Amyloid-beta antibody binding to cerebral amyloid angiopathy fibrils and risk for amyloid-related imaging abnormalities.

Therapeutic antibodies have been developed to target amyloid-beta (Aß), and some of these slow the progression of Alzheimer's disease (AD). However, they can also cause adverse events known as amyloid-related imaging abnormalities with edema (ARIA-E). We investigated therapeutic Aß antibody binding to cerebral amyloid angiopathy (CAA) fibrils isolated from human leptomeningeal tissue to study whether this related to the ARIA-E frequencies previously reported by clinical trials. The binding of Aß antibodies to CAA Aß fibrils was evaluated in vitro using immunoprecipitation, surface plasmon resonance, and direct binding assay. Marked differences in Aß antibody binding to CAA fibrils were observed. Solanezumab and crenezumab showed negligible CAA fibril binding and these antibodies have no reported ARIA-E cases. Lecanemab showed a low binding to CAA fibrils, consistent with its relatively low ARIA-E frequency of 12.6%, while aducanumab, bapineuzumab, and gantenerumab all showed higher binding to CAA fibrils and substantially higher ARIA-E frequencies (25-35%). An ARIA-E frequency of 24% was reported for donanemab, and its binding to CAA fibrils correlated with the amount of pyroglutamate-modified Aß present. The findings of this study support the proposal that Aß antibody-CAA interactions may relate to the ARIA-E frequency observed in patients treated with Aß-based immunotherapies.

A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

The MDM2 Inhibitor Navtemadlin Arrests Mouse Melanoma Growth In Vivo and Potentiates Radiotherapy.

The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of p14ARF. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, WT p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein-protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase II clinical trials. Using mass spectrometry-based proteomics and imaging flow cytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared with their p53 CRISPR-inactivated control cells. In vitro, Navtemadlin induced a significant, p53-dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/MDM4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo. Significance: The MDM2 inhibitor Navtemadlin arrests mouse tumor growth and potentiates radiotherapy. Our results support a threshold model for apoptosis induction that requires a high, prolonged p53 signaling for cancer cells to become apoptotic.

TAp73 represses NF-κB-mediated recruitment of tumor-associated macrophages in breast cancer.

Infiltration of tumor-promoting immune cells is a strong driver of tumor progression. Especially the accumulation of macrophages in the tumor microenvironment is known to facilitate tumor growth and to correlate with poor prognosis in many tumor types. TAp73, a member of the p53/p63/p73 family, acts as a tumor suppressor and has been shown to suppress tumor angiogenesis. However, what role TAp73 has in regulating immune cell infiltration is unknown. Here, we report that low levels of TAp73 correlate with an increased NF-κB-regulated inflammatory signature in breast cancer. Furthermore, we show that loss of TAp73 results in NF-κB hyperactivation and secretion of Ccl2, a known NF-κB target and chemoattractant for monocytes and macrophages. Importantly, TAp73-deficient tumors display an increased accumulation of protumoral macrophages that express the mannose receptor (CD206) and scavenger receptor A (CD204) compared to controls. The relevance of TAp73 expression in human breast carcinoma was further accentuated by revealing that TAp73 expression correlates negatively with the accumulation of protumoral CD163+ macrophages in breast cancer patient samples. Taken together, our findings suggest that TAp73 regulates macrophage accumulation and phenotype in breast cancer through inhibition of the NF-κB pathway.

High intracellular stability of the spidroin N-terminal domain in spite of abundant amyloidogenic segments revealed by in-cell hydrogen/deuterium exchange mass spectrometry.

Proteins require an optimal balance of conformational flexibility and stability in their native environment to ensure their biological functions. A striking example is spidroins, spider silk proteins, which are stored at extremely high concentrations in soluble form, yet undergo amyloid-like aggregation during spinning. Here, we elucidate the stability of the highly soluble N-terminal domain (NT) of major ampullate spidroin 1 in the Escherichia coli cytosol as well as in inclusion bodies containing fibrillar aggregates. Surprisingly, we find that NT, despite being largely composed of amyloidogenic sequences, showed no signs of concentration-dependent aggregation. Using a novel intracellular hydrogen/deuterium exchange mass spectrometry (HDX-MS) approach, we reveal that NT adopts a tight fold in the E. coli cytosol and in this manner conceals its aggregation-prone regions by maintaining a tight fold under crowded conditions. Fusion of NT to the unstructured amyloid-forming Aß40 peptide, on the other hand, results in the formation of fibrillar aggregates. However, HDX-MS indicates that the NT domain is only partially incorporated into these aggregates in vivo. We conclude that NT is able to control its aggregation to remain functional under the extreme conditions in the spider silk gland.

Regulation of Neuronal Na,K-ATPase by Extracellular Scaffolding Proteins.

Neuronal activity leads to an influx of Na⁺ that needs to be rapidly cleared. The sodium-potassium ATPase (Na,K-ATPase) exports three Na⁺ ions and imports two K⁺ ions at the expense of one ATP molecule. Na,K-ATPase turnover accounts for the majority of energy used by the brain. To prevent an energy crisis, the energy expense for Na⁺ clearance must provide an optimal effect. Here we report that in rat primary hippocampal neurons, the clearance of Na⁺ ions is more efficient if Na,K-ATPase is laterally mobile in the membrane than if it is clustered. Using fluorescence recovery after photobleaching and single particle tracking analysis, we show that the ubiquitous α1 and the neuron-specific α3 catalytic subunits as well as the supportive ß1 subunit of Na,K-ATPase are highly mobile in the plasma membrane. We show that cross-linking of the ß1 subunit with polyclonal antibodies or exposure to Modulator of Na,K-ATPase (MONaKA), a secreted protein which binds to the extracellular domain of the ß subunit, clusters the α3 subunit in the membrane and restricts its mobility. We demonstrate that clustering, caused by cross-linking or by exposure to MONaKA, reduces the efficiency in restoring intracellular Na⁺. These results demonstrate that extracellular interactions with Na,K-ATPase regulate the Na⁺ extrusion efficiency with consequences for neuronal energy balance.

Membrane-Depolarizing Channel Blockers Induce Selective Glioma Cell Death by Impairing Nutrient Transport and Unfolded Protein/Amino Acid Responses.

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741-52. ©2017 AACR.

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry.

α-Synuclein (α-syn) is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD). This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively) (doi: 10.15252/embj.201591397[1]). α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively). For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: PXD002256 to PRIDE: PXD002263 and doi: 10.6019/PXD002256 to 10.6019/PXD002263.

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient.

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

Calcium signaling in neocortical development.

The calcium ion (Ca(2+) ) is an essential second messenger that plays a pivotal role in neurogenesis. In the ventricular zone (VZ) of the neocortex, neural stem cells linger to produce progenitor cells and subsequently neurons and glial cells, which together build up the entire adult brain. The radial glial cells, with their characteristic radial fibers that stretch from the inner ventricular wall to the outer cortex, are known to be the neural stem cells of the neocortex. Migrating neurons use these radial fibers to climb from the proliferative VZ in the inner part of the brain to the outer layers of the cortex, where differentiation processes continue. To establish the complex structures that constitute the adult cerebral cortex, proliferation, migration, and differentiation must be tightly controlled by various signaling events, including cytosolic Ca(2+) signaling. During development, cells regularly exhibit spontaneous Ca(2+) activity that stimulates downstream effectors, which can elicit these fundamental cell processes. Spontaneous Ca(2+) activity during early neocortical development depends heavily on gap junctions and voltage dependent Ca(2+) channels, whereas later in development neurotransmitters and synapses exert an influence. Here, we provide an overview of the literature on Ca(2+) signaling and its impact on cell proliferation, migration, and differentiation in the neocortex. We point out important historical studies and review recent progress in determining the role of Ca(2+) signaling in neocortical development.

AmotL2 disrupts apical-basal cell polarity and promotes tumour invasion.

The establishment and maintenance of apical-basal cell polarity is essential for the functionality of glandular epithelia. Cell polarity is often lost in advanced tumours correlating with acquisition of invasive and malignant properties. Despite extensive knowledge regarding the formation and maintenance of polarity, the mechanisms that deregulate polarity in metastasizing cells remain to be fully characterized. Here we show that AmotL2 expression correlates with loss of tissue architecture in tumours from human breast and colon cancer patients. We further show that hypoxic stress results in activation of c-Fos-dependent expression of AmotL2 leading to loss of polarity. c-Fos/hypoxia-induced p60 AmotL2 interacts with the Crb3 and Par3 polarity complexes retaining them in large vesicles and preventing them from reaching the apical membrane. The resulting loss of polarity potentiates the response to invasive cues in vitro and in vivo in mice. These data provide a molecular mechanism how hypoxic stress deregulates cell polarity during tumour progression.

MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division.

The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.

Cxcl12/Cxcr4 signaling controls the migration and process orientation of A9-A10 dopaminergic neurons.

CXCL12/CXCR4 signaling has been reported to regulate three essential processes for the establishment of neural networks in different neuronal systems: neuronal migration, cell positioning and axon wiring. However, it is not known whether it regulates the development of A9-A10 tyrosine hydroxylase positive (TH(+)) midbrain dopaminergic (mDA) neurons. We report here that Cxcl12 is expressed in the meninges surrounding the ventral midbrain (VM), whereas CXCR4 is present in NURR1(+) mDA precursors and mDA neurons from E10.5 to E14.5. CXCR4 is activated in NURR1(+) cells as they migrate towards the meninges. Accordingly, VM meninges and CXCL12 promoted migration and neuritogenesis of TH(+) cells in VM explants in a CXCR4-dependent manner. Moreover, in vivo electroporation of Cxcl12 at E12.5 in the basal plate resulted in lateral migration, whereas expression in the midline resulted in retention of TH(+) cells in the IZ close to the midline. Analysis of Cxcr4(-/-) mice revealed the presence of VM TH(+) cells with disoriented processes in the intermediate zone (IZ) at E11.5 and marginal zone (MZ) at E14. Consistently, pharmacological blockade of CXCR4 or genetic deletion of Cxcr4 resulted in an accumulation of TH(+) cells in the lateral aspect of the IZ at E14, indicating that CXCR4 is required for the radial migration of mDA neurons in vivo. Altogether, our findings demonstrate that CXCL12/CXCR4 regulates the migration and orientation of processes in A9-A10 mDA neurons.

RET PLCγ phosphotyrosine binding domain regulates Ca2+ signaling and neocortical neuronal migration.

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca(2+)) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca(2+) from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca(2+) and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.

Inositol 1,4,5-trisphosphate receptor subtype-specific regulation of calcium oscillations.

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca(2+)) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) are intracellular Ca(2+) release channels that mediate Ca(2+) release from endoplasmic reticulum (ER) Ca(2+) stores. The three IP(3)R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP(3)R by the endogenous modulators IP(3), Ca(2+), protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP(3)R subtype in shaping cytosolic Ca(2+) oscillations.

Noggin and Wnt3a enable BMP4-dependent differentiation of telencephalic stem cells into GluR-agonist responsive neurons.

Early telencephalic development is dependent on the spatially and temporally coordinated regulation by essential signaling factors. For example, members of the Bone Morphogenetic Protein (BMP) family, such as BMP4, are crucial for proper development of dorsal telencephalic structures. Stimulation of multipotent telencephalic neural stem cells (NSCs) with BMP4 induces differentiation primarily into astrocytic and mesenchymal cells. However, BMP4-mediated mesenchymal differentiation is inhibited at certain culture conditions of NSCs, corresponding to in vivo developmental contexts. These inhibitory mechanisms are not fully understood and the terminal fate of non-astrocytic BMP4 treated NSCs under these conditions is unclear. Here we show that secreted factors inhibited BMP4-mediated mesenchymal differentiation of telencephalic NSCs. BMP4 mediated a dramatic and direct up-regulation of endogenous noggin levels, that in turn exerted a concentration-dependent inhibition of BMP4-mediated mesenchymal differentiation of NSCs. Instead, BMP4 exposure of NSCs induced neuronal differentiation in mesenchyme-preventing conditions, whereas treatment with recombinant noggin alone did not. Wnt signaling is known to be essential for the development of neurons derived from the dorsal telencephalon, and co-stimulation of NSCs with BMP4+Wnt3a resulted in a synergistic effect yielding significantly increased number of mature neurons compared to stimulation with each factor alone. Thus whereas only a subset of BMP4-induced neurons derived from telencephalic NSCs, responded to glutamate receptor (GluR) agonists, over 80% of BMP4+Wnt3a-induced neurons responded appropriately to GluR-agonists. Our results increase the understanding of the role for BMP4 in differentiation of telencephalic multipotent progenitors, and reveal novel implications for noggin and Wnt3a in these events.

Dietary sodium modulates the interaction between efferent and afferent renal nerve activity by altering activation of α2-adrenoceptors on renal sensory nerves.

Activation of efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which then reflexively decreases ERSNA via activation of the renorenal reflexes to maintain low ERSNA. The ERSNA-ARNA interaction is mediated by norepinephrine (NE) that increases and decreases ARNA by activation of renal α(1)-and α(2)-adrenoceptors (AR), respectively. The ERSNA-induced increases in ARNA are suppressed during a low-sodium (2,470 ± 770% s) and enhanced during a high-sodium diet (5,670 ± 1,260% s). We examined the role of α(2)-AR in modulating the responsiveness of renal sensory nerves during low- and high-sodium diets. Immunohistochemical analysis suggested the presence of α(2A)-AR and α(2C)-AR subtypes on renal sensory nerves. During the low-sodium diet, renal pelvic administration of the α(2)-AR antagonist rauwolscine or the AT1 receptor antagonist losartan alone failed to alter the ARNA responses to reflex increases in ERSNA. Likewise, renal pelvic release of substance P produced by 250 pM NE (from 8.0 ± 1.3 to 8.5 ± 1.6 pg/min) was not affected by rauwolscine or losartan alone. However, rauwolscine+losartan enhanced the ARNA responses to reflex increases in ERSNA (4,680 ± 1,240%·s), and renal pelvic release of substance P by 250 pM NE, from 8.3 ± 0.6 to 14.2 ± 0.8 pg/min. During a high-sodium diet, rauwolscine had no effect on the ARNA response to reflex increases in ERSNA or renal pelvic release of substance P produced by NE. Losartan was not examined because of low endogenous ANG II levels in renal pelvic tissue during a high-sodium diet. Increased activation of α(2)-AR contributes to the reduced interaction between ERSNA and ARNA during low-sodium intake, whereas no/minimal activation of α(2)-AR contributes to the enhanced ERSNA-ARNA interaction under conditions of high sodium intake.

Biochemistry of calcium oscillations.

Cytosolic calcium (Ca2+) oscillations are vastly flexible cell signals that convey information regulating numerous cellular processes. The frequency and amplitude of the oscillating signal can be varied infinitely by concerted actions of Ca2+ transporters and Ca2+-binding proteins to encode specific messages that trigger downstream molecular events. High frequency cytosolic Ca2+ oscillations regulate fast responses, such as synaptic transmission and secretion, whereas low frequency oscillations regulate slow processes, such as fertilization and gene transcription. Thus, the cell exploits Ca2+ oscillations as a signalling carrier to transduce vital information that controls its behaviour. Here, we review the underlying biochemical mechanisms responsible for generating and discriminating cytosolic Ca2+ oscillations.

Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin.

Current opinion holds that pigment cells, melanocytes, are derived from neural crest cells produced at the dorsal neural tube and that migrate under the epidermis to populate all parts of the skin. Here, we identify growing nerves projecting throughout the body as a stem/progenitor niche containing Schwann cell precursors (SCPs) from which large numbers of skin melanocytes originate. SCPs arise as a result of lack of neuronal specification by Hmx1 homeobox gene function in the neural crest ventral migratory pathway. Schwann cell and melanocyte development share signaling molecules with both the glial and melanocyte cell fates intimately linked to nerve contact and regulated in an opposing manner by Neuregulin and soluble signals including insulin-like growth factor and platelet-derived growth factor. These results reveal SCPs as a cellular origin of melanocytes, and have broad implications on the molecular mechanisms regulating skin pigmentation during development, in health and pigmentation disorders.

The decrease of expression of ryanodine receptor sub-type 2 is reversed by gentamycin sulphate in vascular myocytes from mdx mice.

The mdx mouse, a model of the human Duchenne muscular dystrophy, displays impaired contractile function in skeletal, cardiac and smooth muscles. We explored the possibility that ryanodine receptor (RYR) expression could be altered in vascular muscle. The three RYR sub-types were expressed in portal vein myocytes. As observed through mRNA and protein levels, RYR2 expression was strongly decreased in mdx myocytes, whereas RYR3 and RYR1 expression were unaltered. The use of antisense oligonucleotide directed against RYR sub-types indicated that caffeine-induced Ca(2+) response and Ca(2+) spark frequency depended on RYR2 and RYR1. In mdx mice, caffeine-induced Ca(2+) responses were decreased in both amplitude and maximal rate of rise, and the frequency of Ca(2+) sparks was also strongly decreased. The gentamycin treatment was able to increase both the expression of RYR2 and the caffeine-induced Ca(2+) response to the same level as that observed in wild-type mice. Taken together, these results confirm that both RYR1 and RYR2 are required for vascular Ca(2+) signalling and indicate that inhibition of RYR2 expression may account for the decreased Ca(2+) release from the SR in mdx vascular myocytes. Finally, we suggest that gentamycin can restore the Ca(2+) signalling in smooth muscle from mdx mice by increasing RYR2 and dystrophin expression. These results may help explain the reduced efficacy of contraction in vascular myocytes of mdx mice and Duchenne muscular dystrophy-afflicted patients. Gentamycin treatment could be a good therapeutic tool to restore the vascular function.