Most Cleaved Anti-Müllerian Hormone Binds Its Receptor in Human Follicular Fluid but Little Is Competent in Serum.

CONTEXT: Anti-Müllerian hormone (AMH) is an important clinical marker for diagnosing and assessing the reproductive status and/or disorders in men and women. Most studies have not distinguished between levels of inactive AMH precursor and the cleaved noncovalent complex that binds the AMH type II receptor (AMHRII) and initiates signaling. OBJECTIVE: The objective of the study was to measure the levels of AMH cleavage and bioactivity in human body fluids. DESIGN, SETTING, AND PATIENTS: AMH cleavage levels and bioactivity were measured in the serum of six boys and in the follicular fluid and serum of nine control women and 13 women with the polycystic ovary syndrome (PCOS). MAIN OUTCOME MEASURES: AMH cleavage levels were measured by capturing AMH with an anti-AMH antibody, followed by Western blotting. The bioactivity of cleaved AMH was assessed with an ELISA that measures the levels of AMH capable of binding AMHRII. RESULTS: PCOS women have an elevated level of AMH cleavage in their follicular fluid (24% vs 8% in control women), and most of the cleaved AMH can bind AMHRII. Higher levels of cleavage are observed in female (60%) and male (79%) serum, but very little of the cleaved AMH can bind AMHRII. CONCLUSIONS: These results support an autocrine role for AMH in the pathophysiology of PCOS in the follicle. In addition, they indicate that AMH undergoes interactions or structural changes after cleavage that prevent receptor binding, meaning, unexpectedly, that the level of cleaved AMH in biological fluids does not always reflect the level of bioactive AMH.

JC Polyomavirus Abundance and Distribution in Progressive Multifocal Leukoencephalopathy (PML) Brain Tissue Implicates Myelin Sheath in Intracerebral Dissemination of Infection.

Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath.

Progressive multifocal leukoencephalopathy (PML) development is associated with mutations in JC virus capsid protein VP1 that change its receptor specificity.

Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid-independent binding with brain cells.

Sequencing and analysis of JC virus DNA from natalizumab-treated PML patients.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients. METHODS: Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome. RESULTS: Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement. CONCLUSIONS: These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.

Processing of anti-mullerian hormone regulates receptor activation by a mechanism distinct from TGF-beta.

TGF-ß family ligands are translated as prepropeptide precursors and are processed into mature C-terminal dimers that signal by assembling a serine/threonine kinase receptor complex containing type I and II components. Many TGF-ß ligands are secreted in a latent form that cannot bind their receptor, due to the pro-region remaining associated with the mature ligand in a noncovalent complex after proteolytic cleavage. Here we show that anti-Müllerian hormone (AMH), a TGF-ß family ligand involved in reproductive development, must be cleaved to bind its type II receptor (AMHRII), but dissociation of the pro-region from the mature C-terminal dimer is not required for this initial interaction. We provide direct evidence for this interaction by showing that the noncovalent complex binds to a soluble form of AMHRII in an ELISA format and to AMHRII immobilized on Sepharose. Binding of the noncovalent complex to Sepharose-coupled AMHRII induces dissociation of the pro-region from the mature C-terminal dimer, whereas no dissociation occurs after binding to immobilized AMH antibodies. The pro-region cannot be detected after binding of the AMH noncovalent complex to AMHRII expressed on COS cells, indicating that pro-region dissociation may occur as a natural consequence of receptor engagement on cells. Moreover, the mature C-terminal dimer is more active than the noncovalent complex in stimulating Sma- and Mad-related protein activation, suggesting that pro-region dissociation contributes to the assembly of the active receptor complex. AMH thus exemplifies a new mechanism for receptor engagement in which interaction with the type II receptor promotes pro-region dissociation to generate mature ligand.

Natural mutations of the anti-Mullerian hormone type II receptor found in persistent Mullerian duct syndrome affect ligand binding, signal transduction and cellular transport.

The anti-Müllerian hormone type II (AMHRII) receptor is the primary receptor for anti-Müllerian hormone (AMH), a protein produced by Sertoli cells and responsible for the regression of the Müllerian duct in males. AMHRII is a membrane protein containing an N-terminal extracellular domain (ECD) that binds AMH, a transmembrane domain, and an intracellular domain with serine/threonine kinase activity. Mutations in the AMHRII gene lead to persistent Müllerian duct syndrome in human males. In this paper, we have investigated the effects of 10 AMHRII mutations, namely 4 mutations in the ECD and 6 in the intracellular domain. Molecular models of the extra- and intracellular domains are presented and provide insight into how the structure and function of eight of the mutant receptors, which are still expressed at the cell surface, are affected by their mutations. Interestingly, two soluble receptors truncated upstream of the transmembrane domain are not secreted, unless the transforming growth factor beta type II receptor signal sequence is substituted for the endogenous one. This shows that the AMHRII signal sequence is defective and suggests that AMHRII uses its transmembrane domain instead of its signal sequence to translocate to the endoplasmic reticulum, a characteristic of type III membrane proteins.

A glial cell line-derived neurotrophic factor (GDNF):tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice.

Glial cell line-derived neurotrophic factor (GDNF) has shown robust neuroprotective and neuroreparative activities in various animal models of Parkinson's Disease or amyotrophic lateral sclerosis (ALS). The successful use of GDNF as a therapeutic in humans, however, appears to have been hindered by its poor bioavailability to target neurons in the central nervous system (CNS). To improve delivery of exogenous GDNF protein to CNS motor neurons, we employed chemical conjugation techniques to link recombinant human GDNF to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). The predominant species present in the purified conjugate sample, GDNF:TTC, had a molecular weight of approximately 80 kDa as determined by non-reducing SDS-PAGE. Like GDNF, addition of GDNF:TTC to culture media of neuroblastoma cells expressing GFRalpha-1/c-RET produced a dose-dependent increase in cellular phospho-c-RET levels. Treatment of cultured midbrain dopaminergic neurons with either GDNF or the conjugate similarly promoted both DA neuron survival and neurite outgrowth. However, in contrast to mice treated with GDNF by intramuscular injection, mice receiving GDNF:TTC revealed intense GDNF immunostaining associated with spinal cord motor neurons in fixed tissue sections. That GDNF:TTC provided neuroprotection of axotomized motor neurons in neonatal rats further revealed that the conjugate retained its GDNF activity in vivo. These results indicate that TTC can serve as a non-viral vehicle to substantially improve the delivery of functionally active growth factors to motor neurons in the mammalian CNS.

Quantitative analysis of the activation mechanism of the multicomponent growth-factor receptor Ret.

Cytokines and growth factors signal by modulating the interactions between multiple receptor components to form an activated receptor complex. The quantitative details of the activation mechanisms of this important class of receptors are not well understood. Using receptor phosphorylation measurements in live cells, as well as mathematical modeling and data fitting, we have characterized the multistep mechanism by which the GDNF-family neurotrophin artemin (ART), together with its co-receptor GDNF-family receptor alpha3 (GFRalpha3), brings about activation of the Ret receptor tyrosine kinase through formation of a pentameric signaling complex: ART-(GFRalpha3)(2)-(Ret)(2). By systematically varying the concentrations of ART and cell-surface GFRalpha3, we establish both the sequence of steps by which the signaling complex forms and the affinities of all the steps, including the two-dimensional affinities of the steps involving protein-protein interactions between membrane-bound species. Our results reveal the ways in which the individual binary interactions involved in the activation of a multicomponent receptor govern the receptor's functional properties.

Artemin crystal structure reveals insights into heparan sulfate binding.

Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFRalpha3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangement characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.

Glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFR alpha 1) is highly selective for GDNF versus artemin.

To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.

Multiple actions of systemic artemin in experimental neuropathy.

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.