Abstract

1. BACKGROUND

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the development of a gradually increasing burden of amyloid plaque and tau tangle pathology resulting in the loss of synapses and degeneration of neurons. The disease is characterized by extended pre‐clinical and prodromal (mild cognitive impairment [MCI]) stages prior to dementia—the terminal phase of the disease . ^{1 , 2 , 3} Despite its outsized societal burden, ⁴ therapeutics that slow down or reverse the progression of the disease have been elusive. ⁵

One of the obstacles to drug discovery efforts is the lack of early prognostic biomarkers for AD. Phosphorylated tau (p‐tau) and structural and metabolic brain imaging provide a reliable set of diagnostic biomarkers that strongly correlate with hallmark pathologic changes in the brain and can therefore be used to follow brain pathology only at more advanced stages of AD. ⁶ While amyloid plaque burden is recognized as an early abnormality in the overall trajectory of development of AD, ⁷ it is insufficient to accurately predict the time course for disease progression. ⁸ Also, ≈40% of Alzheimer's Disease Neuroimaging Initiative (ADNI) individuals with MCI did not conform to the National Institute on Aging/Alzheimer's Association (NIA‐AA) framework amyloid/tau/neurodegeneration (AT[N]) classification scheme for defining AD biologically, ⁹ and their cognitive status could therefore be attributed to other factors. ¹⁰ Additional measures that track early pathophysiologic processes involved in the progression of patients classified as MCI are needed to help determine whether AD pathology is truly present, and to estimate with greater accuracy the time of dementia onset. ¹¹ Specifically, early detection of neuro‐dysfunction, such as synapse damage or loss, could provide an improved ability to predict future decline caused by multiple ongoing pathophysiologic processes occurring in the AD brain. ¹²

Most studies of candidate biomarkers that used cerebrospinal fluid (CSF) have examined samples collected at a single point in time, failing to capture the potentially important feature of within‐individual changes in relation to disease progression. Because longitudinal studies could improve our ability to detect early changes in disease pathology, we evaluated the time course of five candidate biomarkers in cognitively normal (CN) patients at baseline and patients with a baseline diagnosis of MCI. The five biomarkers—(1) chromogranin A (CMGA), (2) fatty acid binding protein (FABPH), (3) neuronal pentraxin 2 (NPTX2), (4) secretogranin (SCG2), and (5) neurosecretory protein VGF (VGF)—were selected based on evidence gathered from prior research including a cross‐sectional mass spectrometry (MS)‐based proteomic study of CSF samples from the ADNI cohort using a multiplexed MRM panel, developed with Caprion, and published previously by our team. ¹³ Given the goal of ruling in or out significant rates of change in this group of analytes in MCI subjects with a quantitative MS assay, we did not include the many MCI/AD relevant biomarkers already identified with immunoassays, mainly in cross‐sectional studies, such as neurogranin, Vilip‐1, neurofilament light, and YKL‐40. To the extent that these have been evaluated in longitudinal studies, none have shown marked rates of change that consistently relate to the trajectory of pathology and/or symptoms. ^{11 , 14 , 15}

2. METHODS

3. RESULTS

3.1. Primary results models of longitudinal NPTX2 changes across clinical subsets

The goal of the present study was to examine the rate of change in the concentration of five proteins—CMGA, FABPH, NPTX2, SCG2, and VGF—in the CSF, and to compare these rates between participants stratified on the basis of clinical diagnosis, ratio positivity and clinical progression. Specifically, we fitted three longitudinal linear models to statistically test whether (1) the repeated within‐subject measurements of analyte concentration exhibited robust changes in time across all participants in our analysis set or their pre‐defined subsets; and (2) whether the rate of change in the analyte concentrations was associated with differences in clinical characteristics such as diagnosis, p‐tau₁₈₁/Aβ_1‐42 ratio, and clinical progression.

The results of the longitudinal modeling effort, which allowed us to adjust for important covariates such as each participant's age, gender, APOE ε4 carrier status, and education level, suggested that the concentration of CMGA, NPTX2, SCG2, and VGF proteins in the participants’ CSF tended to decrease over time. These declines were especially evident among participants who were p‐tau₁₈₁/Aβ_1‐42 ratio positive at baseline (Figure 1 and Figure 2B for NPTX2, Table 2, Figures S4‐S7 in supporting information). However, the largest declines, which were also supported by the strongest statistical evidence, were observed for NPTX2. The concentration of NPTX2 among participants with a baseline clinical diagnosis of MCI declined by 0.08 ng/mL per year on average (P < .0001), and by 0.09 ng/mL (P < .0001) among participants with the biomarker profile indicative of AD (baseline p‐tau₁₈₁/Aβ_1‐42 ratio ≥ 0.025; i.e., ratio positive). The declines in NPTX2 concentration were statistically significantly larger among participants classified as MCI compared to CN participants (P = .008; Figure ​Figure2A);2A); as well as between p‐tau₁₈₁/Aβ_1‐42 ratio positive participants at baseline compared to ratio negative participants (P = .001; Figure 2B; see Table 2 for details); that is, among groups of participants that are further along the disease continuum.

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FIGURE 1

Mean yearly rates of change in the cerebrospinal fluid concentration of each of the five studied analytes (columns) in subgroups of study participants defined by baseline diagnosis (Model 1; first row), baseline phosphorylated tau (p‐tau)₁₈₁/amyloid beta(Aβ)_1‐42 ratio positivity (Model 2; second row) and progression (Model 3; third row). CN: cognitively normal (blue); MCI: mild cognitive impairment (red); Ratio+: participants with p‐tau₁₈₁/Aβ_1‐42 ≥ 0.025 (red); Ratio–: participants with p‐tau₁₈₁/Aβ_1‐42 < 0.025 (blue); Prog.: progressors (red), that is, participants who were initially diagnosed as CN (resp. MCI) and who were diagnosed within 4 years and 1 month after the initial diagnosis with MCI or dementia (resp. dementia). Nonprog.: non‐progressors (blue). The x‐axis corresponds to a period of 1 year.

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FIGURE 2

Longitudinal changes from baseline in cerebrospinal fluid concentration of neuronal pentraxin 2 (NPTX2) (raw data from individual participants: thin lines; mean changes resulting from the linear mixed effects (LME) model: thick lines, also shown in Figure 1 under “NPTX2”). A, Participants categorized as cognitively normal (CN) at baseline (blue); participants categorized as mild cognitive impairment (MCI) at baseline (red). B, Participants categorized as phosphorylated tau (p‐tau)₁₈₁/amyloid beta (Aβ)_1‐42 ratio positive at baseline (red); participants categorized as p‐tau₁₈₁/Aβ_1‐42 ratio negative at baseline (blue). C, Participants categorized as progressors (red); participants categorized as non‐progressors (blue)

TABLE 2

Mean yearly rates of change in the CSF concentration of the five analytes under study resulting from the mixed‐effects modeling and their differences among various subsets of study participants

		CMGA	FABPH	NPTX2	SCG2	VGF
Model 1	CN at baseline vs. zero	–0.01 ± 0.01	0.01 ± 0.01	–0.03 ± 0.02	–0.02 ± 0.01	–0.02 ± 0.02
	MCI at baseline vs. zero	–0.04 ± 0.01*	0.02 ± 0.01*	–0.08 ± 0.02‡	–0.02 ± 0.01	–0.04 ± 0.02*
	CN vs. MCI at baseline	0.02 ± 0.01	0.01 ± 0.01	0.05 ± 0.02*	0.01 ± 0.01	0.02 ± 0.02
Model 2	Ratio– at baseline vs. zero	–0.01 ± 0.01	0.02 ± 0.01	–0.03 ± 0.02	0 ± 0.01	–0.01 ± 0.02
	Ratio+ baseline vs. zero	–0.05 ± 0.01†	0.02 ± 0.01	–0.09 ± 0.02‡	–0.04 ± 0.01*	–0.06 ± 0.02‡
	Ratio+ vs. Ratio– at baseline	0.04 ± 0.01†	0 ± 0.01	0.06 ± 0.02†	0.03 ± 0.01*	0.05 ± 0.02‡
Model 3	Non‐progressors vs. zero	–0.02 ± 0.01	0.01 ± 0.01	–0.03 ± 0.02	–0.02 ± 0.01	–0.02 ± 0.02
	Progressors vs. zero	–0.05 ± 0.02*	0.01 ± 0.02	–0.11 ± 0.02‡	–0.03 ± 0.02	–0.06 ± 0.02*
	Progressors vs. Non‐progressors	0.03 ± 0.02	0 ± 0.01	0.08 ± 0.02‡	0.01 ± 0.02	0.04 ± 0.02

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Notes: P‐values (unadjusted for multiplicity) are associated with the tests of the hypothesis that mean rate of change is equal to zero, or the hypothesis that the two rates of change estimated for two participant groups are the same.

Ratio+: participants with p‐tau₁₈₁/Aβ_1‐42 ≥ 0.025. ²⁵

Ratio–: participants with p‐tau₁₈₁/Aβ_1‐42 < 0.025. ²⁵

Progressors: participants who were initially diagnosed as CN (resp. MCI) and who were diagnosed within 4 years and 1 month after the initial diagnosis with MCI or dementia (resp. dementia).

^* < 0.05; ^† < 0.005; ^‡ < 0.0005.

Abbreviations: Aβ, amyloid beta; CMGA, chromogranin A; CN, cognitively normal; CSF, cerebrospinal fluid; FABPH, x fatty acid binding protein; FDG‐PET, fluorodeoxyglucose positron emission tomography; MCI, mild cognitive impairment; NPTX2, neuronal pentraxin 2; p‐tau, phosphorylated tau; SCG2, secretogranin; SUVR, standardized uptake value ratio; VGF, neurosecretory protein VGF.

The magnitude of the declines in NPTX2 concentrations also varied between progressors and non‐progressors (–0.11 vs. –0.03 ng/mL; P = .0004, Figure 2C and Table 2), further validating the association of NPTX2 concentration and clinical prognosis. To put these differences in context, consider that the average baseline concentration of NPTX2 in the study participants was 4.7 ± 0.6 ng/mL (mean ± standard deviation). Thus, over a 5‐year period, a decline of 0.1 ng/mL per year amounts to a cumulative decrease of around 10% or one standard deviation (Figure S8 in supporting information). To validate these findings, we analyzed a restricted dataset involving 110 (59%) participants with at least three measures of NPTX2 concentration in the first 4 years since their first visit to rule out potential undue influence of a small number of measurements from a minority of participants with longer than usual follow‐up. This analysis yielded very similar results to those presented here (data not shown).

3.2. Correlations of the rate of change in NPTX2 concentration with cognitive measures and AD biomarkers

To elucidate the potential role of NPTX2 in the pathophysiologic processes involved in the progression of AD, we examined the relationship between the rates of change in NPTX2 concentration and the rates of change in known biomarkers of neurodegeneration (p‐tau_181, FDG‐PET, hippocampal volume) and measures of cognitive ability (Table S7 in supporting information). These were exploratory post hoc analyses. Among the observed relationships, there was a modest positive relationship between the rates of change in NPTX2 and p‐tau₁₈₁ concentrations in CSF, such that individuals with high rates of decline in NPTX2 had either no change in p‐tau₁₈₁ or slight decreases over the same period (Figure S12 in supporting information). This relationship was especially evident among participants with baseline p‐tau₁₈₁/Aβ_1‐42 ≥ 0.025 (i.e., ratio positive).

Declines in NPTX2 concentration were also correlated with cognitive declines primarily among participants classified as MCI or p‐tau₁₈₁/Aβ_1‐42 ratio positive (Figure 3). These correlations appear fairly robust; even after excluding potentially influential observations (i.e., mean yearly changes in MMSE < —4 and ADAS‐Cog13 > 10) the correlations remained statistically significant (MMSE: P = 0.016, ADAS‐Cog13: P = .012), albeit moderate in strength (MMSE: r = 0.26, ADAS‐Cog13: r = –0.28). The statistically significant correlation observed between NPTX2 concentration and ADAS‐Cog13 cognitive measure and hippocampal volume among CN participants (Table S7), may point to NPTX2 as a potential prognostic marker of impending brain degeneration and cognitive decline.

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FIGURE 3

Correlations between neuronal pentraxin 2 (NPTX2) slopes and Mini‐Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale–Cognitive 13‐item Subscale (ADAS‐Cog13) cognitive measures

4. DISCUSSION

Out of the five studied candidate biomarkers, NPTX2 showed the most significant within‐subject changes over the course of 3 or more years. While within‐subject trajectories of CSF concentrations of the five proteins of interest in ADNI CN and MCI participants were highly variable over a period of 3 or more years, group‐level trajectories based on baseline diagnosis, baseline p‐tau₁₈₁/Aβ_1‐42 ratios in CSF, and clinical progression showed differential patterns of change, with those in NPTX2 standing out. CMGA, NPTX2, SCG2, and VGF all showed steeper declines in ratio‐positive participants, classified as exhibiting p‐tau₁₈₁/Aβ_1‐42 baseline ratio above 0.025. ²⁵ In contrast, a hypothesized decline in FABPH across the subgroups based on earlier cross‐sectional studies comparing control, MCI, and AD participants ¹³ was not observed. This finding highlights the importance of within‐subject longitudinal studies for validating or refuting inferences from cross‐sectional comparisons between diagnostic groups. This study specifically focused on assessing within‐MCI participant changes over time in selected analytes that had not previously been followed longitudinally. Baseline values in MCI participants of neurogranin, VILIP‐1, and others are predictive of subsequent clinical worsening. However, given their very modest (< 5%) annualized rates of change at the group level and small number of participants with large changes, their longitudinal trajectories have not emerged as likely to provide much additional biologically relevant information. ^{14 , 26 , 27 , 28 , 29 , 30 , 31}

From the perspective of identifying a novel biomarker that might apply to drug development, we required a sufficiently accurate and precise assay to interpret individual‐level changes as reflecting biologic processes. Therefore, we developed a quantitative MS assay with labeled analyte internal standards, building on our earlier semi‐quantitative one. ¹³ Moreover, a minimum of three CSF samples at roughly annual or longer intervals allowed for more reliable within‐subject calculations of rates of change. Using this refined methodology, we identified NPTX2 as the analyte that, at a group level, exhibited statistically significant rates of decline in its CSF concentration far greater than any of the other four analytes among MCI participants versus CN; among progressors versus non‐progressors; as well as in participants with positive baseline CSF values of p‐tau₁₈₁/Aβ_1‐42 versus ratio‐negative participants. Second, we observed that NPTX2 declined in 77% (30 out of 39) of progressing and 72% (61 out of 85) of ratio‐positive participants, a substantial proportion (see Figures S10 and S11 in supporting information). These results extend those from cross‐sectional studies, which supported that “baseline” values of NPTX2 concentration in the CSF of MCI participants when added to Aβ₄₂ provided for improved early prognosis compared to Aβ₄₂ alone, and that when added to Aβ₄₂ and p‐tau₁₈₁, ²² this information provided for a more accurate prediction of disease progression using the NIA/AA AT(N) classification scheme. ⁹ The important extension that our longitudinal study provides is the presence of a substantially accelerated clinical progression as indexed by meeting AD diagnostic criteria or by measures of cognitive decline in those individuals in whom NPTX2 decreases regardless of its baseline value.

From the perspective of multiple processes contributing to clinical progression beyond the degree of AD pathologic changes, our findings provide a rationale for exploring modulation of NPTX2 as a therapeutic target for slowing clinical progression in at least a subset of MCI patients who are biologically positive for AD according to the AT(N) classification.

The biological role of NPTX2 has been of wide interest and, at least in animals, one relevant to neuronal function related to cognition based on its involvement in activity‐dependent plasticity. ³² NPTX2 is a member of a family of “long” neuronal pentraxins that traffic to the extracellular surface at excitatory synapses. ³² They have been shown to bind AMPA‐type glutamate receptors and contribute to both developmental and adult synaptic plasticity. ^{33 , 34 , 35 , 36 , 37 , 38 , 39 , 40} Xiao et al. ²¹ found that NPTX2 was downregulated in the post mortem brain of human participants with AD and reduced in the CSF of AD patients, which correlated with diminished cognitive function. Xiao et al. ²¹ interpreted their findings as supporting the hypothesis “that NPTX2 downregulation … represents a previously unrecognized mechanism important for human cognitive dysfunction and progression in Alzheimer's disease.” Furthermore, they noted that because NPTX2 is not downregulated in a widely used model of mouse amyloidosis, ⁴¹ and is distinct from other CSF markers attributed to neurodegeneration including tau and p‐tau, NPTX2 is an indicator of specific rather than general decrease in excitatory synapse function. This stands in contrast to the more general synaptic marker neurogranin, increased concentrations of which are highly correlated with tau and overall pathology. ^{11 , 14 , 26 , 27} A subsequent study in a University of California San Diego cohort showed that the CSF concentration of NPTX2 correlated with cognitive function in MCI participants and was predictive of progression to dementia using the MS data generated by our team and available online. ²² An independent follow‐up analysis of the same cross‐sectional dataset further demonstrated the strong predictive power of decreased NPTX2 concentration in memory decline and medial temporal lobe atrophy. ⁴² Overall, these studies and analyses show that lower values of NPTX2 in the CSF are associated with poor cognitive function consistent with the possibility that NPTX2 concentrations reflect the function of a discrete population of excitatory synapses in the brain. ²¹ Recently, NPTX2 decline has also been reported to be associated with the clinical progression of frontotemporal dementia, ⁴³ which suggests that it taps into a process that may be relevant to interactions with other primary pathophysiologic processes.

Our findings on the relationship of longitudinal changes in NPTX2 to the degree of AD pathology as indexed by the p‐tau₁₈₁/Aβ_1‐42 ratio; stable or progressing MCI; and cognitive function address a related question: To what extent do within‐subject decreases of CSF NPTX2 in MCI participants independent of baseline values predict or correlate with various clinical trajectories? Because within‐subject declines of NPTX2 in the present study correlate with declines in cognitive function of patients beyond what could be explained by Aβ_1‐42, tau, and structural measures, we explored whether these changes could be clearly related to other processes that were documented over time in the same ADNI participants. If the loss of NPTX2 was simply a reflection of the general loss of gray matter (an aspect of AD), then it would not be as useful as a biomarker with direct relevance.

The patterns of change (Figure 2 and Figures S9‐S11 in supporting information) argue strongly against an overall loss of tissue or extent of brain pathology as an explanation for the decline in NPTX2. For instance, a substantial proportion of individuals who progress or have high p‐tau₁₈₁/Aβ_1‐42 ratios did not show a decline in NPTX2 concentration. Furthermore, no correlations were observed between declines in NPTX2 and hippocampal volume among the studied participants.

Unexpectedly, individuals who showed the greatest rates of decline in NPTX2 were those in whom p‐tau₁₈₁ did not change or slightly decreased. This is in the opposite direction of what would be expected if worsening pathology, as indexed by increased p‐tau₁₈₁, were the main determinant of decline in NPTX2. This raises the possibility that in some participants who are positive for AD pathology, a process underlying the declines in NPTX2, accelerates a cognitive decline that is not simply a cascading downstream consequence of AD pathology. Put another way, the present findings indicate that no matter what an MCI participant's starting NPTX2 concentration is in the CSF, if it decreases over a period of several subsequent years, cognitive function will decrease more rapidly, supporting our findings that the presence of AD pathology as indicated by p‐tau₁₈₁/Aβ_1‐42 ratios is not a necessary or sufficient determinant of decreases in NPTX2. This further strengthens the speculation, raised by Xiao et al., ²¹ that NPTX2 could be a therapeutic target for intervention with the added perspective that this might be the case in only a subset of MCI patients.

CONFLICTS OF INTEREST

Susan A. Baker, Angus C. Nairn, and Daniel S. Spellman have nothing to declare. Leslie M. Shaw receives research support from NIH/NIA U19 AG024904, ADNI3 grant; NIH/NIA P30 AG010124, UPENN ADCC grant; and the Michael J. Fox Foundation for Parkinson's Research. He is a consultant for Biogen and Roche Diagnostics and is on the speaker's bureaus for Biogen and Fujirebio. Angus C. Nairn receives research support from NIH/NIA P30 AG066508. Clifford R. Jack Jr. serves on an independent data monitoring board for Roche, has consulted for and served as a speaker for Eisai, and consulted for Biogen, but he does not receive personal compensation from any commercial entities. Additionally, Clifford R. Jack Jr. receives research support from the NIH and the Alexander Family Alzheimer's Disease Research Professorship of the Mayo Clinic. Ondrej Libiger receives salary and company stock as compensation for his employment with Janssen Research & Development. William Z. Potter was previously employed by the National Institute of Mental Health, and he is a stockholder in Merck & Co., Inc.. He is a Co‐Chair Emeritus for the FNIH Biomarkers Consortium Neuroscience Steering Committee. Currently residing in Philadelphia, PA, he serves on DSMBs for AgeneBio and Regenacy and as a consultant for Karuna, Otsuka, Neurocrine, Eliem, and Emerald Lake Safety. Additionally, he receives grant support from the NIA. Nandini Raghavan is employed by Janssen Research & Development and owns stock in Johnson & Johnson. Kelly L. Umaña, Michael C. Biarnes, and Rosa M. Canet‐Avilés were previously employed by the FNIH. Daniel Chelsky and Yannick‐André Breton were previously employed by Caprion Biosciences. Laetitia Cortes and Mark Watson are employed by Caprion Biosciences. The FNIH provided financial support to Caprion Biosciences to perform the submitted work.

COLLABORATORS

Data used in preparation of this article were obtained from the ADNI database (adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A complete listing of ADNI investigators can be found at: http://adni.loni.usc.edu/wp‐content/uploads/how_to_apply/ADNI_Acknowledgement_List.pdf

Supporting information

ACKNOWLEDGMENTS

Notes

Libiger O, Shaw LM, Watson MH, et al., Alzheimer's Disease Neuroimaging Initiative (ADNI), Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium CSF Longitudinal Proteomics Project Team . Longitudinal CSF proteomics identifies NPTX2 as a prognostic biomarker of Alzheimer's disease. Alzheimer's Dement. 2021;17:1976‐1987. 10.1002/alz.12353 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]

Funding information This study, through multi‐institutional collaboration as a project of the Foundation for the National Institute of Health (FNIH) Biomarkers Consortium, Longitudinal Proteomic Changes in Cerebrospinal Fluid from Alzheimer’s Disease Neuroimaging Initiative (ADNI): Towards Better Defining the Trajectory of Prodromal and Early Alzheimer’s Disease, received funding support provided to the FNIH by Genentech, a member of the Roche Group; H. Lundbeck A/S; Janssen Research & Development, LLC; Merck & Co., Inc.; and Takeda Pharmaceutical Company Limited.

[The copyright line for this article was changed on May 26th, 2021 after original online publication.]

REFERENCES