Michelle L. Previtera

Automated Sholl analysis of digitized neuronal morphology at multiple scales: Whole cell Sholl analysis versus Sholl analysis of arbor subregions

The morphology of dendrites and the axon determines how a neuron processes and transmits information. Neurite morphology is frequently analyzed by Sholl analysis or by counting the total number of neurites and branch tips. However, the time and resources required to perform such analysis by hand is prohibitive for the processing of large data sets and introduces problems with data auditing and reproducibility. Furthermore, analyses performed by hand or using course‐grained morphometric data extraction tools can obscure subtle differences in data sets because they do not store the data in a form that facilitates the application of multiple analytical tools. To address these shortcomings, we have developed a program (titled “Bonfire”) to facilitate digitization of neurite morphology and subsequent Sholl analysis. Our program builds upon other available open‐source morphological analysis tools by performing Sholl analysis on subregions of the neuritic arbor, enabling the detection of local level changes in dendrite and axon branching behavior. To validate this new tool, we applied Bonfire analysis to images of hippocampal neurons treated with 25 ng/ml brain‐derived neurotrophic factor (BDNF) and untreated control neurons. Consistent with prior findings, conventional Sholl analysis revealed that global exposure to BDNF increases the number of neuritic intersections proximal to the soma. Bonfire analysis additionally uncovers that BDNF treatment affects both root processes and terminal processes with no effect on intermediate neurites. Taken together, our data suggest that global exposure of hippocampal neurons to BDNF results in a reorganization of neuritic segments within their arbors, but not necessarily a change in their number or length. These findings were only made possible by the neurite‐specific Sholl data returned by Bonfire analysis.

PSD-95 Alters Microtubule Dynamics via an Association With EB3

Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology.

Effects of substrate stiffness and cell density on primary hippocampal cultures.

Previous studies have shown that dendrites are influenced by substrate stiffness when neurons are plated in either pure or mixed cultures. However, because substrate rigidity can also affect other aspects of culture development known to impact dendrite branching, such as overall cell number, it is unclear whether substrate stiffness exerts a direct or indirect effect on dendrite morphology. In this study, we determine whether substrate stiffness plays a critical role in regulating dendrite branching independent of cell number. We plated primary mixed hippocampal cultures on soft and stiff gels, with Youngs moduli of 1 kPa and 7 kPa, respectively. We found that neurons plated on stiffer substrates showed increased branching relative to neurons grown on softer substrates at the same cell number. On the stiff gels, we also observed a cell number-dependent effect, in which increasing initial plating density decreased dendrite branching. This change correlates with an increase in extracellular glutamate. We concluded that both cell number and substrate stiffness play roles in determining dendrite branching, and that the two effects are independent of one another.

Regulation of dendrite arborization by substrate stiffness is mediated by glutamate receptors.

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. To assess how extracellular stiffness affects dendrite morphology, we took biomaterial and biomedical engineering approaches. We cultured mixed and pure hippocampal neurons on hydrogels composed of polyacrylamide (PA) of varying stiffnesses to mimic the effects of extracellular matrix stiffness on dendrite morphology. The majority of investigations of cortical and spinal cord neurons on soft hydrogels examined branching at early time points (days in vitro (DIV) 2–7), an important distinction from our study, where we include later time points that encompass the peak of branching (DIV 10–12). At DIV 12, dendrite branching was altered by stiffness for both pure and mixed neuronal cultures. Furthermore, we treated hippocampal cultures with glutamate receptor antagonists and with astrocyte-conditioned media. Blocking AMPA and NMDA receptors affected the changes in dendrite branching seen at varying rigidities. Moreover, extracellular factors secreted by astrocytes also change dendrite branching seen at varying rigidities. Thus, astrocytes and ionotropic glutamate receptors contribute to mechanosensing.

Cell Growth in Response to Mechanical Stiffness is Affected by Neuron- Astroglia Interactions

Cell adhesion and morphology are affected by the mechanical properties of the extracellular matrix. Using polyacrylamide gels as cell substrates, the cellular response to substrate compliance was investigated in pure neuronal, pure astroglial, or mixed co-cultures. Substrates used spanned a large range of stiffnesses including that of brain tissue. In both pure and mixed cultures, immature (vimentin+) astroglia adhered best to stiffest gels. Mature (GFAP+) astrocyte ad- hesion peaked on intermediate stiffness, while pure GFAP+ astroglial adhesion displayed no intermediate preference and increased with stiffness. Neurite length was constant with stiffness; however, primary dendrite number was lowest on in- termediate gels. Pure neuronal cultures were more adherent to hard gels, while mixed cultures had no stiffness preference. Furthermore, we investigated the role of stiffness in the modulation of the neurotoxic effect of glutamate. Exposure to two glutamate concentrations (500 and 1000 � M) of cultured spinal cord neurons induced cell death. The damage elicited by 500 � m glutamate to neurons in a mixed culture of spinal cord cells is most severe on soft 300 Pa gels. The neurotoxic ef- fect of glutamate on neurons cultured on hard gels where astrocytes are present was strongly attenuated compared with that observed on soft gels, where there is a relatively low number of astrocytes. Our data suggest that mechanical stiffness of the substrate affects the response of both neurons and astroglia, and this response is varied by interaction between the two cell types.

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 substrate elastic modulus on neural precursor cell behavior.

The spinal cord has a limited capacity to self-repair. After injury, endogenous stem cells are activated and migrate, proliferate, and differentiate into glial cells. The absence of neuronal differentiation has been partly attributed to the interaction between the injured microenvironment and neural stem cells. In order to improve post-injury neuronal differentiation and/or maturation potential, cell–cell and cell–biochemical interactions have been investigated. However, little is known about the role of stem cell–matrix interactions on stem cell-mediated repair. Here, we specifically examined the effects of matrix elasticity on stem cell-mediated repair in the spinal cord, since spinal cord injury results in drastic changes in parenchyma elasticity and viscosity. Spinal cord-derived neural precursor cells (NPCs) were grown on bis-acrylamide substrates with various rigidities. NPC growth, proliferation, and differentiation were examined and optimal in the range of normal spinal cord elasticity. In conclusion, limitations in NPC growth, proliferation, and neuronal differentiation were encountered when substrate elasticity was not within normal spinal cord tissue elasticity ranges. These studies elucidate the effect injury mediated mechanical changes may have on tissue repair by stem cells. Furthermore, this information can be applied to the development of future neuroregenerative biomaterials for spinal cord repair.

NOS1AP protein levels are altered in BA46 and cerebellum of patients with schizophrenia.

Dear Editors, Brzustowicz and colleagues (2004) identified significant linkage disequilibrium between schizophrenia and markers within the gene encoding nitric oxide synthase 1 (neuronal; NOS1) adaptor protein (NOS1AP; also termed carboxyl-terminal PDZ ligand of nNOS or CAPON). Quantitative real-time PCR (qRT-PCR) analysis of mRNA from human postmortem dorsolateral prefrontal cortex further revealed that expression of the short isoform of the NOS1AP gene (NOS1AP-S) is significantly increased in patients with schizophrenia (Xu, et al., 2005). More recently, the group also identified a functional risk allele within NOS1AP and showed that this change increased NOS1AP mRNA expression in a cell culture system (Wratten, et al., 2009). Despite these recent reports establishing linkage between NOS1AP and schizophrenia, little is known about NOS1AP protein expression in the brains of affected patients. Previous reports described two distinct NOS1AP isoforms: full-length NOS1AP-L (10 exons, ~75kD) and NOS1AP-S, a C-terminal specific transcript that encodes only the PDZ domain (Jaffrey, et al., 1998; Xu, et al., 2005). We have now identified a novel isoform, NOS1AP-S’ (Figure 1A), in mouse and human tissue using qRT-PCR (data not shown). To evaluate the expression levels of these three NOS1AP isoforms in human brain tissue, postmortem samples from Brodmann’s Area (BA) 46, BA11, Medial Temporal Lobe (MTL), Occipital Lobe (OL), and cerebellum of unaffected patients and those with schizophrenia were obtained from the Human Brain and Spinal Fluid Resource Center (Los Angeles, CA) and subjected to Western blotting with normalization to GAPDH or actin, as previously described (Xu, et al., 2005). Investigators were blinded to all subject information until after statistical analysis. The logarithms of the normalized values for subjects with schizophrenia and unaffected control patients within the same brain region were compared using the standard t-test. Correction for testing of multiple expression levels was made using permutations of case/control labels. Secondary examination of linear models with other covariates was based on the AICc model selection criterion (Burnham, 2002). The L (p = 0.0067; reported p-values are nominal), S´ (p = 0.0082) and S (p = 0.0041) isoforms were increased in BA46 of patients with schizophrenia (Figure 1B) at a nominal significance level, although only the increase in the S isoform was significant (p<0.05) under permutation-based multiple testing adjustment. These data are consistent with previous reports strongly implicating this region in the etiology of schizophrenia (Barch, 2005; Bunney and Bunney, 2000; Xu, et al., 2005). The L (p = 0.0031), S (p = 0.0060), and S´ (p = 0.0156) isoforms were decreased in cerebellum of affected individuals (Figure 1B), although only the decrease of the L isoform was significant under adjustment. While some reports have indicated that schizophrenia may affect the cerebellum, the results are not as consistently observed as in other regions, namely the prefrontal cortex (Avila, et al., 2002; Kapoor, et al., 2006). There were no significant differences in NOS1AP expression between control and affected patients in BA11, the MTL, or the OL (Figure 1B). Additional analysis of NOS1AP expression reveals that no significant changes were evident in BA11, the MTL, or the OL of patients with schizophrenia versus those who are unaffected. While some studies have reported a role for BA11 in schizophrenia, others find no changes in expression of NMDA receptor pathway proteins (Toro and Deakin, 2005). Figure 1 (A) NOS1AP isoforms. The intron/exon boundaries and the predicted transcriptional (arrow) and translational (ATG) start sites for the different NOS1AP isoforms are illustrated. The new NOS1AP isoform is characterized by a unique 5’ exon and transcriptional ... Our data show an alteration of three NOS1AP isoforms in specific regions of the brain for patients diagnosed with schizophrenia. Initially identified in rat, NOS1AP plays a role in the inhibition of glutamate neurotransmission via disruption of NOS1 binding to Postsynaptic Density Protein-95 and -93. This results in uncoupling of NOS1 from the NMDA receptor, and ultimately, inhibition of receptor function (Brzustowicz, 2008; Jaffrey, et al., 1998; Xu, et al., 2005). These data suggest a role for NOS1AP in glutamate receptor hypofunction and manifestation of schizophrenia.

Mechanical Properties of DNA-Crosslinked Polyacrylamide Hydrogels with Increasing Crosslinker Density

Abstract DNA-cross-linked polyacrylamide hydrogels (DNA gels) are dynamic mechanical substrates. The addition of DNA oligomers can either increase or decrease the crosslinker density to modulate mechanical properties. These DNA-responsive gels show promise as substrates for cell culture and tissue-engineering applications, since the gels allow time-dependent mechanical modulation. Previously, we reported that fibroblasts plated on DNA gels responded to modulation in elasticity via an increase or decrease in crosslinker density. To better characterize fibroblast mechanical signals, changes in stress and elastic modulus of DNA gels were measured over time as crosslinker density altered. In a previous study, we observed that as crosslinker density decreased, stress was generated, and elasticity changed over time; however, we had not evaluated stress and elastic modulus measurements of DNA gels as crosslinker density increased. Here, we completed this set of fibroblast studies by reporting stress and elastic modulus measurements over time as the crosslinker density increased. We found that the stress generated and the elastic modulus alterations were correlated. Hence, it seemed impossible to separate the effect of stress from the effect of modulus changes for fibroblasts plated on DNA gels. Yet, previous results and controls revealed that stress contributed to fibroblast behavior.

Glutamate affects dendritic morphology of neurons grown on compliant substrates

Brain stiffness changes in response to injury or disease. As a secondary consequence, glutamate is released from neurons and astroglia. Two types of glutamate receptors, N‐methyl‐d‐aspartate (NMDA) and α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, sense mechanotransduction, leading to downstream signaling in neurons. Recently, our group reported that these two receptors affect dendrite morphology in hippocampal neurons grown on compliant substrates. Blocking receptor activity has distinct effects on dendrites, depending on whether neurons are grown on soft or stiff gels. In the current study, we examine whether exposure to glutamate itself alters stiffness‐mediated changes to dendrites in hippocampal neurons. We find that glutamate augments changes seen when neurons are grown on soft gels of 300 or 600 Pa, but in contrast, glutamate attenuates changes seen when neurons are grown on stiff gels of 3,000 Pa. These results suggest that there is interplay between mechanosensing and glutamate receptor activation in determining dendrite morphology in neurons.