Research in the Waschek laboratory has centered on a class of molecules called neuropeptides. Neuropeptides are short peptides that are classically known to act in the central and peripheral nervous systems as neurotransmitters and neuromodulators. In doing so, they control a wide variety of processes in the brain as well as cardiovascular, pulmonary, gastrointestinal, reproductive, and immune systems. The primary objective of the lab is to better understand the biological roles of neuropeptides, especially their potential action as growth factors and immunomodulators, and the mechanisms controlling their expression. We have focused much of our efforts on two structurally-related neuropeptides: vasoactive intestinal peptide (VIP), and pituitary adenyl cyclase activating peptide (PACAP). Our research falls primarily into the specific areas listed below. As indicated in the more detailed descriptions, in addition to using classical approaches to study their actions, we have generated transgenic and knockout mouse models for PACAP and VIP, Xenopus and zebrafish embryo mRNA injection/expression systems, and viral-based methods to express these peptides in vivo.
VIP and PACAP action in the developing nervous system
PACAP and PACAP Receptor (PAC1) Gene Expression in the embryonic day 10.5 Mouse Neural Tube
Neuropeptides are classically viewed as neurotransmitters or neuromodulators. However, a primary contribution of my laboratory to this field has been our demonstration that PACAP, and perhaps VIP, also act as growth factors to regulate neurogenesis and phenotypic specification. Moreover, they do so at the very earliest stages of nervous system development, i.e. before neural tube closure. Our data suggest that PACAP also acts at later stages to regulate other aspects of brain development, including cerebellar growth, gliogenesis and myelination.
Our work in this area began when we found that the VIP gene is expressed in the mouse brain as early as embryonic day 11 (J Neurochem., 1996). Later, we showed that a functional PACAP ligand receptor signaling system is expressed in the mouse neural tube at the onset of neurogenesis (embryonic day 9.5 to 10.5) (PNAS, 1998). PACAP was found to regulate neuroblast proliferation in cultured cells at this stage, and to inhibit the action of the well-known patterning molecule, sonic hedgehog, which is necessary for the specification of motor neurons (PNAS, 1998). The proliferative action of PACAP in vitro was found to be highly sensitive to the presence of other growth factors. For example, in the absence of other growth factors, PACAP was found to stimulate proliferation, whereas PACAP was found to inhibit proliferation in the presence of fibroblastic growth factor-2 (FGF-2) (J Neurosci Res., 2002), or sonic hedgehog (unpublished). This switch was accompanied by a change in PACAP's signaling pathway form MAP kinase to protein kinase A. This data suggests that PACAP's action in the developing brain might differ significantly from one region to another, depending on the local presence or absence of FGF-2 and other growth factors. In other recent work, we showed that PACAP strongly antagonized the mitotic action of sonic hedgehog in cerebellar granule precursors (J Neurosci., 2002), but stimulated oligodendrocyte progenitor proliferation and delayed myelinogenesis in tissue culture and cerebellar explant models (J Neurosci., 2001). Thus, like many growth factors, cytokines and trophic factors, the actions of PACAP appear to be diverse and context-specific.
Xenopus Embryo mRNA Injection Model
When mRNA is injected into one of the dorsal cells of a four-cell embryo, the encoded protein (GFP in this case) is localized to the injected side. This allows the other side to be used as a control.
To study the developmental actions of PACAP and VIP in vivo, we recently established the Xenopus laevis embryo mRNA injection model, and have created mice with targeted deletions of these peptides. Because Xenopus develops quickly (from a fertilized oocyte to a swimming tadpole in 48h), one can achieve overexpression of a desired gene in the nervous system over the course of development by injecting the encoding mRNA into the dorsal cells of a four cell-stage embryo. We first cloned and characterized the Xenopus cDNAs for PACAP (Endocrinology, 2000a) and the PACAP receptor (PAC1R) (Endocrinology, 2000b), and showed that their early embryonic expression patterns were similar in Xenopus and mice (J Comp Neurol., 2001). In work not yet published, we have shown that PACAP overexpression in the nervous system results in decreased neurogenesis. Moreover, it does this by regulating other known cell patterning molecules. These data provide the first in vivo evidence that PACAP acts at the very earliest stage of development to regulate neurogenesis and aptterning, and represents the first application of the Xenopus model to study the action of neuropeptides in brain development.
The PACAP and VIP knockout mice generated in the lab exhibit a variety of behavioral abnormalities (see below), and in the case of PACAP knockout mice, exhibit altered cerebellar growth. Initial characterizations of our PACAP and VIP knockout mice and descriptions of their circadian rhythm disturbances (see below) have been published (Am J Physiol Regul Integr Comp Physiol., 2003, J Neurophysiol., 2004 and Nat Neurosci., 2005).
PACAP in the pathogenesis of brain tumors
Medulloblastoma detection in live mice by microPET
2-[18F] fluoro-2-deoxy-D-glucose (FDG) signals in a wild-type mouse (left) and a four month-old symptomatic PACAP+/-;ptc+/- double mutant mouse (right). Arrows indicate the location of the cerebellum. High FDG uptake, indicative of high metabolic activity in the tumor, is apparent in the symptomatic mouse on the right. The intense signals in the anterior part of the head comes from the Harderian glands, which typically give very strong signals in FDG PET scans. The bladder and certain tissues with high metabolic rates, such as the heart, also give strong PET signa;s on sections passing through these tissues. On autopsy, the mouse on the right was found to harbor a large medulloblastoma tumor.
Because PACAP appears to act to regulate brain growth during development in part by antagonizing sonic hedgehog signaling, we have hypothesized that this neuropeptide might regulate the pathogenesis of brain tumors such as medulloblastoma, which are associated with overactive hedgehog signaling. As discussed above, we have obtained evidence that PACAP antagonizes the action of sonic hedgehog in the cerebellar external granule cell layer, the germinal zone that gives rise to the tumor. We have determined that PACAP receptors are expressed in freshly-dissected medulloblastoma tumors (obtained from collaborator Dr. Linda Liau, Dept. of Neurosurgery at UCLA). This along with our in vitro data showing an antagonistic action of PACAP on cerebellar granule cell and medulloblastoma cell proliferation suggest that PACAP receptors or downstream signaling components might provide a new therapeutic target for this tumor. To provide evidence for a sonic hedgehog/PACAP interaction in this tumor in vivo, we have bred our PACAP knockout mice with mice harboring a mutation in the patched gene, obtained from Dr. Matthew Scott (Stanford Univ.) Mice with a monoallelic mutation in the patched gene exhibit a 15% incidence of medulloblastoma. Results of breeding studies indicate that the incidence of tumors in patched mutant mice is increased almost three-fold in a PACAP (+/-) background, and that most patched mutant mice die in utero in a PACAP (-/-) background. We have found that these tumors can be detect by microPET (see figure), thereby affording the possibility to detect these tumors in mice early and to monitor their growth. In very recent work, we are collaborating with UCLA investigators Drs. Michael Sofroniew (Dept of Neurobiology) and Harley Kornblum (Dept of Molecular and Medical Pharmacology/Pediatrics) to study cancer stem cells in mouse medulloblastoma tumors.
VIP and PACAP in nerve regeneration
Adenoviral delivery of GFP to brain stem motor neurons by peripheral administration to the facial muscles
These peptides may also play important roles in neuron survival or regeneration after injury. Initially, we showed that PACAP gene expression was strongly upregulated (more than 20-fold) in facial motor neurons in the brainstem after facial nerve axotomy (J Neurosci Res., 1999). We have recently shown that VIP is similarly upregulated (J Neurosci Res., 2003). Motor neuron regeneration is being examined in VIP and PACAP knockout mice, and we have designed a PACAP adenoviral vector expression system to determine if PACAP can rescue motor neurons or promote axonal regeneration. We are also studying the neuroprotective actions of VIP and PACAP in our knockout mice, and are determining the effect of the loss of these peptides on the infiltration and actions of inflammatory cells after nerve injury.
We have also examined mechanisms responsible for neuropeptide induction in nerve injury and have discovered that CD4+ T lymphocytes play a critical role in the injury-induced induction of PACAP (J Neurosci Res., 2003; Neuroscience, 2004; Neuroreport, 2004). Because PACAP is induced in neurons and appears to regulate the immune response after injury, this finding suggests that the regulation is reciprocal.
VIP and PACAP in circadian rhythms
Examples of wheel-running activity records from VIP/PHI+/+ (left), VIP/PHI+/- (middle) and VIP/PHI-/- (right).
Animals were initially entrained to a 12:12-h light-dark (LD) cycle and then maintained in constant darkness (DD). Each horizontal row represents the activity record for a 24-h day that is then double plotted. Successive days are plotted from top to bottom. Gray shaded area represents darkness. The circadian rhythm in VIP-/- exhibits a dramatic eight hour shift after DD is initiated, followed by a decay (shown above) and eventual disappearance of a circadian rhythm.
In normal mice, VIP is expressed at high levels in neurons within the suprachiasmatic nucleus (SCN), the primary circadian timer in mammals. PACAP, on the other hand, is expressed in the retinal ganglion neurons that are known to project to and modulate the effect of light on the circadian clock. The long-term goal of this project is to understand the mechanisms by which environmental signals regulate circadian oscillators as well as how circadian oscillators are coupled to each other, using neurons in the rodent suprachiasmatic nucleus (SCN) as a model system. A major goal of the current work is to understand the mechanisms by which PACAP modulates glutamate-induced signaling in SCN neurons, and how VIP functions in the circadian system. We have an ongoing collaboration with Dr. Christopher Colwell at UCLA to better understand the role of these peptides in circadian rhythms using our VIP and PACAP knockout mice. In this work, we have shown that PACAP knockout mice show a dramatically decreased ability to respond to light with a shift in circadian rhythm (Am J Physiol Regul Integr Comp Physiol., 2004). Electrophysiological and calcium imaging studies show that PACAP likely participates with glutamate to produce the phase shifts by enhancing AMPA currents in SCN neurons. VIP, on the other hand, has an entirely different function in circadian rhythms. VIP knockout were found to exhibit very weak locomotor rhythms, and in some cases no circadian rhythm at all (Am J Physiol Regul Integr Comp Physiol., 2003). The electrophysiological studies indicate that VIP may regulate the rhythm by modulating GABA signaling presynaptically (Am J Physiol Regul Integr Comp Physiol., 2003; J Neurophysiol., 2004). The outcome of this VIP/GABA interaction is synchronization of rhythm in various SCN neurons, which in isolation show randomized 24 rhythms (Nat Neurosci., 2005). Thus, PACAP is critically important in mediating the light resetting of the clock, whereas VIP is important in the maintenance of stable circadian rhythms. In recent work we delivered proteins to retinal ganglion cells using viral vectors (see figure below), and are using homologous recombination in E. coli to engineer green fluorescent protein and other proteins into bacterial artificial chromosomes to generate mice which will express these proteins in VIP-expressing cells in the SCN. This will allow us to perform physiological studies in live retinorecipient cells, and to study the proteins and signaling pathways by which circadian rhythms are entrained by light and other stimuli.
GFP fluorescence in the retinal ganglion layer after intraocular injection of a recombinant adenovirus engineered to express GFP
A subset of retinal ganglion neurons is critically involved in transmitting light signals to the SCN, thereby entraining the clock.
New directions
Neuroimmunology of VIP and PACAP
An abundance of evidence accumulated over the last two decades indicates that VIP and PACAP can potently regulate processes in almost every type of immune cell in vitro via high affinity receptors. For example, we showed several years ago that VPAC1 and VPAC2 receptors were expressed on several immune cell subtypes (Reg Pept., 1995), and that VIP was able to induced alpha-specific immunoglobulin switching in CD40-activated human B cells (J Clin Invest., 1996), suggesting an important role in directing the humoral immune response at mucosal surfaces. The generation of VIP and PACAP knockout mice now affords the possibility of studying the roles of endogenous peptides regulating immune funciton. Following up on the studies of Drs. Rosa Gomariz and Catalina Abad (Complutense University, Madrid) showing that VIP administration ameliorates the symptoms of several immune-based disease animal models, we are currently testing the susceptibility of our VIP and PACAP knockout mice to experimental models of multiple sclerosis and Crohn's disease, and to a cancer model associated with chronic inflammation.
PACAP interaction with the opiate system
We have collaborated with Dr. Chris Evans at UCLA (Dept of Psychiatry) to study the behavior of our PACAP knockout mice with respect to the mesolimbic dopaminergic reward system and sensitivity to pain. The reward system has been examined by measuring locomotor response to morphine. Although PACAP knockout mice were found to exhibit an elevated level of basal locomotor activity, they showed a significantly blunted response to morphine. These findings suggest that endogenous PACAP normally suppresses basal locomotor activity, but facilitates the response to morphine. Pharmacological studies with exogenous PACAP have corroborated these findings. Low doses of PACAP had no effect on basal locomotor activity, but potentiated the locomotor response to morphine. In contrast, high doses of PACAP strongly suppressed basal as well as morphine stimulated morphine activity. To examine the PACAP receptor subtype/s mediating these actions, we obtained mice with a targeted deletion of one of the PACAP receptors (PAC1, from Dr. Phillipe Brabet, UPR CNRS, Montpellier, France). Exogenous PACAP failed to suppress basal motor activity in these mice, but fully maintained the ability to potentiate the response to morphine. Thus, the PACAP action on basal motor activity is mediated by the PAC1 receptor, whereas the response to morphine is mediated by another PACAP receptor. The latter is likely mediated by the VPAC2 receptor, because this PACAP receptor is expressed in the ventral tegmental area and nucleus accumbens.
PACAP may also interact with opiates in pain response. PACAP knockout mice were found to be hypersensitive to pain in the tail flick and hot plate assays. Published studies in which PACAP is administered provide further evidence for an action of PACP on pain sensitivity.
VIP, PACAP and natriuretic peptides: signal transduction
VIP and PACAP classically act by way of seven transmembrane G-protein coupled receptors, and primarily lead to activation of protein kinase A and phosphatidyl inositol pathways. These peptides activate these pathways at concentrations in the dynamic range of receptor binding. However, many of the trophic actions of these peptides occur at much lower concentrations. We recently dissected signal transduction pathways used by VIP-related peptide to regulate neural cell growth using a neuroblastoma tumor cell line model. The data show that VIP-related peptides potently regulate the proliferation of neuroblastoma cells at concentrations as low as 10-13M via the MAP kinase signaling pathways (J Biol Chem., 1998). In addition, the studies have identified a novel growth-related receptor for PHI, a peptide encoded on the same gene as VIP.
This work has led an unexpected finding in the laboratory, that peptides in the VIP family may also act to control growth through a completely different receptor and signaling system. Specifically we have determined that VIP-related peptides also potently act on single transmembrane receptors that classically respond to peptides in the atrial natriuretic peptide (ANP) family (J Biol Chem., 2001). Moreover, the data indicate for the first time that the proliferation of neural cells may be regulated by natriuretic peptides. We have now determined that natriuretic peptides and their receptors are expressed in the mouse neural tube, and that natriuretic peptides regulate proliferation and neurite out growth in cultures of embryonic neuroblasts (Dev Biol., 2004). Natriuretic peptides, like PACAP, may thus play an important role in brain development.
Zebrafish as a model for neurodevelopment
Whole mount in situ1 receptor in zebrafish embryos 48 hours after fertilization
Zebrafish represent a new vertebrate model which has many attributes which are desirable for the study of neurodevelopment, regeneration, cancer and behavior. These include transparent embryos allowing visualization of specific neurons and structures via green fluorescent protein tagging, rapid development, large numbers of offspring, and ability to utilize forward and reverse genetics. Dr. Waschek recently spent nine months sabbatical leave in the laboratory of Dr. Steven Ekker at the University of Minnesota. As a result of this, several new projects have been initiated in the laboratory. Among these are projects to use the zebrafish model to 1) study the developmental actions of PACAP, 2) screen a morpholino library to discover new genes involved in axonal pathfinding, and 3) create a fish model of spinal cerebellar ataxia.
Enhancer trap zebrafish embryo that strongly expresses GFP in primary motor neurons and in a single projection that innervates the ventral myotomes
This line was created in the lab of Dr. Steve Ekker (U of Minnesota), and is being used to screen for genes that regulate axonal pathfinding.