Tau is a microtubule-associated protein that is most abundantly found in neurons and astrocytes within the central nervous system. Tau is known to play a pivotal role in various neurological pathways, interacting with signaling pathways that regulate microtubule stability and axonal transport [1–3]. Dysregulation of tau may disrupt microtubules, interfere with axon transport mechanisms, and be toxic to neurons; thus, the role of tau is central to many human neurodegenerative diseases, such as Alzheimer’s disease (AD), which are collectively referred to as tauopathies [1, 3–5]. An increased level of tau has also been suggested to be an important biomarker for AD [6].

The optic nerve is a part of the central nervous system, and mainly comprises retinal ganglion cell (RGC) axons that project to the optic chiasm and optic tract and ultimately form synapses at the lateral geniculate nucleus. Therefore, the optic nerve could be affected by similar degenerative processes during neurodegeneration. It has been suggested that pathogenic mechanisms similar to those in neurodegenerative diseases may also occur in retinal degenerative diseases such as glaucoma [5, 7–13].

Studies have suggested a possible role of tau protein in glaucomatous optic nerve damage. Yoneda et al. [14] demonstrated an increased level of vitreous tau in patients with retinal disease or those with glaucoma. Gupta et al. [15] showed that abnormal tau protein was present in the human retina with uncontrolled glaucoma. Experimental studies have also suggested that abnormal tau or tau inclusion in mouse RGCs is associated with reduced axonal transport [16], increased excitotoxic RGC degeneration [16], and reduced axonal outgrowth [17], However, the link between the tau protein and the RGC damage remains to be determined.

Glaucoma is a progressive optic neuropathy characterized by the degeneration of RGC axons. Currently, the lamina cribrosa (LC) within the optic nerve head (ONH) is considered the primary site of axonal damage in glaucoma [18–21]. It has been suggested that structural changes of the LC may induce damage of the RGC axons passing through the LC by various mechanisms [21, 22]. On the other hand, the structural differences of the LC are thought to be responsible for different effects of intraocular pressure (IOP) on the RGC axons within the ONH [21, 22]. It has been reported that the LC is thinner in glaucomatous eyes than in healthy eyes [23, 24]. Further, eyes with a thinner LC have also been associated with a worse glaucoma stage [23, 24] and a faster progression [25].

Considering the anatomical proximity between the ONH and retrobulbar CSF space, it is possible that the CSF protein affects the ONH. Given the implication of the LC in glaucoma pathogenesis and the possible influence of tau protein in glaucomatous axonal damage, it would be of interest to know how CSF tau protein is associated with the structural characteristics of the LC. The purpose of the present study was to determine the influence of CSF proteins on the ONH structure, focusing on the relationship between CSF tau and the lamina cribrosa thickness (LCT).

This study is based on the Study for the Usefulness and Standardization of Cerebrospinal Fluid and Plasma Amyloid Biomarkers in Alzheimer’s Disease, which is an ongoing prospective study of patients with AD and healthy participants at the Neurocognitive Behavior Center in collaboration with the Glaucoma Clinic of Seoul National University Bundang Hospital. This study was approved by the Seoul National University Bundang Hospital Institutional Review Board. Informed written consent was obtained from all participants or their legally acceptable representatives in accordance with the Declaration of Helsinki.

Measurement of LCT

The LCT was measured at three locations in each eye (the midhorizontal and the superior and inferior midperipheral regions of the ONH) using thin-slab maximum intensity projection (MIP) images. Thin-slab MIP images were used because they allow a straighter detection of the posterior LC border [29]. The technique of generating thin-slab MIP images is described in detail elsewhere [29]. In brief, three-dimensional volumetric reconstruction of the ONH was performed from the B-scan images by MIP rendering using image-processing software (Amira 5.2.2; Visage Imaging, Berlin, Germany) (Fig. 1a). The thin-slab image was then obtained by selecting two planes (approximately 64 μm apart) inside the three-dimensional volumetric image data; only the data within these two planes were displayed in the thin-slab image (Fig. 1b). The LCT was measured as the distance between the anterior and posterior borders at the central three points (100 μm between each point) in each MIP thin-slab image along an axis perpendicular to the anterior LC surface at the measurement point (Fig. 1c). The measurements obtained from the three thin-slab images were used to calculate the mean LCT of the eye.

The LCT was measured using the manual caliper tool of the Amira 5.2.2 software by two observers (EJL and DSL) who were masked to the clinical information for the subject. All measurements were repeated three times each by the two observers, and the average of the six values was used for the main analysis.

The study initially included 52 subjects (22 AD patients and 30 healthy subjects). Of these, eight subjects were excluded because of a diagnosis of concurrent glaucoma (n = 3), a poor B-scan quality that did not allow delineation of the LC borders (n = 1), poor cooperation during the SD-OCT examination (n = 2), and failure to receive a CSF examination (n = 2), yielding a final sample of 18 AD patients and 26 healthy subjects.

Table 3 presents the factors associated with the LCT. The univariate analysis revealed a significant influence of higher levels of CSF T-tau (P = 0.004) and CSF P-tau_181P (P = 0.027) and a thinner central corneal thickness (P = 0.032) on thinner LCT measurements. In the multivariate analysis, only the CSF T-tau level (P = 0.041) was significantly associated with the LCT (Table 3). The relationship was significant for both a linear model (R ² = 0.179, P = 0.004; Fig. 2a, dotted line) and a nonlinear model (R ² = 0.275, P = 0.001; Fig. 2a, solid line). The linear model explained the relationship between the CSF T-tau and the LCT as the following:

	Univariate analysis			Multivariate analysis			VIF
	β	95 % CI	P value	β	95 % CI	P value
Gender, male	5.769	–15.954, 27.493	0.595
Age, per 1 year older	–1.103	–2.371, 0.165	0.087	–0.572	–1.746, 0.601	0.330	1.086
Aβ1–42, per 1 pg/ml larger	–0.024	–0.100, 0.052	0.521
T-tau, per 1 pg/ml larger	–0.326	–0.543, –0.108	0.004	–0.362	–0.709, –0.016	0.041	2.843
P-tau181P , per 1 pg/ml larger	–0.582	–1.095, –0.069	0.027	0.166	–0.627, 0.958	0.674	2.882
CSF pressure, per 1 mmHg higher	0.593	–3.526, 4.712	0.773
Total MMSE score, per 1 point higher	0.894	–1.108, 2.896	0.373
IOP, per 1 mmHg higher	0.294	–3.737, 4.325	0.884
Spherical error, per 1 D larger	0.024	–5.983, 6.032	0.994
Global RNFL thickness, per 1 μm thicker	0.930	–0.025, 1.886	0.056	0.845	–0.020, 1.709	0.055	1.113
Central corneal thickness, per 1 μm thicker	0.349	0.032, 0.666	0.032	0.216	–0.091, 0.524	0.163	1.145
Axial length, per 1 mm longer	3.516	–7.861, 14.893	0.536

Statistically significant values are shown in bold

Aβ _1–42 amyloid β_1–42, CI confidence interval, CSF cerebrospinal fluid, IOP intraocular pressure, MMSE Mini-Mental Status Examination, P-tau _181P phosphorylated tau_181P, RNFL retinal nerve fiber layer, T-tau total tau, VIF variance inflation factor

$$ y=272.385-0.3255*x $$

where y is the LCT measured in micrometers and x is the level of CSF T-tau measured in pictograms per milliliter.

Regression analysis with an indicator [33] was performed using the diagnostic group (healthy and AD groups) as an indicator variable. This analysis was performed using the R statistical language version 2.15.1 (http://www.R-project.org (accessed 3 December 2014)). This analysis showed that the association between CSF T-tau protein and the LCT was significant in both the healthy and AD groups, with a stronger association found in the healthy group (regression coefficients = –1.098 vs. –0.280, P = 0.018; Fig. 2b).

Representative cases

Figure 3 shows representative cases illustrating the relationship between the CSF level of T-tau and the LCT. The LCT is notably thinner in the eye from the AD group, in which the T-tau protein level in the CSF was 235.2 pg/ml (Fig. 3b), than in the eye from the healthy group, in which the T-tau level was 27.4 pg/ml (Fig. 3a).

The LC comprises connective tissue plates that mainly include extracellular matrix (ECM) (i.e., collagen fibers, elastic fibers, basement membranes, and proteoglycans) and a smaller subset of cellular components (i.e., LC cells, astrocytes, and microglial cells). The LC is thought to provide structural and metabolic support to the RGC axons passing through it.

It has been suggested that eyes with thin LC are more susceptible to mechanical damage of the RGC axons. Thinning of the LC may expose the axon to a steep transition between the IOP and the retrolaminar tissue pressure (translaminar pressure gradient) and significantly influence the axoplasmic flow [22, 34–36], causing further axonal damage. However, it is not fully understood whether some eyes have inherently thin LC, or the LC is thinned in the process of degeneration such as physiologic aging or pathologic neurodegeneration such as glaucoma.

The present study suggests a potential mechanism of LC thinning associated with tau pathology; a higher level of CSF tau was associated with a thinner LC both in healthy individuals and in patients with AD. To the best of the authors’ knowledge, this is the first study to reveal a relationship between CSF proteins and the LC characteristics.

Whether CSF tau is a causal factor for thinning of the LC or a consequence of LC thinning is unclear. Several possibilities may be considered for this issue. First, it is possible that the CSF tau is a causal factor in LC thinning. The cytoplasm of astrocytes contains networks of filamentous proteins, such as the actin cytoskeleton, intermediate filaments, and microtubules that maintain tissue and cell integrity, cell mobility, and cell differentiation [37]. It has been suggested that the accumulation of tau contributes to the collapse of the cytoskeleton and degeneration of astrocytes [38]. When increased CSF tau influences the stability of the cytoskeleton of laminar astrocytes, the astrocytes may become more susceptible to degradation, leading to further thinning of the LC. Second, elevated CSF tau may be a result caused by a neurodegenerative cascade facilitated by the thin LC. A thinner LC is associated with a steeper translaminar pressure gradient that causes the axons passing through the LC to experience an abrupt transition between the high IOP and low retrolaminar pressure. Under these conditions, axonal flow can be interrupted [34–36], and this effect may result in the dysregulation of neurotrophic factors [39, 40], which may in turn increase abnormal tau [41–43]. Finally, however, it is also possible that LC thinning is simply an epiphenomenon accompanied by an elevation in CSF tau associated with neurodegenerative processes. The result of this study does not fully explain the causal relationship between the LCT and CSF tau protein; a long-term investigation of larger samples is required to address this issue.

In the present study, more than half of the subjects were healthy individuals; thus, it is not known whether the non-AD subjects with a thin LC or an elevated CSF tau had subclinical AD. Although data were not presented because of the small number of samples, a subgroup analysis performed within the healthy group revealed a marginal but not significant linear association of a lower total MMSE score with thinner LCT (β = 10.417, P = 0.061). This may indicate that the LCT could also serve as a potential candidate marker representing cognitive function as well as the CSF tau level. A long-term follow-up study is required to verify whether these subjects are more susceptible to developing AD. Additionally, a study that includes subjects with mild cognitive impairment may help to answer this question.

Interestingly, the relationship between the level of CSF tau and the LCT was significant both in the healthy and AD groups, with a stronger relationship found in the healthy group. Although we do not have a clear explanation for this differential correlation between groups, the smaller span of LCT change relative to that of CSF tau level in AD patients might be because of the increased stiffness of the LC tissue in this group, which is associated with older age [44–46]. On the other hand, a relatively larger span of LCT change despite the smaller span of CSF tau level in healthy subjects may indicate that the LCT can serve as a useful marker to monitor subclinical CSF tau increases.

Thus far, the pathogenic influence of tau has mainly been centered on the degeneration of axonal tissue. If this assumption also holds true for RGC degeneration, the CSF tau level should be associated with the number of RGC axons, which is represented by the RNFL thickness. However, in the present study, increased tau was associated only with a smaller LCT and not with a thinner RNFL (data not presented). Instead, a smaller LCT was correlated with both higher CSF tau levels and a thinner RNFL. This finding suggests that CSF tau is more likely associated with glial tissues (LC) than with neural tissues (RGC axons), and the change in the neural tissues might be secondary to that in the glial tissues. This finding supports the assumption that glial cells are more directly associated with neurodegeneration than are neuronal cells [47–49].

In the present study, only the T-tau, not the P-tau_181P, was associated with the LCT. Although we do not have a clear reason for this result, it is possible that the level of P-tau_181P was not sufficiently high to produce a statistical significance. We speculate that other forms of tau phosphorylation could be more highly associated with the LCT. However, the original study (Study for the Usefulness and Standardization of Cerebrospinal Fluid and Plasma Amyloid Biomarkers in Alzheimer’s Disease) did not analyze other types of CSF proteins besides Aβ_1–42 and P-tau_181P. Further study is expected to reveal the precise relationship between the phosphorylated tau and LC characteristics.

In the present study, the Aβ_1–42 level was not associated with the LCT. It is acknowledged that the concentration of Aβ_1–42 in the CSF increases initially but then decreases decades before the expected onset of symptoms [50–52], whereas the concentrations of CSF tau are increased several years before symptoms develop [50–52] and further increase with the disease progression [53, 54]. In this context, the nonassociation between the CSF Aβ_1–42 and the LCT and the association between CSF tau and the LCT may suggest that the LCT could serve as a potential clinical indicator for neurodegeneration in patients with AD or those at risk of developing AD.

This study is limited by the small sample size and its cross-sectional design. Hence, a causal relationship between CSF tau and the LCT and the pathogenic significance of this potential relationship could not be determined. A long-term follow-up study with a larger number of subjects, including subjects with RGC pathology, such as glaucoma, is needed to reinforce the findings of this study.

The present study found that an increased CSF tau level is associated with a thin LC in healthy subjects and in patients with AD. Given the implications of LC thinning in RGC axonal degeneration, it is possible that tau protein contributes to a common pathogenesis of neurodegeneration in the brain and optic nerve. In addition, the LCT could serve as a non-invasive potential biomarker for estimating the level of CSF tau protein and for suspecting the presence of tauopathies. Although the study leaves many questions, its results may provide a better understanding of the mechanisms of tauopathy in neurodegenerative diseases and a basis for future studies investigating the pathogenesis of neurodegeneration in the brain and optic nerve.