The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation.

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 SynTgSod1(-/-) 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.

The positioning and dynamics of organelles depend on membrane-cytoskeleton interactions. Mitochondria relocate along microtubules (MT), but it is not clear whether MT have direct effects on mitochondrial function. Using two-photon microscopy and the mitochondrial fluorescent dyes rhodamine 123 and Rhod-2, we showed that Taxol and nocodazole, which correspondingly stabilize and disrupt MT Interactions between intracellular membranes and microtubules (MT) 1 determine the structure and positioning of subcellular organelles and direct their dynamic movements. MT can associate with the organelles either dynamically or in a stable fashion. Dynamic interactions are required for organelle traffic and involve microtubule motors such as kinesin and dynein (1, 2). Stable interactions are responsible for the positioning and structural maintenance of the endoplasmic reticulum (ER) (3), and mitochondria (4 -9). However, it is not known whether MT can influence the function of mitochondria and ER. Taxol (paclitaxel) and nocodazole, which correspondingly stabilize and disrupt MT, change the structure of the cytoskeleton. In proliferating cells, these drugs prevent normal mitotic spindle formation and cause the cells to halt mitosis and to initiate apoptosis. In comparison with MT-disrupting drugs, Taxol is less toxic and has recently been promoted for the treatment of ovarian, breast, lung, and prostate cancers (10), but it is often accompanied by serious peripheral neuropathies (11), the origin and mechanisms of which are yet unclear. Only one intracellular target has been established for Taxol thus far; it binds to a site located on the inner surface of the MT wall (12) and enhances lateral contacts between tubulin dimers (13). Recent evidence obtained in non-neuronal cells (14, 15) suggests that Taxol can directly target tubulin bound to mitochondria. We show here that Taxol and nocodazole depolarized mitochondria and released previously stored Ca 2ϩ in brain stem pre-Bötzinger complex neurons. Both effects were inhibited by cyclosporin A (CsA) and 2-aminoethoxydiphenyl borate (APB), commonly used blockers of mitochondrial permeability transition pore (mPTP). mPTP opening was validated by using the mPTP-specific calcein/Co 2ϩ imaging technique (16). The effects of Taxol were not mediated by enhanced Ca 2ϩ influx, leading to subsequent overload of mitochondria with Ca 2ϩ or overproduction of reactive oxygen species (ROS), the two main factors that can promote the opening of mPTP with subsequent initiation of apoptosis (17-19). Electron and optical microscopy revealed close interactions of MT with mitochondria in living neurons and isolated mitochondria. We therefore suggest that mPTP induction is triggered by modification of MT interactions with proteins of the outer mitochondrial membrane, e.g. the voltagedependent anion channel (VDAC) (9, 20 -23). In non-neuronal cells mitochondria and ER are closely opposed, and mitochondria can capture Ca 2ϩ released from the ER (21, 24). In neurons, the positions of mitochondria and ER a...

Synaptic plasticity in many regions of the central nervous system leads to the continuous adjustment of synaptic strength, which is essential for learning and memory. In this study, we show by visualizing synaptic vesicle release in mouse hippocampal synaptosomes that presynaptic mitochondria and specifically, their capacities for ATP production are essential determinants of synaptic vesicle exocytosis and its magnitude. Total internal reflection microscopy of FM1-43 loaded hippocampal synaptosomes showed that inhibition of mitochondrial oxidative phosphorylation reduces evoked synaptic release. This reduction was accompanied by a substantial drop in synaptosomal ATP levels. However, cytosolic calcium influx was not affected. Structural characterization of stimulated hippocampal synaptosomes revealed that higher total presynaptic mitochondrial volumes were consistently associated with higher levels of exocytosis. Thus, synaptic vesicle release is linked to the presynaptic ability to regenerate ATP, which itself is a utility of mitochondrial density and 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 which 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±11nM) was slightly above the average Ca2+ affinity of the mitochondria (281±13nM). 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.