A long-acting GDF15 analog causes robust, sustained weight loss and reduction of food intake in an obese nonhuman primate model.

Growth Differentiation Factor-15 (GDF15) is a circulating polypeptide linked to cellular stress and metabolic adaptation. GDF15's half-life is ~3 h and activates the glial cell line-derived neurotrophic factor family receptor alpha-like (GFRAL) receptor expressed in the area postrema. To characterize sustained GFRAL agonism on food intake (FI) and body weight (BW), we tested a half-life extended analog of GDF15 (Compound H [CpdH]) suitable for reduced dosing frequency in obese cynomolgus monkeys. Animals were chronically treated once weekly (q.w.) with CpdH or long-acting GLP-1 analog dulaglutide. Mechanism-based longitudinal exposure-response modeling characterized effects of CpdH and dulaglutide on FI and BW. The novel model accounts for both acute, exposure-dependent effects reducing FI and compensatory changes in energy expenditure (EE) and FI occurring over time with weight loss. CpdH had linear, dose-proportional pharmacokinetics (terminal half-life ~8 days) and treatment caused exposure-dependent reductions in FI and BW. The 1.6 mg/kg CpdH reduced mean FI by 57.5% at 1 week and sustained FI reductions of 31.5% from weeks 9-12, resulting in peak reduction in BW of 16 ± 5%. Dulaglutide had more modest effects on FI and peak BW loss was 3.8 ± 4.0%. Longitudinal modeling of both the FI and BW profiles suggested reductions in BW observed with both CpdH and dulaglutide were fully explained by exposure-dependent reductions in FI without increase in EE. Upon verification of the pharmacokinetic/pharmacodynamic relationship established in monkeys and humans for dulaglutide, we predicted that CpdH could reach double digit BW loss in humans. In summary, a long-acting GDF15 analog led to sustained reductions in FI in overweight monkeys and holds potential for effective clinical obesity pharmacotherapy.

Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aß-Bearing Mice.

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.

Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function.

Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury.

Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury.

Increasing evidence suggests that the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) mediates deleterious effects of astrocytes in progressive neurodegenerative conditions. However, the impact of astrocytic CN/NFAT signaling on neural function/recovery after acute injury has not been investigated extensively. Using a controlled cortical impact (CCI) procedure in rats, we show that traumatic brain injury is associated with an increase in the activities of NFATs 1 and 4 in the hippocampus at 7 d after injury. NFAT4, but not NFAT1, exhibited extensive labeling in astrocytes and was found throughout the axon/dendrite layers of CA1 and the dentate gyrus. Blockade of the astrocytic CN/NFAT pathway in rats using adeno-associated virus (AAV) vectors expressing the astrocyte-specific promoter Gfa2 and the NFAT-inhibitory peptide VIVIT prevented the injury-related loss of basal CA1 synaptic strength and key synaptic proteins and reduced the susceptibility to induction of long-term depression. In conjunction with these seemingly beneficial effects, VIVIT treatment elicited a marked increase in the expression of the prosynaptogenic factor SPARCL1 (hevin), especially in hippocampal tissue ipsilateral to the CCI injury. However, in contrast to previous work on Alzheimer's mouse models, AAV-Gfa2-VIVIT had no effects on the levels of GFAP and Iba1, suggesting that synaptic benefits of VIVIT were not attributable to a reduction in glial activation per se. Together, the results implicate the astrocytic CN/NFAT4 pathway as a key mechanism for disrupting synaptic remodeling and homeostasis in the hippocampus after acute injury. SIGNIFICANCE STATEMENT: Similar to microglia, astrocytes become strongly "activated" with neural damage and exhibit numerous morphologic/biochemical changes, including an increase in the expression/activity of the protein phosphatase calcineurin. Using adeno-associated virus (AAV) to inhibit the calcineurin-dependent activation of the transcription factor NFAT (Nuclear Factor of Activated T cells) selectively, we have shown that activated astrocytes contribute to neural dysfunction in animal models characterized by progressive/chronic neuropathology. Here, we show that the suppression of astrocytic calcineurin/NFATs helps to protect synaptic function and plasticity in an animal model in which pathology arises from a single traumatic brain injury. The findings suggest that at least some astrocyte functions impair recovery after trauma and may provide druggable targets for treating victims of acute nervous system injury.

Early engineering approaches to improve peptide developability and manufacturability.

Downstream success in Pharmaceutical Development requires thoughtful molecule design early in the lifetime of any potential therapeutic. Most therapeutic monoclonal antibodies are quite similar with respect to their developability properties. However, the properties of therapeutic peptides tend to be as diverse as the molecules themselves. Analysis of the primary sequence reveals sites of potential adverse posttranslational modifications including asparagine deamidation, aspartic acid isomerization, methionine, tryptophan, and cysteine oxidation and, potentially, chemical and proteolytic degradation liabilities that can impact the developability and manufacturability of a potential therapeutic peptide. Assessing these liabilities, both biophysically and functionally, early in a molecule's lifetime can drive a more effective path forward in the drug discovery process. In addition to these potential liabilities, more complex peptides that contain multiple disulfide bonds can pose particular challenges with respect to production and manufacturability. Approaches to reducing the disulfide bond complexity of these peptides are often explored with mixed success. Proteolytic degradation is a major contributor to decreased half-life and efficacy. Addressing this aspect of peptide stability early in the discovery process increases downstream success. We will address aspects of peptide sequence analysis, molecule complexity, developability analysis, and manufacturing routes that drive the decision making processes during peptide therapeutic development.

A genetically encoded aza-Michael acceptor for covalent cross-linking of protein-receptor complexes.

Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introducing into a protein an appropriately "tuned" electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, N(ε)-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.

Targeting astrocytes ameliorates neurologic changes in a mouse model of Alzheimer's disease.

Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimer's disease (AD) and most other neurodegenerative disorders, many astrocytes convert to a chronically "activated" phenotype characterized by morphologic and biochemical changes that appear to compromise protective properties and/or promote harmful neuroinflammatory processes. Activated astrocytes emerge early in the course of AD and become increasingly prominent as clinical and pathological symptoms progress, but few studies have tested the potential of astrocyte-targeted therapeutics in an intact animal model of AD. Here, we used adeno-associated virus (AAV) vectors containing the astrocyte-specific Gfa2 promoter to target hippocampal astrocytes in APP/PS1 mice. AAV-Gfa2 vectors drove the expression of VIVIT, a peptide that interferes with the immune/inflammatory calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, shown by our laboratory and others to orchestrate biochemical cascades leading to astrocyte activation. After several months of treatment with Gfa2-VIVIT, APP/PS1 mice exhibited improved cognitive and synaptic function, reduced glial activation, and lower amyloid levels. The results confirm a deleterious role for activated astrocytes in AD and lay the groundwork for exploration of other novel astrocyte-based therapies.

Inhibition of soluble tumor necrosis factor ameliorates synaptic alterations and Ca2+ dysregulation in aged rats.

The role of tumor necrosis factor α (TNF) in neural function has been investigated extensively in several neurodegenerative conditions, but rarely in brain aging, where cognitive and physiologic changes are milder and more variable. Here, we show that protein levels for TNF receptor 1 (TNFR1) are significantly elevated in the hippocampus relative to TNF receptor 2 (TNFR2) in aged (22 months) but not young adult (6 months) Fischer 344 rats. To determine if altered TNF/TNFR1 interactions contribute to key brain aging biomarkers, aged rats received chronic (4-6 week) intracranial infusions of XPro1595: a soluble dominant negative TNF that preferentially inhibits TNFR1 signaling. Aged rats treated with XPro1595 showed improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased protein levels for the GluR1 type glutamate receptor, and lower L-type voltage sensitive Ca(2+) channel (VSCC) activity in hippocampal CA1 neurons. The results suggest that diverse functional changes associated with brain aging may arise, in part, from selective alterations in TNF signaling.

Evaluating the global CpG methylation status of native DNA utilizing a bipartite split-luciferase sensor.

Epigenetic modifications play an essential role in the regulation of gene expression and ultimately cell fate. Methylation of cytosine at CpG dinucleotides (mCpG) is an important epigenetic mark that has been correlated with cancer when present at promoter sites of tumor suppressor genes. To develop a rapid methodology for the direct assessment of global levels of DNA methylation, we first interrogated the methyl-CpG binding domains (MBDs), the Kaiso family of Cys(2)-His(2) zinc fingers, and an SET- and RING-associated domain using a split-luciferase reassembly methodology. We identified MBD1 as the most selective domain for the discrimination between mCpG and CpG sites with over 90-fold selectivity. Utilizing a bipartite strategy, we constructed a purely methylation-dependent bipartite sensor for the direct detection of global levels of DNA methylation by attaching MBD1 domains to each of the split-luciferase halves. This new sensor was validated for the direct determination of genomic DNA methylation levels in in vitro studies without any intervening chemical or enzymatic processing of DNA. Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor.

Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA.

The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50-80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair.

A turn-on split-luciferase sensor for the direct detection of poly(ADP-ribose) as a marker for DNA repair and cell death.

Designed sensors comprising split-firefly luciferase conjugated to tandem poly(ADP-ribose) binding domains allow for the direct solution phase detection of picogram quantities of PAR and for monitoring temporal changes in poly(ADP-ribosyl)ation events in mammalian cells.

Profiling small molecule inhibitors against helix-receptor interactions: the Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2.

We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein-protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.

A general approach for receptor and antibody-targeted detection of native proteins utilizing split-luciferase reassembly.

The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.

Toward a general approach for RNA-templated hierarchical assembly of split-proteins.

The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.

Disparate effects of serum on basal and evoked NFAT activity in primary astrocyte cultures.

In astrocytes, the Ca(2+)-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either approximately 50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5h after treatment with vehicle alone to assess basal NFAT activity, or with Ca(2+) mobilizers and IL-1 beta to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1 beta, TNFalpha, and/or IFN gamma, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.

Interleukin-1beta-dependent signaling between astrocytes and neurons depends critically on astrocytic calcineurin/NFAT activity.

Interleukin-1beta (IL-1beta) and the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, have each been shown to play an important role in neuroinflammation. However, whether these signaling molecules interact to coordinate immune/inflammatory processes and neurodegeneration has not been investigated. Here, we show that exogenous application of IL-1beta (10 ng/ml) recruited calcineurin/NFAT (nuclear factor of activated T cells) activation in primary astrocyte-enriched cultures within minutes, through a pathway involving IL-1 receptors and L-type Ca(2+) channels. Adenovirus-mediated delivery of the NFAT inhibitor, VIVIT, suppressed the IL-1beta-dependent induction of several inflammatory mediators and/or markers of astrocyte activation, including tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor, and vimentin. Expression of an activated form of calcineurin in one set of astrocyte cultures also triggered the release of factors that, in turn, stimulated NFAT activity in a second set of "naive" astrocytes. This effect was prevented when calcineurin-expressing cultures co-expressed VIVIT, suggesting that the calcineurin/NFAT pathway coordinates positive feedback signaling between astrocytes. In the presence of astrocytes and neurons, 48-h delivery of IL-1beta was associated with several excitotoxic effects, including NMDA receptor-dependent neuronal death, elevated extracellular glutamate, and hyperexcitable synaptic activity. Each of these effects were reversed or ameliorated by targeted delivery of VIVIT to astrocytes. IL-1beta also caused an NFAT-dependent reduction in excitatory amino acid transporter levels, indicating a possible mechanism for IL-1beta-mediated excitotoxicity. Taken together, the results have potentially important implications for the propagation and maintenance of neuroinflammatory signaling processes associated with many neurodegenerative conditions and diseases.

Site-specific detection of DNA methylation utilizing mCpG-SEER.

Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site.