Additional file 1: Figure S1. of Slowly progressive dementia caused by MAPT R406W mutations: longitudinal report on a new kindred and systematic review

Additional neuropathology. Additional photomicrographs from three family members. Individuals, immunohistochemistry, and enlargement as indicated. (TIF 11179 kb

Additional file 3: Table S2. of Slowly progressive dementia caused by MAPT R406W mutations: longitudinal report on a new kindred and systematic review

Individual data from previous publications (systematic review). Numbers indicate disease duration at first reported or observed symptom. Data in parentheses are successive or vaguely specified development or data unspecifically described. *Symptoms reported by patient or relative. #Unreliable data owing to possible pharmacological cause (neuroleptics). §Data too imprecisely reported to be included in calculations. AD Alzheimer’s disease, MCI Mild cognitive impairment, Dementia NOS Dementia not otherwise specified, CSF Cerebrospinal fluid, Aβ 42 Amyloid-β 1–42, APOE Apolipoprotein E, ADL Activities of daily living. (PDF 161 kb

Additional file 4: Table S3. of Slowly progressive dementia caused by MAPT R406W mutations: longitudinal report on a new kindred and systematic review

Cerebrospinal fluid examinations. Numbers in parentheses indicate disease duration when cerebrospinal fluid was retrieved. Upward filled triangles = value above laboratory’s reference value for healthy individuals; downward filled triangles = value below laboratory’s reference value for healthy individuals. aCompared with healthy control subjects (157 ± 65 pg/ml, n = 24) and patients with AD (555 ± 248 pg/ml, n = 31). bCompared with healthy control subjects (29 ± 10 pg/ml, n = 21) and patients with AD (86 ± 39 pg/ml, n = 31). N/A Not assessed. (PDF 304 kb

Additional file 2: Table S1. of Slowly progressive dementia caused by MAPT R406W mutations: longitudinal report on a new kindred and systematic review

MAPT haplotype determination based on exome sequencing data. Individuals, residues, and haplotype as indicated. Note that the proband, individual III-2, had Alzheimer’s disease and was excluded from the study. (PDF 181 kb

Additional file 1: Figure S1. of Slowly progressive dementia caused by MAPT R406W mutations: longitudinal report on a new kindred and systematic review

Additional neuropathology. Additional photomicrographs from three family members. Individuals, immunohistochemistry, and enlargement as indicated. (TIF 11179 kb

Substitution of PINK1 Gly411 modulates substrate receptivity and turnover

The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development. Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.</p

Population doubling levels of fibroblast cell lines.

<p>Population doubling levels were calculated for each of the cell lines available in the NINDS repository at the time of cryopreservation. Individual points of the graph correspond to the PDL of individual fibroblast lines, the horizontal line represents the mean PDL for each disease category. PDLs ranged between 2–8 with a mean PDL of ∼5 for both control and disease cell lines. A full list of PDLs for individual cell lines in provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043099#pone.0043099.s001" target="_blank">Table S1</a>.</p

Fibroblast lines generated in this study.

<p>Disease, gene, mutation and mode of inheritance for fibroblast cell lines. The current status of each line (available, submitted but not yet in catalogue) is indicated. Where the status is left blank, this indicates fibroblast lines have been generated but are awaiting submission to the NINDS repository. All variants are heterozygous unless otherwise stated. References indicate where families have been described in the literature. D = autosomal dominant, R = autosomal recessive.</p

Fibroblast cultures express the mesenchymal markers FSP1 and TE7.

<p>Cells generated from skin punch biopsies were verified as fibroblasts by morphological assessment (A) and positive staining with antibodies to fibroblast-specific protein 1 (FSP1) and fibroblast-specific clone TE7 (B, 63×). All fibroblasts examined (n = 6) demonstrated positive staining with both antibodies.</p

Fibroblast morphology and marker expression remain consistent during prolonged culture.

<p>Fibroblast lines were immunostained with antibodies FSP1 and TE7 at multiple consecutive passages (A). Passage numbers are indicated above the panels. Morphology, FSP1 and TE7 staining did not change during five consecutive subculturings (n = 6, representative images from line NM34737, carrying the PSEN1 M146I mutation are shown). FSP1 levels were also detected by western blotting of fibroblast cell lysates (B). FSP1 was detected as a single band at 12 kDa in all fibroblast lines examined (top panel, n = 6). β-actin was used as a loading control (bottom panel). No variation in FSP1 levels was observed between passages or between cell lines.</p