Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain.

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Clusterin in the mouse epididymis: possible roles in sperm maturation and capacitation.

Clusterin (CLU) is known as an extracellular chaperone for proteins under stress, thus preventing them from aggregation and precipitation. We showed herein that CLU, expressed by principal cells of the mouse caput epididymis, was present in high amounts in the lumen. In the cauda epididymis, CLU bound tightly to the sperm head surface and its amount on total sperm was similar to that in the bathing luminal fluid. In both immotile and motile caudal epididymal sperm, CLU was localized over the entire sperm head except at the convex ridge, although in the motile sperm population, the CLU immunofluorescence pattern was distinctively mottled with a lower intensity. However, when motile sperm became capacitated, CLU was relocalized to the head hook region, with immunofluorescence intensity being higher than that on the non-capacitated counterparts. Under a slightly acidic pH of the epididymal lumen, CLU may chaperone some luminal proteins and deliver them onto the sperm surface. Immunoprecipitation of epididymal fluid proteins indicated that CLU interacted with SED1, an important egg-binding protein present in a high amount in the epididymal lumen. In a number of non-capacitated sperm, fractions of SED1 and CLU co-localized, but after capacitation, SED1 and CLU dissociated from one another. While CLU moved to the sperm head hook, SED1 translocated to the head convex ridge, the egg-binding site. Overall, CLU localization patterns can serve as biomarkers of immotile sperm, and non-capacitated and capacitated sperm in mice. The chaperone role of CLU may also be important for sperm maturation and capacitation.

Comparative antioxidant analysis of extracts prepared from Indigofera longiracemosa aerial parts identifies leaf methanolic extract as a promising antioxidant.

Extraction of phytochemicals from the leaves and stems of Indigofera longiracemosa, a previously underexplored dye-yielding plant, was done using four solvents of increasing polarity followed by in vitro assessment of their antioxidant potential. Of the four solvent extracts, methanol extract yielded the highest percentage of phytochemicals. Methanol extracts of leaves and stems also showed the best antioxidant potential in in vitro antioxidant assays. Further, comparison with known standard antioxidant molecules showed lower IC50 values for the extracts tested. Our study thereby validates for the first time the antioxidant potential of I. longiracemosa aerial parts, and identifies an extract enriched in phytochemicals capable of quenching free radicals in vitro at concentrations lower than or comparable to pure antioxidant molecules.

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells.

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.

Primary Sertoli Cell Cultures From Adult Mice Have Different Properties Compared With Those Derived From 20-Day-Old Animals.

Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. This practice is based on the fact that Sertoli cells cease to proliferate and become mature in vivo by 16 to 20 days after birth. However, it is important to verify whether cultured Sertoli cells derived from 20-day-old mice do not proliferate ex vivo and whether they have the same properties as cultured adult Sertoli cells. Herein we described an isolation/culture method of Sertoli cells from 10-week-old adult mice with > 90% purity. Properties of these cultured adult Sertoli cells were then compared with those of cultured Sertoli cells derived from 20-day-old mice (also > 90% purity). By cell counting, bromo-2-deoxyuridine incorporation, and metaphase plate detection, we demonstrated that only adult Sertoli cells did not proliferate throughout 12 culture days. In contrast, Sertoli cells derived from 20-day-old mice still proliferated until Day 10 in culture. The morphology and profiles of intracellular lipidomics and spent medium proteomics of the 2 cultures were also different. Cultured adult Sertoli cells were larger in size and contained higher levels of triacylglycerols, cholesteryl esters, and seminolipid, and the proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including anti-Mullerian hormone, cell division cycle protein 42 (CDC42), and collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice, rather than those from 20-day-old animals, should be used for studies on properties of adult Sertoli cells.

Expression of transgenic PPP1CC2 in the testis of Ppp1cc-null mice rescues spermatid viability and spermiation but does not restore normal sperm tail ultrastructure, sperm motility, or fertility.

Two isoforms of phosphoprotein phosphatase 1, PPP1CC1 and PPP1CC2, are translated from alternatively spliced transcripts of a single gene, Ppp1cc, and differ only at their extreme C-termini. While PPP1CC1 expression is almost ubiquitous, PPP1CC2 is largely restricted to testicular germ cells and mature spermatozoa. Targeted deletion of Ppp1cc leads to sterility of -/- males due to a combination of gross structural defects in developing spermatids resulting in apoptosis and faulty spermiation. Because PPP1CC2 is the only PP1 isoform that demonstrates high-level expression in wild-type meiotic and postmeiotic male germ cells, we have tested whether its loss in Ppp1cc-/- males is largely responsible for manifestation of this phenotype by expressing PPP1CC2 transgenically in the testis of Ppp1cc-/- mice (rescue mice). Herein, we demonstrate that PPP1CC2 expression in the Ppp1cc-/- testis is antiapoptotic, thus reestablishing spermatid development and spermiation. However, because aberrant flagellar morphogenesis is incompletely ameliorated, rescue males remain infertile. Because these results suggest that expression of PPP1CC2 in developing germ cells is essential but insufficient for normal spermatogenesis to occur, appropriate spatial and temporal expression of both PPP1CC isoforms in the testis during spermatogenesis appears to be necessary to produce structurally normal fertility-competent spermatozoa.

Parathyroid Hormone-Related Peptide Elicits Peripheral TRPV1-dependent Mechanical Hypersensitivity.

Bone metastasis in breast, prostate and lung cancers often leads to chronic pain, which is poorly managed by existing analgesics. The neurobiological mechanisms that underlie chronic pain associated with bone-metastasized cancers are not well understood, but sensitization of peripheral nociceptors by tumor microenvironment factors has been demonstrated to be important. Parathyroid hormone-related peptide (PTHrP) is highly expressed in bone-metastasized breast and prostate cancers, and is critical to growth and proliferation of these tumors in the bone tumor microenvironment. Previous studies have suggested that PTHrP could sensitize nociceptive sensory neurons, resulting in peripheral pain hypersensitivity. In this study, we found that PTHrP induces both heat and mechanical hypersensitivity, that are dependent on the pain-transducing transient receptor potential channel family vanilloid, member-1 (TRPV1), but not the mechano-transducing TRPV4 and TRPA1 ion channels. Functional ratiometric Ca2+ imaging and voltage-clamp electrophysiological analysis of cultured mouse DRG neurons show significant potentiation of TRPV1, but not TRPA1 or TRPV4 channel activation by PTHrP. Interestingly, PTHrP exposure led to the slow and sustained activation of TRPV1, in the absence of any exogenous channel agonist, and is dependent on the expression of the type-1 parathyroid hormone receptor (PTH1), as well as on downstream phosphorylation of the channel by protein kinase C (PKC). Accordingly, local administration of specific small-molecule antagonists of TRPV1 to mouse hindpaws after the development of PTHrP-induced mechanical hypersensitivity led to its significant attenuation. Collectively, our findings suggest that PTHrP/PTH1-mediated flow activation of TRPV1 channel contributes at least in part to the development and maintenance of peripheral mechanical pain hypersensitivity, and could therefore constitute a mechanism for nociceptor sensitization in the context of metastatic bone cancer pain.

Convergent phosphomodulation of the major neuronal dendritic potassium channel Kv4.2 by pituitary adenylate cyclase-activating polypeptide.

The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is secreted by both neuronal and non-neuronal cells in the brain and spinal cord, in response to pathological conditions such as stroke, seizures, chronic inflammatory and neuropathic pain. PACAP has been shown to exert various neuromodulatory and neuroprotective effects. However, direct influence of PACAP on the function of intrinsically excitable ion channels that are critical to both hyperexcitation as well as cell death, remain largely unexplored. The major dendritic K(+) channel Kv4.2 is a critical regulator of neuronal excitability, back-propagating action potentials in the dendrites, and modulation of synaptic inputs. We identified, cloned and characterized the downstream signaling originating from the activation of three PACAP receptor (PAC1) isoforms that are expressed in rodent hippocampal neurons that also exhibit abundant expression of Kv4.2 protein. Activation of PAC1 by PACAP leads to phosphorylation of Kv4.2 and downregulation of channel currents, which can be attenuated by inhibition of either PKA or ERK1/2 activity. Mechanistically, this dynamic downregulation of Kv4.2 function is a consequence of reduction in the density of surface channels, without any influence on the voltage-dependence of channel activation. Interestingly, PKA-induced effects on Kv4.2 were mediated by ERK1/2 phosphorylation of the channel at two critical residues, but not by direct channel phosphorylation by PKA, suggesting a convergent phosphomodulatory signaling cascade. Altogether, our findings suggest a novel GPCR-channel signaling crosstalk between PACAP/PAC1 and Kv4.2 channel in a manner that could lead to neuronal hyperexcitability.

Immunocontraceptive approaches in females.

In the female reproductive system, there are multiple points, where immunointerception is feasible. Among these, the most promising are those that interfere immunologically at the level of sperm-egg interaction or neutralize human chorionic gonadotropin (hCG), which is required for the establishment and maintenance of pregnancy. Immunization studies in various animal models including non-human primates with zona pellucida (ZP) glycoproteins have demonstrated curtailment of fertility. To circumvent the ovarian pathology often observed following immunization with ZP glycoproteins, synthetic peptides devoid of 'oophoritogenic' T cell epitopes as immunogens have been proposed. Though of utility for controlling wildlife populations, its application in humans will have to wait till the safety of ZP glycoprotein vaccine is established beyond doubt. Clinical trials in women have established that immunocontraceptive vaccine based on Beta-subunit of hCG inhibits fertility. To make it a practical proposition, novel strategies have to be adopted to increase its immunogenicity so that 'contraceptive' antibody titers are achieved in near to 100% recipients.

Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.