Optogenetic therapies for vision restoration aim to confer intrinsic light sensitivity to retinal ganglion cells when photoreceptors have degenerated and light sensitivity has been irreversibly lost. We combine adaptive optics ophthalmoscopy with calcium imaging to optically record optogenetically restored retinal ganglion cell activity in the fovea of the living primate. Recording from the intact eye of a living animal, we compare the patterns of activity evoked by the optogenetic actuator ChrimsonR with natural photoreceptor mediated stimulation in the same retinal ganglion cells. Optogenetic responses are recorded more than one year following administration of the therapy and two weeks after acute loss of photoreceptor input in the living animal. This in vivo imaging approach could be paired with any therapy to minimize the number of primates required to evaluate restored activity on the retinal level, while maximizing translational benefit by using an appropriate pre-clinical model of the human visual system.
In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1 and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1 (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1 mice were confirmed with fluorescent probes, MitoSOX Red and ROS Brite570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1 in skeletal muscle of wild type mice directly promoted mitochondrial ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1 transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1. Overall, this study reveals a disease-stage-dependent change in mitochondrial function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1 directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.
BackgroundMotor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle.MethodsA transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed “mitoflash.” Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation.ResultsWe found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity.ConclusionsThe loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-017-0123-0) contains supplementary material, which is available to authorized users.
Summary Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX +/tdTomato human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time in vivo . Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye.
While the death of motor neuron is a pathological hallmark of amyotrophic lateral sclerosis (ALS), defects in other cell types or organs may also actively contribute to ALS disease progression. ALS patients experience progressive skeletal muscle wasting that may not only exacerbate neuronal degeneration, but likely has a significant impact on bone function. In our previous published study, we have discovered severe bone loss in an ALS mouse model with overexpression of ALS-associated mutation SOD1 (G93A). Here we further provide a mechanistic understanding of the bone loss in ALS animal and cellular models. Combining mitochondrial fluorescent indicators and confocal live cell imaging, we discovered abnormalities in mitochondrial network and dynamics in primary osteocytes derived from the same ALS mouse model G93A. Those mitochondrial defects occur in ALS mice after the onset of neuromuscular symptoms, indicating that mitochondria in bone cells respond to muscle atrophy during ALS disease progression. To examine whether ALS mutation has a direct contribution to mitochondrial dysfunction independent of muscle atrophy, we evaluated mitochondrial morphology and motility in cultured osteocytes (MLO-Y4) with overexpression of mitochondrial targeted SOD1. Compared with osteocytes overexpressing the wild type SOD1 as a control, the SOD1 osteocytes showed similar defects in mitochondrial network and dynamic as that of the primary osteocytes derived from the ALS mouse model. In addition, we further discovered that overexpression of SOD1 enhanced the expression level of dynamin-related protein 1 (Drp1), a key protein promoting mitochondrial fission activity, and reduced the expression level of optic atrophy protein 1 (OPA1), a key protein related to mitochondrial fusion. A specific mitochondrial fission inhibitor (Mdivi-1) partially reversed the effect of SOD1 on mitochondrial network and dynamics, indicating that SOD1 likely promotes mitochondrial fission, but suppresses the fusion activity. Our data provide the first evidence that mitochondria show abnormality in osteocytes derived from an ALS mouse model. The accumulation of mutant SOD1 protein inside mitochondria directly causes dysfunction in mitochondrial dynamics in cultured MLO-Y4 osteocytes. In addition, the ALS mutation SOD1-mediated dysfunction in mitochondrial dynamics is associated with an enhanced apoptosis in osteocytes, which could be a potential mechanism underlying the bone loss during ALS progression.
Muscle uses Ca2+ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca2+ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca2+ from their surroundings, a process called mitochondrial Ca2+ uptake. Under physiological conditions, Ca2+ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca2+ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca2+ uptake could shape spatio-temporal patterns of intracellular Ca2+ signaling. Malfunction of mitochondrial Ca2+ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca2+ levels. Besides the sudden elevation of Ca2+ level induced by action potentials, Ca2+ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca2+ uptake is fast and big enough to shape intracellular Ca2+ signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca2+ inside mitochondria. This review focuses on characterization of mitochondrial Ca2+ uptake in skeletal muscle and its role in muscle physiology and diseases.
The selective insertion of foreign materials, such as fluorescent markers or plasmids, into living cells has been a challenging problem in cell biology due to the cell membrane's selective permeability. However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies. This problem becomes even more challenging if the study is to be limited to specific cells within a larger population, since other transfection methods, such as viral transfection and lipofection, are not realizable with a high degree of spatial selectivity. Here, we have used a focused femtosecond laser beam to create a small transient hole in the cellular membrane (optoporation) in order to inject nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons). Following optoporation, the dye bound to the intracellular actin network and rise in fluorescence intensity was observed. Theoretical dynamics of the dye's diffusion is discussed, and numerical simulations of diffusion time constants are found to match well with experimental values.
In photodegenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), progressive loss of vision occurs as a result of degeneration of the periphery of the retina and the macula, respectively. Current optogenetic stimulation-based approaches to vision restoration offer the advantages of cellular specificity, high resolution, and minimal invasiveness over electrode arrays; however, the clinical translation of optogenetic activation suffers from the lack of a method for the delivery of opsins into spatially targeted regions of a retina that has degenerated. Non-targeted opsin delivery through viral or non-viral methods to non-photodegenerated retinal areas will perturb these already functioning retinal regions. Furthermore, viral methods are subject to limitations on the delivery of large plasmids, such as fusion constructs of multiple spectrally separated opsins (e.g., channelrhodopsin-2 (ChR2), chimeric opsin variants (C1V1), ReaChR), which can provide higher photo-excitability than can a single narrow-band opsin under ambient light conditions. Here, we report the ultrafast near-infrared laser-based spatially targeted transfection of single and multiple opsins and present a comparison with the opsin expression distribution achieved using another non-viral, but non-targeted, transfection method, lipofection. Functional evaluation of cells transfected with multiple opsins using the laser method revealed a significantly higher white-light-induced photocurrent than in cells expressing a single opsin (ChR2). The laser-assisted targeted delivery of multiple opsin-encoding genes to the peripheral retina/macula is ideal for sensitizing retinal areas that have degenerated, thus paving the way toward the restoration of lost vision in RP/AMD patients.
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