CSF Aβ42 and phospho-tau may remain the quintessential fluid Alzheimer’s disease biomarkers for now, but a growing cadre of other markers are also proving their worth. At the Clinical Trials on Alzheimer’s Disease conference, held December 4–7 in San Diego, California, researchers broadened their horizons with data on CSF markers of glial activation, synaptic dysfunction, and neurodegeneration. Particularly in people with amyloid pathology, a bevy of these new markers tracked closely with CSF p-tau, and sharpened predictions of a person’s impending cognitive decline. The expanded set of markers even debuted as outcome measures in clinical trials.
- In CSF, markers of synaptic dysfunction and neuroinflammation tracked with p-tau.
- Expanded kit of biomarkers sharpen predictions of cognitive decline.
- Biomarkers beyond Aβ and p-tau debut as outcome measures in clinical trials.
Henrik Zetterberg of the University of Gothenburg in Sweden broached the topic of the burgeoning breadth of biomarkers at CTAD by highlighting upward of 20 emerging biomarkers, ranging from those that track core AD pathology Aβ and tau to markers of synaptic dysfunction, axonal damage, neuroinflammation, vascular function, and neurodegeneration. Plasma biomarkers are increasingly soaking up the limelight (see next story), but Zetterberg said that without validating potential biomarkers in CSF first, the field would have little grasp of what plasma markers portend about goings-on inside the brain. This is especially true for neuroinflammatory markers, many of which are also expressed outside the brain, he said. Therefore, validating all AD biomarkers in CSF is critically important.
With so many new markers coming onto the scene, Zetterberg and his Gothenburg colleagues spearheaded a new effort to validate a curated set of CSF markers across multiple cohorts. The Swedish team selected the markers, and Roche developed a standardized master assay—called NeuroToolKit—to measure all of them in CSF. Besides the core AD biomarkers CSF Aβ42, Aβ40, p-tau-181, and total tau, the markers include α-synuclein as a gauge of synaptic dysfunction; S100b, YKL-40, and glial fibrillary acidic protein (GFAP) as markers of astrocyte activation; soluble TREM2 and IL-6 as markers of microglial activation and inflammation; and neurofilament light (NfL) and neurogranin as markers of axonal injury and synaptic dysfunction. The investigators vote new candidate biomarkers into this jointly evaluated set as individual research labs produce promising evidence on them.
At AAIC earlier this year, scientists presented initial NeuroToolKit measurements in the Wisconsin Registry for Alzheimer’s Prevention (WRAP) cohort (Aug 2019 news).
At CTAD, Carol van Hulle of the University of Wisconsin in Madison presented a deeper analysis of correlations among NeuroToolKit markers in 294 CSF samples taken from 202 participants in the WRAP and Wisconsin Aging and Disability Resource Center (WADRC) cohorts that were sent to Gothenburg for analysis. These samples are a subset from WRAP/WADRC’s larger pool of 1,525 samples; they were picked because they represented the full spectrum of AD stages, including 115 people without cognitive deficits, 40 with MCI due to AD, and 47 with AD dementia. The analysis presented at CTAD is cross-sectional, but the scientists are gathering serial samples and many of the participants now have already donated more than one, van Hulle said.
First, the researchers compared each so-called “exploratory biomarker” in the NeuroToolKit to the core AD markers CSF Aβ42 and p-tau-181. The only marker that correlated significantly with Aβ42 was p-tau-181. In contrast, every biomarker in the tool kit, except IL-6, correlated with p-tau-181—indeed, total tau, neurogranin, and α-synuclein moved in lockstep with p-tau-181. As would be expected, both the Aβ42/40 ratio and p-tau/Aβ42 ratio correlated tightly with amyloid-PET measures in the subset of participants who had been scanned. Several of the neurodegenerative and neuroinflammatory biomarkers were higher in people whose p-tau/Aβ42 ratio indicated they had AD pathology than in those who did not. This was true for NfL, neurogranin, α-synuclein, YKL-40, S100b, and GFAP in people with MCI due to AD, and only true for neurogranin in cognitively normal people. These same biomarkers were also higher among people with MCI who later progressed to dementia.
Do additional biomarkers sharpen predictions of a person’s cognitive performance, over p-tau-181/Aβ42 alone? The answer was yes for the neurodegenerative biomarkers α-synuclein, neurogranin, and NfL, which better distinguished between cognitively normal people and those with MCI/AD, and more tightly correlated with performance on the Preclinical Alzheimer’s Cognitive Composite (PACC) than did the core markers alone. However, the glial activation markers YKL-40, sTREM2, GFAP, and S100b did not improve these cognitive correlations over p-tau-181/Aβ42.
Van Hulle suggested that neuroinflammatory markers might track better with clinical progression at later stages of disease. One audience member countered that the opposite may be true—that inflammation might flare up earlier on, and sharpen predictions about progression from normal cognition to MCI. Van Hulle responded that the NeuroToolKit must be tested in larger cohorts. In this sample, only 11 people progressed from being cognitively normal to MCI.
To Zetterberg, the neuroinflammatory markers don’t improve the power of CSF Aβ42/tau to forecast cognitive decline because inflammation is not specific to AD. Inflammation rises with age, in response to vascular problems, infections and, of course, amyloid and tau pathology. “Glial activation markers are simply noisy,” Zetterberg said. For that reason, he expects that they may track with AD later in disease, as inflammation triggered by tau pathology and neurodegeneration surpasses other age-related triggers.
Will other forms of p-tau—namely p-tau-217, which appears to rise even earlier in disease than p-tau-181—be added to the tool kit? Van Hulle said that while she is aware of the new data on p-tau-217, there are not yet plans to add it.
José Luis Molinuevo of Barcelonaβeta Brain Research Center (BBRC) in Barcelona, Spain, uses the NeuroToolKit to understand the interplay between core AD and neuroinflammation and synaptic dysfunction markers in the earliest stages of AD. Molinuevo presented data from 383 participants from ALFA+, a cohort of cognitively normal people aged 45–65 with a family history of AD (Molinuevo et al., 2016). Though 100 people in this cohort had CSF Aβ42/40 ratios indicative of brain amyloid accumulation, only minimal Aβ deposition showed up on their PET scans, suggesting they were in the earliest stages of the disease.
Like van Hulle, Molinuevo reported strong correlations between p-tau and several other biomarkers, most notably neurogranin, while Aβ42 only correlated with p-tau. Connections between p-tau and other biomarkers were strongest in people with amyloid and tau pathology as per their CSF cutoffs. Plotting biomarker levels against the Aβ42/40 ratio, Molinuevo reported that many of the biomarkers started rising just as the cutoff for amyloid positivity was reached, with neurogranin and p-tau shifting most dramatically. NfL, as well as inflammatory markers sTREM2, YKL-40, and GFAP, rose more gradually after the Aβ42/40 threshold was crossed. The findings placed Aβ aggregation at the beginning of the Alzheimer’s cascade, with p-tau, and markers of synaptic dysfunction, neurodegeneration, and neuroinflammation following suit.
Molinuevo reported that YKL-40 correlated with brain shrinkage, while NFL associated with waning glucose metabolism as assessed by FDG-PET.
Together, the Wisconsin and Barcelona findings suggest that very early in the AD continuum—at a time when Aβ is already abnormal according to CSF but not yet on PET—markers of synaptic dysfunction and neuroinflammation are beginning to change.
The most striking finding from the ALFA+ study, according to Zetterberg, was that it validated neurogranin as being specific for AD. This synaptic protein correlated highly with CSF-p-tau, and only rose in people whose Aβ was abnormal. He thinks neurogranin gets released into fluid when synapses malfunction in response to increasing tau hyperphosphorylation, which are themselves triggered by Aβ. NfL, a more general marker of neurodegeneration, rises later as neurons die, along with the neuroinflammatory markers, he added.
Taking It to Trial
Companies developing therapeutics are starting to implement the expanded set of biomarkers as outcome measures in clinical studies. For example, Lindsay Burns from Cassava Sciences in Austin, Texas, presented biomarker data from a tiny Phase 2a trial of PTI-125, a small molecule that reportedly blocks pathological interactions between Aβ and the α7-nicotinic receptor, and TLR-4 (Jul 2012 conference news; Wang et al., 2017). Aβ binding to α7-nicotinic receptors sets off tau phosphorylation and neurodegeneration, while Aβ binding to TLR-4 unleashes neuroinflammation. PTI-125 blocks Aβ associations with both receptors indirectly, by binding to and changing the conformation of filamin A, a scaffolding protein that facilitates Aβ’s receptor interactions. As such, Cassava proposes that its compound could squelch both neurodegeneration and neuroinflammation, and is using biomarkers to track those phenomena.
Cassava used change in a panel of CSF markers as its primary endpoint for this trial, which evaluated a 28-day course of 200 mg daily dose of PTI-125 in 13 people with mild to moderate AD. The panel included Aβ42, neurogranin, NfL, total tau, p-tau-181, YKL-40, IL-6, IL-1β, and TNF-α. By the end of the month on PTI-125, Burns reported, each biomarker had budged, ranging from a more than 30 percent drop in p-tau and neurogranin to dips of 5 to 14 percent in the inflammatory markers. CSF Aβ42 nudged slightly upward, albeit insignificantly. Cassava measured many of these markers in plasma as well, reporting a 41 percent plummet in neurogranin, a 16 percent drop in total tau, and 11 and 4 percent dips in NfL and YLK-40, respectively.
Cassava scientists also took stock of plasma p-tau. Where previous research gauged “p-tau” based on phosphorylation at threonine 181, nowadays researchers are taking a more granular approach, parsing different forms of tau in plasma and CSF. In San Diego, Burns reported declines in the PTI-125 group in p-tau-T181, p-tau-T202, p-tau-T231, and a nitrated form of tau—n-tau-Y29—in plasma. This small study had no placebo group. Burns claimed that every patient responded to treatment across most markers, and that the drug was safe and well-tolerated.
Burns believes the data support the proposed dual mechanism of action, suggesting a change signal in both neurodegenerative and neuroinflammatory biomarkers, as well as the core AD markers. In September 2019, Cassava started a placebo-controlled Phase 2b trial with 60 participants. For more data on plasma biomarkers, see next story.—Jessica Shugart
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- Molinuevo JL, Gramunt N, Gispert JD, Fauria K, Esteller M, Minguillon C, Sánchez-Benavides G, Huesa G, Morán S, Dal-Ré R, Camí J. The ALFA project: A research platform to identify early pathophysiological features of Alzheimer's disease. Alzheimers Dement (N Y). 2016 Jun;2(2):82-92. Epub 2016 Mar 3 PubMed.
- Wang HY, Lee KC, Pei Z, Khan A, Bakshi K, Burns LH. PTI-125 binds and reverses an altered conformation of filamin A to reduce Alzheimer's disease pathogenesis. Neurobiol Aging. 2017 Jul;55:99-114. Epub 2017 Mar 31 PubMed.
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