Christopher Nelson

TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3-IFT-A interaction suggests how these proteins cooperate during neural tube patterning.

PSD-95 Is Required to Sustain the Molecular Organization of the Postsynaptic Density

PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.

Anti-CD22-MCC-DM1 and MC-MMAF Conjugates: Impact of Assay Format on Pharmacokinetic Parameters Determination

CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters determination, we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately determine the ADC PK parameters.

Phosphorylation of Threonine-19 of PSD-95 by GSK-3β is Required for PSD-95 Mobilization and Long-Term Depression

Activity of glycogen synthase kinase-3β (GSK-3β) is required for long-term depression (LTD) via molecular mechanisms that are incompletely understood. Here, we report that PSD-95, a major scaffold protein of the postsynaptic density (PSD) that promotes synaptic strength, is phosphorylated on threonine-19 (T19) by GSK-3β. In cultured rat hippocampal neurons, phosphorylation of T19 increases rapidly with chemical LTD and is attenuated by pharmacologic or genetic suppression of GSK-3β. In organotypic rat hippocampal slices, we find that a nonphosphorylatable PSD-95 mutant (T19A) tagged with photoactivatable green fluorescent protein (PAGFP) shows enhanced stability in dendritic spines versus wild-type PSD-95, whereas the phosphomimetic mutant (PSD-95-T19D) is more readily dispersed. Further, overexpression of PSD-95-T19A, but not WT-PSD-95, impairs AMPA receptor internalization and the induction of LTD. These data indicate that phosphorylation on T19 by GSK-3β destabilizes PSD-95 within the PSD and is a critical step for AMPA receptor mobilization and LTD.

Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB–siRNA conjugates

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody–siRNA complexes provide a possible solution. However, initial reports of antibody–siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody–siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.

Gpr3 stimulates Aβ production via interactions with APP and β-arrestin2.

The orphan G protein-coupled receptor (GPCR) GPR3 enhances the processing of Amyloid Precursor Protein (APP) to the neurotoxic beta-amyloid (Aβ) peptide via incompletely understood mechanisms. Through overexpression and shRNA knockdown experiments in HEK293 cells, we show that β-arrestin2 (βarr2), a GPCR-interacting scaffold protein reported to bind γ-secretase, is an essential factor for GPR3-stimulated Aβ production. For a panel of GPR3 receptor mutants, the degree of stimulation of Aβ production correlates with receptor-β-arrestin binding and receptor trafficking to endocytic vesicles. However, GPR3’s recruitment of βarr2 cannot be the sole explanation, because interaction with βarr2 is common to most GPCRs, whereas GPR3 is relatively unique among GPCRs in enhancing Aβ production. In addition to β-arrestin, APP is present in a complex with GPR3 and stimulation of Aβ production by GPR3 mutants correlates with their level of APP binding. Importantly, among a broader selection of GPCRs, only GPR3 and prostaglandin E receptor 2 subtype EP2 (PTGER2; another GPCR that increases Aβ production) interact with APP, and PTGER2 does so in an agonist-stimulated manner. These data indicate that a subset of GPCRs, including GPR3 and PTGER2, can associate with APP when internalized via βarr2, and thereby promote the cleavage of APP to generate Aβ.

Evidence supporting a role for cathepsin B in the generation of T cell antigenic epitopes of human growth hormone

The elucidation of the enzymatic processing mechanism associated with the formation of antigenic peptide fragments that combine with MHC class II molecules is fundamental to our understanding of the immune system. We have investigated a structurally well defined protein, recombinant human growth hormone (rhGH), as an antigen, and present data supporting the hypothesis that the enzyme cathepsin B can produce peptide fragments bearing T cell epitopes associated with lymphocyte proliferative response to hGH in Balb/c (H-2dhaplotype) mice. Minimal T cell epitopes are not generated; rather the cathepsin cleavage sites flank the three antigenic peptide regions, amino acid residues 31-41, 81-100, and 166-181.

Prospects for implementation of thermoelectric generators as waste heat recovery systems in class 8 truck applications

With the rising cost of fuel and increasing demand for clean energy, solid-state thermoelectric (TE) devices are an attractive option for reducing fuel consumption and CO2 emissions. Although they are reliable energy converters, there are several barriers that have limited their implementation into wide market acceptance for automotive applications. These barriers include: the unsuitability of conventional thermoelectric materials for the automotive waste heat recovery temperature range; the rarity and toxicity of some otherwise suitable materials; and the limited ability to mass-manufacture thermoelectric devices from certain materials. One class of material that has demonstrated significant promise in the waste heat recovery temperature range is skutterudites. These materials have little toxicity, are relatively abundant, and have been investigated by NASA-JPL for the past twenty years as possible thermoelectric materials for space applications. In a recent collaboration between Michigan State University (MSU) and NASA-JPL, the first skutterudite-based 100 W thermoelectric generator (TEG) was constructed. In this paper, we will describe the efforts that have been directed towards: (a) enhancing the technology-readiness level of skutterudites to facilitate mass manufacturing similar to that of Bi2Te3, (b) optimizing skutterudites to improve thermal-to-electric conversion efficiencies for class 8 truck applications, and (c) describing how temperature cycling, oxidation, sublimation, and other barriers to wide market acceptance must be managed. To obtain the maximum performance from these devices, effective heat transfer systems need to be developed for integration of thermoelectric modules into practical generators. [DOI: 10.1115/1.4023097]

Real-time quantification of antibody–short interfering RNA conjugate in serum by antigen capture reverse transcription–polymerase chain reaction

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery.

Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial growth factor and epidermal growth factor-like domain 7

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.