Cerebrospinal fluid surfactant protein-G in neurodegenerative dementia: A comparative study with idiopathic normal-pressure hydrocephalus

Abstract

Background

Surfactant protein G (SP-G) is a recently characterized protein potentially involved in cerebrospinal fluid (CSF) dynamics. Its role in human neurodegenerative dementia remains unknown.

Objective

To investigate the potential clinical relevance of CSF SP-G levels in neurodegenerative dementia (ND; including Alzheimer's disease [AD] and frontotemporal dementia [FTD]), idiopathic normal-pressure hydrocephalus (iNPH), vascular dementia (VaD), and clinical controls (CTR).

Methods

CSF SP-G levels were quantified in 94 individuals: 35 with ND (11 AD + 24 FTD), 19 with iNPH, 13 with VaD, and 27 CTR. Group differences were analyzed using ANOVA with post hoc tests. ANCOVA was performed to adjust for age.

Results

CSF SP-G levels differed significantly among groups in the primary analysis (ANOVA: F(3,90) = 3.862, p = 0.012), with higher levels in iNPH compared with ND (p = 0.012) and CTR (p = 0.026). No significant differences were observed between ND, VaD, and CTR groups. After adjustment for age, the group effect was no longer significant (F(3,89) = 2.253, p = 0.088).

Conclusions

This is the first study to investigate CSF SP-G levels in neurodegenerative dementia. Our findings suggest that SP-G is more closely related to altered CSF physiology in iNPH than to neurodegenerative pathology, although the influence of age indicates that these observations should be interpreted with caution. SP-G may represent a candidate marker of CSF homeostasis and fluid dynamics. Larger, age-matched studies integrating SP-G with established CSF biomarkers and neuroimaging are needed to clarify its diagnostic and pathophysiological significance.

Introduction

Surfactant proteins (SPs) are essential components of the pulmonary surfactant, a lipoprotein complex that reduces alveolar surface tension. ¹ SP-A and SP-D are hydrophilic collectins involved in host defense and immune modulation, whereas SP-B and SP-C are hydrophobic proteins that regulate surface tension during breathing cycles.^2,3 Interestingly, SPs are also expressed in the central nervous system (CNS), potentially acting as regulators of cerebrospinal fluid (CSF) flow. ⁴ Among the SP family, the most recently identified member is surfactant protein-G (SP-G).^5,6 SP-G has recently been identified in the human brain and is implicated in CSF homeostasis,^6–8 with elevated levels observed in conditions involving altered CSF circulation.^4,7,8

To date, only one study by Meinicke and colleagues ⁹ documented an age-dependent increase of SP-G within the brain of a triple-transgenic Alzheimer's disease mouse model (3xTg-AD). Specifically, SP-G immunoreactivity was observed to accumulate in discrete, punctate deposits (colocalized with Reelin) in the hippocampus, and the total SP-G protein content was demonstrably higher in the aged experimental 3xTg-AD mice when compared with wild-type controls. These findings suggest that SP-G may be upregulated in pathology associated with Alzheimer's disease (AD). Nevertheless, to the best of our knowledge, this hypothesis has yet to be explored in human subjects.

Distinguishing idiopathic normal-pressure hydrocephalus (iNPH) from other causes of cognitive impairment in older adults remains a major clinical challenge. The greatest clinical overlap is often observed with vascular cognitive impairment, as both conditions may present with gait dysfunction, psychomotor slowing and executive deficits. In addition, the differential diagnosis between iNPH and neurodegenerative disorders may be complicated by the frequent coexistence of age-related neurodegenerative pathology, the difficulty in distinguishing ventricular enlargement due to hydrocephalus ex vacuo from true iNPH, and the possibility that patients with otherwise typical AD may simultaneously exhibit gait impairment related to concomitant CSF circulation abnormalities. Consequently, biomarkers reflecting CSF dynamics may provide valuable support in the diagnostic evaluation of these patients. Several CSF biomarkers have been investigated in iNPH to improve differential diagnosis with neurodegenerative disorders, particularly AD. Previous studies consistently reported reduced concentrations of total-tau (t-Tau), phosphorylated tau (p-Tau₁₈₁), and amyloid-β protein precursor (AβPP)-derived fragments, including soluble AβPPα (sAβPPα) and soluble AβPPβ (sAβPPβ), in patients with iNPH compared with individuals affected by AD.^10–13 These alterations are thought to reflect impaired CSF turnover, ventricular enlargement and disturbed periventricular metabolism rather than primary neurodegeneration. Reduced concentrations of amyloid-related biomarkers, including amyloid-β 1–42 (Aβ₄₂) and amyloid-β 1–40 (Aβ₄₀), have also been described in iNPH, although findings remain heterogeneous across studies. Importantly, alterations in CSF amyloid concentrations in iNPH should be interpreted with caution, as they may reflect changes in CSF dynamics, turnover, and clearance rather than the presence of primary amyloid pathology. Consequently, increasing attention has been directed towards biomarkers that may more directly reflect CSF circulation and glymphatic dysfunction, which are considered key pathophysiological features of iNPH. Biomarkers reflecting alterations in CSF dynamics could therefore provide valuable support in the diagnostic process.

The novelty of this exploratory study was to provide the first assessment of CSF SP-G levels in human cohorts, with the aim of exploring its potential association with CSF-related pathology in iNPH and neurodegenerative dementia (ND).

Methods

We measured CSF SP-G levels in a sample of individuals diagnosed with ND, specifically probable AD and frontotemporal dementia (FTD). This group was then compared with individuals with iNPH and vascular dementia (VaD), as well as with clinical controls (CTR) representing neurological conditions not primarily involving dementia or CSF circulation abnormalities.

Diagnoses were established independently by the treating clinical teams according to standard diagnostic procedures and internationally accepted criteria adopted at the recruiting center.

Patients with AD were included if they fulfilled the criteria for probable AD according to the National Institute on Aging–Alzheimer's Association criteria (NIA-AA), ¹⁴ based on progressive cognitive decline documented by clinical and neuropsychological assessment and after exclusion of alternative neurological or systemic causes.

Patients with FTD met the revised international consensus criteria for behavioral variant ¹⁵ and for language variants, ¹⁶ requiring progressive deterioration associated with characteristic behavioral and/or language impairment, with supportive neuroimaging findings when available.

Individuals with iNPH were diagnosed according to international guidelines,^17,18 requiring clinical features consistent with the iNPH syndrome, ventriculomegaly on neuroimaging (Evans index ≥ 0.30), and exclusion of secondary causes of hydrocephalus.

Patients with VaD fulfilled the National Institute of Neurological Disorders and Stroke–Association Internationale pour la Recherche et l'Enseignement en Neurosciences (NINDS–AIREN) criteria, ¹⁹ requiring evidence of cognitive impairment associated with cerebrovascular disease and a plausible temporal or anatomical relationship between vascular lesions and cognitive decline.

The CTR group consisted of individuals undergoing lumbar puncture for the diagnostic evaluation of neurological conditions unrelated to neurodegenerative disease (e.g., suspected dysimmune polyneuropathy or multiple sclerosis). They were included only in the absence of cognitive impairment, neurodegenerative disorders, active CNS inflammatory disease, or major structural brain abnormalities.

Across all groups, exclusion criteria included acute neurological illness, CNS infection, major psychiatric disorders, active neoplastic disease involving the CNS, severe systemic illness potentially affecting CSF composition, and incomplete clinical or CSF data.

Participants

The study was approved by the Ethics Committee of the IRCCS Istituto Auxologico Italiano (Italy). It was performed in accordance with the ethical principles of the Declaration of Helsinki. ²⁰ All participants provided written informed consent. The study was conducted at the Division of Neurology and Neurorehabilitation, San Giuseppe Hospital (IRCCS Istituto Auxologico Italiano, Piancavallo, Italy), which is a tertiary neurological and neurorehabilitation center, where patients presenting with a broad spectrum of cognitive, behavioral, and neurodegenerative syndromes routinely undergo comprehensive diagnostic work-up, including CSF analysis when clinically indicated, such as suspected neurodegenerative diseases, cognitive impairment, and disorders of CSF dynamics and immune-mediated disorders. This retrospective study was conducted on archived CSF samples obtained during routine clinical practice. The investigators had access to biological material and final diagnostic categories but not to the complete clinical diagnostic pathway for each participant. Diagnostic classifications reflected routine clinical assessment performed at the recruiting center according to established diagnostic criteria based on clinical assessment, neuroimaging and neuropsychological findings, and CSF biomarker profiles.

Overall, 94 participants aged over 50 years were included in the present study and categorized into four cohorts: (1) individuals diagnosed with ND (n = 35) including probable AD (n = 11) and FTD (n = 24); (2) individuals with iNPH (n = 19); (3) individuals with VaD (n = 13); and (4) individuals with neurological conditions unrelated to dementia who underwent diagnostic CSF analysis for suspected dysimmune polyneuropathy or multiple sclerosis, serving as the CTR group (n = 27). The relative proportion of AD and FTD cases reflects the characteristics of our institutional referral population, which includes patients with heterogeneous cognitive and behavioral syndromes rather than only typical amnestic presentations. Because lumbar puncture cannot be ethically performed solely for research purposes in healthy volunteers, the control group consisted of individuals undergoing clinically indicated CSF examination for neurological conditions unrelated to dementia or CSF circulation disorders.

CSF collection and biochemical analysis

CSF samples were collected by lumbar puncture, performed early in the morning after overnight fasting. ²¹ After collection, CSF samples were aliquoted and stored at −80 °C until analysis. Quantification of CSF SP-G, t-Tau, p-Tau₁₈₁, Aβ₄₂, and Aβ₄₀ levels was performed using high-sensitivity enzyme-linked immunosorbent assays (ELISA) kits, following the manufacturer's instructions. In particular, t-Tau, p-Tau₁₈₁, Aβ₄₂ and Aβ₄₀ levels were measured separately, in duplicate, using commercially available sandwich ELISA kits (Innotest; Innogenetics/Fujirebio, Ghent, Belgium). Intra-assay and inter-assay coefficients of variation were 3.2% and 11.5% for t-Tau, 1.7% and 11.4% for p-Tau₁₈₁, 4.6% and 7.8% for Aβ₄₂, 1.3% and 13.9% for Aβ_40. Quantification of SP-G was performed by a commercially available ELISA kit (SED755Hu, Cloud-Clone Corp, Fernhurst, TX, USA). The analytical sensitivity was defined according to the manufacturer's specifications. The limit of detection (LOD) is 0.055 ng/mL, while the limit of quantitation (LOQ) corresponds to the lowest standard of the calibration curve (0.156 ng/mL). Intra-assay and inter-assay coefficients of variation were <10% and <12%, respectively. All samples were assayed in duplicate, according to the manufacturer's instructions.

For diagnostic purposes, CSF biomarkers were interpreted according to the manufacturer's recommended cut-off values (Fujirebio). The following cutoff values were applied: t-Tau ≤ 404 pg/mL, p-Tau₁₈₁ ≤ 56.5 pg/mL, Aβ₄₂ ≥ 599 pg/mL and Aβ₄₂/Aβ₄₀ ≥ 0.069. Diagnostic classification was based on the integration of clinical assessment, neuroimaging findings and CSF profiles according to established diagnostic criteria for probable AD.

Routine CSF laboratory parameters, including cell count, glucose concentration, and total protein, were evaluated to rule out CNS infections as well as subarachnoid or intraventricular hemorrhage.

Statistical analysis

Statistical analyses were performed using JASP (version 0.95.3; JASP Team, 2025).

Demographic, clinical and CSF biochemical variables were initially compared among diagnostic groups using one-way analysis of variance (ANOVA), followed by Tukey's post hoc tests when appropriate.

All data were tested for normality using Shapiro-Wilk test.

The primary analysis evaluated differences in CSF SP-G concentrations among the four diagnostic groups (iNPH, ND, VaD, and CTR) using one-way ANOVA with Tukey's correction for multiple comparisons. Effect sizes were reported as η².

Based on previous evidence of an intra-subject association between SP-G concentration and age, ⁹ the potential influence of age on CSF SP-G levels was further assessed using an analysis of covariance (ANCOVA), including diagnostic group as a fixed factor and age as a covariate. The homogeneity of regression slopes was tested by evaluating the interaction between group and age.

Spearman's correlation analyses were performed within each diagnostic group to explore the association between SP-G concentration and age.

Finally, exploratory subgroup analyses were performed within the ND cohort to compare CSF SP-G levels and clinical/biochemical characteristics between AD and FTD subgroups. Differences were assessed using the Mann-Whitney U test due to the limited sample size.

All statistical tests were two-sided, and statistical significance was set at p < 0.05.

Results

Demographic and biological descriptive measures for each group are reported in Table 1.

Table 1.

Demographic and clinical descriptive measures.

	Controls mean (SD) [range]	iNPH mean (SD) [range]	ND mean (SD) [range]	VaD mean (SD) [range]	ANOVA (F, p, η2)
	n = 27 m = 17; f = 10	n = 19 m = 13; f = 6	n = 35 m = 18; f = 17	n = 13 m = 10; f = 3
Age (years)	66.24 (9.11) [52.08–83.92]	75.66 (7.67) [53.81–84.72]	70.42 (7.54) [52.08–86.03]	74.43 (7.06) [56.07–83.70]	6.190; <0.001; 0.171
t-Tau (pg/mL)	229.08 (127.23) [75.00–531.00]	200.71 (117.35) [98.00–495.00]	475.20 (352.93) [72.00–1687.00]	289.00 (170.93) [111.00–729.00]	7.279; <0.001; 0.201
p-Tau181 (pg/mL)	45.54 (15.89) [20.00–73.00]	42.12 (38.86) [10.00–181.00]	67.94 (37.38) [20.00–184.00]	49.54 (24.03) [3.00–92.00]	3.868; 0.012; 0.118
Aβ42 (pg/mL)	910.46 (312.85) [319.00–1418.00]	714.23 (289.02) [417.00–1354.00]	797.43 (360.59) [231.00–1577.00]	884.08 (408.10) [437.00–1760.00]	1.337; 0.268; 0.044
Aβ42/Aβ40 ratio	0.12 (0.05) [0.05–0.20]	0.12 (0.074) [0.02–0.22]	0.09 (0.04) [0.03–0.18]	0.11 (0.04) [0.05–0.21]	1.350; 0.263; 0.044
Total CSF protein (mg/dL)	50.13 (21.76) [21.00–111.00]	39.10 (22.44) [17.00–96.00]	48.10 (23.84) [28.50–131.00]	38.91 (8.87) [24.00–52.00]	1.557; 0.205; 0.049
CSF cell count (cells/μL)	2.21 (1.96) [0.60–10.00]	1.38 (1.12) [0.06–4.00]	1.93 (3.05) [0.08–18.00]	3.40 (5.06) [0.04–18.00]	1.323; 0.272; 0.043
Glucose (mg/dL)	63.70 (11.11) [51.00–111.00]	62.32 (6.60) [53.00–79.00]	66.20 (13.53) [53.00–129.00]	63.00 (22.38) [1.00–93.00]	0.430; 0.732; 0.014

Values are expressed as mean, with standard deviation in round brackets and range in square brackets. Bold type indicates the statistically significant result.iNPH: idiopathic normal-pressure hydrocephalus; ND: neurodegenerative dementia; VaD: vascular dementia; m: males; f: females.

No patients exhibited CSF profiles indicative of infectious or inflammatory CNS pathology. Thus, the dataset included all 94 participants.

With respect to age, Tukey's post hoc test revealed differences between controls and both the iNPH and VaD groups, with controls being younger. This likely reflects the earlier onset of the neurological conditions included in the control cohort.

As expected, t-Tau and p-Tau₁₈₁ concentrations differed significantly among the diagnostic groups: post hoc analysis revealed significantly higher t-Tau and p-Tau₁₈₁ levels in the ND group compared with both controls and the iNPH group. This is in accordance with the underlying neurodegenerative pathology, which is characteristically associated with tau dysregulation and neuroaxonal damage.

As shown in Figure 1, for the SP-G concentration, a significant main effect of group emerged [F(3,90) = 3.862; p = 0.012; η² = 0.114], indicating a moderate effect size; post hoc tests showed that SP-G values were significantly higher in iNPH patients when compared with controls (p = 0.026) and the ND group (p = 0.012). The other comparisons were not significant (p ≥ 0.512); in particular, no statistically significant difference in CSF SP-G concentration was found between the ND, VaD, and CTR groups.

Figure 1.

Cerebrospinal fluid concentration of surfactant protein G (SP-G; ng/mL) in the four groups considered in this study. SP-G concentration in idiopathic normal-pressure hydrocephalus (iNPH) was significantly increased compared with clinical controls (CTR; p = 0.026) and compared with patients affected by neurodegenerative dementia (ND; p = 0.012). VD: vascular disease.

Detailed pairwise comparisons from the primary one-way ANOVA model are reported in Table 2. Post hoc analyses corrected for multiple comparisons confirmed that CSF SP-G concentrations were significantly higher in the iNPH group compared with controls and patients with neurodegenerative dementia, while no significant differences were observed among the remaining diagnostic groups.

Table 2.

Post-hoc comparisons of CSF SP-G levels among diagnostic groups. Pairwise comparisons were performed following the primary one-way ANOVA and adjusted for multiple comparisons using Tukey's correction. Mean differences, standard errors (SE), effect sizes (Cohen's d), and adjusted p-values are reported.

		Mean difference	SE	Df	t	Cohen's d	Ptukey
CTR	iNPH	−0.417	0.146	90	−2.865	−0.858	0.026
	ND	0.018	0.124	90	0.143	0.037	0.999
	VaD	−0.175	0.164	90	−1.064	−0.359	0.712
iNPH	ND	0.435	0.138	90	3.139	0.895	0.012
	VaD	0.242	0.175	90	1.386	0.499	0.512
ND	VaD	−0.192	0.158	90	−1.219	−0.396	0.617

CTR: clinical controls; iNPH: idiopathic normal-pressure hydrocephalus; ND: neurodegenerative dementia; VaD: vascular disease.

Given the age differences among diagnostic groups and previous evidence suggesting an age-dependent expression of SP-G, ⁹ an ANCOVA was performed with diagnostic group as the fixed factor and age as a covariate. After adjustment for age, the effect of diagnostic group on CSF SP-G levels was not statistically significant [F(3,89) = 2.253; p = 0.088; η² = 0.067], whereas age significantly contributed to SP-G variability [F(1,89) = 5.052; p = 0.027; η² = 0.050]. To verify the assumption of homogeneity of regression slopes, a group x age interaction term was tested and was not significant [F(4,86) = 0.159; p = 0.923; η² = 0.005].

As shown in Figure 2, there was no significant relationship between age and the SP-G levels in the four groups (Figure 2A–D).

Figure 2.

Correlation plot of age and surfactant protein G (SP-G) levels in the four groups. Within the neurodegenerative dementia (ND) cohort, exploratory analyses comparing Alzheimer's disease (AD) and frontotemporal dementia (FTD) subgroups did not reveal differences in cerebrospinal fluid (CSF) SP-G levels (Supplemental Table 1). As expected, t-Tau concentrations were higher in the AD subgroup (U = 193.0; p = 0.030). VD: vascular disease.

Discussion

To our knowledge, this is the first study investigating CSF SP-G levels in human ND and iNPH. The present findings showed higher CSF SP-G concentrations in patients with iNPH compared with the CTR and ND groups in the unadjusted analyses. However, after accounting for age differences across diagnostic groups, the overall effect of diagnosis was attenuated and no longer reached statistical significance, while age significantly contributed to SP-G variability.

This pattern suggests that age may represent an important source of biological variability in CSF SP-G levels and indicates that the observed between-group differences should not be interpreted as entirely independent of age-related processes. At the same time, the persistence of a moderate effect size after adjustment (η² = 0.067) suggests that diagnostic differences may still contribute to SP-G variation, although the present sample may have been underpowered to detect these effects once age-related variance was taken into account.

Taken together, these findings support a cautious interpretation whereby altered SP-G concentrations in iNPH may reflect the combined contribution of disease-related mechanisms and age-associated biological changes rather than a disease-specific biomarker signature.

Our findings should also be interpreted in the context of the established CSF biomarker profile of iNPH. Several studies have demonstrated that patients with iNPH often exhibit lower CSF concentrations of t-Tau, p-Tau₁₈₁, and AβPP-derived fragments, including soluble sAβPPα and sAβPPβ, compared with patients affected by probable AD. Reduced levels of amyloid-related proteins, including Aβ₄₂ and Aβ₄₀, have also been reported, although results are heterogeneous across studies.^10–13 These alterations are generally considered to reflect impaired CSF turnover, ventricular enlargement, altered periventricular metabolism, and disturbed clearance mechanisms rather than primary amyloid-driven neurodegeneration. Within this framework, SP-G represents a potential additional candidate marker related to CSF homeostasis and fluid dynamics, rather than a replacement for established amyloid and tau biomarkers.

The higher SP-G levels observed in iNPH in the unadjusted analyses may be interpreted in the context of altered CSF circulation and glymphatic system dysfunction. ²² The glymphatic pathway contributes to the clearance of interstitial solutes, including amyloid and tau proteins, through CSF-interstitial fluid exchange, perivascular pathways, and aquaporin-4-dependent water transport.^23,24 Previous studies have reported impaired glymphatic clearance in iNPH, including delayed CSF tracer clearance and alterations of perivascular fluid transport.^22,25,26 Given the expression of SP-G in structures involved in CSF regulation, including the choroid plexus and ependymal epithelium, changes in SP-G concentration may reflect modifications of CSF composition or compensatory responses associated with altered fluid dynamics. ²⁷

An additional relevant consideration is the comparison between iNPH and VaD. These two conditions represent an important diagnostic challenge because they may share overlapping clinical features, including gait impairment, psychomotor slowing, and subcortical executive dysfunction. In the present study, CSF SP-G levels did not significantly differ between iNPH and VaD, suggesting that SP-G may not represent a disease-specific biomarker capable of distinguishing these two conditions. However, this finding may also provide relevant biological insight. Vascular cognitive impairment, particularly when associated with cerebral small vessel disease, has been linked to alterations in perivascular structures, impaired vascular pulsatility, and dysfunction of waste clearance pathways, including the glymphatic system. Increasing evidence suggests that impaired glymphatic clearance and altered CSF–interstitial fluid exchange may contribute to both iNPH and vascular cognitive impairment.^28–30 Therefore, the similar SP-G levels observed in iNPH and VaD may reflect shared abnormalities in CSF homeostasis and perivascular fluid dynamics rather than a mechanism specific to ventricular enlargement. Future studies integrating SP-G measurements with neuroimaging markers of small vessel disease, perivascular spaces, and glymphatic function will be necessary to determine whether SP-G reflects common pathways of fluid dysregulation across different neurological disorders.

The differential diagnosis of iNPH remains challenging for several reasons beyond its overlap with vascular cognitive impairment. In older patients, iNPH frequently coexists with neurodegenerative pathology, particularly AD, making clinical interpretation more challenging. Furthermore, ventricular enlargement may reflect either true CSF circulation abnormalities or hydrocephalus ex vacuo secondary to cerebral atrophy, and distinguishing between these conditions is not always straightforward. In addition, some patients with otherwise typical neurodegenerative syndromes may simultaneously present gait impairment related to concomitant CSF dynamic abnormalities. Within this context, biomarkers reflecting CSF homeostasis and fluid circulation may provide complementary information to conventional neurodegenerative biomarkers and neuroimaging findings.

The ND group showed the expected increase of t-Tau and p-Tau₁₈₁ compared with iNPH and CTR, supporting the presence of neuronal injury and tau-related pathology. In contrast, SP-G levels did not differ between ND, VaD, and CTR, suggesting that CSF SP-G alterations may not be primarily driven by neurodegenerative pathology. Within the ND cohort, exploratory comparisons between AD and FTD did not reveal differences in CSF SP-G levels, whereas higher t-Tau concentrations in AD compared with FTD were consistent with the distinct biological profiles underlying these conditions. These findings further support the hypothesis that SP-G may not primarily reflect neurodegenerative pathology itself.

Although no significant alterations in SP-G were observed in patients with ND in the present study, experimental data from a 3xTg-AD mouse model demonstrate an age-dependent increase of SP-G in the hippocampus, co-localizing with Reelin. ⁹ This co-expression suggests a potential reactive or neuroprotective function of SP-G in response to neuroinflammatory processes and synaptic dysfunction. Clinically, such upregulation may reflect compensatory mechanisms involved in extracellular matrix remodeling or regulation of barrier permeability under neurodegenerative conditions. The association with Reelin may suggest a role for SP-G in neuronal plasticity and circuit remodeling, hinting at a broader functional spectrum beyond CSF regulation.^6,9,31

We observed that CSF SP-G levels in patients with ND were comparable to those in the VaD and CTR groups; overall, these groups seemed to show unaltered CSF levels of this protein when compared with iNPH. Although novel, these results should not be considered conclusive evidence. Rather, they provide a rationale for further translational research in the field.

No CSF/serum albumin ratio (albumin index) was available in the present cohort, as paired serum and CSF albumin measurements were not part of the clinical protocol. Therefore, blood-CSF barrier integrity could not be directly assessed in this study. This remains an important open question, and future studies including simultaneous serum and CSF sampling will be necessary to clarify whether SP-G alterations may also reflect changes in blood-CSF barrier permeability.

The present findings should therefore be considered exploratory. Importantly, the present findings should not be interpreted as demonstrating an incremental diagnostic utility of CSF SP-G beyond currently established clinical, neuroimaging, and biochemical criteria for iNPH. Rather, our results support SP-G as a candidate biomarker reflecting alterations in CSF homeostasis and fluid dynamics, warranting further investigation. Future studies should determine whether SP-G may provide clinically meaningful complementary information in diagnostically challenging conditions, particularly in patients with overlapping neurodegenerative and CSF circulation abnormalities, and whether SP-G measurements may contribute to patient stratification or prediction of treatment response in longitudinal cohorts. If confirmed in larger cohorts, SP-G may represent a candidate biomarker reflecting CSF-related pathophysiological mechanisms, particularly in conditions characterized by altered CSF circulation such as iNPH. Nevertheless, the age-adjusted analysis indicates that larger studies with appropriate matching or longitudinal designs are needed before SP-G can be considered a clinically useful biomarker. Its potential diagnostic value should be investigated in combination with established CSF biomarkers, including tau and Aβ, as well as measures of CSF dynamics.

Several limitations should be acknowledged. First, the sample size of some diagnostic subgroups was relatively small. In particular, the AD subgroup (n = 11) was limited, which significantly reduces statistical power and limits the generalizability of AD-specific findings. Moreover, the imbalance between AD and FTD cases reflects the referral characteristics of our clinical setting and limits the possibility of drawing disease-specific conclusions within the ND group. Therefore, results related to this subgroup should be considered exploratory and interpreted with caution until confirmed by larger independent cohorts. This may have reduced the ability to detect more subtle group differences. Second, the cross-sectional design prevents any inference about longitudinal changes in SP-G levels during disease progression. Third, diagnostic classification was based on clinical assessment supported by CSF biomarkers, but neuropathological confirmation was not available. Furthermore, because of the retrospective design, we had access to archived CSF samples and final diagnostic classifications but were unable to reconstruct the complete diagnostic pathway for each participant. Consequently, analyses relied on the final clinical diagnoses established during routine care rather than on a standardized re-evaluation of the complete diagnostic work-up. Fourth, the CTR group consisted of patients undergoing clinically indicated lumbar puncture for neurological disorders unrelated to dementia, rather than healthy volunteers, since lumbar puncture cannot be ethically performed solely for research purposes. Consequently, the CTR group was younger than the iNPH and VaD groups. Although age was considered in the statistical analysis, residual confounding cannot be completely excluded. In particular, after adjustment for age, the effect of diagnostic group on CSF SP-G levels was attenuated and no longer reached statistical significance, indicating that the present study may have been underpowered to detect diagnostic effects once age-related variability was taken into account. At the same time, the absence of a significant group-by-age interaction and the lack of significant within-group correlations between age and SP-G levels suggest that age alone is unlikely to fully explain the higher SP-G concentrations observed in iNPH. Therefore, larger, prospectively characterized cohorts will be required to clarify whether SP-G alterations remain independently associated with iNPH beyond the effects of aging. In particular, future studies should include diagnostic groups that are closely matched for age, thereby minimizing residual confounding and allowing a more robust assessment of disease-specific differences in CSF SP-G levels.

Conclusion

In this exploratory study, we report the first assessment of CSF SP-G levels in patients with ND and iNPH. In the primary analysis, SP-G concentrations were higher in iNPH compared with the ND and CTR groups, suggesting a possible association between SP-G and altered CSF physiology. However, after adjustment for age, the diagnostic group effect was attenuated, indicating that these findings should be interpreted cautiously.

Although preliminary, these findings suggest that SP-G may provide complementary information to established amyloid- and tau-related biomarkers by reflecting alterations in CSF homeostasis and fluid dynamics, potentially supporting the differential diagnosis between iNPH and ND. Future prospective studies with larger cohorts are needed to validate these findings.

Data availability statement

The data that support the findings of this study are available in Zenodo at the following link: https://doi.org/10.5281/zenodo.18030708