Fisiopatología mitocondrial en la neuropatía de Charcot-Marie-Tooth asociada al gen GDAP1

Las mutaciones en el gen GDAP1, que codifica una proteína localizada en la membrana mitocondrial externa, causan la neuropatía hereditaria de Charcot-Marie-Tooth (CMT), caracterizada por una degeneración progresiva de los nervios periféricos motores y sensitivos. Hoy en día, todavía se desconocen los mecanismos celulares que desencadenan la disfunción neuronal en la enfermedad de CMT relacionada con la deficiencia de GDAP1. En este trabajo, hemos utilizado los cultivos primarios de motoneuronas de la médula espinal de ratones deficientes en GDAP1, para investigar el papel de esta proteína en la biología neuronal y dilucidar los mecanismos implicados en la fisiopatología de la enfermedad. Nuestros resultados muestran que la deficiencia de GDAP1 produce una aberrante morfología mitocondrial, que conlleva a un deterioro en la función bioenergética de las mitocondrias, una alteración en la homeostasis del calcio y un aumento del estrés oxidativo a nivel celular. Las motoneuronas con déficit de GDAP1 también presentan alteraciones en el transporte mitocondrial axonal, lo que puede dificultar la correcta localización de estos orgánulos en los lugares de alta demanda energética, como las sinapsis. En este contexto, hemos demostrado que la pérdida de función de GDAP1 afecta al tamponamiento del calcio por parte de la mitocondria durante la estimulación fisiológica neuronal, ocasionando alteraciones en la señalización del calcio y en el metabolismo energético durante la transmisión sináptica. Además, la deficiencia de GDAP1 disminuye la interacción física entre las mitocondrias y el retículo endoplásmico (RE), produce estrés de RE y afecta al flujo autofágico. Es importante destacar que, todas estas alteraciones celulares se asocian con una disminución en la viabilidad de las motoneuronas. Finalmente, observamos una respuesta inflamatoria crónica en los tejidos diana de la enfermedad en los ratones deficientes en GDAP1, la cual contribuye en la neurodegeneración asociada a la enfermedad de CMT. En conclusión, nuestros resultados demuestran que la pérdida de GDAP1 compromete la función mitocondrial, lo que afecta negativamente a la fisiología y supervivencia neuronal, y ofrecen una visión importante de los mecanismos celulares asociados a la degeneración axonal subyacente a la neuropatía de CMT relacionada con GDAP1

Mitochondria and calcium defects correlate with axonal dysfunction in GDAP1-related Charcot-Marie-Tooth mouse model

Ganglioside-induced differentiation associated protein 1 (GDAP1) gene encodes a protein of the mitochondrial outer membrane and of the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs) and lysosomes. Since mutations in GDAP1 cause Charcot–Marie–Tooth, an inherited motor and sensory neuropathy, its function is essential for peripheral nerve physiology. Our previous studies showed structural and functional defects in mitochondria and their contacts when GDAP1 is depleted. Nevertheless, the underlying axonal pathophysiological events remain unclear. Here, we have used embryonic motor neurons (eMNs) cultures from Gdap1 knockout (Gdap1-/-) mice to investigate in vivo mitochondria and calcium homeostasis in the axons. We imaged mitochondrial axonal transport and we found a defective pattern in the Gdap1-/- eMNs. We also detected pathological and functional mitochondria membrane abnormalities with a drop in ATP production and a deteriorated bioenergetic status. Another consequence of the loss of GDAP1 in the soma and axons of eMNs was the in vivo increase calcium levels in both basal conditions and during recovery after neuronal stimulation with glutamate. Further, we found that glutamate-stimulation of respiration was lower in Gdap1-/- eMNs showing that the basal bioenergetics failure jeopardizes a full respiratory response and prevents a rapid return of calcium to basal levels. Together, our results demonstrate that the loss of GDAP1 critically compromises the morphology and function of mitochondria and its relationship with calcium homeostasis in the soma and axons, offering important insight into the cellular mechanisms associated with axonal degeneration of GDAP1-related CMT neuropathies and the relevance that axon length may have.Peer ReviewedPostprint (published version

Mitochondria and calcium defects correlate with axonal dysfunction in GDAP1-related Charcot-Marie-Tooth mouse model

Ganglioside-induced differentiation associated protein 1 (GDAP1) gene encodes a protein of the mitochondrial outer membrane and of the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs) and lysosomes. Since mutations in GDAP1 cause Charcot–Marie–Tooth, an inherited motor and sensory neuropathy, its function is essential for peripheral nerve physiology. Our previous studies showed structural and functional defects in mitochondria and their contacts when GDAP1 is depleted. Nevertheless, the underlying axonal pathophysiological events remain unclear. Here, we have used embryonic motor neurons (eMNs) cultures from Gdap1 knockout (Gdap1) mice to investigate in vivo mitochondria and calcium homeostasis in the axons. We imaged mitochondrial axonal transport and we found a defective pattern in the Gdap1 eMNs. We also detected pathological and functional mitochondria membrane abnormalities with a drop in ATP production and a deteriorated bioenergetic status. Another consequence of the loss of GDAP1 in the soma and axons of eMNs was the in vivo increase calcium levels in both basal conditions and during recovery after neuronal stimulation with glutamate. Further, we found that glutamate-stimulation of respiration was lower in Gdap1 eMNs showing that the basal bioenergetics failure jeopardizes a full respiratory response and prevents a rapid return of calcium to basal levels. Together, our results demonstrate that the loss of GDAP1 critically compromises the morphology and function of mitochondria and its relationship with calcium homeostasis in the soma and axons, offering important insight into the cellular mechanisms associated with axonal degeneration of GDAP1-related CMT neuropathies and the relevance that axon length may have.Spanish Ministry of Science, Innovation and Universities (grants no. SAF2015–66625-R, [F.P.], no. SAF2017-82560-R [J.S.] and no. SAF2017-88019-C3-3-R [R.B.]), and J.S.]), Instituto de Salud Carlos III (ISCIII, grant no. IR11/TREAT-CMT, [F.P. and J.S.]), the Generalitat de Catalunya & European Regional Development Found (grants no. 2015 FEDER/S-21 and 2017/SRG1308, [F. P.]), and an institutional grant to the CBMSO from the Fundación Ramón Areces

Mitochondria–lysosome membrane contacts are defective in GDAP1-related Charcot–Marie–Tooth disease

Mutations in the GDAP1 gene cause Charcot–Marie–Tooth (CMT) neuropathy. GDAP1 is an atypical glutathione S-transferase (GST) of the outer mitochondrial membrane and the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs). Here, we investigate the role of this GST in the autophagic flux and the membrane contact sites (MCSs) between mitochondria and lysosomes in the cellular pathophysiology of GDAP1 deficiency. We demonstrate that GDAP1 participates in basal autophagy and that its depletion affects LC3 and PI3P biology in autophagosome biogenesis and membrane trafficking from MAMs. GDAP1 also contributes to the maturation of lysosome by interacting with PYKfyve kinase, a pH-dependent master lysosomal regulator. GDAP1 deficiency causes giant lysosomes with hydrolytic activity, a delay in the autophagic lysosome reformation, and TFEB activation. Notably, we found that GDAP1 interacts with LAMP-1, which supports that GDAP1–LAMP-1 is a new tethering pair of mitochondria and lysosome membrane contacts. We observed mitochondria–lysosome MCSs in soma and axons of cultured mouse embryonic motor neurons and human neuroblastoma cells. GDAP1 deficiency reduces the MCSs between these organelles, causes mitochondrial network abnormalities, and decreases levels of cellular glutathione (GSH). The supply of GSH-MEE suffices to rescue the lysosome membranes and the defects of the mitochondrial network, but not the interorganelle MCSs nor early autophagic events. Overall, we show that GDAP1 enables the proper function of mitochondrial MCSs in both degradative and nondegradative pathways, which could explain primary insults in GDAP1-related CMT pathophysiology, and highlights new redox-sensitive targets in axonopathies where mitochondria and lysosomes are involved.Peer ReviewedPostprint (author's final draft

Mitochondrial quantitation and network distribution in cultured MNs and sciatic nerves from WT and <i>Gdap1</i>^<i>-/-</i> mice.

<p>Number of mitochondria <b>(A)</b> and network interconnectivity <b>(B)</b> in cultured MNs are represented. The study was performed in the proximal segments (p) and distal segments (d) of WT (black bars) and <i>Gdap1</i>^<i>-/-</i> (gray bars) axons after 24 hour and 48 hour of cell culture. Error bars represent S.E.M. Student’s <i>t</i> test *p<0.05, **p<0.01 and ***p<0.001 <b>(C)</b> Left panel shows semi-thin cross sections of the sciatic nerve from five months old WT and <i>Gdap1</i>^-/- mice. Mitochondria are clearly visible on higher magnification images of transversal section (right panel). Mitochondrial axonal content was quantified by electron microscopy on proximal and distal cross sections of the sciatic nerve. (n = 4; Error bars represent S.E.M.; asterisks indicate significant differences between WT and <i>Gdap1</i>^<i>-/-</i> mice, Mann-Whitney test, **p<0.01,***p<0.001). <b>(D)</b> Measurement of mitochondrial DNA (mtDNA) copy number in sciatic nerves.</p

Postranscriptional modification of the tubulin cytoskeleton in primary sensory and motor neuron cultures.

<p><b>(A)</b> DRG sensory neurons and <b>(B)</b> embryonic MNs were double-stained for acetylated α-tubulin (acetylated α-tub, green) and β-III tubulin (β-III tub, red). As indicated by respective histograms there is a significant reduction of acetylated α-tubulin in both MN and sensory neurites in <i>Gdap1</i>^<i>-/-</i> mice. Graph represents means and S.E.M of 3 independent culture preparation per genotype. Student’s <i>t</i> test ***p<0.001.</p

Behavioural testing and electrophysiological measurements on <i>Gdap1</i>^<i>-/-</i> mice.

<p><b>(A)</b> Upper panel shows photographs of 3 months-old mice suspended by its tail. WT mice show a characteristic response trying to escape by splaying its hind limbs away from the trunk of its body. In contrast, hind limbs of <i>Gdap1</i>^-/- mice are held tonically against its trunk in an abnormal dystonic posture. Lower panels display a low body position and a dragging tail present in <i>Gdap1</i>^<i>-/-</i> mice as compared to age-matched WT mice. <b>(B)</b> Motor coordination was assessed by rotarod test, (n = 10 for each genotype and at each age group). <b>(C)</b> Representative hind limb walking patterns of 5 months-old WT and <i>Gdap1</i>^-/- mice where the stride length (SL) and stride angle (SA) have been depicted. Footprints revealed that <i>Gdap1</i>^<i>-/-</i> mice walk with an abnormal gait. The scheme of a hindpaw footprint indicating measured parameters (PL: plantar length; TS: toe spreading) has been included. <b>(D)</b> Quantification of various parameters obtained from the gait analysis of WT (black columns) and <i>Gdap1</i>^<i>-/-</i> (grey columns) animals at 5 and 12 months of age. Upper graphs show stride length (left) and stride angle (right). Lower graphs show the quantitative analysis of the hindpaw footprint parameters toe spreading (left) and plantar length (right). Analysis was conducted on 10 clearly visible footprints at 5 animals per genotype. Determination of sciatic nerve compound muscle action potential (CMAP) amplitudes at both distal and proximal <b>(E)</b> as well as motor nerve conduction velocities (MNCV) <b>(F)</b> measured in WT and <i>Gdap1</i>^-/- mice at 2 and 5 months of age (n = 4). Error bars indicate standard error of the mean (S.E.M.). <i>p</i> values were calculated using Student's <i>t</i> test,*p<0.05, **p<0.001, ***p<0.0001.</p

SOCE alteration in <i>Gdap1</i>^<i>-/-</i> embryonic motor neurons <b>(A)</b> Fura-2 [Ca²⁺] signals of embryonic MNs from WT (black) and <i>Gdap1</i>^<i>-/-</i> (grey) mice.

<p>After Ca²⁺ release from cell stores with 5 μM thapsigargin (TG) treatment during 7 min in Ca²⁺ free medium, SOCE was activated by adding 2 mM of CaCl₂. Traces were used to obtain <b>(B)</b> maximum Ca²⁺ peak during SOCE and <b>(C)</b> SOCE Ca²⁺ influx (slope). <b>(D)</b> Fura-2 recordings of 5 μM ionomycin elicited [Ca²⁺]_cyt peak in Ca²⁺-free medium. <b>(E)</b> Maximum [Ca²⁺]_cyt peak obtained in Ca²⁺-free medium represents total amount of cytoplasmic Ca²⁺ after cell stores Ca²⁺ release. Traces were obtained averaging at least 100 cells from each genotype. Error bars represent S.E.M. (***p<0.001, Student’s <i>t</i> test).</p

Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.

<p>Mitochondrial shape descriptors were measured in 20 WT and 30 <i>Gdap1</i>^-/- motorneurons. Student’s t test was performed for normal distributed parameters (number of mitochondria, circularity, roundness and aspect ratio) and Mann-Whitney U test for those that were non-normal distributed (surface area, Feret´s diameter and perimeter). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005115#pgen.1005115.g006" target="_blank">Fig 6</a> for a visual representation.</p><p>*p<0.05,</p><p>**p<0.01.</p><p>Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.</p

Generation of <i>Gdap1</i>^<i>-/-</i> mice.

<p><b>(A)</b> Schematic representation of <i>Gdap1</i>^<i>-/-</i> targeting strategy. Diagram is not to scale. Hatched rectangles represent <i>Gdap1</i> exons 1 to 6, solid line represents mouse chromosome 1. FRT sites are represented by double triangles and <i>lox</i>P sites are right-faced triangles. <b>(B)</b> GDAP1 protein expression was assessed by immunoblotting of selected tissue homogenates prepared from 2 months-old wild-type (WT), <i>Gdap1</i>^<i>+/-</i> (+/-) and <i>Gdap1</i>^-/- (-/-) mice.</p