Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo

ICAR: endoscopic skull‐base surgery

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Investigation of Selective Vulnerability of Motor Neurones in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is a neurodegenerative disease characterised by the loss of motor neurones in the anterior horn of the spinal cord with concomitant muscle weakness. However, a growing body of evidence shows that different motor pools in the anterior horn have a very different vulnerability to SMA. For example, previous studies on mouse have suggested motor neurones innervating three different muscles in the lower leg, tibialis anterior (TA), extensor digitorium longus (EDL) and gastrocnemius (GS) show different levels of axonal degeneration upon Smn loss. It indicated that there were undiscovered factors endeavouring motor neurones with distinct disease resistance. As such, we attempted to discover these disease-modifying factors mainly by investigating the transcriptomes of different motor neurones with differing vulnerability. Further functionality experiments were carried out on both in vivo and in vitro systems to validate the microarray result. Furthermore, various new techniques were also tested to advance research in the future. In general, we identified that higher bioenergetics could be the potential disease modulator, which was further illustrated by the manipulation of this pathway in zebrafish. However, bioenergetics status of the motor neurone was independent of SMN levels and likely to be a result of complex interaction of motor neurone and the surrounding environment in which astrocyte might play an important role. Moreover, the use of DEPArray (dielectrophoresis array) might offer an opportunity to explore this subject ex vivo. Lastly, because axon terminal tends to degenerate prior to the cell body, we extended the concept of selective vulnerability to motor neurone itself and attempted to specifically profile axonal transcriptome using RNAseq combined with the microfluidic device

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

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

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

Primer sequences used in quantitative PCR.

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