Biochemical studies in Normal Pressure Hydrocephalus (NPH) patients: change in CSF levels of amyloid precursor protein (APP), amyloid-beta (Aβ) peptide and phospho-tau

Abstract

Normal Pressure Hydrocephalus (NPH) is one of the causes of dementia of the elderly characterized by impaired mental function, gait difficulties and urinary incontinence. Previously, it was proposed that some of the NPH patients may develop Alzheimer's disease (AD) like pathology. Aim of this study was to compare levels of different CSF biomarkers, including total secreted β-amyloid precursor protein (sAPP), sAPP-alpha form (sAPPα), amyloid-beta (Aβ) peptide, total-tau protein and hyperphosphorylated-tau protein in subjects from NPH and Non-NPH Control (NNC). CSF was collected from 23 NPH patients and 13 Non-NPH controls by lumber puncture. Western blot analysis was performed to measure levels of sAPP-total. ELISA was used separately to determine levels of sAPPα, Aβ peptide, total-tau and phospho-tau proteins. We found a significant decrease in levels of total secreted APP, sAPPα and Aβ (1-42) in the CSF sample of NPH patients vs. NNC. We did not observe any change in levels of total-tau or phospho-tau in NPH vs. NNC subjects. Notably, phospho-tau level was significantly increased in the NPH patients, who were suffering from the disease for more than one year, vs. NNC. Among five biomarkers studied, decreased sAPP, sAPPα and Aβ (1-42) levels in CSF can be molecular markers to distinguish NPH cases from NNC. Disease severity can also be assessed by increased levels of CSF phospho-tau protein and the ratio of phospho-tau to Aβ (1-42), which might be a useful tool for predicting conversion of NPH individuals to other neurodegenerative disorders including Alzheimer's disease (AD).

Fig. 1

a: Effect of dilution of CSF in NPH subjects determined by UV/VIS spectra: To assess whether the CSF samples of NPH are diluted than those of NNC subjects, a wide range of UV/VIS protein spectra (from 220 nm to 750 nm) was recorded from all CSF samples (n = 36) using ‘NanoDrop V2.1.0’ machine. Spectra obtained from the CSF samples of NPH were marked in blue and those of the CSF samples of NNC subjects were marked in black, which showed no significant change in the protein concentrations of the CSF samples of both the groups. b: Effect of dilution of CSF in NPH subjects determined by Bradford protein assay: Total protein in the CSF was also measured by Bradford assay which, likewise, confirmed that total protein contents both NPH and control groups didn’t differ significantly.

Fig. 2

a: Levels of total sAPP: CSF levels of soluble amyloid precursor protein (APP) were determined by semi-quantitative Western blotting technique. An equal volume of CSF sample (15 μl) was loaded onto denaturing polyacrylamide gels (10%). Following PAGE and electrophoretic transfer, the PVDF membrane was probed with APP antibody (clone 22C11), as described in ‘Materials and Methods’. A representative Western blot picture with sAPP protein bands in different lanes was shown from the CSF of 11 NPH and 7 NNC subjects. b: Statistical analysis of APP band density by independent t-test shows a decrease of total sAPP level in the CSF of NPH patients (n = 23) vs. NNC (n = 13), which is statistically significant (p = 0.0013).

Fig. 3

Levels of sAPPα: Measurement of sAPPα with an equal volume (15 μl) of CSF samples from each subject was performed by a specific ELISA as described in ‘Materials and Methods’. Result shows a significantly decrease in sAPPα in the CSF samples of NPH patients. The standard curve with different concentrations of human sAPPα is shown at the left.

Fig. 4

a: Levels of Aβ (1–40): Levels of Aβ (1–40) were assayed in all CSF samples by the sensitive sandwich ELISA method. An equal volume (5 μl) of CSF samples was loaded in the antibody pre-coated 96-well ELISA plate. Samples were incubated overnight and processed as described in ‘Materials and Methods’. Independent t-test showed no change in Aβ (1–40) level in the CSF of NPH patients (p = 0.120). b: Levels of Aβ (1–42): ELISA of Aβ (1–42) with 50 μl of CSF samples was carried out as mentioned in ‘Materials and Methods’ and Fig. 4a. Statistical analysis showed a significant decreased Aβ (1–42) level in the CSF of NPH vs. NNC subjects (p = 0.017). c: Separate analysis showed a further decrease in the magnitude of CSF Aβ (1–42) in NPH patients who are suffering from more than one year, vs. NNC (p = 0.002). d: Relative proportion of Aβ (1–40) and Aβ (1–42): Ratio of levels of Aβ (1–40) and Aβ (1–42) showed no significant change in the CSF samples of NPH patients, vs. NNC (p = 0.197).

Fig. 5

a: Levels of total-tau in the CSF of NNC vs. NPH: Human total-tau ELISA was performed with equal volume of CSF samples which showed no significant change in the CSF of NPH vs. NNC subjects (p = 0.519). b: Levels of phospho-tau in the CSF of NNC vs. NPH: Human phospho-tau ELISA was performed as described in ‘Materials and Methods’ section, which showed no significant change in phospho-tau levels in the CSF of NPH patients vs. NNC subjects (p = 0.153). c: Levels of total-tau in the CSF of NNC vs. NPH patients suffering more than one year: No significant change was observed in the levels of total-tau in the CSF samples of NNC vs. NPH subjects who were suffering from more than one year (p = 0.11). d: Levels of phospho-tau in the CSF of NNC vs. NPH patients suffering more than one year: A significant increase in phospho-tau is observed in the CSF of the patients who are suffering from the disease for more than one year (p = 0.013)(Out of twenty three NPH patients, duration of illness of seven patients was less than one year, nine patients were suffering from more than one year and data from seven patients were not obtained).

Fig. 6

a: Relative proportion of total-tau to Aβ (1–42): Ratio of total-tau and Aβ (1–42) showed an increasing trend in the CSF samples of NPH individuals vs. NNC. However, the increase in the ratio of total-tau/Aβ (1–42) is not statistically significant (p = 0.1). b: Relative proportion of phospho-tau to Aβ (1–42): Relative proportion of phospho-tau and Aβ (1–42) showed an increase (p = 0.004) in the CSF samples of NPH vs. control, indicating more production of phospho-tau and less clearance of Aβ (1–42) in the CSF of NPH individuals vs. NNC.