Patients with FTD were ascertained in an ongoing genetic-epidemiologic study conducted in The Netherlands since 1994 [1, 8, 9], after referral to the out-clinic department of the Erasmus Medical Center, or after a visit in nursing homes and psychogeriatric hospitals by the research physician. Detailed clinical history was obtained from the spouses and first-degree relatives using a checklist of behavioural and cognitive changes, and motor neuron signs.
The age at onset was defined as the age at which the first symptom compatible with FTD diagnosis was observed by a close relative or caretaker [1, 9]. During the neurological examination carried out in all patients, special attention was paid to the presence of extrapyramidal and motor neuron disease signs. Data on family history were provided by a spouse or first-degree relative using a structured questionnaire. Family history was defined as positive if patients had at least one-first-degree relative with dementia, parkinsonism, or MND, irrespective of their age of onset.
Neuropsychological evaluation consisted of tests for memory functions (Rey figure, 15-Word test ), attention and concentration, executive functions (Stroop, Trailmaking A and B, Wisconsin Card Sorting Test, WAIS subtest substitution), language (Boston Naming Test, the Dutch revised version of the Semantic association test ), and visuoconstructive and visuospatial skills (Clock drawing, Block design of the WAIS), and was performed in a subset of patients at our out-clinic department. Due to the variation in test batteries, a detailed comparison between subgroups was not possible. Severity of dementia at ascertainment was assessed using the Clinical Dementia Rating scale .
MRI scans performed between 1990 and 2008 were available for evaluation in 136 out of 387 patients. Available T1-weighted MR, T2-weighted MR, Proton-density (PD) weighted MR, and fluid attenuated inversion recovery (FLAIR) MR images were used for evaluation.
The severity of cortical and caudate atrophy on MRI was evaluated and semi-quantitatively rated according to a three-point scale: (0) normal caudate nucleus; (1) mild or moderate atrophy of the caudate nucleus, either still bulging into the lateral ventricle or with a flat contour; (2) severe atrophy with no visible caudate nucleus. The presence of caudate atrophy was evaluated by a radiologist and neurologist blinded to clinical and pathological findings. Disagreement was solved by consensus.
Mutation screening of all exons and exon–intron regions of MAPT, GRN and CHMP2B genes was performed in all patients with a positive family history, and in a subset of patients with sporadic FTD (n = 72) as previously described [1, 9].
Two hundred thirty-two patients died during a follow up period of 14 years, of which 74 patients underwent brain autopsy. Brain autopsy was carried out within four hours of death according to the Legal and Ethical Code of Conduct of The Netherlands Brain Bank. Macroscopic inspection of the brain included the frontal, temporal, parietal, and occipital lobes, cerebellum, hippocampus, basal ganglia and substantia nigra.
Tissue blocks were taken from all cortical areas, hippocampus, amygdala, basal ganglia, substantia nigra, pons, medulla oblongata, cerebellum, and cervical spinal cord. They were embedded in paraffin blocks and subjected to routine staining with haematoxylin-eosin, Bodian, methenamine-silver, and Congo red. The severity of neuron loss was scored as absent, mild, or moderate-severe.
Immunohistochemistry was performed with antibodies directed against: hyperphosphorylated tau (AT-8, Innogenetics, Ghent, Belgium; 1:400); ubiquitin (anti-ubiquitin, DAKO, Glostrup, Denmark; 1:500, following 80°C antigen retrieval); β-amyloid protein (anti-beta amyloid, DAKO, Glostrup, Denmark; 1:100, following formic acid pre-treatment); α-synuclein (anti-α-synuclein; Zymed Laboratories, San Francisco, California, USA; undiluted, following formic acid pre-treatment); poli-ubiquitin-binding protein p62 (BD Biosciences Pharmingen, San Diego, CA, USA; 1:200, following pressure cooking); TDP-43 (Biotech, Chicago, IL, USA; 1:100, following pressure cooking); and α–internexin (anti-alpha-internexin, Invitrogen, Camarillo, CA, USA, 1:100, following pressure-cooking), FUS (Sigma-Aldrich anti-FUS; 1:25–1:200 with initial overnight incubation at room temperature, following pressure cooking).
Antigen retrieval was done either for 30 min in 0.1 M sodium citrate buffer at 80°C and pH 7.7 or using pressure cooking in 0.1 M sodium citrate buffer (pH 6) for 5 min. Pre-treatment with 70% formic acid was done for 5 min (α-synuclein and β-amyloid).
Primary antibodies were incubated overnight at 4°C. Endogenous peroxidase activity was inhibited by incubation for 30 min in PBS-hydrogen peroxide-sodiumazide solution (100 ml 0,1 M PBS + 2 ml 30% H2O2 + 1 ml natriumazide). The Histostain-Plus broad-spectrum kit DAB (Zymed, San Francisco, California, USA) was used and slides were counterstained with Mayer’s haematoxylin and mounted in entellan.
Neuropathological examination of brains from the 74 autopsied patients with clinical FTD revealed 70 brains with FTLD (FTLD-U in 37, FTLD-tau in 32 and FTLD with no inclusions (FTLD-ni) in one), whereas familial Creutzfeldt-Jakob disease (fCJD) was diagnosed in two brains, and Alzheimer’s disease (AD) in the remaining two.
The study was approved by the Medical Ethical Committee at the Erasmus MC-University Medical Center Rotterdam. For each patient, a spouse or first-degree relative of the patient gave written informed consent.