Gregory T. Macleod

Neuron-specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD-knockout mice

Deletion of copper‐zinc superoxide dismutase (CuZnSOD) in Sod1–/– mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle‐specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1–/– mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1–/– mice). SynTgSod1–/– mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1–/– mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTg‐Sod1–/– mice compared with control animals, whereas muscles of age‐matched Sod1–/– mice displayed 30–40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1–/– mice were absent in SynTgSod1–/– mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1–/– mice but not in SynTgSod1–/– mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1–/– mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.—Sakellariou, G. K., Davis, C. S., Shi, Y., Ivannikov, M. V., Zhang, Y., Vasilaki, A., Macleod, G. T., Richardson, A., Van Remmen, H., Jackson, M. J., McArdle, A., Brooks, S. V. Neuron‐specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD‐knockout mice. FASEB J. 28, 1666‐1681 (2014). www.fasebj.org

Presynaptic Mitochondria in Functionally Different Motor Neurons Exhibit Similar Affinities for Ca2+ But Exert Little Influence as Ca2+ Buffers at Nerve Firing Rates In Situ

Mitochondria accumulate within nerve terminals and support synaptic function, most notably through ATP production. They can also sequester Ca2+ during nerve stimulation, but it is unknown whether this limits presynaptic Ca2+ levels at physiological nerve firing rates. Similarly, it is unclear whether mitochondrial Ca2+ sequestration differs between functionally different nerve terminals. We addressed these questions using a combination of synthetic and genetically encoded Ca2+ indicators to examine cytosolic and mitochondrial Ca2+ levels in presynaptic terminals of tonic (MN13-Ib) and phasic (MNSNb/d-Is) motor neurons in Drosophila, which, as we determined, fire during fictive locomotion at ∼42 Hz and ∼8 Hz, respectively. Mitochondrial Ca2+ sequestration starts in both terminals at ∼250 nm, exhibits a similar Ca2+-uptake affinity (∼410 nm), and does not require Ca2+ release from the endoplasmic reticulum. Nonetheless, mitochondrial Ca2+ uptake in type Is terminals is more responsive to low-frequency nerve stimulation and this is due to higher cytosolic Ca2+ levels. Since type Ib terminals have a higher mitochondrial density than Is terminals, it seemed possible that greater mitochondrial Ca2+ sequestration may be responsible for the lower cytosolic Ca2+ levels in Ib terminals. However, genetic and pharmacological manipulations of mitochondrial Ca2+ uptake did not significantly alter nerve-stimulated elevations in cytosolic Ca2+ levels in either terminal type within physiologically relevant rates of stimulation. Our findings indicate that presynaptic mitochondria have a similar affinity for Ca2+ in functionally different nerve terminals, but do not limit cytosolic Ca2+ levels within the range of motor neuron firing rates in situ.

Cytosolic Calcium Coordinates Mitochondrial Energy Metabolism with Presynaptic Activity

Most neurons fire in bursts, imposing episodic energy demands, but how these demands are coordinated with oxidative phosphorylation is still unknown. Here, using fluorescence imaging techniques on presynaptic termini of Drosophila motor neurons (MNs), we show that mitochondrial matrix pH (pHm), inner membrane potential (Δψm), and NAD(P)H levels ([NAD(P)H]m) increase within seconds of nerve stimulation. The elevations of pHm, Δψm, and [NAD(P)H]m indicate an increased capacity for ATP production. Elevations in pHm were blocked by manipulations that blocked mitochondrial Ca2+ uptake, including replacement of extracellular Ca2+ with Sr2+ and application of either tetraphenylphosphonium chloride or KB-R7943, indicating that it is Ca2+ that stimulates presynaptic mitochondrial energy metabolism. To place this phenomenon within the context of endogenous neuronal activity, the firing rates of a number of individually identified MNs were determined during fictive locomotion. Surprisingly, although endogenous firing rates are significantly different, there was little difference in presynaptic cytosolic Ca2+ levels ([Ca2+]c) between MNs when each fires at its endogenous rate. The average [Ca2+]c level (329 ± 11 nm) was slightly above the average Ca2+ affinity of the mitochondria (281 ± 13 nm). In summary, we show that when MNs fire at endogenous rates, [Ca2+]c is driven into a range where mitochondria rapidly acquire Ca2+. As we also show that Ca2+ stimulates presynaptic mitochondrial energy metabolism, we conclude that [Ca2+]c levels play an integral role in coordinating mitochondrial energy metabolism with presynaptic activity in Drosophila MNs.

Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

Both constitutive secretion and Ca2+-regulated exocytosis require the assembly of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

The Lack of CuZnSOD Leads to Impaired Neurotransmitter Release, Neuromuscular Junction Destabilization and Reduced Muscle Strength in Mice

Elevated reactive oxygen species (ROS) production and ROS-dependent protein damage is a common observation in the pathogenesis of many muscle wasting disorders, including sarcopenia. However, the contribution of elevated ROS levels to –a breakdown in neuromuscular communication and muscle atrophy remains unknown. In this study, we examined a copper zinc superoxide dismutase [CuZnSOD (Sod1)] knockout mouse (Sod1 −/−), a mouse model of elevated oxidative stress that exhibits accelerated loss of muscle mass, which recapitulates many phenotypes of sarcopenia as early as 5 months of age. We found that young adult Sod1 −/− mice display a considerable reduction in hind limb skeletal muscle mass and strength when compared to age-matched wild-type mice. These changes are accompanied by gross alterations in neuromuscular junction (NMJ) morphology, including reduced occupancy of the motor endplates by axons, terminal sprouting and axon thinning and irregular swelling. Surprisingly however, the average density of acetylcholine receptors in endplates is preserved. Using in vivo electromyography and ex vivo electrophysiological studies of hind limb muscles in Sod1 −/− mice, we found that motor axons innervating the extensor digitorum longus (EDL) and gastrocnemius muscles release fewer synaptic vesicles upon nerve stimulation. Recordings from individually identified EDL NMJs show that reductions in neurotransmitter release are apparent in the Sod1 −/− mice even when endplates are close to fully innervated. However, electrophysiological properties, such as input resistance, resting membrane potential and spontaneous neurotransmitter release kinetics (but not frequency) are similar between EDL muscles of Sod1 −/− and wild-type mice. Administration of the potassium channel blocker 3,4-diaminopyridine, which broadens the presynaptic action potential, improves both neurotransmitter release and muscle strength. Together, these results suggest that ROS-associated motor nerve terminal dysfunction is a contributor to the observed muscle changes in Sod1 −/− mice.

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (ΔΨm), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pHm) and ΔΨm were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]m was 0.22 ± 0.04 μM, but it rose to ~26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]m coincided with a rapid elevation in pHm and was followed by an after-stimulus ΔΨm hyperpolarization. However, pHm decreased and no ΔΨm hyperpolarization was observed in response to lower levels of [Ca²⁺]m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]m are required to stimulate presynaptic mitochondrial energy metabolism.

Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo

•  Changes in pH occur within neurons during nerve activity and in response to hypoxic insult. •  Many aspects of neurophysiology are potentially influenced by intracellular pH changes. •  At the fruit fly larval neuromuscular junction, fluorescent genetically encoded pH‐indicators (GEpHIs) revealed significant cytosolic acidification of presynaptic termini during nerve activity. •  GEpHIs revealed that presynaptic pH changes occur in live intact larvae, indicating for the first time that such pH changes are not an artifact of experimental conditions. •  The pH changes in presynaptic termini are substantial and are likely to influence synaptic function.

Differential Control of Presynaptic CamKII Activation and Translocation to Active Zones

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca2+ mobilization from the endoplasmic reticulum (ER) and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca2+ release. Here these issues are addressed in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. We report that neuronal excitation acts with ER Ca2+ stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca2+ provided by different presynaptic ER Ca2+ release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca2+ in the terminal, while translocation to AZs requires Ca2+ microdomains generated by IP3Rs.

A TRPV Channel in Drosophila Motor Neurons Regulates Presynaptic Resting Ca2+ Levels, Synapse Growth, and Synaptic Transmission

Presynaptic resting Ca(2+) influences synaptic vesicle (SV) release probability. Here, we report that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca(2+)] by promoting Ca(2+) release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. We find that Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. We also show that the absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, our findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [Ca(2+)].