Firestein Laboratory




Cypin is a positive regulator of dendrite patterning

The major finding of this research is that localized microtubule assembly regulates dendrite branching.  Microtubules act as the cytoskeleton, and it is this cytoskeleton that creates the morphology of dendrites, including their branching patterns. It was previously thought that microtubules were transported into dendrites to increase growth and branching; however, we found that a protein termed cypin promotes localized microtubule assembly in the dendrite during arborization. Specifically, 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 (Akum et al., 2004; Fernandez et al., 2008; Fernandez et al., 2009) and that the suppression of cypin protein expression decreases the number of dendrites and dendrite branches.  Cypin binds directly to tubulin heterodimers, thereby promoting microtubule assembly.  Furthermore, we found a novel mechanism by which the small GTPase, RhoA, regulates dendritogenesis through a cypin-dependent pathway (Chen and Firestein, 2007). We found that activated RhoA acts to inhibit cypin protein expression, via a translation-dependent mechanism, and by doing so, decreases dendrite number. Since RhoA protein expression itself is locally translated, our data suggest that by regulating the expression of cypin, RhoA itself serves to regulate local microtubule assembly.

PSD-95 is a negative regulator of dendrite patterning

The major finding of this research is that PSD-95, an essential protein for synaptic function, acts in a nonsynaptic manner to regulate microtubule dynamics and regulate dendrite branching. Cypin’s interaction with PSD-95 is essential for stabilizing newly formed dendrite branches (Charych et al., 2006).  When PSD-95 is overexpressed, dendrite arborization is significantly decreased (Charych et al., 2006; Sweet et al., 2011a,b). Conversely, when PSD-95 expression is decreased in these neurons, the number of dendrite branches increases.  More recently, we identified a mechanism by which PSD-95 decreases dendrite number (Sweet et al., 2011a,b).  We found that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3, a protein that regulates the growth and catastrophe of microtubules. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites, representing altered microtubule dynamics, less organized microtubules at dendritic branch points, and decreased dendritogenesis.  Our studies suggest a new and exciting mechanism for the regulation of the cytoskeleton and dendrite branching in hippocampal neurons by PSD-95.

Another regulator of dendritic patterning, NOS1AP, is linked to schizophrenia

The major finding of this research is that NOS1AP, a protein with altered expression in brain of patients with schizophrenia, alters dendritogenesis.  Previously, Dr. Linda Brzustowicz of the Department of Genetics at Rutgers University 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). We began to collaborate with Dr. Brzustowicz when she showed that there are two forms of NOS1AP (L, long and S, short) and that expression of NOS1AP-S mRNA is significantly increased in the dorsolateral prefrontal cortex of patients with schizophrenia (Xu, et al., 2005).  In line with this report, we have recently found an alteration of NOS1AP isoforms in specific regions of the brain for patients diagnosed with schizophrenia (Hadzimichalis et al., 2010). Since there are reports suggesting that schizophrenia is a developmental disorder, we analyzed whether altering NOS1AP levels in developing neurons affects their development. We found that overexpression of NOS1AP during hippocampal neuron development in culture results in decreased dendrite number (Carrel et al., 2009). We identified carboxypeptidase E (CPE) as a binding partner for the middle region of NOS1AP and showed that NOS1AP signals through CPE to regulate dendrite number. Taken together, our results suggest that NOS1AP plays an important role in the initiation, outgrowth, and maintenance of dendrites during development and that overexpression of NOS1AP may play a role in the pathophysiology of schizophrenia.

Substrate stiffness influences CNS cell growth and dendritogenesis

The major finding of this research is that neurons sense the stiffness of the substrate on which they are grown via glutamate receptor-mediated signaling to influence dendrite branching.  This is a new and exciting area of focus for my laboratory. Normally, the development of neurons and glia are studied using cells grown on glass or plastic both of which have stiffness on the order of GPa. Brain tissue is much less stiff than these materials, and it changes in response to disease or injury. To address whether stiffness has an effect on dendrite morphology, we grew cells on bis-acrylamide crosslinked hydrogels popularized by Wang and Pelham. We found that neurons grown on stiffer substrates have more dendrites than those grown on softer substrates and that this effect is due, in part, to factors secreted by astrocytes (Previtera et al., 2010a,b). We found that cultures plated on stiffer substrates release more glutamate and that blocking AMPA and NMDA receptors on neurons eliminated differences in dendrite number in neurons grown on gels of different stiffnesses (Previtera et al., 2010b). Thus, we provided the first mechanism by which dendrites are changed in response to stiffness: the fact that astrocytes and ionotropic glutamate receptors contribute to mechanosensing.  Our studies provide an understanding of the mechanisms by which CNS cells, and neurons in particular, sense differences in substrate rigidity, providing insight into what may occur during development or disease states.

Effects of glutamate on neuronal connectivity

The major finding of this research is that loss of action potential synchronization is dependent on the initial amount of synchronization prior to injury.  Microelectrode arrays have been used to study the pharmacological and toxicological response of neuronal networks to numerous compounds. The large number of electrodes evenly spaced in a grid provides information on the response of neurons to pharmacological treatments throughout the network.  Additionally, neuronal cultures grown on MEAs respond to compounds in the same concentration ranges that result in functional changes in vivo. In our work, we use MEAs to record the electrical activity of cortical neurons to analyze the functional changes that occur after exposure to different glutamate concentrations.  Until now, we have primarily used MAP2 immunostaining or LDH assays to determine neuronal death in response to excess glutamate.  However, these assays do not address whether compounds that neuroprotect also preserve neuronal circuitry. Our data show that treatment with different concentrations of glutamate results in different injury profiles and that the initial level of synchronized firing between regions of a neuronal network affects how they respond to injury (Kutzing et al., 2011).  We also analyzed the use of memantine for protection of neuronal connectivity from glutamate-induced injury.  We found that the timing of memantine treatment is important for conferring neuroprotection against glutamate-induced neurotoxicity (Kutzing et al., 2012).

Using iPSC technology to study the role of NOS1AP in schizophrenia

Neuronal dendrites are branched cellular processes with bulbous surface structures called spines, required for receiving synaptic connections between neurons. When aberrations in these processes occur, cognitive disorders, such as schizophrenia, result. How the dendrite is patterned and shaped during development determines neuronal circuitry and information processing needed for normal brain function. We have chosen to study nitric oxide synthase 1 adaptor protein (NOS1AP). NOS1AP is a known regulator of NMDA receptor and nNOS activity, both of which have been shown to regulate dendrite morphology.  In addition, NOS1AP has been identified as a susceptibility locus for schizophrenia in genetic studies. We have found that NOS1AP is expressed at higher levels in the dorsolateral prefrontal cortex of postmortem brains from patients with schizophrenia.  In addition, we found that overexpression of NOS1AP results in decreased dendrite number in primary cultures of hippocampal neurons. We hypothesize that the alteration of NOS1AP protein that occurs in genetic variants associated with schizophrenia results in aberrant neuronal development.  Until now, we have used cultured rat hippocampal neurons as our model system.  Although this model system is quite useful for elucidating the role that NOS1AP plays in neuronal development, the use of a more physiologically relevant system – human neurons – will help us to understand NOS1AP function in the context of clinically-diagnosed schizophrenia samples. We will use iPSC technology to make human neurons that have altered levels of NOS1AP by transfection of appropriate plasmids or from patients with schizophrenia and the associated NOS1AP risk allele. We will then characterize these neurons for differences in dendrite number and patterning and in neural circuitry. Our long-term goal is to establish a human neuronal culture model of the altered dendritic function in schizophrenia in order to discover mechanisms and to screen for novel therapeutic strategies.

Regulation of Neural Circuitry by Cypin

In the mammalian central nervous system, individual neurons have elaborate dendrite networks, reflecting the density and complexity of incoming presynaptic contacts. The extent of dendrite branching is influenced by learning, which increases branching, or injury, which decreases branching. The proposed work tests the hypothesis that the enzyme cytosolic PSD-95 interactor (cypin) is an essential mediator of learning and memory. Rutgers’ research team will use genetic models and pharmacological delivery in mice to identify the role cypin plays in memory acquisition and recall. Using compounds that we have identified as affecting multiple aspects of cypin activity, we will test whether altering cypin affects function and activity of neural circuits in vitro by microelectrode array (MEA) and Ca2+ imaging analyses. We will also administer the compounds to mice and measure neural dynamics with two-photon in vivo imaging and neurobehavior. These experiments will significantly add to our understanding of how neural circuitry at the cellular level in the animal as it translates into cognitive function.

mTOR/Akt signaling in traumatic brain injury: Progression and therapeutic approaches to improve outcome

We have chosen a relatively unexplored therapeutic pathway for treating TBI.  The mTOR/Akt is a central regulating pathway involved in metabolism, cell growth and proliferation, differentiation, and cellular decisions that include apoptosis and autophagy.  Unlike other receptor and channel antagonists tested for treating TBI, the mTOR/Akt pathway does not interrupt normal neurotransmission.  However, as an emerging set of studies points to the central importance of this pathway in regulating dendritic protein synthesis and synaptic plasticity, this pathway could be involved in shaping the recovery of neural circuits after TBI.  In addition, some limited evidence of mTOR directly playing a role in TBI is now appearing in the literature.

Our broad aims in this multi-investigator proposal answer three general questions:

  • Is the mTOR/Akt pathway consistently activated following either mild or moderate TBI?
  • Does this pathway contribute to the repair of neural circuit wiring and function after TBI?
  • Is there an optimal therapeutic approach to control the activation of mTOR/Akt signaling and improve outcome after TBI?



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