Gross and macroscopic neuropathologic assessment was performed by standardized procedures. Formalin-fixed, paraffin-embedded tissue samples from the primary motor and visual cortices, inferior parietal lobule, mid-frontal gyrus, superior temporal gyrus, amygdala, and posterior hippocampus were cut at 5 µm thickness, mounted on glass slides and stained with H&E. Thioflavin-S fluorescent microscopy was performed to evaluate SP (10× objective) and NFT densities (40× objective). Primitive, neuritic, and cored type plaques were included in the SP counts and were truncated at 50, which is twice the number required to meet Khachaturian’s criteria for AD diagnosis [20 (link)]. NFT distribution was assessed to determine Braak stage. Intracellular and extracellular NFT counts from two hippocampal regions (CA1 and subiculum) and three association cortices (inferior parietal, mid-frontal, and superior temporal) were used to determine AD subtypes. Additional sections previously underwent immunohistochemical staining and were processed using the DAKO Autostainer (DAKO Auto Machine Corporation, Carpinteria, CA, USA) with DAKO Envision+ HRP System. The posterior hippocampus was stained with an antibody that detects the 25 kDa C-terminal fragment of TDP-43 (a generous gift from Leonard Petrucelli, Mayo Clinic, FL, USA). Assessment of TDP-43 pathology was performed as previously described [2 (link)]. Cerebrovascular disease was assessed using a simple scheme proposed by Jellinger and Attems [18 ], as previously reported. Lewy body disease (LBD) pathology was assessed by use of immunohistochemistry [34 (link)], as previously described.
For genotyping, genomic DNA was extracted from frozen brain by standard procedures. Genotyping for MAPT H1/H2 (SNP rs1052554 A/G, A = H1, G = H2), APOE alleles (SNP rs429358 C/T and rs7412 C/T), GRN (SNP rs5848 C/T), and TMEM106B (SNP rs1990622 C/T) was performed using a Taqman SNP genotyping assay (Applied Biosystems, Carlsbad, CA, USA). Genotype calls were obtained with SDS v2.2 software (Applied Biosystems). Although there is overlap in the HpScl-AD group between previous Genetic studies [9 (link), 32 (link)], Genetic information on the HpScl group has not been previously reported. Genotyping availability can be found in Supplementary methods.
Clinical reports were reviewed blind to pathologic diagnosis to collect education, family history, age of onset, disease duration, and Mini Mental State Examination (MMSE) scores. Family history was noted as positive if at least one first-degree relative had dementia. Age of onset was recorded as the age of initial cognitive abnormalities, as opposed to age of diagnosis. Disease duration was ascertained as the number of years elapsed between age of death and age of onset. Longitudinal decline was calculated as a slope of three or more MMSE scores, where the MMSE score was the dependent variable and elapsed years between testing and death were the independent variable. Antemortem clinical diagnosis of probable AD, possible AD and mild cognitive impairment was considered an amnestic diagnosis. Availability of clinical information can be found in Supplementary methods.