Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy

Peer reviewe

Impairment of Drosophila orthologs of the human orphan protein C19orf12 induces bang sensitivity and neurodegeneration

Mutations in the orphan gene C19orf12 were identified as a genetic cause in a subgroup of patients with NBIA, a neurodegenerative disorder characterized by deposits of iron in the basal ganglia. C19orf12 was shown to be localized in mitochondria, however, nothing is known about its activity and no functional link exists to the clinical phenotype of the patients. This situation led us to investigate the effects of C19orf12 down-regulation in the model organism Drosophila melanogaster. Two genes are present in D. melanogaster, which are orthologs of C19orf12, CG3740 and CG11671. Here we provide evidence that transgenic flies with impaired C19orf12 homologs reflect the neurodegenerative phenotype and represent a valid tool to further analyze the pathomechanism in C19orf12-associated NBIA

Loss of melusin is a novel, nNOS/FoxO3-independent master switch of unloading-induced muscle atrophy.

Background: Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Since interventions against known atrophy initiators, such as oxidative stress and nNOS redistribution, are only partially effective, we investigated the involvement of melusin, a muscle-specific integrin-associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes. Methods: Muscle atrophy was induced in the rat soleus by tail-suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail-suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size and atrogene expression after AAV infection. In vivo interference of exogenous melusin with dominant-negative kinases and other atrophy attenuators (Grp94 cDNA; 7-nitroindazole) on size of unloaded rat myofibers was also explored. Results: Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6h in the tail-suspended rat (P<0.001), and to about 35% after 8-day bed rest in humans (P<0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of \uf0621D-integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7-day unloaded rat myofibers (-31%,) compared to controls (-48%, P=0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin-expressing AAV ameliorated contractile properties of 7-day unloaded muscles (P<0.05) and relieved myofiber atrophy (- 33%) by reducing Atrogin-1 and MurF-1 transcripts (P<0.002), despite of a two-fold increase in FoxO3 protein levels (P=0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK or FAK kinase activity, since it persisted after co-transfection with dominant-negative kinase forms (P<0.01). Conversely, melusin cDNA transfection, combined with 7-nitroindazole treatment or with cDNA transfection of the nNOS-interacting chaperone Grp94, abolished 7-day-unloaded myofiber atrophy. Conclusions: Disuse/unloading-induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy

Expression of <i>CG3740</i> and <i>CG11671</i> in heads from 10 days-old flies.

<p>The <i>Ribosomal Protein 49</i> has been used as endogenous control to normalize <i>CG3740</i> and <i>CG11671</i> expression level. (<b>A</b>) <i>CG3740</i> and <i>CG11671</i> have been measured in single RNAi flies (<i>CG3740</i> RNAi and <i>CG11671</i> RNAi) and in double RNAi flies (<i>CG3740</i>; <i>CG11671</i> RNAi) using the same number of males and females (n = 4). The level of <i>CG3740</i> and <i>CG11671</i> in control flies has been set to 1 and the expression of both genes in down-regulated flies expressed relatively. (<b>B</b>) <i>CG3740</i> and <i>CG11671</i> have been measured in single heterozygous deletion flies (<i>Df(1)</i>BSC589/+ and <i>Df(3L)</i>BSC579/+) and in double heterozygous deletion flies (<i>Df(1)</i>BSC589/+; <i>Df(3L)</i>BSC579/+) using female flies (n = 4). The level of <i>CG3740</i> and <i>CG11671</i> in control flies has been set to 1 and the expression of both genes in heterozygous deletion flies expressed relatively. Data refer to the average of three independent experiments ± SD.</p

Alignment of the fly C19orf12 orthologs (CG3740, CG11671) with human C19orf12 protein.

<p>Proteins have been aligned using Clustal 2.1 multiple alignment tool. Stars (*) indicate identities and dots indicate a higher (:) and a lower (.) degree of similarity. The two <i>D. melanogaster</i> proteins are 63% and 55% similar to C19orf12 and share 72% similarity with each other. The transmembrane domains predicted by PolyPhobius are marked in yellow.</p

Prussian blue staining for detection of iron deposits.

<p>The staining was performed on paraffin-embedded sections of 28 days-old flies. No iron deposits were detected in the medulla or retina of either controls or double RNAi flies. Clear iron inclusions were present in paraffin-embedded sections of mouse spleen (positive control).</p

Bang test in young (1 day-old) and aged (1 month-old) control (w¹¹¹⁸ and <i>act</i>-GAL4/+) and double RNAi males.

<p>A) Flies have been vortexed twice (Bang I and II) with 10 minutes in between and the time needed to upright recorded. A) Representative trajectories are reported for aged control (green), double RNAi (red) and for double heterozygous (blue) flies.</p

Expression of <i>CG3740</i> and <i>CG11671</i> in head, thorax and abdomen of <i>D. melanogaster</i>.

<p>The analysis has been performed on total RNA extracted from adult w¹¹¹⁸ flies using the same number of males and females (n = 4). The <i>Ribosomal Protein 49</i> has been used as endogenous control to normalize <i>CG3740</i> and <i>CG11671</i> expression level. The level of <i>CG11671</i> in the abdomen has been set to 1 and the expression of <i>CG3740</i> and <i>CG11671</i> in the other tissues expressed relatively.</p

Detection of vacuoles in ultrathin Epon plastic sections.

<p>A) Horizontal head sections from 28 days-old control (w¹¹¹⁸) and down-regulated (<i>CG3740</i>; <i>CG11671</i> RNAi) flies. B) Number and size of vacuoles have been quantified with Image J running the applications “Find Edges” and “Analyze Particles” on thresholded 8-bit pictures. Particle size was imposed bigger than 20 pixels² and with a circularity factor between 0,5 and 1. Detected vacuoles were displayed using “Overlay Mask”. Data were manually validated to exclude artifacts. Brains analyzed for each fly strain: n = 3. Scale bar: 100 µm.</p