Targeting of Neuronal Proteins and Dendrite Branching

Our research focuses on two processes that are important for proper communication to occur in the brain. The first is the mechanism underlying proper patterning of dendrites on a neuron.  Since the way that a neuron receives information is determined by the branching pattern of its dendrites, understanding this process will help us to understand why disorders occur in which dendrites are patterned incorrectly. The second process that we study is how signaling molecules, such as neurotransmitter receptors and enzymes, are targeted to synaptic sites on the dendrites of neurons.  Again, a number of disorders occur when there is improper targeting of these proteins in neurons. 

Our laboratory has found that a common molecule in these two processes is a family of proteins called MAGUKs (membrane-associated guanylate kinases).  These proteins contain domains, such as the PDZ domain, to which other proteins bind.  As such, the MAGUKs act as scaffolds to bring together receptors and their signaling partners at the synapse.  We use primary cultures of hippocampal neurons to study the function of MAGUKs and their binding partners and have identified novel functions for these proteins.  Our results are of therapeutic value for cognitive disorders.

Identification of cypin (cytosolic PSD-95 interactor)

 Driven by an interest in understanding how receptors and their effectors are trafficked to distinct sites on polarized cells, we decided to use hippocampal neuron cultures as a model system and to use molecular approaches to address these questions.  Since one particular MAGUK, the postsynaptic density protein-95 (PSD-95), is almost exclusively localized to synaptic sites in these neurons, we are interested in understanding how this precise localization occurs. Dr. Firestein identified a novel protein, called cypin (cytosolic PSD-95 interactor, which acts to decrease PSD-95 localization at synaptic sites. Binding of cypin to PSD-95 is necessary but not sufficient to cause this decrease.  Furthermore, no other known PSD-95 binding partner can decrease PSD-95 localization, and thus, cypin was the first protein shown to regulate MAGUK localization.  Since PSD-95 is thought to be a major player in pathways underlying learning and memory, this work, which was published in Neuron, is significant for understanding how learning and memory is regulated.

The role of cypin in dendrite patterning

In addition to being a PSD-95 binding protein, cypin also acts as an enzyme that breaks down guanine into xanthine and ammon ia.  Until now, the role of this enzymatic activity in neuronal function has been poorly understood.  To begin to elucidate the role of guanine breakdown by cypin in neurons, we constructed mutants of cypin protein and first expressed them in a non-neuronal cell line (COS-7 cells).  We found that cypin’s enzymatic activity is dependent on two functional domains found within the cypin protein: a zinc-binding domain and a domain that has high homology to CRMP, a protein involved in axonal outgrowth (Akum et al., 2004). In contrast, we found that cypin’s enzymatic activity is not dependent on its binding to PSD-95.  Since cypin is expressed early in development of hippocampal neurons and has homology to CRMP, we hypothesized that cypin may play a role in dendrite patterning.  Indeed, we found that overexpression of wildtype (or normal) cypin and not mutant forms of cypin in hippocampal neurons results in an increase in the number of dendrites and dendrite branches.  This increase correlates directly with cypin’s enzymatic activity.  In addition, we found that the suppression of cypin protein expression decreases the number of dendrites and dendrite branches.  Moreover, we have found that cypin binds directly to tubulin heterodimers, thereby promoting microtubule assembly.  Microtubules act as the cytoskeleton, and it is this cytoskeleton that underlies the morphology of dendrites, including their branching patterns.  Thus, cypin acts to regulate dendrite branching by breaking down guanine and by promoting microtubule assembly (Akum et al., 2004).  Our studies provide the first direct link between guanine metabolism and dendrite morphology, two functions that go awry in a host of cognitive disorders.

The role of PSD-95 in dendrite patterning

Our further analysis of the role of cypin in regulating dendrite number revealed that cypin’s interaction with PSD-95 is essential for stabilizing newly formed dendrite branches.  To further analyze the role of PSD-95 in dendrite patterning, we assayed dendrite number by altering PSD-95 protein levels in cultured hippocampal neurons. When PSD-95 is overexpressed, the number of dendrite branches, but not primary dendrites, decreases (Charych et al., 2006). Conversely, when PSD-95 expression is decreased in these neurons, the number of dendrite branches, but not the number of primary dendrites , increases.   These studies were performed at a developmental stage when the majority of PSD-95 clusters are not synaptic. In addition, we found that PSD-95 decreases branching independently of a form of neuronal activity that has also been shown to affect dendrite branching. Thus, we have found a nonsynaptic function of PSD-95 that may be activity-independent.

The observations described above lead to a model whereby PSD-95 intrinsically acts as a stop signal for dendrite branching.  In elucidating the mechanism by which this occurs, we have found that when overexpressed in COS-7 cells, PSD-95 disrupts the organization of the microtubule cytoskeleton (Charych et al., 2006).  Similarly, studies in progress suggest that PSD-95 also disrupts microtubule organization in neurons.  We have also found that the other three PSD-95 family members (SAP-102, SAP-97, and PSD-93) appear to have no effect on the microtubule cytoskeleton.  Our studies suggest a new and exciting mechanism for the regulation of the cytoskeleton and dendrite branching in hippocampal neurons by PSD-95.  

Snapin as a binding partner for cypin: role in dendritic patterning

We have also performed a yeast two-hybrid screen to identify binding partners that may regulate cypin function.  I previously screened a rat brain library with a cypin mutant lacking a sequence motif that binds to PDZ domains, protein-protein interaction domains found on proteins such as PSD-95.  Using this strategy, I identified 11 putative interacting proteins.  Our laboratory is currently focusing on one of these proteins, snapin.  Although originally identified as a regulator of SNAP-25-mediated synaptic vesicle fusion, there is some debate about the function of this protein. Using biochemical techniques, we have found that snapin binds to the CRMP homology domain of cypin and competes with tubulin for binding to this region. (Chen et al., 2005)  As a result of this competition, snapin binding to cypin significantly slows cypin-promoted microtubule assembly. By immunocytochemical assay, we have found that snapin protein is concentrated in hippocampal neuron cell bodies in developing neurons and that snapin expression increases as primary dendrite development slows down.  Consistent with these data, overexpression of snapin results in a decrease in primary dendrite number in hippocampal neurons.  Taken together, our data support a model whereby snapin binds to cypin in the cell body, attenuating cypin-promoted microtubule assembly and primary dendrite formation.  In the dendrites, where there is little snapin, cypin can continue to promote secondary dendrite branching unencumbered.  Thus, we have found a postsynaptic function for a protein that has previously been characterized solely as a presynaptic molecule. 

Study of DLG-1, a MAGUK protein in C. elegans

Since C. elegans affords easy manipulation of protein levels using genetics, I collaborated with Dr. Chris Rongo to assess the role of MAGUK proteins and their partners in establishing cellular polarity.  We found that DLG-1, a PSD-95 family member, and its putative binding partner cypin localize to adherens junctions in epithelial cells (Firestein and Rongo, 2001; Köppen et al., 2001).  Furthermore, knocking out DLG-1 disrupted both adherens junction formation and cypin localization.  Our studies showed a new role at the adherens junction for MAGUK proteins and their binding partners.

Cypin and sec8 compete for PSD-95 binding

Our studies with C. elegans yielded valuable insight into the role of the PSD-95 family of proteins and cypin in maintaining cellular polarity.  We complemented these studies by examining how cypin may regulate MAGUK localization in mammalian cells.  Cypin binds to the first two PDZ domains of PSD-95, which localize PSD-95 to the synapse.  We thus hypothesized that cypin may act to compete the binding of a targeting or chaperone molecule to PSD-95 to block trafficking of PSD-95 to the synapse.  To identify proteins that may be involved in PSD-95 localization, we searched for PDZ binding motifs in proteins known to be involved in protein trafficking. We found that two members of the exocyst family, sec8 and exo84, contain consensus sequences for PDZ binding (Riefler et al., 2003).  We found that both of these subunits bind to the first and second PDZ domains of PSD-95, and that sec8 and PSD-95 colocalize in PC12 cells, a model system for neurons.  Furthermore, we have also observed that cypin competes with sec8 for PSD-95 binding.  Our results suggest that the exocyst complex plays a role linking PSD-95 to microtubules or to vesicles, two mechanisms previously shown to be involved in PSD-95 targeting.  Subsequent to the initial interaction, cypin can compete sec8 binding, thereby releasing PSD-95 from the microtubules or vesicles needed for localization.  Our findings demonstrate a potential mechanism for regulating the trafficking of PSD-95 to the synapse, a process that is tightly controlled during learning and memory as well as during development.

A novel role for the PDZ domain of nNOS: localization of CtBP

Although the majority of the projects in our laboratory focus on the function of MAGUK proteins and their binding partners, a different but highly related project that we have worked on focuses on the role of the PDZ domain of neuronal nitric oxide synthase (nNOS) in the localization of other proteins in neurons.  Using affinity chromatography, we identified the transcriptional co-repressor CtBP as a binding partner of nNOS (Riefler et al., 2001).  Using biochemical techniques, we have shown that CtBP binds to the peptide-binding site of the PDZ domain of nNOS.  Furthermore, this interaction provides a novel function for nNOS.  In neurons, a portion of nNOS is localized to the synapse via its interaction with PSD-95.  As such, it is poised to respond to activation of the NMDA receptor by glutamate.  The nNOS that interacts with CtBP is not synaptically localized; it is cytosolic.  Our results suggest that nNOS traps CtBP in the cytosol, blocking the interaction of CtBP with its nuclear targets for co-repression.  Thus, we have found a novel nonsynaptic function for the PDZ domain of nNOS. 

Glial-neuronal interactions:  Uric acid protects neurons from glutamate-toxicity

We have just begun to develop a project aimed at identifying mechanisms by which spinal cord neurons can be protected after injury.  It has been reported that uric acid can reduce damage to neurons elicited by oxidative stress. Since uric acid binds directly to peroxynitrite, a free radical generated during injury, it is believed that this is the mechanism by which uric acid confers protection.  In contrast, our studies utilizing cultures derived from embryonic rat spinal cord indicate that an astroglia-mediated mechanism is involved in the effects of uric acid to protect neurons from glutamate toxicity (Du et al., in press).   The damage elicted by glutamate to neurons in a mixed culture of spinal cord cells can be reversed by uric acid. Furthermore, addition of uric acid after the termination of glutamate exposure suggests that uric acid plays an active role in mediating neuroprotection rather than purely binding peroxynitrite, as previously thought.  Importantly, in pure neuron cultures from the same tissue, uric acid does not protect against glutamate toxicity.  Addition of astroglia to the pure neuron cultures restores the ability of uric acid to protect the neurons from glutamate-induced toxicity. Our results also suggest that glia provide EAAT-1 and EAAT-2 glutamate transporters to protect neurons from glutamate and that functional EAATs may be necessary to mediate the effects of UA. Thus, we have found an active role for glia in neuronal recovery after spinal cord injury.