Additional file 1 of Motoneurons innervation determines the distinct gene expressions in multinucleated myofibers

Additional file 1: Figure S1. Difference of nucleus between SR and NSR at NMJs. A. Whole-mount staining of diaphragm muscles from 2-month-old C57BL/6J mice. Note that several nuclei are enriched at R-BTX-labeled synaptic sites (indicated with dotted lines). Up, diaphragm muscles. Down, a single muscle fiber. B. Quantification of number of nucleus in the synaptic region (SR) or the non-synaptic region (NSR) in A. n =16 myofibers. Results were from three mice. C. Representative images of isolated SR and NSR from soleus muscles. Red, R-BTX. D. Immunoblot showing the expression of Tom20 and H3K4me2 in SR and NSR of diaphragm muscles. E. Immunoblot showing the expression of Tom20 and Kdm1a in SR and NSR of diaphragm muscles during development. F. ATP levels in SR and NSR of diaphragm muscles. Unless otherwise specified, three independent experiments were performed; the mean ± SEM is shown. t-test in B, and E, *p < 0.05 and ***p < 0.001

Primer sequences used in quantitative PCR.

<p>Primer sequences used in quantitative PCR.</p

Microarray analysis of differentially vulnerable motor neuron pools reveals fundamental differences in their basal molecular composition.

<p>(A) Schematic illustration of experimental design (TA, tibialis anterior; EDL, extensor digitorum longus; GS, gastrocnemius; Vul, vulnerable MNs, Res, resistant MNs; Int, intermediate phenotype MNs). (B) Volcano plots of differentially expressed transcripts in resistant compared to vulnerable MN pools, intermediate compared to vulnerable MN pools, and resistant compared to intermediate MN pools. (C) Ratio trending analysis: transcripts that were significantly changed (p<0.05) between resistant (EDL) and vulnerable (TA) groups with a differential trending value in the intermediate (GS) group were first identified after which the data set underwent enrichment analysis to reveal enriched biological pathways. Graph shows an example of genes in one enriched biological pathway (mitochondrial electron transport chain genes). Note that transcripts showed highest expression levels in resistant (EDL) neurons, with a decreasing level of expression as the vulnerability status of the groups increased (GS through to TA). (D) qPCR validation for 3 distinct mitochondrial genes confirming up-regulation in disease-resistant MN pools (N = 3), Unpaired two tailed student <i>t-test</i> (* P<0.05). (E) Bar chart (mean & s.e.m.) showing a reduction in ATP in the spinal cord of early and late-symptomatic SMA mice compared to littermate controls using an ATP assay (N = 3 spinal cords per genotype).</p

Pgk1 expression is pathologically relevant in mouse and zebrafish models of SMA.

<p>(A) Expression of PGK1 protein in the spinal cord, skeletal muscle, sciatic nerve and heart of late-symptomatic P8 SMA mice. Protein levels were quantified and normalized to an appropriate loading control. (B) Bar chart (mean & s.e.m.) showing a significant reduction in PGK1 protein levels in SMA mouse spinal cord and sciatic nerve. N = 6 SPC per genotype. N = 3 muscle per genotype. N = 7 sciatic nerves per genotype. N = 3 hearts per genotype (C) Knockdown of Pgk1 in zebrafish induced an axonal outgrowth phenotype (middle panel arrow) similar to smn knockdown (arrow bottom panel) and also produced swellings in the tips of outgrowing axons indicative of axonal transport deficiencies. Scale bars = 50 μM (D) Quantification of axonal outgrowths showed a significant increase in truncated motor axons in pgk1 and smn morphants compared to controls. (E) Efficiency of <i>pgk1</i> knockdown in embryos was shown by western blot embryos normalized to an appropriate loading control (N = 3 per group, batches of 30 pooled zebrafish embryos per lane). N = 20 embryos per group. Unpaired two-tailed students <i>t-test</i> * p<0.05, ** p<0.01 *** p<0.001 **** p<0.0001.</p

Enrichment analysis of microarray data reveals biological pathways changed between disease-resistant and vulnerable motor neuron pools.

<p>Enrichment analysis of microarray data reveals biological pathways changed between disease-resistant and vulnerable motor neuron pools.</p

Overexpression of necdin ameliorates the motor axon outgrowth phenotype in <i>smn</i> morphant zebrafish.

<p>(A) Western blotting of cytochrome C, an electron transport chain protein showed an increase in NDN overexpressing embryos suggesting an increase in mitochondrial biogenesis. (B) Cyt C protein levels were quantified relative to a loading control. (C) Representative confocal micrographs of motor neuron axons exiting the spinal cord in control (top), <i>smn</i> morphant (middle) and <i>smn</i> morphant over-expressing Ndn (bottom) Tg(<i>hb9</i>:GFP) zebrafish embryos. Note the presence of the axonal outgrowth phenotype associated with <i>smn</i> knockdown (arrow heads) is reduced in the Ndn expressing animals. Scale bars = 50 μM. (D) Bar chart (mean & s.e.m.) showing a significant increase in the number of normal MNs, and a concomitant significant decrease in the number of severely affected MNs, in co-injected <i>smn</i> MO and Ndn mRNA embryos compared to single <i>smn</i> MO injected embryos at 30 hpf. Unpaired two-tailed student <i>t-tests</i>; * p<0.05, ** p<0.01 *** p<0.001. N = 20 embryos per experimental group.</p

Mitochondrial dysfunction occurs in smn morphant zebrafish.

<p>(A) Levels of ATP5A protein, a subunit of mitochondrial membrane ATP synthase, were significantly reduced in <i>smn</i> morphant zebrafish. (B) Levels were quantified using fluorescent Western blotting and normalized to COXIV loading control (N = 3 per group, batches of 30 pooled zebrafish embryos per lane). (C) Mitochondrial oxygen consumption rates (OCR) of control and <i>smn</i> morphant 24 hpf zebrafish analyzed using the Seahorse XF24 analyser showed mitochondrial bioenergetic defects (D) Basal respiration was significantly reduced in <i>smn</i> morphants compared to controls. (E) ATP linked respiration was significantly reduced in <i>smn</i> morphants compared to controls. (F) Mitochondrial proton leak was also reduced in <i>smn</i> morphants compared to controls. N = 14 per group) Unpaired two-tailed student <i>t-test</i> * P<0.05, ** p<0.01 *** p<0.001.</p

Overexpression or pharmacological activation of pgk1 rescues motor neuron phenotypes in smn morphant zebrafish.

<p>(A) Representative confocal micrographs of primary motor neuron axons exiting the spinal cord in control (top), <i>smn</i> morphant (middle) and <i>smn</i> morphant over-expressing <i>pgk1</i> (bottom) Tg(<i>hb9</i>:GFP) zebrafish embryos. Note the presence of an axonal outgrowth/branching phenotype associated with <i>smn</i> knockdown (arrow heads) that is reduced in the <i>pgk1</i> over-expressing animals. Scale bars = 50 μM. (B) Overexpression of Pgk1 in <i>smn</i> morphant zebrafish at 30 hpf led to a significant increase in normal motor axons and significant decrease in severe axonal outgrowth phenotypes compared to single <i>smn</i> MO injected embryos. (C) Representative confocal micrographs of motor neuron axons exiting the spinal cord in control (top), <i>smn</i> morphant (middle) and <i>smn</i> morphant treated with 2.5 μM terazosin (bottom) Tg(<i>hb9</i>:GFP) zebrafish embryos. Note how the presence of the axonal outgrowth/branching phenotype associated with <i>smn</i> knockdown (arrow heads) is reduced in the terazosin-treated animals. (D) Bar chart (mean & s.e.m) showing activation of Pgk1 by treatment with 2.5 μM terazosin in <i>smn</i> morphant zebrafish at 30 hpf led to a significant increase in normal motor axons and significant decrease in severe axonal outgrowth phenotypes compared to untreated <i>smn</i> MO injected embryos. Unpaired two-tailed student <i>t-tests</i> * p<0.05, ** p<0.01 *** p<0.001. n = 20 embryos per group.</p

PGK1 is enriched in disease-resistant motor neuron pools, expressed in neuronal cells cellular and axonal compartments <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Gene expression profile graph showing transcripts trending across differentially vulnerable motor neuron pools, with PGK1 highlighted. Note that <i>Pgk1</i> was 5-fold higher expressed in the EDL disease-resistant motor neuron pool compared to the TA vulnerable motor neuron pool, with expression levels trending through the GS intermediate pool. (B) Representative confocal micrographs showing expression of PGK1 in the cytoplasm of motor neurons (MN) in mouse spinal cord. Scale bars = 20 μM. (C) Expression of PGK1 was also detected in the majority of axons in the sciatic nerve (SN), being localised alongside neurofilament (H-NF; upper panels) but not co-localising with glial S100 label (lower panels). Scale bars = 5 μM (D) <i>In vitro</i> analysis showed expression of PGK1 in the cell body and axonal processes of mouse cortical neurons (CtxN). Scale bars = 30 μM. (E) Expression of pgk1 was detected in the axonal nerve terminals of mouse cortical neurons (CtxN). Scale bars = 15 μM. (F) Expression of PGK1 was also found in mouse primary motor neuron (MN) cell bodies and axonal compartments (arrow). Scale bars = 30 μM. (G) Expression of Pgk1 in the axonal terminals/growth cones (arrow) of mouse primary motor neurons (MN). Scale bars = 15 μM.</p

Summary model showing ATP-generating pathways likely to influence the vulnerability status of MNs in SMA.

<p>Efficient ATP generation through different processes maintains levels in energy demanding MNs, critical for their function and survival during disease. Certain motor neuron pools possess inherently higher bioenergetic capacities that provide protection during cellular insult both at the cell body and at the NMJ. Efficient ATP generated from mitochondria allows cellular homeostasis to be maintained. During hypoxia, glycolytic pathways are employed to meet acute ATP demand, particularly at the NMJ, critical for vesicular recycling and synaptic transmission. Mitochondrial transport along the axon provides local ATP to maintain axonal integrity. Glycolytic machinery present along the axon allows for fast vesicular transport down to the NMJ to deliver packaged proteins for pre-synaptic function.</p