Multireceptor fingerprints in progressive supranuclear palsy

Brains were obtained from patients with PSP (aged 72 ± 7 years, 9 male, 7 female) recruited in a nationwide study on PSP between 2000 and 2009. Brain autopsy was conducted by The Netherlands Brain Bank according to its Legal and Ethical Code of Conduct. Control subjects consisted of age- and sex-matched subjects (aged 76 ± 10 years old, 9 male, 5 female) without a history of neurological or psychiatric diseases.

Patients were examined after referral to the outpatient department of the Erasmus University Medical Center and by visiting patients in nursing homes as part of a large longitudinal study [2, 16] approved by the medical ethics committee of the Erasmus University Medical Center. All participants or their first-degree relatives signed informed consent forms. All patients were examined by a research physician (W.Z.C. or L.D.K.) or a neurologist (A.J.W.B. or J.C.v.S.). A detailed clinical history was obtained from patients and their family members and by reviewing medical records. The neurological examination was videotaped according to a standardized protocol. Structural neuroimaging of patients was reviewed to exclude other disease causes. Family history was considered positive when at least one first-degree relative had dementia or parkinsonism. The possibility of postmortem examination was discussed with patients and their relatives. Relevant medication used in the last 3 months of life was recorded. Clinical diagnosis of patients was established in a consensus meeting according to the National Institute of Neurological Diseases and Stroke/Society for Progressive Supranuclear Palsy criteria [1]. Neuropathological diagnosis of PSP was established according to international criteria [17].

At The Netherlands Brain Bank, the right hemispheres of all brains are processed for routine staining and immunohistochemistry against several antibodies: AT8 (1:40; Innogenetics, Ghent, Belgium), ubiquitin (1:500; Dako, Glostrup, Denmark), three-repeat tau isoform (1:3000; Upstate Biotechnology, Charlottesville, VA, USA), four-repeat tau isoform (1:100; Upstate Biotechnology), p62 (1:200, following 80 °C antigen retrieval; BD Biosciences Pharmingen, San Diego, CA, USA), trans-activation response DNA-binding protein 43 (TDP-43, 1:100 following pressure-cooking; Proteintech, Chicago, IL, USA), β-amyloid (anti-β-amyloid, 1:100 following formic acid pretreatment; Dako), and α-synuclein (anti-α-synuclein, undiluted following formic acid pretreatment; Zymed Laboratories, South San Francisco, CA, USA). Slides were incubated overnight at 4 °C. Endogenous peroxidase activity was inhibited by 30-minute incubation in a PBS-hydrogen peroxide-sodium azide solution (100 ml of 0.1 M PBS, 2 ml of 30% H₂O₂, 1 ml of NaN₃). The Histostain-Plus broad-spectrum immunohistochemistry kit with 3,3′-diaminobenzidine (Zymed Laboratories) was used as a detection system. Slides were counterstained with Mayer’s hematoxylin and mounted in Entellan medium (EMD Millipore, Billerica, MA, USA).

A separate semiquantitative assessment of tau pathology for area 24′ and the caudate nucleus was carried out by two raters (W.Z.C. and J.C.v.S.) using a 2-point grading scale, which is an adaptation of the visual guide proposed by Williams et al. [18]. We defined Williams’s grades 1 and 2 as mild and grades 3 and 4 as moderate to severe.

Probes from midcingulate area 24′ and the caudate putamen were taken from the left hemisphere, frozen in isopentane at −40 °C with a postmortem delay of 6 ± 1 h (patients with PSP) and 8 ± 1 h (control subjects), and serially sectioned at −20 °C in 10-μm-thick sections with a cryostat. Alternating sections were processed for the visualization of 20 transmitter receptors according to standard protocols (Table 1) comprising a preincubation to remove endogenous ligands and external substances such as medication, a main incubation to label binding sites with a tritiated ligand in the presence (nonspecific binding) or absence (total binding) of a nonlabeled displacer, and a rinsing step to eliminate unbound radioactivity [14, 19]. Nonspecific binding was less than 5% of total binding for all examined binding sites and thus was ignored in the present study. All sections intended for the visualization of a given receptor type were incubated in the same radioactive solution.

Radioactively labeled sections were coexposed against tritium-sensitive films (Amersham Hyperfilm®; GE Healthcare Life Sciences, Braunschweig, Germany) with plastic [³H]-standards of known concentrations of radioactivity (Amersham Microscales®; GE Healthcare Life Sciences). Upon purchase, Microscales® were calibrated with the aid of brain homogenate standards for which total protein content had been determined by means of the Bradford assay [20]. Resulting autoradiographs were processed by densitometry with a video-based image-analyzing technique [19]. The Microscales® were used to compute a calibration curve, which, together with the parameters specific for each binding experiment (i.e., specific activity, dissociation constant, and concentration of the ligand), enabled transformation of grayscale values in the autoradiographs of samples into a binding site density per unit of protein (femtomoles per milligram of protein). Mean densities were thus obtained for a series of three or four sections per receptor type in the area 24′ and caudate nucleus probe of each case.

IBM SPSS Statistics version 21.0 for Windows software (IBM, Armonk, NY, USA) was used for analysis. Demographic features were analyzed by independent samples t test or chi-square test. Discriminant analyses were performed separately for data from area 24′ and the caudate nucleus to visualize the multivariate distance between control subjects and patients with PSP and between the two groups into which the patients could be subdivided on the basis of tau pathology severity or frontal versus nonfrontal presentation. Only in the case of a significant result did we perform post hoc tests (univariate F-tests) to reveal which receptor types differed between control subjects and patients with PSP or PSP subgroups. These p values were not corrected for multiple comparisons. Significance levels were set at p < 0.05 for the omnibus tests and p < 0.01 for the post hoc tests. The discriminant analysis was chosen as a global test because it offers several advantages over the procedures classically used to test group differences [21], the most important of which are that it is nonparametric and that it supports the analysis of multivariate datasets with more dependent variables (receptor densities, comprising 20 in this study) than cases (individuals in this study, comprising 14 control subjects and 16 patients with PSP). This is accomplished by reducing the receptor densities to a smaller number of discriminant scores (two in this study) for statistical testing and graphing.

On the basis of discriminant analyses of receptor densities, classification of patients with PSP and control subjects was significant in the caudate nucleus (Wilks’ lambda = 0.103, chi-square = 34.127, p = 0.025) and in area 24′ (Wilks’ lambda = 0.071, chi-square = 47.592, p < 0.001). Post hoc univariate F-tests revealed significantly higher densities of peripheral benzodiazepine (pBZ) and adenosine receptor type 1 (A₁) receptors, but lower densities of kainate receptors and of nicotinic cholinergic receptors of the α₄/β₂ type (nACh) in the caudate nucleus of PSP brains than in brains of control subjects (Table 4, Fig 1a). In contrast to this, significantly higher N-methyl-d-aspartate (NMDA), γ-aminobutyric acid receptor type B (GABA_B), pBZ, and serotonin receptor type 2 (5-HT₂) receptor densities were found in area 24′ of PSP patient brains than in control brains (Table 5, Fig. 1d).

Because patients with PSP displayed high interindividual variability in receptor density alterations (see large SD in Fig. 1a and d), we subdivided the cohort on the basis of the clinical parameters presence of frontal presentation and severity of tau pathology in the examined regions, and we tested separately for data obtained from the caudate nucleus and area 24′ whether these factors were associated with receptor density alterations in patients with PSP.