deficient mice. EMBO Rep. 2020 Oct 5;21(10):e50241. Epub 2020 Sep 14 PubMed.

Other Citations

1. PR006

Further Reading

No Available Further Reading