23 Aug 2024

A new multiplex diagnostic method appears poised to resculpt the biomarker landscape. NULISA, aka NUcleic acid Linked Immunosorbent Assay, can detect Aβ peptides, p-tau isoforms, and other potential markers of neurodegeneration with sensitivities 10- to 20-fold greater than some single-molecule array (SIMOA) immunoassays currently used to measure AD markers, according to Henrik Zetterberg, University of Gothenburg, Sweden.

As reported at AAIC last month in Philadelphia, and in recent publications, Alzheimer’s disease labs have already started using this technology. So far, the results look promising. NULISA identifies people with brain amyloid as well as commercially available plasma tests do. Since it can detect hundreds of targets simultaneously in one sample, it is already starting to identify new potential markers.

The “NU” Proteomics. NULISA uses antibody pairs to detect minute quantities of antigen in fluid samples. Tails on the antibodies allow for a two-step capture and purification process. Proximity ligation amplifies the signal. [Courtesy of Alamar Biosciences.]

NULISA brings into apposition pairs of nucleic-acid tagged antibodies that recognize different epitopes of the same target. Enzymes ligate the tags, which can then be quantified by polymerase chain reaction or next-generation sequencing amplification. Specific tag sequences identify specific antibodies, allowing multiple targets to be quantified simultaneously. The technology was developed by scientists at Alamar Biosciences, Fremont, California (Feng et al., 2023). Because the antibodies have polyA or biotinylated tails, the antibody-antigen complexes can be captured sequentially on polyT or streptavidin matrices, allowing for a two-step purification.

“The upshot is you get really pure complexes and exquisite sensitivity,” said Zetterberg.

NULISA measures protein targets down to the attomolar level. That’s 18 zeroes, or 10^-18 molar; for those of us who are not used to dealing in such infinitesimal quantities, that equates to just over 1 molecule in a 0.2 mL sample.

What Else Can NULISA Do?
Scientists have begun to use this technology to measure known AD markers in multiplex fashion—and to search for new ones. At AAIC, Xiao-Jun Ma and colleagues at Alamar introduced a suite of antibodies that quantify 120 proteins previously linked to neurodegenerative diseases. They include Aβ peptides, p-tau isoforms, and a range of other targets involved in immune responses, microglial signaling, and synapse maintenance, to name a few (table below). Most of these are present above the limit of detection in plasma, and more than 85 percent are detectable in dried blood spot samples (see previous story). In a small pilot study of 13 people with AD and 13 controls, Ma found 30 markers upregulated in AD and four downregulated, hinting at an AD proteomic fingerprint.

Targets Galore? In multiplex analysis, NULISA simultaneously detects almost 120 proteins linked to neurodegenerative diseases. [Courtesy of Alamar Biosciences.]

Also in Philadelphia, Andrea Benedet, U Gothenburg, reported on NULISA analysis of samples that had already been tapped for a previous round-robin comparison of different AD markers run by Ashton (see Nov 2023 conference news). These amply studied samples came from 20 people with AD and 20 controls.

As expected, NULISA detected more p-tau181, p-tau217, p-tau231, and GFAP in the plasma and/or CSF or the AD group than in controls, and less Aβ42. In fact, based on plasma p-tau217, NULISA identified people with amyloid pathology with the same accuracy as did the immunoassays and mass spectrometry assays used in the round robin.

In another head-to-head study, scientists at Oskar Hansson’s lab at Lund University, Sweden, have compared the NULISA p-tau217 tests against commonly used immunoassay- and mass-spectrometry-based tests in a subset of 463 samples from the BioFinder-2 cohort in Sweden, and 97 samples from the Knight ADRC at Washington University, St. Louis (see Part 12 of this series). In the former, NULISA p-tau217 was almost as good at distinguishing people who had amyloid in the brain as the %p-tau217 in plasma, yielding an AUC of 0.93 versus 0.96 for the latter. In the U.S. samples there was no difference.   

Work from Pedro Rosa-Neto’s lab at McGill University, Montreal, supports the validity of this new technology. Yi-Ting Tina Wang and colleagues compared the performance of NULISA to that of SIMOA immunoassays for p-tau181 and p-tau231 run at UGothenburg, and commercial immunoassays for p-tau217 from Janssen and ALZpath. Among 397 volunteers in the Translational Biomarkers in Aging and Dementia, aka TRIAD, cohort in Montreal, NULISA analysis of plasma p-tau217 was the top performer, identifying people who were amyloid-positive with an AUC of 0.918. NULISA slightly outperformed the AlzPath and Janssen tests. Among the subset of people who were cognitively unimpaired, NULISA also did best. The Janssen test for p-tau217 best identified those who had positive tau PET scans, with NULISA a close second. The differences between these p-tau217 tests were negligible, hence NULISA appears about as accurate as immunoassays in discriminating amyloid- and tangle-positive individuals. 

Benedet wants to use NULISA to find new markers. After all, the current crop captures only part of the pathology wrecking the AD brain. She studies later disease stages where additional markers might pop up. Among samples from TRIAD and BioFinder, she found more than a dozen changes in the CSF of people with known tau pathology. More abundant were the neurodegeneration marker neurofilament light, the fatty acid binding protein FABP3, the synaptic protein neurogranin, and the myeloid cell receptor TREM1. More depleted were synaptic neuropentraxins, interleukin 13, and interferon-γ. Some of these differences also emerged in plasma. Benedet said her pilot studies confirm that NULISA can detect known markers and highlight its potential to find and ones.

The Benedet lab has also begun to deploy NULISA in frontotemporal and Lewy body dementias. At AAIC, Joel Smirén reported more than a dozen proteins that are up in the plasma of 18 symptomatic progranulin mutation carriers compared to plasma of 20 noncarriers. They ranged from markers of neuronal injury, such as α-synuclein, neuropentraxin 2, SNAP-25 and NfL to markers of lysosomal dysfunction, such as SQSTM1, aka p62. The panel also spotted protein concentration increases in presymptomatic carriers, albeit without statistical significance. These will be re-examined in larger cohorts, Smiren said.

Meanwhile, Bárbara Gomes reported that in TRIAD, NULISA detected protein reductions in the CSF of 112 people who had Lewy body pathology as judged by seed amplification assay of α-synuclein protofibrils. Those included the transcription factor REST, which has been linked to neuroprotective responses (Lu et al., 2014), RUVBL2, a helicase involved in DNA repair, corticotrophin releasing hormone, the cytokine IL16, and the transmitter neuropeptide Y.

Other labs are also jumping on this new technology. Scientists in Thomas Karikari’s lab at the University of Pittsburgh are using Alamar’s CNS panel and an inflammation panel that measures 250 proteins. They are hunting for signs of neuronal injury, vascular damage, inflammation, and synucleinopathy in AD. As Xuemei Zeng of the University of Pittsburgh noted in her AAIC talk, these processes are recognized in the revised diagnostic criteria as important pathologies in AD, but no specific markers are yet available (Nov 2023 conference news).

Zeng has tested plasma from 113 volunteers in the Monongahela Youghiogheny Healthy Aging Team-Neuroimaging cohort. This population-based study characterizes mild cognitive impairment in economically distressed southwestern Pennsylvania. This population has a high level of comorbidities, such as diabetes, hypertension, and kidney disease. Participants had a clinical dementia rating of below 1 at baseline; at which point a quarter had amyloid pathology. Thus far, about half have had their second-year follow-up, by which point a third of them tested positive for amyloid.

In this MYHAT-NI cohort, too, NULISA levels of plasma p-tau217, p-tau181, p-tau231, NFL, and Aβ42 all correlated tightly with levels determined by SIMOA. Plasma p-tau217 and p-tau231 associated with amyloid, as did downregulation of superoxide dismutase 1, the tricarboxylic acid cycle enzyme MDH1, and the extracellular matrix protein TIMP3.

Could NULISA markers predict amyloid accumulation? Zeng correlated protein level at baseline with subsequent annual PET changes. As might be expected, the higher a person’s plasma p-tau217 at baseline, the more amyloid they accumulated. Interestingly, levels of the chemokines CCL13, CCL17, CCL26, CXCL1, and CXCL8 correlated with less amyloid accumulation over time. “This could mean that those chemokines are neuroprotective,” said Zeng.

She also found proteins that correlated with neurofibrillary tangles. Unsurprisingly, the former included plasma p-tau 181, 217, and 231, but also SFRPI, aka secreted frizzled-related protein 1, and 14-3-3g. SFRP1, an endogenous inhibitor of α-secretase, has been linked to amyloid pathology (Esteve et al., 2019). Zeng found it to be suppressed in tangle-positive volunteers, hinting that it might be contributing to the amyloid cascade.

14-3-3γ is a jack-of-all-trades that binds various signaling molecules, such as kinases, phosphatases, and membrane receptors. It was up in the blood of tau-positive people. Recently, in an unbiased proteomics analysis of ADNI, scientists reported that 14-3-3γ was the most upregulated in AD versus controls among more than 6,000 proteins (Guo et al., 2024).

At AAIC, 14-3-3γ, also called YWHAG, cropped up elsewhere, too. Scientists from Tony-Wyss Coray’s lab at Stanford University, California, reported that it is elevated in AD CSF. Among people in ADNI, at the Knight Alzheimer’s Disease Research Center at Washington University, and at Stanford, the higher the YWHAG:NPTX2 ratio, the faster a person’s cognition declined over the next one to 15 years. This ratio better predicted decline than did p-tau181/Aβ42, ApoE4, or level of baseline cognitive impairment. Markers of disease progression top many an Alzheimerologist’s wish list.   

For her part, Zeng found that 17 markers in the NULISA panel changed over time in the MYHAT-NI cohort. Glial markers, including CHIT1 and CHI3L1, waned on follow-up in people who had tangles, as did the cytokines IL17A, SCF2, CXCL1, and markers of vascular integrity, such as PDGFRB and VEGFA. Curiously, no marker seemed to increase over that time. One marker, sequestosome 1, correlated with neurodegeneration over two years.

All told, NULISA and dried blood/plasma analysis (see previous story) appear set to reshape biomarker research analysis, and perhaps even routine clinical testing, for neurodegenerative diseases.

Some people at AAIC wondered if the time has come when scientists will finally find functional markers for cognitive decline. Benedet thinks the field is not quite there yet. So does Sterling Johnson, University of Wisconsin. “I think our field is getting there, but it will take more iteration,” he said. “What we saw with these new multiplex panels is pretty incredible. I think it is that kind of approach that is going to help.”—Tom Fagan

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References

News Citations

1. A Finger-Prick Test for Alzheimer’s Disease? 21 Aug 2024
2. Plasma p-Tau-217 Assays Work Well, But No Home Run for Diagnosis 30 Nov 2023
3. In Head-to-Head Testing, P-Tau217/Tau217 Comes Out on Top. By a Hair. 21 Aug 2024
4. No REST for Weary Neurons: Protective Factor Stems Cognitive Decline 19 Mar 2014
5. New Alzheimer’s Diagnostic Criteria Remain ‘Research Only’ 28 Nov 2023

Paper Citations

1. Feng W, Beer JC, Hao Q, Ariyapala IS, Sahajan A, Komarov A, Cha K, Moua M, Qiu X, Xu X, Iyengar S, Yoshimura T, Nagaraj R, Wang L, Yu M, Engel K, Zhen L, Xue W, Lee CJ, Park CH, Peng C, Zhang K, Grzybowski A, Hahm J, Schmidt SV, Odainic A, Spitzer J, Buddika K, Kuo D, Fang L, Zhang B, Chen S, Latz E, Yin Y, Luo Y, Ma XJ. NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing. Nat Commun. 2023 Nov 9;14(1):7238. PubMed.
2. Esteve P, Rueda-Carrasco J, Inés Mateo M, Martin-Bermejo MJ, Draffin J, Pereyra G, Sandonís Á, Crespo I, Moreno I, Aso E, Garcia-Esparcia P, Gomez-Tortosa E, Rábano A, Fortea J, Alcolea D, Lleo A, Heneka MT, Valpuesta JM, Esteban JA, Ferrer I, Domínguez M, Bovolenta P. Elevated levels of Secreted-Frizzled-Related-Protein 1 contribute to Alzheimer's disease pathogenesis. Nat Neurosci. 2019 Aug;22(8):1258-1268. Epub 2019 Jul 15 PubMed.
3. Guo Y, Chen SD, You J, Huang SY, Chen YL, Zhang Y, Wang LB, He XY, Deng YT, Zhang YR, Huang YY, Dong Q, Feng JF, Cheng W, Yu JT. Multiplex cerebrospinal fluid proteomics identifies biomarkers for diagnosis and prediction of Alzheimer's disease. Nat Hum Behav. 2024 Oct;8(10):2047-2066. Epub 2024 Jul 10 PubMed.

Further Reading

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