ANT1 overexpression models: Some similarities with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by progressive muscle weakness. Adenine nucleotide translocator 1 (ANT1), the only 4q35 gene involved in mitochondrial function, is strongly expressed in FSHD skeletal muscle biopsies. However, its role in FSHD is unclear. In this study, we evaluated ANT1 overexpression effects in primary myoblasts from healthy controls and during Xenopus laevis organogenesis. We also compared ANT1 overexpression effects with the phenotype of FSHD muscle cells and biopsies. Here, we report that the ANT1 overexpression-induced phenotype presents some similarities with FSHD muscle cells and biopsies. ANT1-overexpressing muscle cells showed disorganized morphology, altered cytoskeletal arrangement, enhanced mitochondrial respiration/glycolysis, ROS production, oxidative stress, mitochondrial fragmentation and ultrastructure alteration, as observed in FSHD muscle cells. ANT1 overexpression in Xenopus laevis embryos affected skeletal muscle development, impaired skeletal muscle, altered mitochondrial ultrastructure and led to oxidative stress as observed in FSHD muscle biopsies. Moreover, ANT1 overexpression in X. laevis embryos affected heart structure and mitochondrial ultrastructure leading to cardiac arrhythmia, as described in some patients with FSHD. Overall our data suggest that ANT1 could contribute to mitochondria dysfunction and oxidative stress in FSHD muscle cells by modifying their bioenergetic profile associated with ROS production. Such interplay between energy metabolism and ROS production in FSHD will be of significant interest for future prospects.

Treatments for spinal cord injury: is there hope in neurosteroids?

In this review, we describe the current therapeutic strategies to find a cure for paralysis. We use the example of DHEA, a neurosteroid normally produced in the developing neural tube, to raise the hypothesis that such a class of molecules, capable of modulating proliferation of committed neural precursors, could serve as an environmental cue in the adult injured spinal cord to promote re-population of CNS lesion with endogenous dormant precursor cells. Such mechanism may be a part of the natural response to heal the injured CNS and promote recovery of function, suggesting that neurosteroid-treatment could be a promising and novel therapeutic avenue for SCI. We will review pertinent biological activities of DHEA supporting this hypothesis, demonstrate that such activities, dependent on an intact sonic-hedgehog pathway, are responsible for the motor and bladder functional recovery observed after DHEA-treatment in the adult injured spinal cord. We will also raise the current limitations to further development of DHEA- or other neurosteroid-treatments as drug candidates, including the urgent need to further document DHEA long-term safety in CNS indications.

Myelomeningocele: characterization of a surgically induced sheep model and its central nervous system similarities and differences to the human disease.

OBJECTIVE: The purpose of this study was to determine how closely the surgically induced sheep myelomeningocele (MMC) model resembles the central nervous system derangements seen in human disease, and identify which aspects of MMC are the result of the early neuronal developmental defect, and which are secondary to the cerebrospinal fluid (CSF) drainage. STUDY DESIGN: An MMC-like lesion was created surgically in 16 fetal sheep at 75 days' gestation: 5 died in utero, 7 underwent no fetal repair, 4 were repaired (2-layer closure or biological glue) at 100 days' gestation. MMC sheep were delivered at term and allowed to survive up to 17 days for analysis of behavioral status and feeding behavior. Animals not repaired in utero were repaired at birth. All lambs were sacrificed and analyzed for hindbrain herniation, hydrocephalus, and other CNS derangements. RESULTS: Hindbrain herniation was observed in 43% of animals not repaired in utero, and in 1 lamb repaired with Bioglue. No animal developed hydrocephalus or other CNS derangements. CONCLUSION: Although this sheep MMC-like model reproduces the CSF leak, but not the developmental defect seen in humans, it suggests CSF leak contributes to hindbrain herniation seen in humans. This model may be useful to develop new minimally invasive techniques to halt CSF leak in utero.

Transcriptional regulation of P450scc gene expression in the embryonic rodent nervous system.

Steroid hormones are synthesized in adrenals, gonads, placenta, and the central and peripheral nervous systems (neurosteroids). Neurosteroidogenesis, like conventional steroidogenesis, begins with the conversion of cholesterol to pregnenolone, catalyzed by mitochondrial P450 side-chain cleavage enzyme (P450scc). Transcription of the P450scc gene in the adrenals and gonads requires steroidogenic factor-1, which is not expressed in the nervous system cells that express P450scc. A crucial transcriptional regulatory region of the rat P450scc gene is at -130/-94. We have purified two nuclear proteins (70 and 86 kDa) from rat glial C6 cells that specifically bind to the -130/-94 region of the rat P450scc promoter and identified them as the DNA-binding subunits of autoimmune antigen Ku. Ku colocalized with P450scc in several regions of the nervous system, but its overexpression in C6 cells did not augment transcription from a -130/-94 Luciferase construct. Members of the Sp family of transcription factors also bind to the same DNA sequence as Ku. Sp4 and Sp2 colocalize with P450scc in the nervous system early in development, whereas Sp1 and Sp4 colocalize later in development. Sp1 robustly increased transcription from this element in Sp-deficient Drosophila SL2 cells, and Ku synergistically enhanced this Sp1-stimulated transcription. Thus, members of the Sp transcription family play a role in activating P450scc gene transcription in the nervous system, and Ku may further augment this activation.

Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1alpha-hydroxylase gene expression in the proximal renal tubule.

Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1alpha-hydroxylase activity and the renal expression of P450c1alpha and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1alpha-hydroxylase activity and renal P450c1alpha mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1alpha-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman's capsule, thick ascending limb of Henle's loop, distal tubule, and collecting duct. In mice fed a phosphorus-restricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman's capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1alpha gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1alpha mRNA and protein. The increase in P450c1alpha gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1alpha gene.

Bladder function after incomplete spinal cord injury in mice: quantifiable outcomes associated with bladder function and efficiency of dehydroepiandrosterone as a therapeutic adjunct.

OBJECT: The authors conducted a study to establish outcomes associated with bladder function in a mouse model of spinal cord injury (SCI) and to assess the sensitivity of these outcomes in determining the efficacy of pharmacological treatments. METHODS: A mouse model of moderate contusive SCI was used. Outcome parameters included physiological, behavioral, and morphological measurements. To test the sensitivity of these outcomes, the authors used a dehydroepiandrosterone (DHEA) treatment that they had previously shown to promote neurological recovery effectively after SCI. A behavioral scale was used to identify the day at which autonomic function of the bladder was recovered. The reduction in the daily volume of urine during the period of functional recovery paralleled this scale. They then determined the day postinjury at which the functional differences between the vehicle- and DHEA-treated mice exhibited the maximal amplitude. Changes were measured in the composition of the extracellular matrix relative to collagen expression in the layer muscularis of the detrusor at this time point. They found that SCI increases the ratio of collagen type III to collagen type I in the detrusor. Moreover, in the DHEA-treated group, this ratio was similar to that demonstrated in sham-operated mice, establishing the sensitivity of this outcome to assess therapeutic benefits to the bladder function. They next examined the relationship between measurements of neurological recovery and controlled voiding by using cluster analysis. CONCLUSIONS: The authors found that early recovery of controlled voiding is predictive of motor recovery.

Dehydroepiandrosterone biosynthesis, role, and mechanism of action in the developing neural tube.

Dehydroepiandrosterone (DHEA) is synthesized from cholesterol by activity of P450scc and P450c17, enzymes that we previously characterized in the developing nervous system. We describe the localization of P450c17 in the differentiated field of the ventral spinal cord in different motor neuron subtypes. We show that, during organogenesis, P450c17 activity is regulated along the antero/posterior axis of the spinal cord concomitantly with the gradient of neurogenesis. To examine whether DHEA may modulate this process, we measured proliferation and differentiation of ventral neural precursors in primary and explant cultures. Our results showed that DHEA-induced the expression of class II protein Nkx6.1, motor neuron precursor Olig-2, and definitive motor neuron marker Isl-1/2. DHEA also promoted proliferation of ventrally committed precursors in isolated spinal cord precursor cultures and in whole spinal cord explants. Both the proliferative and inductive effects of DHEA were dependent on sonic hedgehog signaling. The possibilities that the effects observed with DHEA were due to its metabolism into androgens or to activation of NMDA receptors were excluded. These results support the hypothesis that the tight regulation of DHEA biosynthesis may be a biologic clock restricting the period of ventral neuronal-precursor proliferation, thus controlling the number of pre-committed neurons in the developing neural tube.

Suitability of allogeneic sertoli cells for ex vivo gene delivery in the injured spinal cord.

Cell-based gene delivery for gene therapy offers the advantages of long-term stable expression of proteins without the safety concerns associated with viral vectors. However, issues of immune rejection prevent the widespread use of allogeneic cell implants. In this study, we determine if Sertoli cells, known for their immune privileged status, are suitable vehicles for allogeneic cell-based gene delivery into the injured spinal cord. As proof of concept, Sertoli cells were modified with recombinant adenovirus expressing enhanced green fluorescent protein (eGFP) or a human trophic factor, neurotrophin-3 (hNT-3), and eGFP. Genetically modified Sertoli cells retained their immunosuppressive ability in vitro, based upon lymphocyte proliferation assays, and were capable of generating biologically relevant levels of NT-3. Similarly, modified, allogeneic cells, implanted into the acutely injured spinal cord, reduced the early inflammatory response while producing significant levels of hNT-3 for at least 3 days after grafting. Moreover, these cells survived for at least 42 days after implantation in the injured cord. Together, these results demonstrate that Sertoli cells function in immunomodulation, can be engineered to produce bioactive molecules, and show long-term survival after implantation into the hostile environment of the acutely injured spinal cord. Such long-term survival represents an important first step toward developing an optimal cell-based delivery system that generates sustained expression of a therapeutic molecule.