Alzheimer-Associated Neuronal Thread Protein: Research Course and Prospects for the Future

Abstract

Alzheimer’s disease (AD) is the leading cause of dementia. With aging societies, the prevalence of AD is increasing dramatically worldwide. The onset of AD is often not identified, and currently no available treatments are capable of stopping the disease process and its effect on cognitive decline. Thus, well-validated biomarkers of the preclinical stages of AD are needed. Alzheimer-associated neuronal thread protein (AD7c-NTP) is a member of the neuronal thread protein family and has a molecular weight of approximately 41 kD. AD7c-NTP has been identified as a biomarker for its specifically elevated levels in putative brain domains, cerebrospinal fluid (CSF), and the urine of AD and mild cognitive impairment (MCI) patients. Since the urine test is non-invasive, easy to perform, and patients accept it more easily than other methods, the urinary AD7c-NTP concentration has been recommended as a practical diagnostic tool for diagnosing AD and MCI. AD7c-NTP has undergone nearly 25 years of research course from its initial discovery to pathological verification, multi-center clinical evaluation, improvement of detection methods, epidemiological investigation, and combined application with other biomarkers. However, as a fluid biomarker, AD7c-NTP can be detected in urine instead of the traditional biomarker sources—CSF or blood, which has made the use of AD7c-NTP as a biomarker controversial. In this article, we review the research course of AD7c-NTP and suggest directions for future research.

Keywords

Alzheimer’s disease Alzheimer-associated neuronal thread protein biomarker urine

As findings regarding the pathophysiological, mo-lecular, and structural changes in Alzheimer’s disease (AD) increased, some biomarkers have been developed as diagnostic tools; this has shifted the diagnosis of AD from qualitative symptoms to quantifiable dynamic biological alterations [1, 2]. The early in vivo detection of AD requires non-invasive assays that are highly sensitive via relatively specific biomarkers that reflect AD-related pathophysiological abnormalities. Alzheimer-associated neuronal thread protein (AD7c-NTP) is a novel cDNA that encodes an approximately 41 kD membrane-spanning phosphoprotein [3]. AD7c-NTP is overexpressed in AD brains and its corresponding protein is accumulated in neurons with AD progression. Since 1996, when AD7c-NTP was first discovered and reported by de la Monte et al., for 25 years of research on AD7c-NTP has yielded improvement of detection methodologies, multi-center clinical validation, investigation of population-based epidemiology, and combined application with other indicators [4]. AD7c-NTP has been identified as a biomarker for its specifically elevated levels in putative brain domains, cerebrospinal fluid (CSF), and urine of AD patients. Since the urine test is non-invasive, easy to perform, and patients accept it more easily than other methods, the urinary AD7c-NTP concentration has been recommended as a practical tool for diagnosing AD. However, AD7c-NTP is a fluid biomarker detected in urine instead of the traditional biomarker sources—CSF or blood, which has made the use of AD7c-NTP as a biomarker controversial. Furthermore, many questions remain regarding AD7c-NTP. How is AD7c-NTP involved in AD pathology? How is AD7c-NTP secreted into the urine and remain stable? What are the unresolved issues and potential avenues for future research on AD7c-NTP? In this article, we review AD7c-NTP from its initial discovery to clinical applications, during which we attempt to answer the above questions.

THE DISCOVERY OF AD7c-NTP (1989–1997)

AD is a multifactorial disease with “multiple-hit” underlying mechanisms, including conclusive genetic factors (such as family early-onset mutations in amyloid precursor protein [APP], presenilin 1 [PSEN1], and presenilin 2 [PSEN2], as well as sporadic late-onset mutations in many different genes, including the homozygous E4 allele of apolipoprotein E [APOE]), neuroinflammation, impaired energy metabolism, mitochondrial dysfunction, and chronic oxidative stress [5 –7]. The pathological features of AD are senile plaques deposited by amyloid-β (Aβ) protein extracellularly, neurofibrillary tangles (NFTs) formed by abnormal phosphorylated of tau protein intracellularly, and neuron loss. One such top area of research has focused on impaired brain metabolism and, in particular, the role of insulin resistance (IR)-related mechanisms [8, 9]. Based on this research, together with findings regarding pancreatic thread protein (PTP)—a secretory protein derived from pancreatic acinar cells that is insoluble at physiologic pH and appears as fibrils under electron microscopy—de la Monte et al. found that PTP may be associated with Aβ neuritic plaques and NFTs underlying AD [10]. However, the physiologic function of PTP was previously unknown. Another study used a full-length cloned bovine PTP cDNA and synthetic oligonucleotides corresponding to human PTP cDNA to perform hybridization studies as well as immunolabeling assays; this study found abnormal accumulation of protein expression in AD and the developing human brain [11]. This protein shares three identical antigenic epitopes with PTP, namely NTP.

NTPs comprise a family of molecules expressed in the brain and in primitive neuroectodermal tumor cell lines, and their expression levels are increased in neurons during proliferation, differentiation, and brain development, as well as during neurodegeneration caused by AD [12 –14]. The nature and distribution of NTP expression have been characterized by brain-derived polyclonal and monoclonal antibodies, and several distinct NTP-immunoreactive molecules (42 kD, 39 kD, 26 kD, 21 kD, 17–18 kD, and 15 kD) have been identified [14]. Prior to a novel Alu sequence-containing cDNA—designated as AD7c-NTP—being isolated from an end-stage AD brain cDNA library and verified in both postmortem and clinical CSF specimens, AD7c-NTP has previously been confirmed to be a potential biomarker of AD [4]. The AD7c-NTP gene is composed of 1,442 bp, and the open reading frame contains 1,125 nucleotides; the translated 375 amino-acid sequence encodes an approximately 41 kD membrane-spanning phosphoprotein [9]. In autopsy brain tissues, the concentration of AD7c-NTP in CSF of definitive AD samples (9.2±8.2 ng/ml) was significantly higher than that in age-matched control samples (1.6±0.9 ng/ml). Additionally, in clinical specimens, the concentration of AD7c-NTP in CSF of AD samples (4.6±3.4 ng/ml) was also significantly higher than that in aged-control samples (1.2±0.7 ng/ml) and the non-AD neurological-disease control samples (1.0±0.9 ng/ml) [4]. So far, the discovery course of AD7c-NTP has gone through the PTP-NTP-AD7c-NTP process, including the following: from gene to protein levels, from post-mortem specimens to live specimens; and from brain tissues to CSF.

THE RELATIONSHIP BETWEEN AD7c-NTP AND AD PATHOLOGY (1996–2003)

AD7c-NTP mRNA transcripts have been detected in the frontal and temporal lobes of postmortem brain tissues (via northern blotting and reverse transcription-polymerase chain reaction [RT-PCR]), but not in the pancreas, kidney, liver, spleen, gastrointestinal tract (various regions), ovaries, fallopian tubes, uterus, thyroid, lung, skeletal muscle, testis, or thymus [4]. Meanwhile, AD7c-NTP protein has also been demonstrated to be expressed in the frontal and temporal lobes (western blotting), and quantitative analysis of AD7c-NTP has also been detected in postmortem CSF by western blotting and enzyme-linked immunosorbent assays (ELISAs) [4]. These findings illustrated that AD-related overexpression of AD7c-NTP is relatively specific of the brain and that elevated levels in CSF may reflect the severity of AD (data are reported in the previous section). Moreover, the cellular localization of AD7c-NTP mRNA (in frontal and temporal cortical neurons) and protein (in neurons, neuropil fibers, white-matter fibers, and irregular neuritic processes) have been shown to exhibit higher expression levels in AD brains compared to those in control brains, as demonstrated by in-situ hybridization and immunohistochemical, respectively [4]. Further research via double-labeled paraffin sections has revealed that AD7c-NTP is co-localized with phosphorylated tau (p-tau) and phosphorylated-neurofilament in neuronal perikarya, abnormal neurites, and swollen axons of frontal and temporal tissues from AD brains [14]. However, double-labeled cells with non-uniform patterns suggest that there may be sequential but overlapping waves of altered immunoreactivity of AD7c-NTP. In addition to the presence of extracellular NFTs, the phenomenon that increased AD7c-NTP expression is most prominent in neurons that are either cytologically intact or slightly degenerated, rather than in apoptosis cells, provide some evidence that AD7c-NTP may provide early diagnostic clues before irreversible neurodegeneration occur [14].

In vitro studies have shown that over-expression of AD7c-NTP gene in transfected post-mitotic primary rat neuronal cells causes cell death and neuritic sprouting, which are two prominent abnormalities associated with AD [15]. These results provide evidence that aberrantly increased AD7c-NTP expression may play a role in AD-type neurodegeneration. Further research in human neuronal cells has demonstrated that overexpression of AD7c-NTP increases apoptosis-mediated cell death, impairs mitochondrial function, and increases cellular levels of p53 and CD95 pro-apoptosis gene products that are known to occur in AD [16]. In addition, overexpression of AD7c-NTP is associated with increased levels of p-tau protein, but not Aβ, which is consistent with in vivo co-localized results [14, 16]. Mechanistic research has shown that in human SH-SY5Y neuronal cells, AD7c-NTP expression is regulated by insulin and IGF-1 stimulation and is phosphorylated by GSK-3β on serine residues [17]. Furthermore, in PNET2 human central nervous system (CNS)-derived neuronal cells transfected with AD7c-NTP cDNA, insulin and IGF-1 stimulation is associated with reduced viability with increased levels of p53, p21/Waf-1, phospho-JNK, and phospho-tau, as well as reduced levels of Bcl-2 and phospho-Erk MAPK [18]. These results suggest that overexpression of AD7c-NTP and its association with reduction in cellular viability are mediated by impaired insulin/IGF-1 signaling. Since CNS neurons are abundant with insulin and IGF-1 receptors, they may be particularly vulnerable to the adverse effects of AD7c-NTP. In recent years, a wealth of data has pointed to a potential role of the insulin/IGF-1 pathway in the pathogenesis of AD. Compelling evidence has indicated that insulin and IGF-1 have direct effects on metabolism, clearance of Aβ, and the development of NFTs [19, 20]. These findings indicate that the insulin/IGF-1 signaling pathway may represent a novel and promising therapeutic target for the treatment of AD. Therefore, changes in AD7c-NTP levels may be useful as an objective indicator to detect the efficacies of drugs or interventions targeting the insulin/IGF-1 signaling pathway.

ASSAYS FOR DETECTING AD7c-NTP (1998–2016)

The relationship between AD7c-NTP and AD pathology has been confirmed at the levels of both gene and protein expression. The detection methods have included immunoradiometric assay [4, 12], in-situ hybridization [4], RT-PCR [4], northern blotting [4], western blotting [4 , 14], and immunohistochemistry [4 , 12–14]. However, results of autopsies are meaningless for clinical diagnoses, and biopsies of brain tissue are not feasible in living patients. Based on the structure of AD7c-NTP with a hydrophobic leader sequence and myristoylation motif, de la Monte et al. have speculated that it may be secreted into CSF and ventricular fluid, and further verified its presence in postmortem AD and aged-control samples via western blotting and ELISAs [9]. Nevertheless, lumbar punctures are required to obtain CSF samples. Unfortunately, this procedure is associated with potentially serious complications, and patients with probable AD or mild cognitive impairment (MCI) usually have no indication of lumbar puncture in clinical practice.

Subsequently, Ghanbari et al. demonstrated that AD7c-NTP in urine (24 h collection) has the same molecular mass as that in CSF and cerebral tissue [21, 22]. Their results verified that the levels of urinary AD7c-NTP in AD patients (2.5 ng/mL) were significantly higher than those of the control group (0.8 ng/mL); furthermore, taking 1.5 ng/mL as the cutoff value, the specificity and sensitivity were comparable to AD7c-NTP levels in CSF [22]. Under normal circumstances, the blood-brain barrier (BBB) keeps neurotoxic plasma-derived components, cells, and pathogens out of the brain. However, early BBB breakdown and/or dysfunction have been shown in AD before dementia, neurodegeneration, and/or brain atrophy occur [23]. AD7c-NTP can be detected in urine may be a result of the damaged BBB, but the specific process mediating translocation of AD7c-NTP from CSF/blood to urine is still unknown. We speculate that urinary AD7c-NTP may be derived from the consequence of neuronal death, and the early BBB breakdown and/or dysfunction of AD may accelerate the release of AD7c-NTP.

Since the urine test for AD7c-NTP is non-invasive and easy to perform, many laboratories in different countries and regions have developed their own urine AD7c-NTP diagnostic kit to verify the specificity and sensitivity of AD7c-NTP as an AD biomarker for clinical diagnosis (description detailed in the next section) [24 –29]. However, clinical application and research of AD7c-NTP still face many problems in terms of issues with detection methods and procedures. Previous studies of AD7c-NTP in urine have been based on specimens from first-morning urine/24 h urine, stored immediately at 2–8°C and tested within 24 h [22 , 24–29]. Notably, in clinical practice, immediate analysis of fresh specimens or collection of the first-morning urine/24 h urine is sometimes impractical. Wang et al. verified that AD7c-NTP can be tested with random urine instead of the first-morning urine; additionally, they found that if the specimen cannot be tested in time, boric acid functions as a preservative when stored at 4°C insofar as the sample is tested within 5 days [30]. This result represents a minor step of AD7c-NTP toward a reliable and readily available laboratory test. Moreover, a new technology named “urimem” has been developed by Gao et al. that can absorb urinary protein onto a membrane, which may also be a feasible method to assist in clinical detection [31].

In the process of biomarker development, the establishment of standards for sample collection and the unified calibration of specific instrumentation are necessary to avoid multicenter variability. However, it has remained unclear as to how AD7c-NTP may be kept stable from CSF to urine. Based on evidential extrapolations, AD7c-NTP is the product of brain degeneration, which the CSF discards to maintain homeostasis [23, 32]. Drastic changes in protein levels are always removed from the CSF and blood by homeostatic mechanisms to maintain all cells in a stable internal environment [33]. By contrast, urine has no such homeostatic mechanism, it derives from blood, and can accumulate and tolerate considerable changes without causing harm to the body. This property makes urine a better source of biomarker compared to that from other body fluids [33]. AD7c-NTP is a 41 kD protein with an isoelectric point of 9.89. Based on the amphoteric ionization properties of proteins, AD7c-NTP has a positive polarity in the blood at a pH between 7.35–7.45, while plasma proteins are negatively charged with an isoelectric point of 4–6, such that they can be combined closely and be kept stable. Next, the ability of different substances to pass through the glomerular filtration membrane depend on the size of the substance and the charge it carries. AD7c-NTP can be filtered through the kidney due to its low molecular weight of 41 kD and positive polarity, as the Glomerular basement membrane is negatively charged, and allows only low molecular-weight substances (below 60 kD) to pass through. We speculate that high concentrations of AD7c-NTP in the blood enter into the renal tubules due to differences in concentration and then reabsorb back into the blood due to a potential difference. This is a “double-balanced” steady state process (as shown in schematic Fig. 1).

Fig. 1

The “double-balance” of concentration and charge in the medullary loop of AD7c-NTP. BBB, blood-brain barrier; GBM, glomerular basement membrane; RT, renal tubules.

THE MULTI-CENTER CLINICAL VERIFICATION OF AD7c-NTP (2002–2016)

As previously mentioned, based on the detection of postmortem and clinical CSF samples, de la Monte et al. initially reported that AD7c-NTP is a potential biomarker of AD [4]. Since then, Ghanbari et al. developed an ELISA, known as the AD7c test, to detect AD7c-NTP in urine [21]. Since urinary AD7c-NTP has the same molecular weight and the antibody-binding properties as those of AD7c-NTP in CSF, these discoveries have prompted multiple laboratories from different countries and regions to verify the diagnostic efficiency of urinary AD7c-NTP as an AD biomarker in recent years. The diagnostic characteristics of urinary AD7c-NTP in representative studies are shown in Table 1.

Table 1

The diagnostic characteristics of urinary AD7c-NTP in these representative studies included in the review (searched in PubMed)

Study ID	Patients (n)	Controls (n)	Diagnostic criteria	Multi-centered	Double-blinded	Detection assay	Cut-off value	Sensibility (%)	Specificity (%)
Munzar et al. [24]	Possible/Probable AD (82)	Non-AD demented^a (13)	not controlled	Yes	Yes	Competitive ELISA	18.0 RU/mL	89.0	90.0
		Non-demented (27)
Levy at al. [25]^b	Probable AD (7)	Normal (7)	NINCDS-ADRDA	Yes	Yes	Competitive ELISA	—	—	—
Goodman et al. [26]	Probable AD (35)	Non-AD (43)	NINCDS-ADRDA	Yes	Yes	Competitive	22.0 g/mL	91.4	90.7
	Possible AD (53)		NINCDS-ADRDA			Affinity	22.0 g/mL
	MCI (37)		AAN			Assay	22.0 g/mL
Youn et al. [27]	Probable AD (49)	PD (20) Healthy (22)	NINCDS-ADRDA	Yes	Yes	Urine NTP Assay	21.6 g/mL	81.6	78.6
Ma et al. [28]	Probable AD (49)	Healthy (118)	NINCDS-ADRDA	Yes	Yes	ELISA	1.5 ng/mL	89.3	84.7
Ma et al. [29]^c	Probable AD (45) MCI (60)	Normal (65)	NINCDS-ADRDA CDR, MoCA et al.	No	Yes	ELISA	—	—	—

^aNon-AD demented included corticobasal degeneration, delirium, depression, epilepsy, hyperparathyroidism, Lewy body disease, metabolic encephalopathy, multi-infarct dementia, multiple sclerosis, pseudodementia, and psychosis. ^bThe reference [25] was a follow-up study. ^cThe purpose of [29] was to compare the urinary AD7c-NTP levels in MCI with AD groups with those in the control group. PD, Parkinson disease; NINCDS-ADRDA, the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related Disorders Association criteria; AAN, American Academy of Neurology. CDR, clinical dementia rating; MoCA, Montreal cognitive assessment.

These studies have commonly revealed that in comparison to the normal control group [24 –29], non-AD demented group [24], and Parkinson’s disease group [27], urinary AD7c-NTP levels are significantly increased in AD and MCI patients. Due to the different detection assays used in various laboratories and the inconsistencies in AD diagnostic criteria adopted, the resulting diagnostic sensitivities (81.6%–91.4%) and specificities (78.6%–90.7%) have not been consistent across studies. However, these studies have provided evidence-based evaluations of the clinical application of urinary AD7c-NTP.

COMBINED APPLICATION OF AD7c-NTP WITH OTHER INDICATORS (2000–2020)

The current diagnostic methods for AD could include the neuropsychological testing, genetic screening, imaging examination, and detection of fluid biomarkers. The combinatorial use of different indicators may eventually achieve the diagnostic accuracy necessary to identify patients at the earliest stages of AD, when interventions are most promising [34, 35]. Murphy et al. measured CSF levels of AD7C-NTP and tau in AD, Parkinson’s disease, and elderly healthy-control groups; the combined evaluation of the two biomarkers raised the specificity to 93%at a 63%sensitivity level, and CSF AD7c-NTP showed a small but significant inverse correlation with Mini-Mental State Examination (MMSE) scores of AD patients [36]. Zhang et al. analyzed the correlation between urine AD7c-NTP and Aβ deposition via [¹¹C]-labeled Pittsburgh compound B (PiB)-PET in AD and MCI patients. The results showed that set 1.46 ng/ml as the cut-off value, urinary AD7c-NTP had a high specificity (92.9%) and moderate sensitivity (68.8%) in predicting Aβ deposition among patients with cognitive impairment. Furthermore, urinary AD7c-NTP levels were strongly correlated with symptoms of agitation in patients with AD and MCI [37]. Zheng et al. found that a testing strategy combining the detection of serum brain derived neurotrophic factor (BDNF) and urinary AD7c-NTP in MCI patients who harbored the APOE ɛ4 allele yields a positive predictive value (PPV) of 98%and a negative predictive value (NPV) of 90%[38]. Li et al. further confirmed urinary AD7c-NTP level are significantly higher in subjects with APOE ɛ3/4 and 4/4 than those without APOE ɛ4, and urinary AD7c-NTP levels also positively correlated with APOE genotype grade [39]. Additionally, some other studies have reported that a combination of urinary AD7c-NTP with plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) and peripheral blood CD45RA+T- lymphocyte subsets yield an effective improvement in the detection efficacy [40, 41].

EPIDEMIOLOGICAL INVESTIGATION OF AD7c-NTP (2014–2020)

To verify potential confounding factors that may affect urinary AD7c-NTP results, the distributions of demographic characteristics of urinary AD7c-NTP, and its associations with other common chronic diseases were investigated in an epidemiological study consisting of 1,805 participants [42]. The results were in accordance with a previous study involving 294 healthy participants, which revealed that urinary AD7c-NTP in the Chinese population had a trend of increasing with age, and females exhibited higher levels than did males [43]. This phenomenon is consistent with the increase in the incidence of AD with age and that women are at higher risk than are men [44]. However, previous multicenter clinical studies have not found this trend of AD7c-NTP in terms of gender and age. This discrepancy may mainly be attributed to small sample sizes, and/or different detection methods. Meanwhile differences in urinary proteins based on gender and age in humans and rats have also been revealed by proteomics [45, 46]. Based on the above results, we suggest that future AD7c-NTP or other urine biomarker investigations should consider gender as a crucial factor in experimental design and data analysis, and reference intervals by gender should be estimated.

Additionally, in a population-based survey, the distribution of AD7c-NTP was found to not be affected by education, occupation, body mass index, place of residence, family history of dementia, hypertension, stroke, anemia, diabetes, dyslipidemia, renal insufficiency, cancer, chronic lung disease, chronic liver disease, thyroid dysfunction, subjective cognitive decline, or depression symptoms [42, 47]. However, there were significant differences in urinary AD7c-NTP levels between APOE allele carriers with and without a history of coronary heart disease or diabetes [39]. Additionally, individuals with amnestic MCI (aMCI) have higher levels of urinary AD7c-NTP than those with non-amnestic MCI (naMCI) [48]. Although the above-mentioned common diseases in old age do not seem to affect urinary AD7c-NTP levels, the combined presence of cognitive dysfunction is associated with increased AD7c-NTP levels. A cross-sectional study demonstrated that the level of urinary AD7c-NTP was elevated in older hypertensive patients with lower cognitive function, and that IR may be involved in this process [49]. A case-control study revealed that in comparison to patients with only late-life depression (LLD), patients with LLD and cognitive impairment showed significantly higher urinary AD7c-NTP levels [50]. Thus, urinary AD7c-NTP levels may represent a potential biomarker for early identification of cognitive impairment in LLD patients.

COMPREHENSIVE EVALUATION OF AD7c-NTP ANG FUTURE DIRECTIONS

This review systematically summarized the re-search course of AD7c-NTP (as shown in Fig. 2). The discovery of AD7c-NTP initially originated from research on IR-related AD pathogenesis, as well as its similarity with PTP in terms of insolubility and morphology. AD7c-NTP was then isolated from an end-stage AD brain cDNA library. In vivo studies have shown that AD7c-NTP expression is elevated in select brain tissues, as well as in CSF and urine, in AD patients. In vitro studies have further revealed AD7c-NTP-mediated neuronal apoptosis and mitochondrial damage. Since urinary AD7c-NTP detection is non-invasive and easily accessible, multi-center clinical research has validated its diagnostic sensitivity and specificity as an early screening biomarker of AD/MCI. However, in clinical practical applications, due to various confounding factors, the sensitivity and specificity of AD7c-NTP vary considerably across practices. Nevertheless, before lumbar punctures or expensive imaging tests, we recommend urinary AD7c-NTP as an initial tool to screen for AD/MCI. Additionally, progress in detection methods has improved the practicality of AD7c-NTP assays in clinical applications. Moreover, population-based epidemiologic studies have provided more comprehensive elucidation of AD7c-NTP. Taken together, the results described here illustrate that urinary AD7c-NTP is moving closer toward the development of a reliable and accurate tool that may contribute to early diagnosis of AD and MCI.

Fig. 2

Timeline of the research course of Alzheimer-associated neuronal thread protein (AD7c-NTP). Orange box, discovery process; blue box, technical developments; purple box, pathophysiological findings; green box, clinical diagnostic verification; golden box, epidemiological investigation. PTP, pancreatic thread protein; NTP, neuronal thread protein; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay.

However, there are still some shortcomings and caveats regarding findings of AD7c-NTP related to AD. First, there is a discrepancy between the AD7c-NTP cDNA sequence and genomic sequences in humans. Additionally, findings have been limited to post-mortem samples from AD patients, whereas previous pathogenic studies have not been fully verified. We speculate that AD7c-NTP remains stable from CSF to urine due to the concentration and potential “double-balanced” theory, but no experimental findings have verified this speculation. Furthermore, AD7c-NTP detection assays exhibit considerable variability and have not yet been standardized for use across different clinical centers, and different AD7c-NTP cutoff values by age and gender still need to be established. Lastly, combining AD7c-NTP with other novel urine AD biomarkers in clinical research may be represent a promising direction.

Footnotes

ACKNOWLEDGMENTS

This work was supported by funding from the Capital Health Research and Development of Special (grant no.2020-2Z-1034), the National Key R&D Program of China (2018YFA0108503), and the National Key R&D Program of China (grant no.2016YFC1306300). The authors also wish to thank Dr. Xin Lin for his help.

Authors’ disclosures available online ().

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