28 Mar 2024

With people now being treated with amyloid immunotherapy in the clinic, reducing the risk of ARIA has taken on a new sense of urgency. This inflammatory side effect occurs in people who have amyloid in small to medium-sized blood vessels of the brain, aka cerebral amyloid angiopathy. However, CAA cannot be easily detected, making it hard to screen patients for the condition. To better manage amyloid immunotherapy, scientists are seeking fluid biomarkers for vascular amyloid, as well as ways to mitigate the risk of microhemorrhages that underlie ARIA. They would also like better mouse models for both CAA and ARIA. At the International Conference on Alzheimer’s and Parkinson’s Diseases, held March 5 to 9 in Lisbon, Portugal, progress in all these areas impressed attendees.

Shinobu Kitazume of Fukushima Medical University, Japan, debuted a mouse model with extensive vascular amyloid deposits that could be used to study CAA, while Thierry Bussiere of Biogen described mice that develop ARIA-like vascular lesions. On the biomarker front, Marcel Verbeek of Radboud University Medical Center, Nijmegen, The Netherlands, suggested the protein TIMP4, aka tissue inhibitor of metalloprotease-4, as a possible CAA biomarker. Regarding mitigation, Anna Bonaterra-Pastra of the Vall d'Hebron Research Institute, Spain, reported that intravenous injections of Apolipoprotein J, aka clusterin, in aged amyloidosis mouse models cut the number of microhemorrhages in half, perhaps by suppressing matrix metalloproteases that damage blood vessels.

The talks attracted a lot of interest. Costantino Iadecola of Weill Cornell Medical College, New York, believes the two mouse models will advance research. “Both may tell us more about the dynamics of Aβ vascular accumulation and clearance, and help us better understand the impact that Aβ antibodies may have on vascular amyloid,” he told Alzforum.

Mice With CAA? APP knock-in mice (bottom) have parenchymal amyloid (green) and none in blood vessels (red), but when crossed with mice expressing endothelial APP (top), amyloid preferentially deposits in vessels (overlay appears yellow, right). [Courtesy of Shinobu Kitazume, Fukushima Medical University.]

Are Endothelial Cells the Key to Vascular Amyloid?
Recent data suggest that before anti-amyloid antibodies begin removing Aβ from plaques, they first interact with amyloid in the vasculature, triggering inflammation that leads to damaged blood vessels, edema, and microhemorrhages (Aug 2023 conference news). Autopsies of people who died with ARIA jibe with this, showing inflamed blood vessels in people with severe CAA (Jan 2024 news). It has been difficult to study CAA and ARIA in mice, however, since they do not develop much vascular amyloid.

In Lisbon, Kitazume suggested that this species difference comes down to endothelial cells. In people, endothelial cells make a version of amyloid precursor protein, APP770, that is longer than the one neurons make—APP695. Kitazume and colleagues generated an antibody specific for this longer version, which contains two additional domains near the N-terminal end totaling 75 amino acids (see image above at right). Immunostaining revealed APP770 coating blood vessels in the human cortex (Kitazume et al., 2010; Kitazume et al., 2012; Miura et al., 2020). This implies that endothelial cells might be a primary source of vascular amyloid, Kitazume noted. The secreted α/β cleavage product sAPP770 is abundant in human blood, with sAPP770α predominating, but nearly absent from cerebrospinal fluid, in keeping with an endothelial origin. By contrast, rodents have almost no sAPP in their blood, hinting that their endothelial cells make little APP.

Could boosting endothelial APP production in mice promote vascular amyloidosis? To test this, Kitazume and colleagues generated mice that expressed human APP770 specifically in their endothelial cells. These EC-APP770+ mice pumped out high levels of serum sAPP770. At two years of age, they had about six times as much Aβ40 in their blood as did wild-type mice. However, they did not deposit cerebrovascular amyloid.

To spur amyloidosis, the researchers crossed the mice with APP^NL-F knock-ins. That did the trick. By 15 months of age, the offspring had developed extensive vascular deposits in the cortex, particularly in meningeal blood vessels (see CAA image above; Tachida et al., 2022). When the researchers crossed EC-APP770+ mice with APP^NL-G-F knock-ins, which have more aggressive amyloidosis, vascular amyloid formed by 7 months of age.

“I think these might be great models to study ARIA,” noted Cynthia Lemere at Brigham and Women’s Hospital, Boston, one of the ADPD organizers. For example, in the EC-APP770+/ APP^NL-F crosses, endogenous IgG antibodies coated vascular deposits, suggesting the potential for an inflammatory response. EC-APP770+ mice are available from RIKEN.

Mouse ARIA Resembles Human
Bussiere took a different approach to model ARIA. Biogen researchers treated 5xFAD mice with weekly injections of aducanumab, gantenerumab, or 3D6, the mouse version of bapineuzumab, for up to 12 weeks. The researchers assessed ARIA using 7 or 9.4 T MRI scans.

ARIA incidence varied with dose and antibody. With all mice on 5 or 10 mg/kg developing edema, 3D6 produced the most. On 50 mg/kg aducanumab, half the mice got ARIA, and on 50 mg/kg gantenerumab, about a third did. On MRIs, the researchers saw bright spots indicating vascular lesions, as well as more diffuse patches of edema. As in people, edema either resolved with time, or worsened and caused blood vessels to spring leaks. As with human ARIA, large hemorrhages were rare.

Histochemical analysis revealed damage to meningeal arterioles, including the presence of enlarged macrophages containing amyloid, and patches of the plasma protein fibrin in degenerating vessel walls. These morphological changes correlated with MRI signals. Overall, vascular changes in 3D6-treated mice resembled those in people with severe ARIA, Bussiere noted (Solopova et al., 2023; Castellani et al., 2023). The model reproduces many of the characteristics of human ARIA and could be useful for research, he concluded.

Iadecola agreed. “I find the data of interest because they used MRI to assess brain injury and edema, which is relevant to the way ARIA is diagnosed in humans,” he told Alzforum. Lemere noted the importance of being able to detect both ARIA-E and ARIA-H in mouse models. Her group has seen ARIA-H on MRI, but so far not ARIA-E. “It was nice to see it can be done,” she told Alzforum.

Fluid Biomarkers for CAA
In the clinical setting, detecting CAA would help identify people who are not good candidates for amyloid immunotherapy (Aug 2023 conference news). In a meta-analysis of 170 studies covering 73,000 people, Verbeek found that almost a quarter of people over the age of 55 have moderate to severe CAA on postmortem analysis, and most of those cases are missed on MRI. The majority are people with cognitive impairment, of whom many would be eligible for anti-amyloid immunotherapy (Jäkel et al., 2021). 

What fluid markers correlate with CAA? Numerous studies have flagged matrix metalloproteases and their inhibitors. This large family of proteins chews up extracellular matrix, aiding tissue repair. In CAA, expression and activity of several MMPs, particularly 2 and 9, rises, correlates with degradation of the extracellular matrix and damage to blood vessels (Jung et al., 2003; Zhao et al., 2015). MMP inhibitor expression also goes up, perhaps in compensation. One MMP inhibitor, TIMP4, caught Verbeek’s eye as a potential CAA marker because it is specifically expressed in brain and heart, and rises in the plasma in people with vascular dementia.

Microhemorrhage Marker? Metalloprotease inhibitor TIMP4 (brown) is absent from healthy blood vessels (left), elevated in cerebral amyloid angiopathy (middle), and higher still in CAA vessels with microhemorrhages (right). [Courtesy of Lieke Jäkel, Radboud University.]

First, Verbeek investigated whether TIMP4 was expressed in vessels with CAA. His group examined human postmortem occipital lobe sections from 39 people with CAA, 18 with CAA and intracerebral hemorrhages (ICH), and 42 unaffected controls. Their average age was 78. As he had hypothesized, people with CAA had more TIMP4 in cerebral blood vessels than did controls. Those with CAA-ICH had even more (image above). TIMP4 associated with neither plaques nor tangles, nor with superficial siderosis, i.e., the leakage of blood on the surface of the brain.

To find out if TIMP4 would make a good fluid biomarker, the researchers measured it in CSF and in serum from 38 people with CAA and from 37 controls. The CAA cohort had more TIMP4 in the serum, and less in the CSF. The worse the CAA, the lower CSF TIMP4 was. This pattern resembles that of Aβ, which drops in CSF as the protein deposits in plaques. In fact, TIMP4 correlated with Aβ40 in CSF, albeit weakly, with an r value of 0.3.

The ratio of CSF/serum TIMP4 distinguished CAA and control groups better than did CSF or plasma TIMP4 alone, but the overlap between the groups was large. TIMP4 could be combined with other markers to create a more specific measure, Verbeek suggested. He previously reported that several Aβ peptides, including Aβ38, Aβ40, Aβ42, and Aβ43, are lower in CAA CSF than in CSF from AD patients or controls (De Kort et al., 2023; van den Berg et al., 2024). “Combined with TIMP4, these might constitute a specific panel to identify CAA and predict ARIA,” he suggested in Lisbon.

Protection Against Blood Vessel Damage?
Vascular breakdown and larger hemorrhages are the most dangerous aspect of ARIA. Could these be prevented? Bonaterra-Pastra and Mar Hernández-Guillamon, also at the Universitat Autònoma, have proposed ApoJ as a candidate therapy because this chaperone co-deposits with vascular amyloid, and is elevated in the plasma of CAA patients (Camacho et al., 2019; Bonaterra-Pastra et al., 2023). Hernández-Guillamon and colleagues previously found that treating 14-month-old APP23 mice with recombinant human ApoJ prevented the accumulation of plaques and vascular amyloid (Fernández de Retana et al., 2019). 

To find out if ApoJ could ameliorate established CAA, Bonaterra-Pastra moved to 21-month-old mice, which develop extensive vascular amyloid and spontaneous microhemorrhages. She injected 1 mg/kg recombinant human ApoJ twice weekly into mouse veins for three months. This cut the number of microhemorrhages from an average of 12 per mouse to six, as seen by MRI. For larger bleeds, those greater than 300 μm in diameter, there was an even bigger drop, to about a sixth as many as in untreated mice.

In addition, ApoJ-treated mice had half as many damaged, fibrinogen-positive blood vessels in their cortex, and more healthy smooth muscle actin cells, compared to untreated controls, indicating less damage to vessel walls. There was no difference in the amount of vascular amyloid, however, indicating the treatment was not clearing it.

How, then, did ApoJ protect mice? Bonaterra-Pastra found less MMP12 in treated animals. The amount of MMP12 correlated with the number and volume of microhemorrhages in both treated and untreated mice, at r=0.6 to 0.8. Other studies have linked MMP12 to blood-brain barrier breakdown as well (Power et al., 2003; Wells et al., 2005; Chelluboina et al., 2015). In Lisbon, Lemere likewise reported that MMP12, along with the complement protein C3, were the two most upregulated proteins in mice that developed microhemorrhages after treatment with 3D6.

To find out if the same might be happening in people, Bonaterra-Pastra analyzed MMP12 levels in plasma from 22 volunteers with lobar ICH, 18 with hemorrhages in deeper brain regions, and 17 healthy controls. Lobar ICH associates with CAA, while deeper bleeds do not. She found no consistent difference in MMP12 levels between groups. However, in the lobar ICH group, the amount of MMP12 correlated with larger, irregularly shaped hemorrhages. That, in turn, correlated with a worse prognosis. Data presented by Ines Hristovska of Lund University, Malmö, Sweden, supported this idea. She showed that in the BioFinder study MMP12 is elevated in people who have microbleeds or white-matter lesions.

Bonaterra-Pastra thinks ApoJ could protect against microhemorrhages by lowering MMP12, and that it would be worth testing if giving patients ApoJ before amyloid immunotherapy could lessen the risk of ARIA.

“Her data look exciting, both in terms of a potential mitigation strategy for ARIA and for blood-based biomarkers for CAA, both of which are sorely needed,” Lemere told Alzforum.—Madolyn Bowman Rogers

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References

News Citations

1. Is ARIA an Inflammatory Reaction to Vascular Amyloid? 9 Aug 2023
2. Brain of Woman Who Died on Leqembi Shows Worst-Case Scenario 26 Jan 2024
3. Wanted: Fluid Biomarkers for CAA, ARIA 10 Aug 2023

Research Models Citations

1. APP NL-F Knock-in
2. APP NL-G-F Knock-in
3. APP23

Therapeutics Citations

1. Aduhelm
2. Gantenerumab
3. Bapineuzumab

Paper Citations

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4. Tachida Y, Miura S, Muto Y, Takuwa H, Sahara N, Shindo A, Matsuba Y, Saito T, Taniguchi N, Kawaguchi Y, Tomimoto H, Saido T, Kitazume S. Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice. J Biol Chem. 2022 Jun;298(6):101880. Epub 2022 Mar 31 PubMed.
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11. van den Berg E, Kersten I, Brinkmalm G, Johansson K, de Kort AM, Klijn CJ, Schreuder FH, Gobom J, Stoops E, Portelius E, Gkanatsiou E, Zetterberg H, Blennow K, Kuiperij HB, Verbeek MM. Profiling amyloid-β peptides as biomarkers for cerebral amyloid angiopathy. J Neurochem. 2024 Jul;168(7):1254-1264. Epub 2024 Feb 16 PubMed.
12. Camacho J, Moliné T, Bonaterra-Pastra A, Ramón Y Cajal S, Martínez-Sáez E, Hernández-Guillamon M. Brain ApoA-I, ApoJ and ApoE Immunodetection in Cerebral Amyloid Angiopathy. Front Neurol. 2019;10:187. Epub 2019 Mar 13 PubMed.
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14. de Retana SF, Marazuela P, Solé M, Colell G, Bonaterra A, Sánchez-Quesada JL, Montaner J, Maspoch D, Cano-Sarabia M, Hernández-Guillamon M. Peripheral administration of human recombinant ApoJ/clusterin modulates brain beta-amyloid levels in APP23 mice. Alzheimers Res Ther. 2019 May 10;11(1):42. PubMed.
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External Citations

1. RIKEN

Further Reading

News

1. Unlocking Blood-Brain Barrier Boosts Immunotherapy Efficacy, Lowers ARIA 11 Nov 2023
2. Human Blood-Brain Barrier Model Blames Pericytes for CAA 15 Jun 2020
3. Diagnosis for Inflammation in Cerebral Amyloid Angiopathy 8 Jan 2016