A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans.

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury.

Ground Control to Major Tom: The Cell Body Signals Axon Degeneration.

Axon degeneration in response to trophic deprivation was thought to be locally restricted to the axon. However, increasing evidence points to a requirement for the cell body in the degenerative program. Now, Simon et al. identify the pro-apoptotic protein Puma as a key factor in this cell body-derived signal.

Identification of spinal circuits transmitting and gating mechanical pain.

Pain information processing in the spinal cord has been postulated to rely on nociceptive transmission (T) neurons receiving inputs from nociceptors and Aß mechanoreceptors, with Aß inputs gated through feed-forward activation of spinal inhibitory neurons (INs). Here, we used intersectional genetic manipulations to identify these critical components of pain transduction. Marking and ablating six populations of spinal excitatory and inhibitory neurons, coupled with behavioral and electrophysiological analysis, showed that excitatory neurons expressing somatostatin (SOM) include T-type cells, whose ablation causes loss of mechanical pain. Inhibitory neurons marked by the expression of dynorphin (Dyn) represent INs, which are necessary to gate Aß fibers from activating SOM(+) neurons to evoke pain. Therefore, peripheral mechanical nociceptors and Aß mechanoreceptors, together with spinal SOM(+) excitatory and Dyn(+) inhibitory neurons, form a microcircuit that transmits and gates mechanical pain. PAPERCLIP:

Artemin, a glial cell line-derived neurotrophic factor family member, induces TRPM8-dependent cold pain.

Chronic pain associated with injury or disease can result from dysfunction of sensory afferents whereby the threshold for activation of pain-sensing neurons (nociceptors) is lowered. Neurotrophic factors control nociceptor development and survival, but also induce sensitization through activation of their cognate receptors, attributable, in part, to the modulation of ion channel function. Thermal pain is mediated by channels of the transient receptor potential (TRP) family, including the cold and menthol receptor TRPM8. Although it has been shown that TRPM8 is involved in cold hypersensitivity, the molecular mechanisms underlying this pain modality are unknown. Using microarray analyses to identify mouse genes enriched in TRPM8 neurons, we found that the glial cell line-derived neurotrophic factor (GDNF) family receptor GFRα3 is expressed in a subpopulation of TRPM8 sensory neurons that have the neurochemical profile of cold nociceptors. Moreover, we found that artemin, the specific GFRα3 ligand that evokes heat hyperalgesia, robustly sensitized cold responses in a TRPM8-dependent manner in mice. In contrast, GFRα1 and GFRα2 are not coexpressed with TRPM8 and their respective ligands GDNF and neurturin did not induce cold pain, whereas they did evoke heat hyperalgesia. Nerve growth factor induced mild cold sensitization, consistent with TrkA expression in TRPM8 neurons. However, bradykinin failed to alter cold sensitivity even though its receptor expresses in a subset of TRPM8 neurons. These results show for the first time that only select neurotrophic factors induce cold sensitization through TRPM8 in vivo, unlike the broad range of proalgesic agents capable of promoting heat hyperalgesia.

A sensory-labeled line for cold: TRPM8-expressing sensory neurons define the cellular basis for cold, cold pain, and cooling-mediated analgesia.

Many primary sensory neurons are polymodal, responding to multiple stimulus modalities (chemical, thermal, or mechanical), yet each modality is recognized differently. Although polymodality implies that stimulus encoding occurs in higher centers, such as the spinal cord or brain, recent sensory neuron ablation studies find that behavioral responses to different modalities require distinct subpopulations, suggesting the existence of modality-specific labeled lines at the level of the sensory afferent. Here we provide evidence that neurons expressing TRPM8, a cold- and menthol-gated channel required for normal cold responses in mammals, represents a labeled line solely for cold sensation. We examined the behavioral significance of conditionally ablating TRPM8-expressing neurons in adult mice, finding that, like animals lacking TRPM8 channels (Trpm8(-/-)), animals depleted of TRPM8 neurons ("ablated") are insensitive to cool to painfully cold temperatures. Ablated animals showed little aversion to noxious cold and did not distinguish between cold and a preferred warm temperature, a phenotype more profound than that of Trpm8(-/-) mice which exhibit only partial cold-avoidance and -preference behaviors. In addition to acute responses, cold pain associated with inflammation and nerve injury was significantly attenuated in ablated and Trpm8(-/-) mice. Moreover, cooling-induced analgesia after nerve injury was abolished in both genotypes. Last, heat, mechanical, and proprioceptive behaviors were normal in ablated mice, demonstrating that TRPM8 neurons are dispensable for other somatosensory modalities. Together, these data show that, although some limited cold sensitivity remains in Trpm8(-/-) mice, TRPM8 neurons are required for the breadth of behavioral responses evoked by cold temperatures.

Characterization of behavioral and neuromuscular junction phenotypes in a novel allelic series of SMA mouse models.

A number of mouse models for spinal muscular atrophy (SMA) have been genetically engineered to recapitulate the severity of human SMA by using a targeted null mutation at the mouse Smn1 locus coupled with the transgenic addition of varying copy numbers of human SMN2 genes. Although this approach has been useful in modeling severe SMA and very mild SMA, a mouse model of the intermediate form of the disease would provide an additional research tool amenable for drug discovery. In addition, many of the previously engineered SMA strains are multi-allelic by design, containing a combination of transgenes and targeted mutations in the homozygous state, making further genetic manipulation difficult. A new genetic engineering approach was developed whereby variable numbers of SMN2 sequences were incorporated directly into the murine Smn1 locus. Using combinations of these alleles, we generated an allelic series of SMA mouse strains harboring no, one, two, three, four, five, six or eight copies of SMN2. We report here the characterization of SMA mutants in this series that displayed a range in disease severity from embryonic lethal to viable with mild neuromuscular deficits.

Pharmacological blockade of TRPM8 ion channels alters cold and cold pain responses in mice.

TRPM8 (Transient Receptor Potential Melastatin-8) is a cold- and menthol-gated ion channel necessary for the detection of cold temperatures in the mammalian peripheral nervous system. Functioning TRPM8 channels are required for behavioral responses to innocuous cool, noxious cold, injury-evoked cold hypersensitivity, cooling-mediated analgesia, and thermoregulation. Because of these various roles, the ability to pharmacologically manipulate TRPM8 function to alter the excitability of cold-sensing neurons may have broad impact clinically. Here we examined a novel compound, PBMC (1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate) which robustly and selectively inhibited TRPM8 channels in vitro with sub-nanomolar affinity, as determined by calcium microfluorimetry and electrophysiology. The actions of PBMC were selective for TRPM8, with no functional effects observed for the sensory ion channels TRPV1 and TRPA1. PBMC altered TRPM8 gating by shifting the voltage-dependence of menthol-evoked currents towards positive membrane potentials. When administered systemically to mice, PBMC treatment produced a dose-dependent hypothermia in wildtype animals while TRPM8-knockout mice remained unaffected. This hypothermic response was reduced at lower doses, whereas responses to evaporative cooling were still significantly attenuated. Lastly, systemic PBMC also diminished cold hypersensitivity in inflammatory and nerve-injury pain models, but was ineffective against oxaliplatin-induced neuropathic cold hypersensitivity, despite our findings that TRPM8 is required for the cold-related symptoms of this pathology. Thus PBMC is an attractive compound that serves as a template for the formulation of highly specific and potent TRPM8 antagonists that will have utility both in vitro and in vivo.

Scraping through the ice: uncovering the role of TRPM8 in cold transduction.

The proper detection of environmental temperatures is essential for the optimal growth and survival of organisms of all shapes and phyla, yet only recently have the molecular mechanisms for temperature sensing been elucidated. The discovery of temperature-sensitive ion channels of the transient receptor potential (TRP) superfamily has been pivotal in explaining how temperatures are sensed in vivo, and here we will focus on the lone member of this cohort, TRPM8, which has been unequivocally shown to be cold sensitive. TRPM8 is expressed in somatosensory neurons that innervate peripheral tissues such as the skin and oral cavity, and recent genetic evidence has shown it to be the principal transducer of cool and cold stimuli. It is remarkable that this one channel, unlike other thermosensitive TRP channels, is associated with both innocuous and noxious temperature transduction, as well as cold hypersensitivity during injury and, paradoxically, cold-mediated analgesia. With ongoing research, the field is getting closer to answering a number of fundamental questions regarding this channel, including the cellular mechanisms of TRPM8 modulation, the molecular context of TRPM8 expression, as well as the full extent of the role of TRPM8 in cold signaling in vivo. These findings will further our understanding of basic thermotransduction and sensory coding, and may have important implications for treatments for acute and chronic pain.

TRPM8: from cold to cancer, peppermint to pain.

Temperature perception is vital for cellular and metabolic homeostasis, avoidance, and survival. In the primary afferent nerve terminal, select members of the transient receptor potential (TRP) family of ion channels reside and convert thermal stimuli into neuronal activity. The cold and menthol receptor, TRPM8, is the predominant thermoceptor for cellular and behavioral responses to cold temperatures. Remarkably, this single molecular sensor of cold, that responds at a discrete thermal threshold in vitro (approximately 28°C), enables sensory afferents to respond to distinct, yet varied thermal thresholds (approximately 28 to <5°C). Thus, unlike other thermally-gated TRP channels which are activated at either innocuous or noxious temperatures, TRPM8 provides perception of both pleasantly cool and painfully cold. In addition to this diversity in sensory signaling, TRPM8 has an emerging role in a variety of biological systems, including thermoregulation, cancer, bladder function, and asthma. Here we summarize some key points related to TRPM8 and its potential as a drug target to treat a wide variety of physiological conditions. Nonetheless, it remains to be seen how this single "cool" molecule can serve in such a multitude of biological processes.

TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo.

Somatosensory neurons detect environmental stimuli, converting external cues into neural activity that is relayed first to second-order neurons in the spinal cord. The detection of cold is proposed to be mediated by the ion channels TRPM8 and TRPA1. However, there is significant debate regarding the role of each channel in cold-evoked pain, complicating their potential as drug targets for conditions such as cold allodynia and hyperalgesia. To address this debate, we generated mice lacking functional copies of both channels and examined behaviors and neural activity in response to painful cold and noxious cooling compounds. Whereas normal mice display a robust preference for warmth over cold, both TRPM8-null (TRPM8(-/-)) and TRPM8/TRPA1 double-knockout mice (DKO) display no preference until temperatures reach the extreme noxious range. Additionally, in contrast to wildtype mice that avoid touching cold surfaces, mice lacking TRPM8 channels display no such avoidance and explore noxious cold surfaces, even at 5 degrees C. Furthermore, nocifensive behaviors to the cold-mimetic icilin are absent in TRPM8(-/-) and DKO mice, but are retained in TRPA1-nulls (TRPA1(-/-)). Finally, neural activity, measured by expression of the immediate-early gene c-fos, evoked by hindpaw stimulation with noxious cold, menthol, or icilin is reduced in TRPM8(-/-) and DKO mice, but not in TRPA1(-/-) animals. Thus our results show that noxious cold signaling is exclusive to TRPM8, mediating neural and behavioral responses to cold and cold-mimetics, and that TRPA1 is not required for acute cold pain in mammals.

Intracellular MUC1 peptides inhibit cancer progression.

PURPOSE: During cancer progression, the oncoprotein MUC1 binds beta-catenin while simultaneously inhibiting the degradation of the epidermal growth factor receptor (EGFR), resulting in enhanced transformation and metastasis. The purpose of this study was to design a peptide-based therapy that would block these intracellular protein-protein interactions as a treatment for metastatic breast cancer. EXPERIMENTAL DESIGN: The amino acid residues responsible for these interactions lie in tandem in the cytoplasmic domain of MUC1, and we have targeted this sequence to produce a MUC1 peptide that blocks the protumorigenic functions of MUC1. We designed the MUC1 inhibitory peptide (MIP) to block the intracellular interactions between MUC1/beta-catenin and MUC1/EGFR. To allow for cellular uptake we synthesized MIP adjacent to the protein transduction domain, PTD4 (PMIP). RESULTS: We have found that PMIP acts in a dominant-negative fashion, blocking both MUC1/beta-catenin and MUC1/EGFR interactions. In addition, PMIP induces ligand-dependent reduction of EGFR levels. These effects correspond to a significant reduction in proliferation, migration, and invasion of metastatic breast cancer cells in vitro, and inhibition of tumor growth and recurrence in an established MDA-MB-231 immunocompromised (SCID) mouse model. Importantly, PMIP also inhibits genetically driven breast cancer progression, as injection of tumor-bearing MMTV-pyV mT transgenic mice with PMIP results in tumor regression and a significant inhibition of tumor growth rate. CONCLUSIONS: These data show that intracellular MUC1 peptides possess significant antitumor activity and have important clinical applications in the treatment of cancer.

Diversity in the neural circuitry of cold sensing revealed by genetic axonal labeling of transient receptor potential melastatin 8 neurons.

Sensory nerves detect an extensive array of somatosensory stimuli, including environmental temperatures. Despite activating only a small cohort of sensory neurons, cold temperatures generate a variety of distinct sensations that range from pleasantly cool to painfully aching, prickling, and burning. Psychophysical and functional data show that cold responses are mediated by both C- and A delta-fibers with separate peripheral receptive zones, each of which likely provides one or more of these distinct cold sensations. With this diversity in the neural basis for cold, it is remarkable that the majority of cold responses in vivo are dependent on the cold and menthol receptor transient receptor potential melastatin 8 (TRPM8). TRPM8-null mice are deficient in temperature discrimination, detection of noxious cold temperatures, injury-evoked hypersensitivity to cold, and nocifensive responses to cooling compounds. To determine how TRPM8 plays such a critical yet diverse role in cold signaling, we generated mice expressing a genetically encoded axonal tracer in TRPM8 neurons. Based on tracer expression, we show that TRPM8 neurons bear the neurochemical hallmarks of both C- and A delta-fibers, and presumptive nociceptors and non-nociceptors. More strikingly, TRPM8 axons diffusely innervate the skin and oral cavity, terminating in peripheral zones that contain nerve endings mediating distinct perceptions of innocuous cool, noxious cold, and first- and second-cold pain. These results further demonstrate that the peripheral neural circuitry of cold sensing is cellularly and anatomically complex, yet suggests that cold fibers, caused by the diverse neuronal context of TRPM8 expression, use a single molecular sensor to convey a wide range of cold sensations.