TDP-43 and FUS transgene constructs.

<p>(A) Full-length wild type human TDP-43 and the clinical mutation A315T were cloned into a vector for expression in motor neurons by the <i>unc-47</i> promoter and injected into <i>C. elegans</i>. (B) Full-length wild type human FUS and the clinical mutation S57Δ were cloned into the <i>unc-47</i> expression vector and injected into <i>C. elegans</i>. RRM (RNA Recognition Motif), Q/G/S/Y (Glutamine-Glycine-Serine-Tyrosine-rich region), R/G (Arginine-Glycine-rich region), NLS (Nuclear localization signal).</p

TDP-43 and FUS transgenes do not affect lifespan.

<p>Beginning at Day 1 of adulthood we tested the lifespans of wild type non-transgenic N2 worms and transgenics expressing (A) wtTDP-43 and mTDP-43 as well as (B) animals expressing wtFUS and mFUS. Animals expressing TDP-43 or FUS transgenes had lifespans indistinguishable from N2 worms.</p

Decreased GABA staining in mutant TDP-43 and FUS worms.

<p>(A) Fluorescent micrograph of a fixed <i>unc-47p::GFP;mTDP-43</i> worm stained with a GABA antibody revealed neurodegeneration in motor neurons that mirrors the loss of GFP signals. Scale bar represents 10 µm for all photos. (B) Staining of <i>unc-47p::GFP;mFUS</i> worms also showed loss of GABA signals similar to the loss of GFP in the motor neurons.</p

Mutant TDP-43 and FUS cause adult-onset, age-dependent paralysis in <i>C. elegans</i>.

<p>Transgenics were monitored from the adult stage and scored daily for paralysis. (A) mTDP-43 worms show a rate of progressive paralysis that is greater than transgenics expressing wtTDP-43 (P<0.001). (B) Transgenics expressing mFUS become paralysed significantly sooner than wtFUS control transgenics (P<0.001). (C) Transgenic worms expressing GFP in motor neurons show low levels of paralysis.</p

Mutant TDP-43 and FUS aggregate in vivo.

<p>(A) Representative image of a fixed <i>unc-47p::GFP;mTDP-43</i> worm stained with a human TDP-43 antibody. The green channel shows GFP labelled motor neurons. Antibody staining (red signal) revealed aggregation of TDP-43 signals in motor neurons. Staining of motor neuron nuclei with DAPI (blue signal) revealed that TDP-43 is both cytoplasmic (single arrowhead) and nuclear (double arrowhead). Scale bar represents 10 µm for all photos. (B) Staining of <i>unc-47p::GFP;mFUS</i> worms with a human FUS antibody (red signal) and DAPI (blue signal) revealed cytoplasmic (single arrowhead) and nuclear (double arrowhead) accumulations in motor neurons.</p

Accelerated paralysis phenotypes for TDP-43 and FUS transgenics in liquid culture.

<p>(A) Paralysis phenotypes resolve over a number of hours for wtTDP-43 and mTDP-43 worms grown in liquid culture. mTDP-43 worms have a faster rate of paralysis compared to wtTDP-43 transgenics (P<0.001). (B) Transgenic mFUS worms show motility defects and become paralysed at a rate faster than wtFUS controls (P<0.001). (C) <i>unc-47p::GFP</i> transgenics have an increased rate of paralysis compared to non-transgenic N2 worms (P<0.001).</p

Mutant TDP-43 and FUS are highly insoluble.

<p>Shown are representative images from western blotting of the soluble supernatant and insoluble pellet fractions of protein extracts from transgenic TDP-43 and FUS strains. (A) Blotting against TDP-43 shows that a large proportion of the TDP-43 signal resides in the insoluble fraction for mTDP-43 worms, while the signal is largely soluble for the wtTDP-43 samples. (B) Immunoblotting with a human FUS antibody revealed that mFUS proteins primarily resided in the insoluble fractions while wtFUS proteins were exclusively soluble. Immunoblotting for actin was used as the loading control.</p

Mutant TDP-43 and FUS impair synaptic transmission.

<p>(A) Cholinergic neuronal transmission was measured by determining the onset of paralysis induced by the cholinesterase inhibitor aldicarb. <i>unc-47(e307)</i> mutants and mTDP-43 transgenics were hypersensitive to aldicarb-induced paralysis compared to either wtTDP-43 transgenics or N2 worms (P<0.001 for <i>unc-47</i> or mTDP-43 compared to N2 or wtTDP-43 worms). (B) mFUS transgenics and <i>unc-47(e307)</i> mutants were more sensitive to aldicarb induced paralysis compared to either wtFUS transgenics or N2 controls (P<0.001). (C) <i>unc-47</i> mutants grown on regular worm plates showed age-dependent progressive paralysis.</p

Mutant TDP-43 causes motor neuron degeneration.

<p>Shown are representative photos of living, adult <i>unc-47p::GFP</i>, <i>unc-47p::GFP;TDP-43</i>, and <i>unc-47p::GFP;FUS</i> transgenics. (A) Image of an entire <i>unc-47p::GFP</i> worm showing the GABAergic motor neurons. Scale bar represents 50 µm. (B) High-magnification of the framed area from (A) showing wild type morphology of motor neurons. Scale bar represents 20 µm. High magnification of motor neurons labelled with <i>unc-47p::GFP</i> in (C) wtTDP-43, (D) mTDP-43, (E) wtFUS and (F) mFUS transgenics showing gaps along neuronal processes (arrows). Scale bar represents 10 µm for photos (C) to (F). (G) Quantification of neurodegeneration in transgenic worms at days 1, 5 and 9 of adulthood. * wtTDP-43 and wtFUS have a higher rate of neurodegeneration compared to <i>unc-47p::GFP</i> controls at days 1 and 5 of adulthood (P<0.05). †mTDP-43 transgenics have a higher rate of neurodegeneration at days 5 and 9 compared to wtTDP-43 transgenics (P<0.001). ‡mFUS transgenics show an enhanced rate of neurodegeneration at days 5 and 9 of adulthood in compared to wtFUS transgenics (P<0.001).</p

Additional file 2: Table S2. of DNA hypomethylation of Synapsin II CpG islands associates with increased gene expression in bipolar disorder and major depression

Mixed Model ANOVA results for SYN1, SYN2, and SYN3 promoter CpG methylation. A. SYN1: Type III Tests of Fixed Effects. B. SYN2: Type III Tests of Fixed Effects. C. SYN3: Type III Tests of Fixed Effects. (XLSX 11 kb