Linking tuberous sclerosis complex, excessive mTOR signaling, and age-related neurodegeneration: a new association between TSC1 mutation and frontotemporal dementia

Author(s): Olney, Nicholas T; Alquezar, Carolina; Ramos, Eliana Marisa; Nana, Alissa L; Fong, Jamie C; Karydas, Anna M; Taylor, Joanne B; Stephens, Melanie L; Argouarch, Andrea R; Van Berlo, Victoria A; Dokuru, Deepika R; Sherr, Elliott H; Jicha, Gregory A; Dillon, William P; Desikan, Rahul S; De May, Mary; Seeley, William W; Coppola, Giovanni; Miller, Bruce L; Kao, Aimee W

The protocol for this study was approved by the Institutional Review Board of the University of California, San Francisco. After informed consent was obtained, subjects underwent neurological evaluation, neuropsychological testing, informant interview, blood draw and neuroimaging.

Sequencing
The proband was screened by Sanger sequencing for mutations in the following genes: APP, PSEN1, PSEN2, MAPT, PGRN, FUS, and TARDBP. The hexanucleotide repeat length of C9orf72 was screened as previously described [1].Whole exome regions of the proband and sibling were captured using the SeqCap EZ Human Exome Kit v3 and sequenced on an Illumina HiSeq2500 sequencer with an average of 115x depth of coverage. Sequence reads were mapped to the GRCh37/hg19 reference genome and variants joint-called using the GATK Haplotype Caller according to GATK Best Practices recommendations [2]. Ingenuity Variant Analysis was used for variant annotation and filtering. A series of filtering steps were applied to prioritize variants, as previously described [3]. PCR amplification from genomic DNA, followed by Sanger sequencing was performed to validate candidate variants.
SH-SY5Y cells were infected with virus particles and, after the antibiotic selection, individual clones with enlarged cell size were isolated, expanded, checked by PCR and sequenced.

Cell area measurement
Micrographs from control and TSC1 mutant cells (passage 3) were taken using a Carl Zeiss Axio Ver.A1 inverted microscope with 20x magnification. Measurement of cell area of 300 independent cells was performed using Image J. Statistical significance was estimated by one-way analysis of variance (ANOVA) followed by the Bonferroni's test for multiple comparisons. A value of p<0.05 was considered significant.

Cell differentiation
SH-SY5Y cell lines were differentiated into neurons by treatment with 10µM of retinoic acid (RA) for 6 days in EMEN/F12 media supplemented with 10% of FBS and 1% Penicillin-Streptomycin, followed by another 4 days of treatment with 50ng/mL of BDNF in EMEN/F12 media supplemented only with 1% Penicillin-Streptomycin [7].