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Introduction. In vitro data demonstrate that estrogens improve differentiation of myoblasts deriving from FSHD patients, counteracting muscle differentiation impairment caused by the homeobox protein DUX4. Objectives. We aim to assess in vivo estrogen activity on regenerative potential of muscle precursor cells (perivascular cells, PVCs) derived from healthy individuals and engineered to express DUX4, or derived from FSHD patients. Methods. Cherry-expressing PVCs were implanted into injured hindlimb-tibial muscle of NSG female mice treated with 17?-estradiol (E2) or fulvestrant. Animals were monitored by fluorescence emission and by functional treadmill test, and molecularly by IHC, gene and protein expression. Results: Human PVCs are able to participate to muscle regeneration of injured muscle. Preliminary data show that DUX4 reduces the performance of implanted PVCs whereas 17?-estradiol is able to recover it. Conclusions: These data indicate the usefulness of PVCs to study in vivo muscle differentiation of FSHD and suggest the potential protective function of estrogen.

Up-regulation of the dystrophin-related gene utrophin represents a promising therapeutic strategy for the treatment of Duchenne muscular dystrophy (DMD). The strategy is based on the capacity of utrophin to functionally replace defective dystrophin. In order to re-program the utrophin expression level in muscle, we engineered artificial zinc finger transcription factors (ZFATFs) on Zif268/Egr1 backbone. Using selective amino acid substitutions, the natural Zif268 transcription factor was specifically re-directed to the human and mouse conserved regions of the utrophin promoter ''A''. Furthermore, these minimally modified ZF-ATFs were designed to minimize any host immune-response against these proteins. Our present focus is the delivery of ZFATFs using recombinant Adeno-associated viral vectors engineered to express the therapeutic genes under the control of the human alpha-actin muscle specific promoter (mAAV-vector), combined with the use of the muscle-targeting AAV-serotype 8. Upon intraperitoneal injection of mAAV8- ZF-ATFs into dystrophin-deficient (mdx) mice, a significant amelioration of the pathological phenotype was observed. Histological and physiological analysis revealed a reduction of fiber necrosis and cell infiltration, as well as the functional recovery in muscle contractile force. Since utrophin is also a key determinant of the neuromuscular junction (NMJ), ongoing studies will characterize the NMJ in mAAV8-ZF-ATF treated mice. In summary, the development of ZF-ATF-re-directed technology, coupled with the mAAV delivery, highlights the potential of this novel therapeutic strategy for treatment of DMD.

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445–461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary beta1b, beta2e, beta3a, and beta4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463-474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four beta subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the beta subtype. The type of beta subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; beta3a promotes the fast mode, whereas beta4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a beta subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.

Processing of Abeta-precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the (682) YENPTY(687) motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Y(682) to Gly (APP(YG/YG) mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP-like protein 2 KO background. This evidence prompted us to characterize further the APP(YG/YG) mice. Here, we show that APP(YG/YG) mice develop aging-dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP(YG/YG) mice. The profound deficits caused by the Y(682) mutation underscore the biological importance of APP and indicate that APP(YG/YG) are a valuable mouse model to study APP functions in physiological and pathological processes.