Introduction
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide. The major pathological hallmarks of AD include extracellular depositions of β-amyloid (Aβ) peptides as well as intracellular neurofibrillary tangles consisting of hyperphosphorylated tau, loss of synapses, and neuroinflammation [
1,
2]. The earliest pathophysiological events are expected to occur 10–20 years before the onset of dementia [
3]. Changes in cerebrospinal fluid (CSF) biomarkers reflecting amyloid pathology (Aβ
42) and neurodegeneration [total tau (t-tau) and phosphorylated tau (p-tau)] occur early in the course of AD and are increasingly implicated in the early and predictive diagnosis of AD [
4,
5]. The accuracy of diagnosis based on these core AD biomarkers is high, as long as markers of neurodegeneration and amyloidosis are altered concordantly [
6]. However, in a proportion of patients, biomarker results may be contradictory, leading to lower diagnostic accuracy [
7]. Additionally, Aβ
42, t-tau, and p-tau allow no conclusions about cognitive performance and only a limited prediction of cognitive decline to be made, a feature that is especially important for clinical trials [
8]. Therefore, additional biomarkers reflecting further aspects of AD pathophysiology, such as synaptic degeneration and neuroinflammation, are needed. Loss of synapses is an early event in the course of AD, and the correlation between synapse density and performance on neuropsychiatric tests such as the Mini Mental State Examination (MMSE) and verbal fluency tests is well established [
9‐
12]. Neurogranin is a postsynaptic protein expressed in the neocortex, amygdala, caudate nucleus, putamen and hippocampus in the rodent brain [
13]. In the human brain, expression is highest in associative cortical areas [
14], suggesting a link with cognition. It is concentrated in dendritic spines of principal excitatory synapses, and its translocation to dendritic spines is impaired in AD [
15‐
17]. Neurogranin levels are reduced in the hippocampus and cortex in AD, indicating a loss of dendrites [
2].
Synaptic proteins, including neurogranin, have been shown to be present in the CSF [
18]. A first pilot study using immunoprecipitation (IP) and Western blot analysis showed a marked increase in CSF neurogranin levels in AD [
19]. In a later study, using both IP-mass spectrometry and a newly developed immuno-based assay, researchers verified elevated levels of CSF neurogranin in a larger cohort of patients with AD [
20,
21]. Importantly, high CSF neurogranin levels were also found in prodromal AD cases, and the degree of increase correlated with the rate of future cognitive decline [
21].
Neuroinflammation is another common feature of AD pathology, and several epidemiological studies suggest a decrease in risk for AD after long-term administration of nonsteroidal anti-inflammatory drugs [
22]. YKL-40, a 39 kDa glycoprotein homologue to chitinase, is a marker for macrophage and microglial differentiation and activation [
23‐
25]. Elevated CSF levels were shown in several infectious and noninfectious disorders of the central nervous system (CNS) [
26]. Also, in AD, YKL-40 seems to be elevated in CSF [
27‐
29]. The aim of this study was to investigate whether neurogranin as a marker for synaptic loss reflects cognitive disturbances and, together with YKL-40, shows aspects of AD pathophysiology complementary to amyloid pathology and neurodegeneration.
Discussion
We have shown that the synaptic protein neurogranin and YKL-40 are elevated in the CSF of patients with AD. Even though both markers were significantly increased, they did not correlate with each other in AD.
In the diagnosis of cognitive disturbances, biochemical markers as indicators of the disease are increasingly implicated. Unfortunately, biochemical markers reflecting cognitive decline are still sparse [
8]. It has long been known that the number of synapses is well correlated with the degree of cognitive disturbances [
10,
34,
35]. Therefore, it is expected that biomarkers indicating synaptic integrity would be well suited to reflect cognitive decline. In our study, CSF neurogranin levels were elevated in AD. However, we found no difference in the levels of neurogranin in the dementia stage versus MCI. In addition, there was no correlation between neurogranin levels and MMSE scores. Thus, our results are in line with previous reports of elevated levels of neurogranin in AD [
19,
20,
36,
37]. In contrast to our present study, Thorsell et al. did not distinguish between MCI due to AD and MCI due to other diseases, and they measured neurogranin levels in the MCI group between that of controls and that of patients with AD [
19]. In their study, Kvartsberg et al. included a neuropsychological follow-up investigation which showed that high CSF levels of neurogranin at baseline predicted a more rapid decline in cognition [
20]. This might indicate that neurogranin reflects not the synaptic density but rather the intensity of current synaptic destruction.
In line with previous studies, we have shown that neurogranin differentiated well between AD and other neurodegenerative diseases. Established core biomarkers (i.e., Aβ
1–42, t-tau, and p-tau) have high diagnostic accuracy in discriminating individuals with AD from subjects without cognitive disturbances, but their diagnostic performance in differentiating AD from other dementias is far from optimal [
38]. Interestingly, CSF neurogranin was not elevated in our cohort of patients with other neurodegenerative diseases. However, the cohort of non-AD-D patients was small and comprised especially patients with frontotemporal lobar degeneration. Further research is necessary to clarify whether the elevation of neurogranin is specific for AD.
The stronger correlation of neurogranin and tau/p-tau in non-AD patients as compared with patients with AD and the missing elevation of neurogranin in non-AD-D patients also points to a mechanism of neurodegeneration in AD distinct from the physiological dying of neurons and distinct from other neurodegenerative diseases. Most likely, it shows a degeneration of synapses that is weakly related to the axonal damage indicated by tau [
39]. The exact mechanism by which neurogranin is released is unclear.
Elevated levels of CSF YKL-40 in early stages of AD have been demonstrated in two independent studies, but there are also contradictory data [
28,
32,
40]. In our study, we confirmed that YKL-40 is elevated early in the course of AD and that the levels do not change during disease progression. In addition, YKL-40 levels in other dementias did not differ from those with MCI not due to AD. This suggests that neuroinflammation in AD pathology differs from that in other dementias. In accordance with the concept of inflammaging, introduced by Franceschi et al., we found a correlation of YKL-40 with age.
Inflammaging describes a low-grade, chronic upregulation of inflammatory responses during aging as a risk factor for several age-dependent diseases [
41,
42]. Accumulating evidence shows a similar alteration in the CNS of the elderly as a prodrome of AD [
43]. In part, this increased immune reactivity in the aged brain might be derived from primed microglia. Primed microglia are in a preactivated state and tend to react in a prolonged manner and by secretion of higher amounts of proinflammatory signals [
44]. Excessive inflammatory responses by primed microglia aggravate neurodegeneration, impair synaptic plasticity, and lead to cognitive decline [
45]. However, we did not find a correlation between YKL-40 and MMSE. Yet, as a marker for microglial activation, YKL-40 seems well suited to reflect these aspects of AD pathophysiology.
Even though a link between microglial activation and synaptic degeneration can be postulated, we found no correlation between neurogranin and YKL-40 in our study. As detailed above, YKL-40 is a rather unspecific marker that is highly influenced by patients’ comorbidities. This might also explain why data on YKL-40 correlations are somewhat contradictory. Two studies showed a correlation with p-tau and t-tau, whereas a third did not find any correlation with CSF tau levels [
27,
29,
40]. Data on correlations with MMSE are likewise conflicting [
29,
40]. The missing correlation between neurogranin and YKL-40 suggests that these two markers reflect two different aspects of neurodegeneration in AD. Whereas YKL-40 might represent Aβ-mediated activation of microglia and neuroinflammation, elevated levels of neurogranin might indicate synaptic damage of another origin, such as direct Aβ-mediated neurotoxicity via soluble oligomers, disturbances in calcium homeostasis, or mitochondrial damage [
46‐
52].
To evaluate neurogranin and YKL-40 as potential biomarkers for AD, we determined ROC curves for both markers alone and a combination of both markers by multiplication. With an AUC of 0.85, the diagnostic performance of neurogranin is in the reported range of the isolated core biomarkers. The combination of Aβ, tau, and neurogranin might therefore improve diagnostic performance considerably. A comparison with core biomarkers was not possible in our study, as patients were selected according to these markers. To further evaluate the potential of neurogranin as a diagnostic biomarker, further studies including patients not stratified by established biomarkers are needed. The additional benefit of YKL-40 as biomarker for AD is limited, with an AUC of 0.66, and is a rather unspecific marker. However, YKL-40 might be useful for patient stratification and monitoring of drugs targeting microglial activation.
Competing interests
KB has served on advisory boards for Eli Lilly and Company, IBL International, and Roche Diagnostics and has given lectures for Fujirebio Europe. KB and HZ are cofounders of Brain Biomarker Solutions AB (Gothenburg, Sweden), a GU Ventures–based platform company at the University of Gothenburg. PL has received consultation and lecture honoraria from Innogenetics, IBL International, AJ Roboscreen, Beckman Coulter, and Roche and holds the position of visiting professor at the Medical University of Białystok (Białystok, Poland). KH, HK, EP, UA, TJO, JK, JMM, and PS declare that they have no competing interests.
Authors’ contributions
KH, PL, KB, JMM, HZ, and PS designed the study. PL, JMM, TJO, JK, and PS investigated the patients and collected the samples. KH and HK carried out the experiments. EP, UA, KB, PL, and HZ supervised and substantially supported the acquisition of data based on their vast experience. KH, EP, KB, UA, HZ, and PS carried out statistical analysis. KH and PS drafted the manuscript. All authors reviewed the manuscript critically and provided constructive comments to improve the quality of the manuscript. All authors read and approved the final manuscript.