Additional file 1 of Increased TMEM106B levels lead to lysosomal dysfunction which affects synaptic signaling and neuronal health
Bibliographic record
Abstract
Additional file 1: Figure S1. TMEM106B overexpression leads to increased LAMP1 and PGRN levels in MEFs. (A) Immunoblot and (B) quantification of LAMP1, PGRN, and TMEM106B protein levels indicate elevated levels of LAMP1 and PGRN in MEFs derived from TMEM106B overexpression mice which confirms that increased levels of TMEM106B induces lysosomal dysfunction. Data represented as mean ± SEM. One-way ANOVA (n=3/genotype). *, P < 0,05; **, P < 0,01. The 15kDa band represents an N-terminal fragment of TMEM106B of unknown significance which appears to be human-specific but is otherwise uncharacterized. Figure S2. TEM images of wild-type and TMEM106B(+) neurons showing large cytoplasmic vacuoles. Ultrastructural examination with TEM confirmed the presence of numerous cytoplasmic vacuoles with variable content, sizes and shapes in hTMEM106B(+) neurons. While in few wild-type neurons similar structures could be observed, these were far less abundant and generally much smaller in size. Scale bars (5 µm). Figure S3. Ultrastructural characterization of aberrant vacuoles in TMEM106B overexpressing neurons. The electron-lucent cytoplasmatic vacuoles are enclosed by single membranes. Many vacuoles are largely empty, or contain only small irregularly shaped cytosolic components A,B,C). Cytoplasmic invaginations can penetrate the vacuoles (C,D), in rare cases resembling a network of cytoplasm stretches (E). Some vacuoles contain mostly degraded material (F), while others mostly hold a build-up of membranes and vesicles (G, H). Scale bars (1µm). Figure S4. The vacuoles do not colocalize with the autophagosome marker LC3. Representative images of wild-type and hTMEM106B(+) stained for LAMP1 and LC3. Neurons were treated with rapamycin (500nM) and/or bafilomycin (50nM) for 4 h to induce autophagosome formation. The majority of the vacuoles do not colocalize with LC3. Only few vacuoles were associated with autophagosomes. Scale bars (20µm). Figure S5. TMEM106B overexpression does not lead to apparent lysosomal or neurological changes in 21-months-old mice. (A) Immunoblot and (B) quantification of hTMEM106B and total TMEM106B protein levels in hemibrain lysates of 21-month old animals. (C) Immunoblot and (D) quantification of lysosomal proteins indicate normal levels of lysosomal proteins in the brain. (E) Immunoblot and (F) quantification of neural markers GFAP, SYN, NeuN, and IBA1 indicate normal levels of neural cell types. Data represented as mean ± SEM. One-way ANOVA. *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001. Figure S6. TMEM106B overexpression does not lead to gliosis in 21-month-old mice. Representative images of (A) GFAP and (B) IBA1 immunostaining of sagittal brain sections of wild-type, hTMEM106B(+), and hTMEM106B(++) mice. Scale bar (2 mm). Quantitative intensity measurements of (C) GFAP and (D) IBA1 (n = 4-6/genotype). Data represented as mean ± SEM. One-way ANOVA. Figure S7. TMEM106B overexpression mice do not have TDP- 43 aggregates. (A) Immunoblot and (B) quantification of TDP- 43 protein levels in RIPA- and UREA-soluble fractions of hemibrain lysates of 21-month-old animals does not show increased amounts of TDP- 43 in the insoluble fraction. Data represented as mean ± SEM. One-way ANOVA (n = 3/genotype). (C) Immunohistochemistry with phosphorylation-independent TDP- 43 (12B4) antibody reveals predominant nuclear staining in wild-type and hTMEM106B(+) mice as illustrated in different brain regions. (D) Immunohistochemistry with S409/410 phosphorylation-dependent TDP- 43 (1D3) antibody. No specific immunoreactivity was observed in wild-type and hTMEM106B(+) mice as illustrated in various brain regions. Insert shows positive control for 1D3 immunohistochemistry with nuclear cytoplasmic inclusions in human postmortem FTLD-TDP case. Scale bar 50 µm. Figure S8. Wild-type and TMEM106B overexpression mice do not generate TMEM106B fibrils. (A) We evaluated the presence of TMEM106B fibrils in 21-month-old mice using the same methodology as we used in human studies [82, 83]. (B) Immunoblot of the insoluble fraction does not show TMEM106B immunoreactive pathology in either wild-type or hTMEM106B overexpression mice. (C) Representative images of immunostainings of the cortex of 21-month-old Tmem106b knock-out, wild-type, and hTMEM106B(+) animals using VIB SB0051 fibril-specific antibody. All cases showed a similar pattern with staining of vascular endothelial cells and some small glia with morphology of microglia, that was interpreted as non-specific. None of the animals in any of the groups showed pathological accumulation of TMEM106B CTF, similar to what is seen in human cases.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.022 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.002 | 0.004 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.002 | 0.001 |
| Insufficient payload (model declined to judge) | 0.912 | 0.182 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; the direct Gemma label and the distilled Codex classifier agree on what is shown here.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".