Ethics statement

This study was reviewed and approved by the ethics committees of Juntendo University Hospital and Kuopio University Hospital (KUH) and conforms to the World Medical Association Declaration of Helsinki. All patients included in the study or their relatives gave informed consent for participation in the study. If the clinician suspected that dementia would significantly affect the capacity of the patient to consent, the next of kin or guardian consented on behalf of the patient. When consent was obtained from a participant’s proxy, the patient’s opinion was asked and considered, and no patients were recruited against their will.

Study design

In the first part of the study, we evaluated two cohorts of patients with probable iNPH: a Japanese cohort for analysis and a Finnish cohort for validation. We measured and compared the differences in the concentrations of CSF protein biomarkers for differential diagnosis among four groups: Japanese patients with probable iNPH (Japanese iNPH group), cognitively normal healthy individuals, hereafter referred to as the normal control (NC) group, patients with Alzheimer’s disease (AD) and patients with Parkinson’s disease (PD; Fig. 1). We validated the results for the NC group and patients with iNPH (Finnish iNPH group) in the Finnish cohort. Next, we intended to clarify the role and significance of PTPRQ in acquired (secondary) normal-pressure hydrocephalus (NPH; in which symptoms appeared subsequent to a preceding neurological injury, e.g., subarachnoid haemorrhage, meningitis) and congenital/developmental aetiologies, which present with panventriculomegaly, wide foramen of Magendie and large cisterna magna [19].

In the second part of the study, immunostaining and mRNA expression analyses were performed in autopsied brain specimens of NCs and patients with iNPH.

Study population

This study included two cohorts: one from Japan and the other from Finland. Patients who had at least one component of the NPH triad (gait disturbance, dementia, and urinary incontinence), and those with ventriculomegaly on computed tomography (CT)/magnetic resonance imaging (MRI) were enrolled as possible iNPH patients. In accordance with the Japanese guidelines for the management of iNPH, probable iNPH was defined as the presence of at least one component of the NPH triad with tight high convexity on CT/MRI or improvement of symptoms after CSF tap test or drainage [6]. CSF shunt surgeries were performed in patients with probable iNPH. A retrospective analysis of the detailed course of clinical symptoms was performed. Evaluation items included Mini Mental State Examination (MMSE) scores [19, 20] and iNPH Grading Scale (iNPHGS) scores [21].

The first cohort in Japan comprised the Japanese NC group, which consisted of 10 subjects (median age 73 years; five men); these patients were aged ≥60 years and had no known brain diseases or subjective declines in cognition and MMSE scores ≥26; the Japanese iNPH group consisted of 30 consecutive patients with iNPH (median age 74.5 years; 23 men) who received a lumboperitoneal shunt intervention between February 2013 and April 2015; the AD group consisted of 12 patients with AD (median age 74.5 years; six men), diagnosed by a neurologist according to the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer’s Disease and Related Disorders Association criteria for probable AD [22]; finally, the PD group consisted of 12 patients with PD (median age 72 years; six men), diagnosed by a neurologist according to the Movement Disorder Society Clinical Diagnostic Criteria for PD [23] (Table 1). An additional survey was conducted to compare iNPH with non-idiopathic NPH: 11 NPH patients with acquired aetiologies (median age 72 years; two men) and five NPH patients with congenital/developmental aetiologies (median age 67 years; three men; Table 2). The whole Japanese cohort was recruited from the Department of Neurology and Neurosurgery, Juntendo University, Tokyo, Japan.

The KUH Neurosurgery Department provides full-time acute and elective neurosurgical services for the KUH patient population from a recruitment area of Eastern Finland. The Kuopio NPH registry comprises all presumed iNPH patients from the KUH patient population and contains clinical baseline and follow-up data, diagnoses from other hospitals, medications and causes of death from national registries, and neuropathological findings of these patients [24, 25]. The Finnish cohort included the Finnish NC group and patients with probable iNPH. The Finnish NC group consisted of 30 patients (median age 71 years; 13 men) who were scheduled for knee or hip replacement surgeries at the KUH. The Finnish iNPH group consisted of 30 consecutive patients (median age 73 years;19 men) who consented to a lumbar puncture with CSF removal. The Finnish iNPH group had undergone ventriculoperitoneal shunt with a right frontal cortical biopsy [24, 26] at KUH between December 2013 and February 2015. Patients in the Finnish cohort who underwent biopsy at the time of CSF shunting were classified into two groups based on the presence or absence of amyloid and/or tau deposition in the frontal lobe cortex following histological examination (Table S1) [27-30].

Expression analysis using autopsied brains was performed during the second part of the study. Immunohistochemical staining was performed to identify the expression levels of PTPRQ in the frontal lobe cortex, cingulate gyrus at the level of mamillary bodies, hippocampus and thalamus, including the paraventricular system of the autopsied brains of patients with iNPH (10 patients: Japanese-iNPH [n = 3], Finnish iNPH [n = 7]) and NC subjects (n = 6) without significant AD pathology [28-30] (Table S2). All patients were clinically followed up until death, and the development of symptoms was documented by neurologists, neurosurgeons, and general practitioners.

Patients were diagnosed with dementia according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria [31]. Cognitive status was evaluated at the time of surgery and retrospectively at the time of death. Cognition was classified as normal, mild cognitive impairment, mild dementia, moderate dementia, or severe dementia (bedridden).

All clinical data from the hospitals in Juntendo University Hospital or the KUH catchment areas were collected.

Collection of cerebrospinal fluid

Cerebrospinal fluid was collected via lumbar puncture with the patient in the recumbent position (sitting position for the Finnish iNPH group). All CSF samples were centrifuged to remove cells and debris, aliquoted, and immediately stored in polypropylene tubes at −80°C until biochemical analysis.

Enzyme-linked immunosorbent assay

We measured the concentration of PTPRQ in the CSF of patients with probable iNPH and in NCs using an enzyme-linked immunosorbent assay (ELISA; Cloud-Clone Corp., Houston, TX, USA) according to the manufacturer’s protocol.

Additionally, we used Innotest assays for the quantitative determination of other CSF biomarkers, including p-tau (NIPRO, Osaka, Japan or Fujirebio, Ghent, Belgium), t-tau (NIPRO or Fujirebio), and Aβ42 (Fujirebio), which are markers of AD pathology. We also analysed the sensitivity and specificity of PTPRQ for the auxiliary diagnosis of probable iNPH.

Immunohistochemistry and histological evaluation

Post-mortem brain tissues were fixed in 10% neutral formalin for at least 1 week and then cut into 1-cm-thick coronal slices. The brain specimens were taken from 16 standard regions [25], embedded in paraffin, and cut into 6-µm sections. The sections were dewaxed using xylene, rehydrated in a graded series of alcohol to water, and subjected to antigen retrieval using Dako Target Retrieval Solution, Citrate pH 6 (×10; Agilent Technologies, Inc., Santa Clara, CA, USA) in an autoclave at 121°C for 10 min. Endogenous peroxidase was blocked by incubation of the brain sections with 0.3% hydrogen peroxide for 30 min. The sections were blocked with 5 × SEA BLOCK Blocking Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% donkey serum phosphate-buffered saline (PBS) at 25°C for 30 min to prevent non-specific antibody binding.

The sections were incubated with rabbit polyclonal IgG anti-PTPRQ (immunized with amino acids 2208–2299; 1:50 dilution; Cloud-Clone Corp.) overnight at 4°C. The following day, the sections were incubated with EnVision System Labelled Polymer (DAKO, Glostrup, Denmark), which was used as the secondary antibody, for 30 min at room temperature. Following each treatment, the slides were washed with PBS (three times for 5 min). Finally, the sections were stained with 3,3′-diaminobenzidine (DAB) and counterstained with Mayer’s haematoxylin, dehydrated, cleared and mounted.

Protein tyrosine phosphatase receptor type Q staining was viewed under an E800 microscope (Nikon, Tokyo, Japan), and images were captured with an AxioCam HRc CCD camera using AxioVison Rel 4.7.2.0 imaging processing software (Carl Zeiss, Microimaging GmbH, Jena, Germany). Whole-slide images of Aβ42, tau, and haematoxylin and eosin stains were captured with a NanoZoomer Digital slide scanner (Hamamatsu Photonics K.K., Hamamatsu City, Japan) [32, 33]. An experienced pathologist evaluated all the slides without knowledge of the patient’s outcome.

Immunofluorescence staining

The sections were blocked using a method similar to that used for single immunohistochemical staining, and stained with rabbit polyclonal IgG anti-PTPRQ (1:50) overnight at 4°C. The following day, the sections were incubated with secondary antibodies, including goat anti-rabbit IgG (H + L) Alexa Fluor™Plus488 (A32731; diluted 1:100, Thermo Fisher Scientific Inc.), for 60 min at room temperature. Next, the nuclei in the sections were stained with Hoechst 33342, trihydrochloride, trihydrate (H3570; Thermo Fisher Scientific Inc.) for 2 min at room temperature, and a glass coverslip was mounted onto each section using VECTASHIELD Mounting Medium (H-1000; Vector Laboratories Inc., Burlingame, CA, USA). Finally, the sections were examined under a confocal scanning microscope (Leica TCS-SP5) using a Leica Application SuiteX 3.4.2.18368 (Leica Microsystems, GmbH, Wetzlar, Germany).

In situ hybridization

In situ hybridization was performed on formalin-fixed, paraffin-embedded sections of periventricular samples in the iNPH and NC groups using the GeneticLab QuantiGene ViewRNA in situ hybridization tissue evaluation kit (Thermo Fisher Scientific Inc.). After deparaffinization, the sections were boiled in pretreatment solution for 10 min, digested with protease for 20 min, and then hybridized with designed probes against PTPRQ (VA1-3017466). Fast Red substrates were used to produce signals. The sections were examined under an E800 microscope (Nikon), and images were captured with an AxioCam HRc CCD camera using AxioVison Rel 4.7.2.0 imaging processing software (Carl Zeiss, Microimaging GmbH).

Data analyses and statistics

Descriptive statistics were used to summarize the data. The Shapiro–Wilk test and QQ plots were used to assess the normality of distributions. In each comparison, we used Tamhane’s T2 multiple comparison test after a one-way analysis of variance for continuous variables and Kruskal–Wallis’s H test for categorical variables. The Mann–Whitney U-test with Bonferroni correction was used to compare among the different groups. Wilcoxon signed-rank test was used for within-group comparisons of MMSE and iNPHGS scores before and 3 months after CSF shunting. Pearson correlation coefficient was estimated for finding linear association between two continuous variables. A chi-squared test was conducted to compare proportions.

The receiver-operating characteristic (ROC) curve was calculated to evaluate the goodness-of-fit for predictors of probable iNPH versus healthy neurological state. Statistical analyses were performed using IBM Statistical Package for the Social Sciences Version 25.0 (SPSS, Cary, NC, USA) for Windows. For all analyses, P < 0.05 was considered statistically significant.

Differentiation of iNPH from other neurodegenerative diseases

We confirmed that the preoperative CSF concentrations of p-tau, t-tau, and Aβ42 did not differ significantly between the Japanese iNPH and the NC groups (Table 1). Conversely, CSF PTPRQ concentrations were significantly higher in the Japanese and Finnish iNPH groups (mean [SD] 619 [398] pg/ml and 762 [570] pg/ml, respectively) compared to the respective NC groups (296 [94] pg/ml, **P = 0.001; and 371 [94] pg/ml, ***P < 0.001, respectively) and in the PD group (338 [230] pg/ml, *P = 0.041). However, the Japanese iNPH and AD groups showed no significant difference in CSF PTPRQ concentrations (619 [398] pg/ml vs. 365 [225] pg/ml; P = 0.075 [Table 1, Fig. 2]).

Using a cut-off PTPRQ concentration of 313 pg/ml, iNPH was detected with a sensitivity of 83.3%, specificity of 80%, and an area under the ROC curve (AUC) of 0.860 in the Japanese iNPH group. In the validation analysis, using a cut-off PTPRQ concentration of 555 pg/ml, iNPH was detected with a sensitivity of 63.3%, specificity of 100%, and an AUC of 0.849 in the Finnish iNPH group (Fig. S1).

Difference between iNPH with and without Alzheimer’s disease comorbidity

Analysis of the pathological tissues obtained by cortical biopsy at the time of CSF shunting showed that there were significant differences in age (*P = 0.01), MMSE scores at biopsy (*P = 0.012), MMSE scores after shunt (**P = 0.003), iNPHGS scores at biopsy (*P = 0.04), iNPHGS scores after shunt (*P = 0.018), concentrations of Aβ42 (**P = 0.004) and p-tau (*P = 0.048) between the group with amyloid deposits and/or tau deposits (n = 16) and the group without deposits (n = 14). However, the concentration of PTPRQ and t-tau did not differ significantly between the two groups (P = 0.257 and P = 0.525, respectively; Table S1).

Difference of PTPRQ between shunt responders and non-responders

We confirmed that there were no statistically significant differences between the Japanese and Finnish iNPH groups. The Japanese and Finnish iNPH groups were thus combined to create a single probable iNPH group, and no significant differences were found in PTPRQ concentrations between the shunt responder group (n = 46) and shunt non-responder group (n = 14; P = 0.432 [Table S3]).

Differences between iNPH and non-idiopathic NPH

The patients with non-idiopathic NPH (acquired + congenital/developmental) had significantly dilated ventricles compared to those with iNPH (*P = 0.011; Table 2). CSF PTPRQ concentrations were significantly higher in non-idiopathic NPH (mean [SD] 1704 [861] pg/ml) compared to the iNPH group (***P < 0.001; Fig. 3). There was a positive correlation between the Evans Index and CSF PTPRQ concentrations in NPH (correlation coefficient = 0.384, *P = 0.013).

Validation of PTPRQ expression in the brain tissues

Immunohistochemistry was performed to examine the localization of PTPRQ in the brain. We performed an absorption test to identify the specificity of PTPRQ immunostaining, and the results demonstrated that the addition of the antigen peptide to the primary antibody solution completely abolished PTPRQ immunostaining. The choroid plexus (Fig. 4a,b) and ependymal cells in patients with iNPH (Fig. 4c–e) were positively stained with the anti-PTPRQ antibody, as was the surface area of the ventricle that was directly in contact with the CSF in both iNPH and NC subjects.

Immunostaining and in situ hybridization also revealed that PTPRQ was expressed in the ependymal cells (Fig. 4f) and choroid plexus (Fig. 4g) of iNPH subjects.