Terminal Schwann Cells Are Essential for Neuromuscular Junction Function and Recovery after Nerve Injury.

BACKGROUND: Terminal Schwann cells (tSCs), nonmyelinating glial cells at the neuromuscular junction (NMJ), are integral to NMJ development, function, remodeling, and response to injury. It is essential to understand their requirement for NMJ function. In this study, the authors assessed consequences of immune-mediated tSC ablation in adult S100 -GFP mice of both sexes in homeostasis and after nerve injury. METHODS: The authors examined NMJ morphology and function in the extensor digitorum longus muscle during homeostasis at post-tSC ablation days 3, 14, and 42 and after peroneal nerve transection and immediate repair at 3 and 6 weeks after nerve injury and tSC ablation (postinjury and ablation). RESULTS: tSC ablation resulted in significant decreases ( P < 0.05) in tSC numbers per NMJ and end plate fragmentation. NMJ innervation and EDL tetanic force were significantly decreased at post-tSC ablation day 14 ( P < 0.05) and tSCs reestablished their NMJ coverage at post-tSC ablation day 42. After nerve injury, motor end plate fragmentation increased ( P < 0.01) with tSC ablation compared with injured control mice. NMJ reinnervation and extensor digitorum longus tetanic force were significantly reduced ( P < 0.001), even at 6 weeks postinjury and ablation, compared with control mice. CONCLUSION: These results add to the understanding that tSCs, with their proregenerative potential, help maintain NMJ integrity in homeostasis and are necessary for NMJ reinnervation after peripheral nerve injury. CLINICAL RELEVANCE STATEMENT: Terminal Schwann cells are integral for efficient NMJ recovery after nerve injury. This cell population may provide a novel therapeutic target to improve outcomes for patients with nerve injuries; additional investigation is warranted.

T-box transcription factor 21 is expressed in terminal Schwann cells at the neuromuscular junction.

INTRODUCTION/AIMS: Terminal Schwann cells (tSCs) are nonmyelinating Schwann cells present at the neuromuscular junction (NMJ) with multiple integral roles throughout their lifespan. There is no known gene differentiating tSCs from myelinating Schwann cells, making their isolation and investigation challenging. In this work we investigated genes expressed within tSCs. METHODS: A novel dissection technique was utilized to isolate the tSC-containing NMJ band from the sternomastoid muscles of S100-GFP mice. RNA was isolated from samples containing: (a) NMJ bands (tSCs with nerve and muscle), (b) nerve, and (c) muscle, and microarray genetic expression analysis was conducted. Data were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescent staining. To identify genes specific to tSCs compared with other NMJ components, analysis of variance and rank-order analysis were performed using the Partek Genomic Suite. RESULTS: Microarray analysis of the tSC-enriched NMJ band revealed upregulation (by 4- to 12-fold) of several genes unique to the NMJ compared with muscle or nerve parts alone (P < .05). Among these genes, Tbx21 (or T-bet) was identified, which showed a 12-fold higher expression at the NMJ compared with sciatic nerve (P < .002). qRT-PCR analysis showed Tbx21 mRNA expression was over ninefold higher (P < .05) in the NMJ relative to muscle and nerve. Tbx21 protein colocalized with tSCs and was not noted in myelinating SCs from sciatic nerve. DISCUSSION: We found TBX21 to be expressed in tSCs. Additional studies will be performed to determine the functional significance of TBX21 in tSCs. These studies may enhance the investigative tools available to modulate tSCs to improve motor recovery after nerve injury.

Macrophage roles in peripheral nervous system injury and pathology: Allies in neuromuscular junction recovery.

Peripheral nerve injuries remain challenging to treat despite extensive research on reparative processes at the injury site. Recent studies have emphasized the importance of immune cells, particularly macrophages, in recovery from nerve injury. Macrophage plasticity enables numerous functions at the injury site. At early time points, macrophages perform inflammatory functions, but at later time points, they adopt pro-regenerative phenotypes to support nerve regeneration. Research has largely been limited, however, to the injury site. The neuromuscular junction (NMJ), the synapse between the nerve terminal and end target muscle, has received comparatively less attention, despite the importance of NMJ reinnervation for motor recovery. Macrophages are present at the NMJ following nerve injury. Moreover, in denervating diseases, such as amyotrophic lateral sclerosis (ALS), macrophages may also play beneficial roles at the NMJ. Evidence of positive macrophages roles at the injury site after peripheral nerve injury and at the NMJ in denervating pathologies suggest that macrophages may promote NMJ reinnervation. In this review, we discuss the intersection of nerve injury and immunity, with a focus on macrophages.

Macrophage-Derived Vascular Endothelial Growth Factor-A Is Integral to Neuromuscular Junction Reinnervation after Nerve Injury.

Functional recovery in the end target muscle is a determinant of outcome after peripheral nerve injury. The neuromuscular junction (NMJ) provides the interface between nerve and muscle and includes non-myelinating terminal Schwann cells (tSCs). After nerve injury, tSCs extend cytoplasmic processes between NMJs to guide axon growth and NMJ reinnervation. The mechanisms related to NMJ reinnervation are not known. We used multiple mouse models to investigate the mechanisms of NMJ reinnervation in both sexes, specifically whether macrophage-derived vascular endothelial growth factor-A (Vegf-A) is crucial to establishing NMJ reinnervation at the end target muscle. Both macrophage number and Vegf-A expression increased in end target muscles after nerve injury and repair. In mice with impaired recruitment of macrophages and monocytes (Ccr2-/- mice), the absence of CD68+ cells (macrophages) in the muscle resulted in diminished muscle function. Using a Vegf-receptor 2 (VegfR2) inhibitor (cabozantinib; CBZ) via oral gavage in wild-type (WT) mice resulted in reduced tSC cytoplasmic process extension and decreased NMJ reinnervation compared with saline controls. Mice with Vegf-A conditionally knocked out in macrophages (Vegf-Afl/fl; LysMCre mice) demonstrated a more prolonged detrimental effect on NMJ reinnervation and worse functional muscle recovery. Together, these results show that contributions of the immune system are integral for NMJ reinnervation and functional muscle recovery after nerve injury.SIGNIFICANCE STATEMENT This work demonstrates beneficial contributions of a macrophage-mediated response for neuromuscular junction (NMJ) reinnervation following nerve injury and repair. Macrophage recruitment occurred at the NMJ, distant from the nerve injury site, to support functional recovery at the muscle. We have shown hindered terminal Schwann cell (tSC) injury response and NMJ recovery with inhibition of: (1) macrophage recruitment after injury; (2) vascular endothelial growth factor receptor 2 (VegfR2) signaling; and (3) Vegf secretion from macrophages. We conclude that macrophage-derived Vegf is a key component of NMJ recovery after injury. Determining the mechanisms active at the end target muscle after motor nerve injury reveals new therapeutic targets that may translate to improve motor recovery following nerve injury.

Gpr126/Adgrg6 contributes to the terminal Schwann cell response at the neuromuscular junction following peripheral nerve injury.

Gpr126/Adgrg6 is an adhesion G protein-coupled receptor essential for Schwann cell (SC) myelination with important contributions to repair after nerve crush injury. Despite critical functions in myelinating SCs, the role of Gpr126 within nonmyelinating terminal Schwann cells (tSCs) at the neuromuscular junction (NMJ), is not known. tSCs have important functions in synaptic maintenance and reinnervation, and after injury tSCs extend cytoplasmic processes to guide regenerating axons to the denervated NMJ. In this study, we show that Gpr126 is expressed in tSCs, and that absence of Gpr126 in SCs (SC-specific Gpr126 knockout, cGpr126) results in a NMJ maintenance defect in the hindlimbs of aged mice, but not in young adult mice. After nerve transection and repair, cGpr126 mice display delayed NMJ reinnervation, altered tSC morphology with decreased S100ß expression, and reduced tSC cytoplasmic process extensions. The immune response promoting reinnervation at the NMJ following nerve injury is also altered with decreased macrophage infiltration, Tnfα, and anomalous cytokine expression compared to NMJs of control mice. In addition, Vegfa expression is decreased in muscle, suggesting that cGpr126 non-cell autonomously modulates angiogenesis after nerve injury. In sum, cGpr126 mice demonstrated delayed NMJ reinnervation and decreased muscle mass following nerve transection and repair compared to control littermates. The integral function of Gpr126 in tSCs at the NMJ provides the framework for new therapeutic targets for neuromuscular disease.

What is Normal? Neuromuscular junction reinnervation after nerve injury.

INTRODUCTION: In this study we present a reproducible technique to assess motor recovery after nerve injury via neuromuscular junction (NMJ) immunostaining and electrodiagnostic testing. METHODS: Wild-type mice underwent sciatic nerve transection with repair. Hindlimb muscles were collected for microscopy up to 30 weeks after injury. Immunostaining was used to assess axons (NF200), Schwann cells (S100), and motor endplates (α-bungarotoxin). Compound motor action potential (CMAP) amplitude was used to assess tibialis anterior (TA) function. RESULTS: One week after injury, nearly all (98.0%) endplates were denervated. At 8 weeks, endplates were either partially (28.3%) or fully (71.7%) reinnervated. At 16 weeks, NMJ reinnervation reached 87.3%. CMAP amplitude was 83% of naive mice at 16 weeks and correlated with percentage of fully reinnervated NMJs. Morphological differences were noted between injured and noninjured NMJs. DISCUSSION: We present a reproducible method for evaluating NMJ reinnervation. Electrodiagnostic data summarize NMJ recovery. Characterization of wild-type reinnervation provides important data for consideration in experimental design and interpretation.

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age-related morphological changes.

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age-related changes. Aging effects on the NMJ components-motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)-have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild-type mice, all NMJ components showed age-associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain-specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic-specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age-related changes via sympathetic innervation.

Clinical relevance of terminal Schwann cells: An overlooked component of the neuromuscular junction.

The terminal Schwann cell (tSC), a type of nonmyelinating Schwann cell, is a significant yet relatively understudied component of the neuromuscular junction. In addition to reviewing the role tSCs play on formation, maintenance, and remodeling of the synapse, we review studies that implicate tSCs in neuromuscular diseases including spinal muscular atrophy, Miller-Fisher syndrome, and amyotrophic lateral sclerosis, among others. We also discuss the importance of these cells on degeneration and regeneration after nerve injury. Knowledge of tSC biology may improve our understanding of disease pathogenesis and help us identify new and innovative therapeutic strategies for the many patients who suffer from neuromuscular disorders and nerve injuries.

Effect of time and dose of recombinant follicle stimulating hormone agonist on the superovulatory response of sheep.

The objective of this study was to determine the superovulatory potential of a single-chain analog of human FSH (Fcα) when administered to ewes either 3 days before, or coincident with, simulated luteolysis (pessary removal [PR]). A total of 40 animals were randomly assigned to receive Fcα at doses of 0.62, 1.25, or 2.5 IU/kg of body weight (bwt) 3 days before PR or 0.31, 0.62, 1.25, or 2.5 IU/kg of bwt at PR. Control ewes received protein without FSH activity. Blood samples were collected during the periovulatory period and ovarian tissue was collected 11 days after PR. Ovulation rate did not differ from the control group in ewes receiving the smallest doses of Fcα (0.31 and 0.62 IU/kg). However, a significant superovulatory response was noted in sheep receiving Fcα at doses of 1.25 and 2.5 IU/kg and this response was comparable in animals receiving the largest dose levels of Fcα at, or 3 days before, PR. The interval between PR and the LH surge was significantly extended and the LH surges were less synchronous in animals receiving Fcα at PR when compared with animals receiving the potent FSH agonist 3 days before PR. Taken together, these data indicate that the human single-chain gonadotropin with FSH activity promotes superovulation in ewe lambs in the breeding season. A single injection of the recombinant gonadotropin 3 days before luteolysis synchronizes the LH surge. The use of the single-chain analog of FSH in assisted reproduction for domestic animals is likely to be of practical significance as an alternative to conventional gonadotropins in superovulation protocols in livestock species.

Effect of single-chain ovine gonadotropins with dual activity on ovarian function in sheep.

We examined the half-life and biological activity of two single-chain proteins that combined portions of ovine FSH and LH. We proposed the hypothesis that these chimeric proteins would display LH and FSH activities and would promote follicle maturation in ewes. Estrus activity was synchronized using progestogen-impregnated vaginal pessaries. To negate the impact of endogenous LH and FSH, animals received serum-containing antibodies against GNRH 1 day before pessary removal (PR). At PR sheep (five animals per group) received a single injection (10 IU/kg, i.v.) of either the ovine-based (oFcLcα) gonadotropin analog, an ovine-based analog containing oLHß truncated at the carboxyl terminus (oFcL(ΔT)cα), or a human-based gonadotropin analog (hFcLcα). Control animals received a comparable amount of gonadotropin-free protein. Ovulation was induced 3 days after PR using human chorionic gonadotropin (1000 IU, i.v.). Ovaries were collected 11 days after PR. Neither estradiol (E2) or progesterone (P4) production, development of preovulatory follicles or corpora lutea (CL) were noted in control animals receiving gonadotropin-free protein. Significant increase in the synthesis of E2 and P4 was noted in sheep receiving the dually active gonadotropin analogs. The number of CLs present 11 days after PR was significantly increased in sheep receiving the chimeric glycoproteins compared with control animals. The magnitude of the secretory and ovarian responses did not differ between hFcLcα and oFcLcα or between oFcLcα and oFcL(ΔT)cα. Immunoactivity of LH and FSH was low in control animals, but was significantly elevated in sheep receiving the gonadotropin analogs. In conclusion, ovine-based gonadotropin analogs are functionally active in sheep and a single injection is adequate to induce the development of multiple ovulatory follicles.

Redirecting intracellular trafficking and the secretion pattern of FSH dramatically enhances ovarian function in mice.

FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes.

A novel carboxyl-terminal heptapeptide initiates the regulated secretion of LH from unique sub-domains of the ER.

The coordinated secretion of LH and FSH are critical for reproductive functions. After translocation into the endoplasmic reticulum (ER), their biosynthetic routes diverge at a determinative step prior to sorting in the regulated (LH) and constitutive (FSH) secretion pathways. Recently, we identified a C-terminal heptapeptide sequence, present only in the LHß subunit, as a critical signal for entry of the LH dimer into the regulated pathway. We showed that an LHß mutant lacking the heptapeptide (LHßΔT) assembled more efficiently with the α subunit than wild-type LHß subunit, and this LHΔT dimer was secreted constitutively. Thus, an association exists between the presence of the C-terminal heptapeptide and sorting of the LH heterodimer to the regulated pathway. To study how this delayed LHß subunit assembly is related to the trafficking of LH, we exploited the single subunit transfection model in rat somatotrope-derived GH3 cells with the use of immunofluorescence confocal microscopy. The LHß subunit showed a distinct immunofluorescent localization as compared to the FSHß subunit and LHß mutants. The wild-type LHß subunit exhibited a perinuclear staining corresponding to the ER/nuclear envelope region. In contrast, the wild-type FSHß subunit and the mutants LHßΔT and LHßL119A displayed no detectable perinuclear staining; only peripheral ER puncta were observed. Also, no perinuclear fluorescence was detected in cells expressing the LH heterodimer. We propose that the C-terminal heptapeptide is responsible for delayed heterodimer assembly within an ER sub-domain of the nuclear envelope, as an early partitioning event necessary for the entrance of LH into the regulated secretory pathway, whereas FSHß does not traverse the nuclear envelope region. These data suggest that, at least for LH, the molecular decision to enter the regulated secretory pathway is a pre-Golgi event controlled by the novel C-terminal heptapeptide.

A dileucine determinant in the carboxyl terminal sequence of the LHß subunit is implicated in the regulated secretion of lutropin from transfected GH3 cells.

LH and FSH are essential for control of gonadal function. They are synthesized in the same gonadotrope but differ in their mode of secretion. LH release is regulated, while FSH is secreted constitutively. One unique feature of LHß is a carboxyl terminal hydrophobic heptapeptide. We demonstrated that deleting the heptapeptide diverted the truncated LH dimer to the constitutive pathway in vitro. To examine if the residues of this heptapeptide play a role in LH sorting, leucines 118-119 were substituted with alanine (L118A and L119A, respectively). The intracellular pool of the L118A mutant protein decreased with a corresponding increase in constitutive secretion. Moreover, immunofluorescence microscopy revealed that the L118A mutant exhibited fewer puncta as compared to wild-type LH. L119A behaved similar to wild-type LH, indicating that a single leucine residue at position 118, rather than a dileucine motif, contributes to the process that sorts LH into the regulated pathway.

Rerouting of a follicle-stimulating hormone analog to the regulated secretory pathway.

LH and FSH are produced by the same gonadotrope cells of the anterior pituitary but differ in their mode of secretion. This coordinated secretion of LH and FSH is essential for normal follicular development and ovulation in females and for spermatogenesis in males. The structural signals encoded in the LH and FSH subunits that govern the intracellular sorting of LH through the regulated secretory pathway and FSH through the constitutive pathway are largely unknown. Our laboratory recently identified the seven amino acid carboxy tail of LH beta as a sorting signal for LH in GH(3) cells. Here we compared the morphological features of GH(3) cells expressing an FSH analog containing the heptapeptide (FL7AA) with wild-type FSH using confocal microscopy. These experiments were performed to develop a rerouting model for examining structure-function links between secretion pathways of FSH/LH and their biological action. Both FSH- and LH-expressing cells exhibit a fluorescence pattern of randomly dispersed cytoplasmic puncta. FL7AA expressing cells have more intracellular accumulation compared with wild-type FSH and display a unique halo pattern of fluorescence near the plasma membrane. Such a pattern was not observed in cells expressing FSH or LH. Our results demonstrate that this FSH analog containing the carboxy heptapeptide of LH beta is rerouted to the regulated secretory pathway in GH(3) cells. This rerouted gonadotropin provides a unique model to study the trafficking, regulation, and function of LH and FSH.

Secretory trafficking signal encoded in the carboxyl-terminal region of the CGbeta-subunit.

Although the LHbeta- and chorionic gonadotropin-beta- (CGbeta) subunits share a high degree of sequence identity (>85%) in the first 114 amino acids, there is considerable sequence divergence at their carboxy ends. The CGbeta-subunit terminates with a unique carboxyl-terminal extension (115-145; carboxyl-terminal peptide), which contains four O-linked oligosaccharides, whereas the LHbeta-subunit bears a hydrophobic heptapeptide (115-121) at its carboxy terminus. LH is released through the regulated pathway in the pituitary, whereas CG is secreted constitutively from the placenta. We previously demonstrated in rat somatotroph-derived GH(3) cells that the LH is associated primarily with a regulated routing, and although the majority of CG was released constitutively from the cells, there was a fraction that was segregated through the regulated pathway. Moreover, we showed that the LHbeta heptapeptide is a determinant for the regulated secretion of LH. Given that the primary evolutionary change between LHbeta and CGbeta occurred at the carboxy terminus, these data suggested that the presence of the CGbeta carboxyl-terminal peptide region is responsible for the constitutive secretion of CG. A CG114 mutant (CGDeltaT) was constructed and expressed in GH(3) cells. Steady-state labeling and pulse-chase experiments demonstrated that the CGDeltaT entered the regulated pathway resulting in over 4-fold increase in the intracellular pool. The secretagogue, forskolin, stimulated CGDeltaT release over 3-fold, which was accompanied by a parallel intracellular decrease, and only marginal stimulation of CG was seen. Immunofluorescence demonstrated a unique membrane pattern of staining for CGDeltaT compared with dispersed cytoplasmic puncta for CG. Stimulation with forskolin caused a significant reduction in the relative fluorescence of CGDeltaT cells compared with a minor reduction for CG. These data show that the CGDeltaT analog resembles LH in its intracellular trafficking, further supporting the hypothesis that determinants at the carboxyl-terminal end of the CGbeta-subunit evolved from the LHbeta-subunit primarily to overcome the slow release and intracellular storage of LH resulting in rapid secretion of CG from the placenta.

Single-chain human gonadotropin analogs induce follicle development in sheep.

The biopotency of single-chain analogs of human hFSH, human chorionic gonadotropin (hCG), and a dually active gonadotropin construct (FcCGbetaalpha) was examined. Sheep (bwt=61.4+/-1.1 kg; n=6 ewes/treatment) received a single injection (5 IU/kg, i.v.) of the hFSH analog (Fcalpha), the hCG analog (CGbetaalpha), FcCGbetaalpha, or Fcalpha and CGbetaalpha. Control animals received conditioned media. Ovulation was induced 3 days after analog administration using hCG (1000 IU, i.v.). Basal serum concentrations of estradiol (E(2)) were maintained in control animals. Neither Fcalpha nor CGbetaalpha alone induced significant E(2) production during the pre-hCG period. Conversely, serum concentrations of E(2) were increased (P<0.05) 2 days after administration of FcCGbetaalpha or Fcalpha+ CGbetaalpha. Although P(4) concentrations were maintained at basal levels in control animals, significant increase was noted in all other treatment groups during the post-hCG period. Final ovarian weight was significantly increased (P<0.05) in animals receiving Fcalpha, Fcalpha+ CGbetaalpha, or FcCGbetaalpha, but not CGbetaalpha alone. Most of the ovarian enlargement was attributed to the formation of corpora lutea. Collectively, these observations demonstrate that the single-chain analogs of the human gonadotropins are active in sheep. The construct with singular FSH activity supports follicle development but not E(2) production. Conversely, the construct that incorporates beta-domains from both CG and FSH has dual activity. The long-lived nature of the single-chain constructs suggests that these recombinant gonadotropins may be effective alternatives to pituitary- or placenta-derived gonadotropins in out-of-season breeding and/or superovulation protocols.

A carboxyl-terminal sequence in the lutropin beta subunit contributes to the sorting of lutropin to the regulated pathway.

Although synthesized in the same pituitary gonadotropes, the secretion profiles of lutropin (LH) and follitropin (FSH) differ. LH is secreted through a regulated pathway and associated with a bolus release at mid-estrous cycle. In contrast, the majority of FSH is secreted constitutively with an incremental increase until ovulation. Both share an identicalalpha subunit, and thus thebeta subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl-terminal heptapeptide of the LHbeta subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSHbeta subunit, influences the intracellular behavior of the LH dimer. To test the hypothesis that the peptide contributes to differential sorting, we monitored the fates of LH and LHDeltaT (LHbeta subunit lacking the carboxyl-terminal seven amino acids) dimers in the rat somatotrope-derived GH(3) cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHDeltaT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release 3-fold, which was accompanied by a parallel decrease of intracellular LH; only marginal forskolin stimulation of LHDeltaT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHDeltaT compared with LH, consistent with increased constitutive secretion of LHDeltaT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSHbeta subunit resulted in an increased regulated secretion of this FSH analog compared with wild-type FSH. These data are the first to identify a novel structural determinant responsible for the sorting of a member of the glycoprotein hormone family into the regulated secretory pathway.

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH).

To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo.

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.

The LHbeta gene of several mammals embeds a carboxyl-terminal peptide-like sequence revealing a critical role for mucin oligosaccharides in the evolution of lutropin to chorionic gonadotropin in the animal phyla.

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) beta to the beta-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHbeta gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHbeta gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHbeta gene (boCTP) would be sufficient to generate the LHbeta species of a ruminant with properties typical to the CGbeta subunit. The mutated bovine LHbeta-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGbeta CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGbeta subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGbeta-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGbeta subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHbeta to CGbeta evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHbeta gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.