UEV-1 Is an Ubiquitin-Conjugating Enzyme Variant That Regulates Glutamate Receptor Trafficking in C. elegans Neurons

The regulation of AMPA-type glutamate receptor (AMPAR) membrane trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the factors that mediate these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR trafficking in vivo. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in the accumulation of GLR-1 in elongated accretions in neuron cell bodies and along the ventral cord neurites. Mutants also have a corresponding behavioral defect—a decrease in spontaneous reversals in locomotion—consistent with diminished GLR-1 function. The localization of other synaptic proteins in uev-1-mutant interneurons appears normal, indicating that the GLR-1 trafficking defects are not due to gross deficiencies in synapse formation or overall protein trafficking. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants, and that receptors arrive at these endosomes independent of clathrin-mediated endocytosis. UEV-1 homologs in other species bind to the ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in most tissues. Nevertheless, ubc-13 mutants are similar in phenotype to uev-1 mutants, suggesting that the two proteins do work together to regulate GLR-1 trafficking. Our results suggest that UEV-1 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking

The effects of extracellular matrix compliance and protein expression on neurons:

Extracellular and intracellular cues affect neuronal morphology and contribute to brain diseases, such as schizophrenia, and injury. First, we examined how extracellular cues influence branching parameters of hippocampal neurons. Brain injury or disease can initiate changes in local or global stiffness of brain tissue. While stiffness of the extracellular environment is known to affect the morphology and function of many cell types, little is known about how the dendrites of neurons respond to changes in brain stiffness. We cultured hippocampal neurons on hydrogels composed of polyacrylamide of varying rigidities to mimic the effects of extracellular matrix stiffness on dendrite morphology. At 12 days in vitro, dendrite branching was altered by stiffness; i.e. branching peaked in neurons grown on gels of intermediate stiffness (8 kPa). Furthermore, we found that ionotropic glutamate receptors play roles in regulating dendrite morphology. AMPA receptors play a role in dendritc arborization for harder stiffness, >2kPa, at all distances from the cell body. NMDA receptors play a role in dendritic arborization for a range of rigidities (1-25 kPa), at only proximal and intermediate distances from the cell body. However, a caveat to these studies is that cell adhesion is affected by the rigidity of these substrates. Addressing this caveat is of great importance because cell density affects dendrite branching. Thus, we also determined whether substrate stiffness plays a critical role in determining dendrite branching independent of cell density. We concluded that substrate stiffness does play a crucial role in determining dendrite branching patterns independent of cell number; however, the density of cells plated on substrates also influences the dendrite branching pattern of neurons. In the second chapter of my thesis, we looked at how intracellular proteins in different sections of the human brain are affected in schizophrenia. By Western blotting, we examined human, postmortem brain samples. NOS1AP protein expression increased in the dorsal lateral prefrontal cortex of patients with schizophrenia and not in the occipital lobe, medial temporal lobe, or cerebellum. Thus, this thesis demonstrates how extracellular and intracellular cues affect disease states, such as brain injury and schizophrenia.Ph.D.Includes bibliographical references (p. 101-119)by Michelle L. Previter

Substrate Stiffness Regulates Proinflammatory Mediator Production through TLR4 Activity in Macrophages.

Clinical data show that disease adversely affects tissue elasticity or stiffness. While macrophage activity plays a critical role in driving disease pathology, there are limited data available on the effects of tissue stiffness on macrophage activity. In this study, the effects of substrate stiffness on inflammatory mediator production by macrophages were investigated. Bone marrow-derived macrophages were grown on polyacrylamide gels that mimicked the stiffness of a variety of soft biological tissues. Overall, macrophages grown on soft substrates produced less proinflammatory mediators than macrophages grown on stiff substrates when the endotoxin LPS was added to media. In addition, the pathways involved in stiffness-regulated proinflammation were investigated. The TLR4 signaling pathway was examined by evaluating TLR4, p-NF-κB p65, MyD88, and p-IκBα expression as well as p-NF-κB p65 translocation. Expression and translocation of the various signaling molecules were higher in macrophages grown on stiff substrates than on soft substrates. Furthermore, TLR4 knockout experiments showed that TLR4 activity enhanced proinflammation on stiff substrates. In conclusion, these results suggest that proinflammatory mediator production initiated by TLR4 is mechanically regulated in macrophages

Comparison of proinflammatory mediator concentrations in media between US (<i>white bars</i>) and TNF–α–stimulated (<i>grey bars</i>) BMM.

<p>BMMs were grown on soft <i>(left graphs)</i> and stiff <i>(right graphs)</i> substrates. N_wells≥7. Data were assessed using Mann–Whitney U test. N_wells≥ 4.</p

MyD88-dependent TLR4 signal transduction.

<p><i>(A)</i> TLR4–deficient BMMs grown on gels. Media from US (<i>white bars</i>) and stimulated (<i>grey bars</i>) TLR4–deficient (<i>TLR4</i>^–/–) and WT BMMs grown on 230 kPa gels were subjected to ELISAs and Griess assays. Graphs show concentrations of (<i>from top to bottom</i>) IL–1β, IL–6, and NO. Data from ELISAs were assessed using Kruskal–Wallis followed by Dunn’s multiple comparisons test and data from Griess assays were assessed using one–way ANOVA followed by Tukey’s multiple comparisons test. N_wells≥ 21. <i>(B)</i> Representative MyD88 (<i>top</i>) and IFN–β (<i>middle</i>) immunoblots. Lysates from US and stimulated BMM grown on 0.3, 47, and 230 kPa gels were subjected to Western blotting. Blots were probed with (<i>from top to bottom</i>) anti–MyD88, anti–IFN–β, and anti–GAPDH antibodies. <i>(C)</i> Densitometry. Data are a percentage of 230 kPa band intensities. Data were assessed using one–way ANOVA followed by Tukey’s multiple comparisons test. Densitometry for IFN–β was not performed because no visual changes were observed. Three or more samples were subjected to Western blotting.</p

TLR4 signal transduction on PA gels.

<p><i>(A)</i> Representative TLR4 immunoblot (<i>top</i>) and densitometry (<i>bottom</i>). Lysates from US and stimulated BMMs grown on 0.3, 47, or 230 kPa gels were subjected to Western blotting. Blots were probed with (<i>from top to bottom</i>) rabbit anti–TLR4 or anti–GAPDH antibodies. For densitometry, data are a percentage of 230 kPa band intensities. Data were assessed using one–way ANOVA followed by Tukey’s multiple comparisons test. <i>(B)</i> Representative IκBα immunoblot (<i>top</i>) and densitometry (<i>bottom</i>). Blots were probed with (<i>from top to bottom</i>) anti–p–IκBα, anti–IκBα, or anti–GAPDH antibodies. For densitometry, data are a ratio of p–IκBα/IκBα. Data were assessed using unpaired t test with Welch’s correction. <i>(C)</i> Representative NF–κB (<i>top</i>) immunoblot and densitometry (<i>bottom</i>). Blots were probed with (<i>from top to bottom</i>) anti–p–NF–κB, anti–NF–κB and anti–GAPDH antibodies. For densitometry, data are a ratio of p–NF–κB/NF–κB. Data were assessed using unpaired t test with Welch’s correction. <i>(D)</i> Representative p–NF–κB immunoblot for cytosolic (C) and nuclear (N) fractions (<i>top</i>) and densitometry (<i>bottom</i>). Blots were probed with anti–p–NF–κB antibody. For densitometry, data are a percentage of 230 kPa band intensities. Data were assessed using unpaired t test with Welch’s correction. Three or more samples were subjected to Western blotting for each antibody.</p

Comparison of proinflammatory mediator concentrations between US (<i>white bars</i>) and LPS–stimulated (<i>grey bars</i>).

<p>BMMs were grown on soft <i>(0</i>.<i>3 kPa</i>, <i>left graphs)</i> and stiff <i>(230 kPa</i>, <i>right graphs)</i> substrates. Data were assessed using Mann–Whitney U test. N_wells≥7.</p

Comparison of proinflammatory mediator concentrations in media between US (<i>white bars</i>) and stimulated (<i>grey bars</i>) BMMs.

<p>BMMs were grown on collagen–or laminin–functionalized soft (<i>left graphs)</i> and stiff <i>(right graphs)</i> gels. Data were assessed using Mann–Whitney U test. N_wells≥ 4.</p