Additional file 2: Figure S2. of Evidence for an early innate immune response in the motor cortex of ALS

MCP1+ cells and CCR2+ cells are present in the motor cortex and they are in close proximity to CSMN in the MCP1-CCR2-hSOD1G93A mice. (a) Representative image showing very few MCP1+ cells in the motor cortex of MCP1-CCR2-WT mice. (b) Representative image showing increased numbers of MCP1+ cells in the motor cortex of MCP1-CCR2-hSOD1G93A mice. MCP1+ cells express microglia marker CD11b. Insets enlarged to the right (b’-b”’). (c) Experimental design depicting retrograde transduction of CSMN approach using AAV-eGFP in the MCP1-CCR2-WT and MCP1-CCR2-hSOD1G93A mice. AAV2-eGFP was injected into the CST of mice at P30, and tissue was collected at P60. (d, e) Representative images show MCP1+ cells near transduced CSMN (eGFP+) in the layer V of the motor cortex (d, e) and in the layer II/III of the motor cortex (f, g) in MCP1-CCR2-hSOD1G93A mice. (h, j) Representative images show MCP1+ cells expressing phagocytic marker CD68 and their interaction with transduced CSMN in the layer V of motor cortex in the MCP1-CCR2-hSOD1G93A mice. (k-n) Representative image showing CCR2+ cells in layer II/III of motor cortex co-localizing with monocyte marker CD45 and infiltrating monocyte marker Ly6C. Scale bar:s: a,b,d-g =20 μm; k-n = 10 μm. (PDF 1521 kb

Additional file 3: Figure S3. of Evidence for an early innate immune response in the motor cortex of ALS

MCP1+ cells express neither Arginase 1 (Arg1) nor inducible nitric oxide synthase (iNOS) in the MCP1-CCR2-hSOD1G93A mice. (a) Representative images of Arg1+ cells (arrowheads) and MCP1+ cells (arrows) in the liver of MCP1-CCR2- hSOD1G93A mice 6 h post LPS I.P. injection (positive control). (b) Representative images of 2° only for Arg1 (negative control) and MCP1+ cells (arrows) in the liver of MCP1-CCR2- hSOD1G93A mice 6 h post LPS I.P. injection. (c) Representative images of MCP1+ cells (arrows) in the spleen of MCP1-CCR2- hSOD1G93A mice 6 h post LPS I.P. injection (positive control) show co-localization with iNOS (arrows). (d) Representative images of 2° only for iNOS (negative control) and MCP1+ cells (arrows) in the spleen of MCP1-CCR2- hSOD1G93A mice 6 h post LPS I.P. injection. (e) Experimental design depicting retrograde transduction of CSMN approach using AAV-eGFP in the MCP1-CCR2-WT and MCP1-CCR2-hSOD1G93A mice. AAV2-eGFP was injected into the CST of mice at P30, and tissue was collected at P60. (f-g) Representative images of the layer II/III of motor cortex show lack of co-localization of MCP1+ cells with Arg1 in MCP1-CCR2-WT mice (f) and MCP1-CCR2- hSOD1G93A mice (g). (h-i) Representative images of the layer II/III of motor cortex show lack of co-localization of MCP1+ cells with iNOS in MCP1-CCR2-WT mice (h) and MCP1-CCR2- hSOD1G93A mice (i). Scale bar = 10 μm. (PDF 961 kb

Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

<div><p>We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from <i>Ehd1</i>-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining <i>Ehd1</i>-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in <i>Ehd1</i>-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, <i>Ehd1</i>-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. <i>Ehd1</i>-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.</p></div

EHD1 modulates BIN1 mediated tubule formation <i>in vivo</i>.

<p>Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A & B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.</p

Misexpression of triad proteins and expansion of the T-tubule compartment in <i>Ehd1-</i>heterozygous muscle.

<p>(A) BIN1 and DHPR levels are increased +2.25 and +5.4 fold in <i>Ehd1</i>+/- quadriceps muscle compared to WT correlating with the increase in T-tubule structures. Gel code bands are shown as a loading control (LC). (B) Junctophilin 2 (JP2) protein levels were decreased 13-fold in <i>Ehd1+/-</i> quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (C) Junctophilin 1 (JP1) protein levels were similar in <i>Ehd1+/-</i> quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (D) Ultrastructural analysis reveals ectopic (dotted arrow) and elongated (arrow) T-tubules in 8-week-old <i>Ehd1-</i>heterozygous muscle (<i>Ehd1+/-</i>) stained with potassium ferricyanide to color the T-tubule structures black. (E) <i>Ehd1-</i>heterozygous muscle contains duplicated triads containing 2 T-tubules (black arrows) and 3 sarcoplasmic reticulum (SR) in 1 triad unit. Scale 0.5μm. (F) <i>Ehd1-</i>heterozygous muscle stained with potassium ferricyanide, outlines duplicated T-tubule structures (two black arrows). Scale 0.5μm. (G) Ultrastructural analysis of 2-D images reveals increased tubule abnormalities in <i>Ehd1-</i>heterozygous muscle, 12.5%, compared to 1.7% in control muscle (n>400 structures per genotype, p = 0.04).</p

<i>Ehd1-</i>heterozygous mice have elevated creatine kinase levels.

<p>(A) Serum creatine kinase (CK) levels are highly elevated in <i>Ehd1-</i>heterozygous (<i>Ehd1+/-</i>) mice at birth (n > 10 of each genotype, p<0.0001). (B) CK levels are consistently elevated in <i>Ehd1+/-</i> mice at 8wk, 6m, and 14m compared to WT controls (n ≥ 6 of each genotype, p<0.0002). (C) Evans blue dye uptake was measured from excised tissues (absorbance per mg tissue). Graph expressed as an average of all tissues. Evans blue dye uptake (measured as absorbance) is similar in <i>Ehd1+/-</i> and WT (n>6 animals per genotype, ns, nonsignificant).</p

Disordered T-tubules in <i>Ehd1-</i>heterozygous muscle.

<p>Myofibers were immunostained with anti-BIN1 (red) and anti-DHPR (green) antibodies. Representative myofibers are shown. <i>Ehd1-</i>heterozygous (<i>Ehd1+/-</i>) fibers displayed disorganized (white arrowhead) and aggregated (white arrow) T-tubule structures in 27% of myofibers, marked by DHPR, also evidenced in DIC images compared to 0% in control fibers. <i>Ehd1</i>-heterozygous muscle with extensive BIN1 fluorescence extending beyond DHPR staining (yellow arrowhead). Scale 5μm.</p

Reduction in EDL force production in <i>Ehd1-</i>heterozygous muscle.

<p>(A) Representative traces from 8-week male WT and <i>Ehd1-</i>heterozygous (<i>Ehd1+/-)</i> EDL muscles with stimulation pulses marked below the force traces. <i>Ehd1+/-</i> muscle has reduced force. (B) <i>Ehd1+/-</i> EDL muscle has reduced twitch force (n > 5 per genotype, p<0.05). (C) <i>Ehd1+/-</i> EDL muscle has reduced maximum tetanic force (n > 5 per genotype, p = 0.05).</p

Genomic clusters of misexpressed genes.

<p>Shown are genes that are misexpressed in the LMNA mutant heart that are colocalized in the same genomic interval. The chromosome position is shown on the left. The two genes within each interval are indicated in the subsequent columns.</p

Model of chromatin positioning and gene expression.

<p>In the case of the <i>LMNA</i> E161K mutation, two distinct loci on chromosome 13 were displaced to a more intranuclear position (right). We hypothesize that loss of interaction with the lamina (blue) prevents interaction with active chromatin complexes (black) and reduces gene expression.</p