Neurotrophins Meeting Summary
Growth and Transmitter Functions of Neurotrophic Factors: Roles for Therapeutic Interventions Both Old and New
C. Anthony Altar, Ph.D.
Otsuka America Pharmaceuticals, Inc.
Neurotrophic factors are known to play roles in mature and developing nervous systems and to span the temporal, spatial, and functional domains previously reserved for neurotransmitters. This new understanding of these widely signaling molecules suggests new areas of applied research. Such areas include whether neurotrophins mediate actions of antidepressants, antipsychotics, or analgesics or contribute to learning and memory, the rewarding components of substance abuse, or even the benefits that enriched environments or exercise can provide to the brain. A key challenge for the future is to solidify the initial links that have been forged between function and the levels or turnover of neurotrophins or their receptors and to exploit such links by developing therapeutics that augment endogenous neurotrophins in brain and peripheral targets.
GDNF - A Trophic Factor for Multiple Subpopulations of
Barry J. Hoffer, M.D., Ph.D.
National Institute on Drug Abuse
Trophic factors, once thought to be important only for development in the mammalian central nervous system (CNS), are now recognized to have important roles in adult CNS plasticity and responses to injury. My talk will focus on glial cell line-derived neurotrophic factor (GDNF) and other members of this remote subfamily of TGF-ß superfamily molecules. Actions of GDNF family members on midbrain dopamine neurons, cerebral cortex, spinal cord neurons, and GABAergic neurons will be presented.
NGF Signaling in CNS Cholinergic Neurons
William C. Mobley, M.D., Ph.D. (Contributors: Charles Howe, Jon Cooper, Josh Kilbridge, Jane Chua-Couzens)
Nerve growth factor acts on cholinergic neurons of the basal forebrain and striatum. We will review the trophic dependencies of these neurons and the important features of nerve growth factor (NGF) actions. Data suggesting that failed NGF signaling causes degeneration of basal forebrain cholinergic neurons in a mouse model of Down syndrome will be discussed.
Recent Developments in the GDNF Family of Trophic Factors and Their Receptors
Eugene M. Johnson, Jr., Ph.D.
Washington University Medical School
The glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors comprises three molecules (GDNF, neurturin, and persephin) that act via the tyrosine kinase receptor protein RET. RET does not directly bind the factors; rather, binding is conferred by glycosylphosphatidylinositol-linked coreceptors. The coreceptors display relative, but not absolute, selectivity for the ligands. These factors act on several neuronal populations, including dopaminergic neurons, motor neurons, and several peripheral neurons. The current status of this rapidly advancing field will be discussed.
Mechanisms of Gene Induction After Chronic Cocaine and Voluntary Running:
A Role for Neuropeptides and Trophic Factors
Stefan Brené, Ph.D. (Contributors:
Martin Werme, Johan Widenfalk, Peter Thorén, Lars Olson)
It has been suggested that genetic factors influence behaviors such as stress and addiction. We analyzed the effect of chronic voluntary running in running wheels on the mRNA levels encoding the striatal neuropeptides dynorphin, enkephalin, and substance P in Lewis and Fischer rats. To compare the effects of chronic running, we also analyzed the effects of chronic cocaine and morphine treatment on the levels of neuropeptide mRNAs. In a manner similar to their addictive behaviors, the drug-prone Lewis rats developed a higher preference for excessive voluntary running compared with Fischer rats, which may suggest a common mechanism in the neurobiological control of excessive running and addiction. We then demonstrated a similar induction by chronic cocaine and running on dynorphin mRNA in the dorsomedial striatum. The running-induced effect on dynorphin mRNA levels was blocked by the opioid receptor antagonist naloxone, which suggests that running induces dynorphin mRNA in the dorsomedial striatum by a mechanism that involves endogenous opioids. In addition, we analyzed the effect of chronic running on levels of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor TrkB in hippocampus. Running induced, whereas running withdrawal decreased, the levels of BDNF and TrkB mRNAs in hippocampal subfields. These results may have implications for the roles of neuropeptides and trophic factors in controlling addictive and excessive running behavior.
Functional Recovery in GDNF-Treated Parkinsonian Monkeys
Don M. Gash, Ph.D.
University of Kentucky
Glial cell line-derived neurotrophic factor (GDNF) promotes functional recovery in both rodent and nonhuman primate models of Parkinson's disease. Studies from our laboratory and others have demonstrated that GDNF induces neuroprotective changes in central dopamine neurons as well as promotes recovery from injury. GDNF does not cross the blood-brain barrier, necessitating intraparenchymal delivery of the trophic factor. Our recent studies (Grondin, Zhang, Gerhardt, Gash) have focused on the antiparkinsonian effects of chronic infusion of GDNF into the rhesus monkey lateral ventricle.
Neurotrophins and Activity-Dependent Synaptic Plasticity in the Hippocampus
Susan L. Patterson, Ph.D. (Contributors: Eric Kandel, Kelsey Martin, Alexei Morozov)
The experience-dependent modification of the strength of functional connections in the brain is thought to underlie a variety of important mental processes, including refinement of synaptic connections during development, acquisition of learned behaviors, and storage of memories. Synaptic plasticity also appears to play a role in the pathophysiology and treatment of several mental disorders, including depression, anxiety, schizophrenia, and drug addiction. Synaptic plasticity, like memory, has at least two forms: (1) a short-term form that involves the modification of existing proteins and (2) a long-lasting form that requires gene transcription and the synthesis of new proteins. Using a combination of pharmacological and genetic approaches, we demonstrated that the neurotrophin brain-derived neurotrophic factor (BDNF) is an important modulator of early long-term potentiation (LTP) at the Schaffer collateral synapse in hippocampal slices. More recently, we found that BDNF also plays a role in the late phase of LTP.
Targeting Peptides Into the Central Nervous System
Laszlo Prokai, Ph.D. (Contributor: James W. Simpkins)
University of Florida
The microvasculature of the central nervous system (CNS) is characterized by tight junctions between the endothelial cells and, thus, behaves as a continuous lipid bilayer that prevents the passage of polar and lipid-insoluble substances such as peptides. Highly active enzymes expressed in the morphological components of the microcirculation also represent a metabolic component that contributes to the homeostatic balance of the CNS. Peptides generally cannot enter the brain and spinal cord from the circulating blood because they are highly polar and lipid insoluble and metabolically unstable; active transport systems exist for only very few of them in this membranous barrier separating the systemic circulation from the interstitial fluid of the CNS. This blood-brain barrier (BBB) is, therefore, the major obstacle to peptides and peptide-based drugs that may be useful for combating diseases affecting the brain and the spinal cord. This presentation briefly reviews and evaluates invasive and biological carrier-based approaches and focuses on the development of chemical-enzymatic (pro-drug and chemical delivery/targeting) strategies to overcome the BBB for centrally active peptides that are attractive as potential neuropharmaceuticals.
Noninvasive Methods of Delivering Neurotrophic Therapy Across the BBB
Raymond T. Bartus, Ph.D.
The blood-brain barrier (BBB) restricts many types of therapeutically useful agents, including proteins, from entering the brain following vascular injection. Two completely different approaches to overcoming this obstacle posed by the BBB have shown promise: (1) use of endogenous active transport systems (such as transferrin) to piggyback large conjugated or fusion proteins, such as nerve growth factor, glial cell line-derived neurotrophic factor, and so forth, into the central nervous system and
(2) use of the bradykinin agonist Cereport (lobradimil) to transiently increase permeability of the BBB, permitting diffusion of smaller molecules (e.g., potential neurotrophic modulators) into the brain. Recent data demonstrating proof of principle, as well as future prospects and current limitations associated with each approach, will be reviewed.
CNS Gene Transfer To Modify Learning
Howard J. Federoff, M.D., Ph.D.
University of Rochester Medical Center
Somatic mosaic analysis has been used to express nerve growth factor (NGF) intrahippocampally in mice. Permanent gain of NGF function achieved by somatic mosaic gene activation results in a time-dependent reorganization of septohippocampal circuitry, enhanced learning, and experience-augmented alteration in the neural substrate underlying learning.
Astrocyte-Derived Neurotrophic Factor Expression During Development and Following Neuronal Injury
Joan P. Schwartz, Ph.D. (Contributors:
Vivian W. Wu, Takuo Nomura, Mojca Krzan)
National Institute of Neurological Disorders and Stroke
Injury to the adult brain transforms resting astrocytes to a reactive state, and a variety of studies have shown increased expression of neurotrophic factors by the reactive astrocytes. In general, neurotrophic factors are thought to support regeneration of neurons, and the ability to utilize endogenous neurotrophic factor production by reactive astrocytes as a treatment for injured neurons would be useful. However, that requires a complete understanding of the properties of the reactive astrocytes. One method of simplifying the complexities of the brain has been to study individual cells in culture, although many would argue that the properties of the cells change too drastically to be relevant. Results obtained using a culture model of reactive astrocytes, prepared from adult rats injected with 6-hydroxydopamine
(6-OHDA) unilaterally in the substantia nigra, demonstrate that many of the biochemical properties of reactive astrocytes in culture are similar to those of neonatal astrocytes, whereas the properties of normal adult cells are strikingly different. Of particular interest is the difference in expression of neurotrophic factors. For example, levels of nerve growth factor (NGF) mRNA and protein show large differential expression: The astrocytes from 6-OHDA-lesioned rat brain contain tenfold to twentyfold more mRNA and NGF than normal adult astrocytes, whereas neonatal astrocyte levels are intermediate. Treatment with the cytokines interleukin-1ß, tumor necrosis factor-", or interferon-( leads to dramatic increases in NGF mRNA and protein in the reactive astrocytes as well as the neonatal astrocytes, whereas no changes are observed in astrocytes from either normal or saline-injected adult rats. Similar differences are seen for responses to the
ß-adrenergic agonist isoproterenol, although it is capable of elevating cyclic AMP in all types of cells. Regulation of expression of other neurotrophic factors, including glial cell line-derived neurotrophic factor, is currently being analyzed to determine whether synthesis of all neurotrophic factors is depressed in normal adult astrocytes in culture. More importantly, the results from the cultures will be compared with in vivo results, since this serves as a verification that the culture model can be used to understand the properties of reactive astrocytes.
Regulation of Growth Factor Expression in the Rat Brain by Hormones and Stress
Mark A. Smith, M.D., Ph.D.
DuPont Pharmaceuticals Company
Neurotrophic factor expression in the central nervous system is regulated by a variety of small molecules, including stress hormones and antidepressants. Both glucocorticoids and thyroid hormone can modulate growth factor expression in rat brains. In addition, stress can upregulate and downregulate growth factor expression in the brains of neonate, adult, and aged rats. In contrast, antidepressant drugs modulate growth factor expression in a manner opposite to stress. Thus, neurotrophic factors are regulated by a variety of substances, which suggests that some forms of adult neural plasticity may be mediated by growth factors.
Protection Against the Neurotoxic Effects of Methamphetamine and
6-Hydroxydopamine by GDNF
Wayne A. Cass, Ph.D.
University of Kentucky
The repeated administration of methamphetamine to animals can induce long-lasting decreases in dopamine release, uptake, and content in the striatum. Glial cell line-derived neurotrophic factor (GDNF) can prevent these decreases when given 1 day prior to neurotoxic doses of methamphetamine. GDNF can also partially protect dopamine neurons against a more complete lesion induced by
6-hydroxydopamine (6-OHDA). When injected into the nigra prior to 6-OHDA, GDNF can completely prevent loss of nigral dopamine levels and partially prevent loss of evoked overflow of dopamine in the striatum. Experiments are under way to determine whether GDNF can accelerate recovery from the neurotoxic effects of methamphetamine.
Regulation of Neurotrophic Factor Signaling Pathways in the Mesolimbic Dopamine System by Drugs of Abuse
David S. Russell, M.D., Ph.D.
Chronic exposure to drugs of abuse elicits a number of changes in the mesolimbic dopaminergic nuclei, including the levels of expression of tyrosine hydroxylase and neurofilament proteins, neuronal morphology, and others. These changes are suggestive of alterations in neurotrophic support, but little evidence exists for significant regulation of neurotrophic factors or their receptors within these regions following chronic drug exposure. We have found, however, that the signaling pathway proteins that are utilized by these receptors are significantly, and differentially, regulated. For instance, phospholipase C-(, the major tyrosine kinase-coupled phospholipase C in brain, and the phosphotidylinositol-3-kinase pathways are differentially regulated following chronic morphine exposure. These alterations in the signaling pathway proteins would be expected to affect biochemical and functional neuronal features under neurotrophic factor control. This may represent a novel form of plasticity that could contribute to the changes seen in the drug-addicted state and suggests novel therapeutic protein targets.
Neurotrophic Factors and Drugs of Abuse
Eric J. Nestler, M.D., Ph.D.
Chronic exposure to drugs of abuse produces long-term biochemical and morphological changes in the mesolimbic dopamine system. Some of these changes can be prevented by the administration of certain neurotrophic factors directly into the mesolimbic dopamine system. In addition, these changes, as well as behavioral plasticity to drugs of abuse, are abnormal in mutant mice that are deficient in these neurotrophic factors. Together, these findings support the view that some of the long-term effects of drug exposure on the mesolimbic dopamine system may be achieved via perturbation of endogenous neurotrophic factor signaling pathways, which raises the possibility that medications aimed at these pathways could be novel treatment agents for addictive disorders.
A Paracrine Paradigm for In Vivo Gene Therapy in the Central Nervous System: Treatment of Chronic Pain
Alan A. Finegold, Ph.D.1 (Contributors: Andrew J. Mannes and Michael J. Iadarola)
National Institute of Dental and Craniofacial Research
University of Pennsylvania
A limitation of current gene therapy efforts aimed at central nervous system disorders is distribution of vectors directly injected into neural tissue. We circumvented this problem by transferring genes to the meninges surrounding the spinal cord, achieving an in vivo gene transfer paradigm for treating chronic pain. The therapeutic vector consisted of a recombinant adenovirus encoding a secreted form of the potent endogenous opioid ß-endorphin. In an inflammation model of persistent pain, administration of the vector into the cerebrospinal fluid surrounding the spinal cord transduced meningeal pia mater cells, resulting in increased
ß-endorphin secretion that attenuated inflammatory hyperalgesia. This demonstration of a gene transfer approach to pain treatment can be generalized to neurodegenerative disorders in which broad spatial distribution of therapeutic effect is critical.
Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP38)-Induced Neurite Outgrowth in PC12 Cells is PKC and ERK Dependent
Donald W. Fink, Jr., Ph.D. (Contributors: Philip Lazarovici and Hao Jiang)
Food and Drug Administration
Hebrew University of Jerusalem
National Institute of Child Health and Human Development
PACAP38 elicits a robust outgrowth of neurites in cultured PC12 cells. Dominant negative Src and Ras variant PC12 cells, as well as selective protein kinase pharmacologic antagonists, have been utilized to identify elements of the signal transduction cascade responsible for PACAP38-induced neuritogenesis. Our findings indicate that PACAP38-stimulated neurite outgrowth requires protein kinase C and activation of extracellular-regulated kinase, but is independent of protein kinase A, the nerve growth factor receptor tyrosine kinase, Ras, and Src.
Behavioral and Structural Effects of Ganglioside Treatment for Nerve Grafting in the Rat
James M. Kerns, Ph.D.1 (Contributors: James K. Sobeski, John F. Safanda, Mark H. Gonzalez)
Rush Medical College
University of Illinois at Chicago
We examined the influence of GM-1 ganglioside on peripheral nerve regeneration through vascular or avascular nerve grafts in rat sciatic nerve during 5 months of postoperative recovery. The following evaluation methods were used: locomotion behavior (SFI), nerve conduction velocity (NCV), toe-chewing, gastrocnemius wet weight, and nerve histology. The average NCV of the graft groups was 45 percent (avascular) to 57 percent (vascular) of the normal nerve (52.7 m/sec), but for the graft groups treated with GM-1, the NCV was 62 to 64 percent. Toe-chewing was less evident in the treatment groups, but other measures showed no major differences. We conclude that the addition of GM-1 ganglioside enhances only certain aspects (possibly sensory) of the regeneration in grafted nerves.