FSH, bone, belly and brain.

The pituitary gland orchestrates multiple endocrine organs by secreting tropic hormones, and therefore plays a significant role in a myriad of physiological processes, including skeletal modeling and remodeling, fat and glucose metabolism, and cognition. Expression of receptors for each pituitary hormone and the hormone itself in the skeleton, fat, immune cells, and the brain suggest that their role is much broader than the traditionally attributed functions. FSH, believed solely to regulate gonadal function is also involved in fat and bone metabolism, as well as in cognition. Our emerging understanding of nonreproductive functions of FSH, thus, opens potential therapeutic opportunities to address detrimental health consequences during and after menopause, namely, osteoporosis, obesity, and dementia. In this review, we outline current understanding of the cross-talk between the pituitary, bone, adipose tissue, and brain through FSH. Preclinical evidence from genetic and pharmacologic interventions in rodent models, and human data from population-based observations, genetic studies, and a small number of interventional studies provide compelling evidence for independent functions of FSH in bone loss, fat gain, and congnitive impairment.

Gene-Dose-Dependent Reduction Fshr Expression Improves Spatial Memory Deficits in Alzheimer's Mice.

Alzheimer's disease (AD) is a major progressive neurodegenerative disorder of the aging population. High post-menopausal levels of the pituitary gonadotropin follicle-stimulating hormone (FSH) are strongly associated with the onset of AD, and we have shown recently that FSH directly activates the hippocampal Fshr to drive AD-like pathology and memory loss in mice. To establish a role for FSH in memory loss, we used female 3xTg;Fshr+/+, 3xTg;Fshr+/- and 3xTg;Fshr-/- mice that were either left unoperated or underwent sham surgery or ovariectomy at 8 weeks of age. Unoperated and sham-operated 3xTg;Fshr-/- mice were implanted with 17ß-estradiol pellets to normalize estradiol levels. Morris Water Maze and Novel Object Recognition behavioral tests were performed to study deficits in spatial and recognition memory, respectively, and to examine the effects of Fshr depletion. 3xTg;Fshr+/+ mice displayed impaired spatial memory at 5 months of age; both the acquisition and retrieval of the memory were ameliorated in 3xTg;Fshr-/- mice and, to a lesser extent, in 3xTg;Fshr+/- mice- -thus documenting a clear gene-dose-dependent prevention of hippocampal-dependent spatial memory impairment. At 5 and 10 months, sham-operated 3xTg;Fshr-/- mice showed better memory performance during the acquasition and/or retrieval phases, suggesting that Fshr deletion prevented the progression of spatial memory deficits with age. However, this prevention was not seen when mice were ovariectomized, except in the 10-month-old 3xTg;Fshr-/- mice. In the Novel Object Recognition test performed at 10 months, all groups of mice, except ovariectomized 3xTg;Fshr-/- mice showed a loss of recognition memory. Consistent with the neurobehavioral data, there was a gene-dose-dependent reduction mainly in the amyloid ß40 isoform in whole brain extracts. Finally, serum FSH levels < 8 ng/mL in 16-month-old APP/PS1 mice were associated with better retrieval of spatial memory. Collectively, the data provide compelling genetic evidence for a protective effect of inhibiting FSH signaling on the progression of spatial and recognition memory deficits in mice, and lay a firm foundation for the use of an FSH-blocking agent for the early prevention of cognitive decline in postmenopausal women.

Development and biophysical characterization of a humanized FSH-blocking monoclonal antibody therapeutic formulated at an ultra-high concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the development of a unique formulation for our first-in-class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, confirmed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) Spectroscopy. Three rapid freeze-thaw cycles at -80 °C/25 °C or -80 °C/37 °C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (Tm) for formulated MS-Hu6 increased by >4.80 °C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Development and Biophysical Characterization of a Humanized FSH-Blocking Monoclonal Antibody Therapeutic Formulated at an Ultra-High Concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the creation of a unique formulation for our first-in- class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, conformed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. Three rapid freeze-thaw cycles at -80°C/25°C or -80°C/37°C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (T m ) for formulated MS-Hu6 increased by >4.80°C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Optimizing a therapeutic humanized follicle-stimulating hormone-blocking antibody formulation by protein thermal shift assay.

Biopharmaceutical products are formulated using several Food and Drug Administration (FDA) approved excipients within the inactive ingredient limit to maintain their storage stability and shelf life. Here, we have screened and optimized different sets of excipient combinations to yield a thermally stable formulation for the humanized follicle-stimulating hormone (FSH)-blocking antibody, MS-Hu6. We used a protein thermal shift assay in which rising temperatures resulted in the maximal unfolding of the protein at the melting temperature (Tm ). To determine the buffer and pH for a stable solution, four different buffers with a pH range from 3 to 8 were screened. This resulted in maximal Tm s at pH 5.62 for Fab in phosphate buffer and at pH 6.85 for Fc in histidine buffer. Upon testing a range of salt concentrations, MS-Hu6 was found to be more stable at lower concentrations, likely due to reduced hydrophobic effects. Molecular dynamics simulations revealed a higher root-mean-square deviation with 1 mM than with 100 mM salt, indicating enhanced stability, as noted experimentally. Among the stabilizers tested, Tween 20 was found to yield the highest Tm and reversed the salt effect. Among several polyols/sugars, trehalose and sucrose were found to produce higher thermal stabilities. Finally, binding of recombinant human FSH to MS-Hu6 in a final formulation (20 mM phosphate buffer, 1 mM NaCl, 0.001% w/v Tween 20, and 260 mM trehalose) resulted in a thermal shift (increase in Tm ) for the Fab, but expectedly not in the Fc domain. Given that we used a low dose of MS-Hu6 (1 µM), the next challenge would be to determine whether 100-fold higher, industry-standard concentrations are equally stable.

Bone circuitry and interorgan skeletal crosstalk.

The past decade has seen significant advances in our understanding of skeletal homeostasis and the mechanisms that mediate the loss of bone integrity in disease. Recent breakthroughs have arisen mainly from identifying disease-causing mutations and modeling human bone disease in rodents, in essence, highlighting the integrative nature of skeletal physiology. It has become increasingly clear that bone cells, osteoblasts, osteoclasts, and osteocytes, communicate and regulate the fate of each other through RANK/RANKL/OPG, liver X receptors (LXRs), EphirinB2-EphB4 signaling, sphingolipids, and other membrane-associated proteins, such as semaphorins. Mounting evidence also showed that critical developmental pathways, namely, bone morphogenetic protein (BMP), NOTCH, and WNT, interact each other and play an important role in postnatal bone remodeling. The skeleton communicates not only with closely situated organs, such as bone marrow, muscle, and fat, but also with remote vital organs, such as the kidney, liver, and brain. The metabolic effect of bone-derived osteocalcin highlights a possible role of skeleton in energy homeostasis. Furthermore, studies using genetically modified rodent models disrupting the reciprocal relationship with tropic pituitary hormone and effector hormone have unraveled an independent role of pituitary hormone in skeletal remodeling beyond the role of regulating target endocrine glands. The cytokine-mediated skeletal actions and the evidence of local production of certain pituitary hormones by bone marrow-derived cells displays a unique endocrine-immune-skeletal connection. Here, we discuss recently elucidated mechanisms controlling the remodeling of bone, communication of bone cells with cells of other lineages, crosstalk between bone and vital organs, as well as opportunities for treating diseases of the skeleton.

FSH-blocking therapeutic for osteoporosis.

Pharmacological and genetic studies over the past decade have established the follicle-stimulating hormone (FSH) as an actionable target for diseases affecting millions, namely osteoporosis, obesity, and Alzheimer's disease. Blocking FSH action prevents bone loss, fat gain, and neurodegeneration in mice. We recently developed a first-in-class, humanized, epitope-specific FSH-blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a Good Laboratory Practice (GLP)-compliant platform, we now report the efficacy of MS-Hu6 in preventing and treating osteoporosis in mice and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labeled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone and bone marrow. The MS-Hu6 displayed a ß phase t½ of 7.5 days (180 hr) in humanized Tg32 mice. We tested 217 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. The MS-Hu6 showed the same level of "humanness" as human IgG1 in silico and was non-immunogenic in ELISpot assays for IL-2 and IFN-γ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable, and manufacturable, and is therefore poised for future human testing.

Glycerol 3-phosphate dehydrogenase regulates heat shock response in Saccharomyces cerevisiae.

The aim of this work was to identify elements of adaptive regulatory mechanism for basal level of yeast histone deacetylase Sir2. Heat shock response (HSR) was altered in the absence of the NAD-dependent glycerol 3-phosphate dehydrogenase (Gpd1). Increase in HSR was lower in ΔGpd1 cells than wild-type cells. An inverse correlation existed between Gpd1 and Sir2; Sir2-deleted cells showed higher expression of Gpd1 while deletion of Gpd1 led to higher expression of Sir2. In the absence of Gpd1, basal activity of Sir2 promoter was higher and was increased further upon heat shock, suggesting higher Sir2 levels. No interaction between Gpd1 and Sir2 was detected without or with heat shock using immunoprecipitation. The results show that Gpd1 regulates HSR in yeast cells and likely blocks its uncontrolled activation. As uncontrolled stress adversely affects the cellular adaptive response, Gpd1 may be a component of the cell's catalogue to ensure a balanced response to unmitigated thermal stress.

Lysine245 plays a crucial role in stability and function of glycerol 3-phosphate dehydrogenase (Gpd1) in Saccharomyces cerevisiae.

Glycerol 3-phosphate dehydrogenase (Gpd1 isoform) catalyzes the rate limiting step of glycerol synthesis and is a critical component of the osmo-responsive machinery in yeast. The three-dimensional structure of the enzyme is similar to the enzyme from many other organisms, including humans. A recent study with the human enzyme has proposed K120 (K152 in yeast) to be in the correct orientation for catalysis; K204 (K245 in yeast) is out of plane and is not a participant in the catalytic cycle. The current work was carried out to establish the role of K245 in the catalytic cycle of yeast Gpd1. K245A mutant was found to possess lower catalytic activity. Osmotically stressed cells expressing Gpd1 (K245A) showed no change in intracellular glycerol as compared with wild-type cells which showed ~60% increase. Fluorescence microscopy, native polyacrylamide gel electrophoresis (PAGE) analysis, fluorescence spectroscopy, and Thioflavin T spectrofluorimetry showed a relatively unstable, aggregation- and degradation-prone conformation for the mutant. In silico studies showed an aggregation "hotspot" around K245. This study establishes the requirement of K245 for conformational stability and functional adaptation of Gpd1 in Saccharomyces cerevisiae.

Does N-terminal huntingtin function as a 'holdase' for inhibiting cellular protein aggregation?

Proteolytic cleavage of huntingtin gives rise to N-terminal fragments. While the role of truncated mutant huntingtin is described in Huntington's disease (HD) pathogenesis, the function of N-terminal wild-type protein is less studied. The yeast model of HD is generated by the presence of FLAG tag and absence of polyproline tract as flanking sequences of the elongated polyglutamine stretch. We show that the same sequence derived from wild-type huntingtin exon1 is able to inhibit the aggregation of proteins in vitro and in yeast cells. It is able to stabilize client proteins as varied as luciferase, α-synuclein, and p53 in a soluble but non-native state. This is somewhat similar to the 'holdase' function of small heat shock proteins and 'nonchaperone proteins' which are able to stabilize partially unfolded client proteins in a nonspecific manner, slowing down their aggregation. Mutagenesis studies show this property to be localized at the N17 domain preceding the polyglutamine tract. Distortion of this ordered segment, either by deletion of this segment or mutation of a single residue (L4A), leads to decreased stability and increased aggregation of client proteins. It is interesting to note that the helical conformation of the N17 domain is also essential for aggregation of the N-terminal mutant protein. Our results provide evidence for a novel function for the amphipathic helix derived from exon1 of wild-type huntingtin.