Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster

Circadian clocks are highly conserved transcriptional regulators that control 24-hour oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcription properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks; we found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. This result suggests that the presumed dominance of the brain clock in regulating peripheral clocks needs to be re-examined. This result further demonstrates that LABL allows rapid, affordable, and direct real-time monitoring of clocks in vivo.

TACAN is a novel regulator of PKD2

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in membrane receptor PKD1 or cation channel PKD2. TACAN (also named TMEM120A), recently reported as an ion channel in neuron cells for mechano and pain sensing, is also distributed in diverse non-neuronal tissues such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes we found that TACAN inhibited the channel activity of PKD2 gain-of-function mutant F604P. The first and last transmembrane domains of TACAN were found to interact with the PKD2 C- and N-terminal portions, respectively. We showed that the TACAN N-terminus acted as a blocking peptide and that TACAN inhibits the PKD2 function through the PKD2/TACAN binding. By patch clamping in mammalian cells, we found that TACAN inhibits both the single channel conductance and open probability of PKD2 and mutant F604P. Further, PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechano sensitivity of the PKD2/TACAN channel complex.

An Evolutionary Perspective on Hox Binding Site Preferences in Two Different Tissues

Transcription factor (TF) networks define the precise development of multicellular organisms. While many studies focused on TFs expressed in specific cell types to elucidate their contribution to cell specification and differentiation, it is less understood how broadly expressed TFs perform their precise functions in the different cellular contexts. To uncover differences that could explain tissue-specific functions of such TFs, we analyzed here genomic chromatin interactions of the broadly expressed Drosophila Hox TF Ultrabithorax (Ubx) in the mesodermal and neuronal tissues using bioinformatics. Our investigations showed that Ubx preferentially interacts with multiple yet tissue-specific chromatin sites in putative regulatory regions of genes in both tissues. Importantly, we found the classical Hox/Ubx DNA binding motif to be enriched only among the neuronal Ubx chromatin interactions, whereas a novel Ubx-like motif with rather low predicted Hox affinities was identified among the regions bound by Ubx in the mesoderm. Finally, our analysis revealed that tissues-specific Ubx chromatin sites are also different with regards to the distribution of active and repressive histone marks. Based on our data, we propose that the tissue-related differences in Ubx binding behavior could be a result of the emergence of the mesoderm as a new germ layer in triploblastic animals, which might have required the Hox TFs to relax their binding specificity.

rab-27 acts in an intestinal pathway to inhibit axon regeneration in C. elegans

Injured axons must regenerate to restore nervous system function, and regeneration is regulated in part by external factors from non-neuronal tissues. Many of these extrinsic factors act in the immediate cellular environment of the axon to promote or restrict regeneration, but the existence of long-distance signals regulating axon regeneration has not been clear. Here we show that the Rab GTPase rab-27 inhibits regeneration of GABAergic motor neurons in C. elegans through activity in the intestine. Re-expression of RAB-27, but not the closely related RAB-3, in the intestine of rab-27 mutant animals is sufficient to rescue normal regeneration. Several additional components of an intestinal neuropeptide secretion pathway also inhibit axon regeneration, including NPDC1/cab-1, SNAP25/aex-4, KPC3/aex-5, and the neuropeptide NLP-40, and re-expression of these genes in the intestine of mutant animals is sufficient to restore normal regeneration success. Additionally, NPDC1/cab-1 and SNAP25/aex-4 genetically interact with rab-27 in the context of axon regeneration inhibition. Together these data indicate that RAB-27-dependent neuropeptide secretion from the intestine inhibits axon regeneration, and point to distal tissues as potent extrinsic regulators of regeneration.

Protein Kinase A in Human Retina: Differential Localization of Cβ, Cα, RIIα, and RIIβ in Photoreceptors Highlights Non-redundancy of Protein Kinase A Subunits

Protein kinase A (PKA) signaling is essential for numerous processes but the subcellular localization of specific PKA regulatory (R) and catalytic (C) subunits has yet to be explored comprehensively. Additionally, the localization of the Cβ subunit has never been spatially mapped in any tissue even though ∼50% of PKA signaling in neuronal tissues is thought to be mediated by Cβ. Here we used human retina with its highly specialized neurons as a window into PKA signaling in the brain and characterized localization of PKA Cα, Cβ, RIIα, and RIIβ subunits. We found that each subunit presented a distinct localization pattern. Cα and Cβ were localized in all cell layers (photoreceptors, interneurons, retinal ganglion cells), while RIIα and RIIβ were selectively enriched in photoreceptor cells where both showed distinct patterns of co-localization with Cα but not Cβ. Only Cα was observed in photoreceptor outer segments and at the base of the connecting cilium. Cβ in turn, was highly enriched in mitochondria and was especially prominent in the ellipsoid of cone cells. Further investigation of Cβ using RNA BaseScope technology showed that two Cβ splice variants (Cβ4 and Cβ4ab) likely code for the mitochondrial Cβ proteins. Overall, our data indicates that PKA Cα, Cβ, RIIα, and RIIβ subunits are differentially localized and are likely functionally non-redundant in the human retina. Furthermore, Cβ is potentially important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.

Biphalin—A Potent Opioid Agonist—As a Panacea for Opioid System-Dependent Pathophysiological Diseases?

Biphalin, one of the opioid agonists, is a dimeric analog of enkephalin with a high affinity for opioid receptors. Opioid receptors are widespread in the central nervous system and in peripheral neuronal and non-neuronal tissues. Hence, these receptors and their agonists, which play an important role in pain blocking, may also be involved in the regulation of other physiological functions. Biphalin was designed and synthesized in 1982 by Lipkowski as an analgesic peptide. Extensive further research in various laboratories on the antinociceptive effects of biphalin has shown its excellent properties. It has been demonstrated that biphalin exhibits an analgesic effect in acute, neuropathic, and chronic animal pain models, and is 1000 times more potent than morphine when administered intrathecally. In the course of the broad conducted research devoted primarily to the antinociceptive effect of this compound, it has been found that biphalin may also potentially participate in the regulation of other opioid system-dependent functions. Nearly 40 years of research on the properties of biphalin have shown that it may play a beneficial role as an antiviral, antiproliferative, anti-inflammatory, and neuroprotective agent, and may also affect many physiological functions. This integral review analyzes the literature on the multidirectional biological effects of biphalin and its potential in the treatment of many opioid system-dependent pathophysiological diseases.

Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.

DAF-2/insulin IGF-1 receptor regulates mobility during ageing by integrating opposite inputs from muscle and neuronal tissues

During ageing, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In C. elegans, mutants of the insulin receptor DAF-2 represent a paradigm of healthy ageing, as their increased lifespan is accompanied by a delay in age-related loss of mobility. However, these animals are less mobile than wild-type animals in early adulthood. Here we investigated the DAF-2-dependent relationship between longevity and mobility using an auxin-inducible degron to trigger tissue-specific degradation of endogenous DAF-2. As previously reported, inactivation of DAF-2 in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline or muscle was not. However, neither intestinal nor neuronal depletion of DAF-2 prevented the age-related loss of mobility. In 1-day-old adults, DAF-2 depletion in neurons reduced mobility, while muscle depletion had no effect. By contrast, DAF-2 depletion in the muscle of middle-age animals improved their mobility. The combination of neuronal and muscle effects thus mimics the global locomotor phenotype of daf-2 mutants. Yet, we observed that neuronal or muscle DAF-2 depletion both preserved the mitochondria network in ageing muscle. Overall, these results show that the mobility pattern of daf-2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue-specific contribution of insulin-like signaling in integrated phenotypes at the whole organism level.

Review of Post-embedding Immunogold Methods for the Study of Neuronal Structures

The post-embedding immunogold (PI) technique for immunolabeling of neuronal tissues utilizing standard thin-section transmission electron microscopy (TEM) continues to be a prime method for understanding the functional localization of key proteins in neuronal function. Its main advantages over other immunolabeling methods for thin-section TEM are (1) fairly accurate and quantifiable localization of proteins in cells; (2) double-labeling of sections using two gold particle sizes; and (3) the ability to perform multiple labeling for different proteins by using adjacent sections. Here we first review in detail a common method for PI of neuronal tissues. This method has two major parts. First, we describe the freeze-substitution embedding method: cryoprotected tissue is frozen in liquid propane via plunge-freezing, and is placed in a freeze-substitution instrument in which the tissue is embedded in Lowicryl at low temperatures. We highlight important aspects of freeze-substitution embedding. Then we outline how thin sections of embedded tissue on grids are labeled with a primary antibody and a secondary gold particle-conjugated antibody, and the particular problems encountered in TEM of PI-labeled sections. In the Discussion, we compare our method both to earlier PI methods and to more recent PI methods used by other laboratories. We also compare TEM immunolabeling using PI vs. various pre-embedding immunolabeling methods, especially relating to neuronal tissue.

Mitochondrial Protein PGAM5 Emerges as a New Regulator in Neurological Diseases

As mitochondrial dysfunction has increasingly been implicated in neurological diseases, much of the investigation focuses on the response of the mitochondria. It appears that mitochondria can respond to external stimuli speedy fast, in seconds. Understanding how mitochondria sense the signal and communicate with cytosolic pathways are keys to understand mitochondrial regulation in diseases or in response to trauma. It was not until recently that a novel mitochondrial protein, phosphoglycerate mutase family member 5 (PGAM5) has emerged to be a new regulator of mitochondrial homeostasis. Although controversial results reveal beneficial as well as detrimental roles of PGAM5 in cancers, these findings also suggest PGAM5 may have diverse regulation on cellular physiology. Roles of PGAM5 in neuronal tissues remain to be uncovered. This review discusses current knowledge of PGAM5 in neurological diseases and provides future perspectives.