Michael E. Greenberg, PhD
Associate Director, MRRC
Professor of Neurology
Harvard Medical School
F.M. Kirby Director, Division of Neuroscience
Children's Hospital

MRRC Project(s)

R01 CA43855-14
Nerve Growth Factor Regulation of Gene Expression

R37 NS28829-12
Electrical Stimulation of Immediate Early Genes

P01 HD24926-11
Signal Transduction in Embryo Development -PI, Prj 2: Signal Transduction Pathways that Suppress Apoptosis and Promote Cell Survival

Research Description

The objectives of our research are to define the signal transduction pathways that control the development and function of the mammalian nervous system. It is our expectation that by defining the signaling pathways that allow nerve cells to respond to changes in their environment we will gain insight into the mechanisms that control neural development and the complex functions of the mature nervous system that underlie behavioral responses. The characterization of neural signal transduction pathways also has the potential to reveal the ways in which disruptions of these normal processes can lead to devastating neuronal degenerative diseases and such neurological disorders as seizures, mental retardation, stroke and cancers of the nervous system.

Our research over the last decade has focused on identifying the mechanisms by which extracellular stimuli trigger cellular responses that are critical for the proliferation, differentiation, and survival of cells in the developing nervous system, and for the adaptive responses of neurons in the mature nervous system. Towards this end we began by characterizing a class of genes termed immediate early genes (IEGs) whose transcription is activated rapidly and transiently in response to a variety of extracellular stimuli, including neurotrophic factors and neurotransmitters. Our more recent work has delineated signaling pathways that promote neuronal survival and inhibit apoptotic cell death.

Highlights of Major Accomplishments

• Delineation of mechanisms by which neuronal activity regulates gene expression, e.g.,
  • identification of the critical regulatory elements within the c-fos gene that mediate the stimulation of c-fos transcription by activation of L-type voltage-sensitive Ca²⁺ channels (L-VSCCs),
  • identification of the several distinct kinase cascades involved in the stimulation of c-fos transcription by activation of L-VSCCs,
  • identification of the molecular mechanisms by which activation of L-VSCCs leads to transcription of brain-derived neurotrophic factor (BDNF).

Major Results

1. Investigations of the importance of IEGs and in particular fos family members during development and in neuronal adaptive responses

2. Neurotrophins promote the survival of neurons and their withdrawal can trigger cell death by apoptosis

3. Elucidation of the mechanisms by which neuronal activity regulates gene expression

1. To examine the importance of IEGs and in particular fos family members during development and in neuronal adaptive responses

To accomplish this goal, we have generated cells and mice in which the function of particular members of the Fos family is disrupted. Mice that bear a mutation that inactivates the c-fos gene are viable but suffer significant developmental abnormalities, including osteopetrosis and immune defects. We generated mice in which the FosB gene is disrupted. FosB mutant mice exhibit normal morphological development and are normal with respect to a number of cognitive and sensory functions. However, FosB mutant mice are profoundly deficient in their ability to nurture young animals. This nurturing defect appears to be due to the absence of FosB in the preoptic area (POA), a region of the hypothalamus that is critical for nurturing. Nurturing behavior in mice can be studied outside the context of pregnancy and parturition. Young female and male mice can be induced to engage in nurturing behaviors upon repeated exposure to newborn pups. Under these circumstances the nurturing response has the hallmarks of an adaptive neuronal response. Although the first exposure to newborn pups does not elicit a response, repeated exposure to pups will result in nurturing. The nurturing response is induced in young animals by a protein- dependent mechanism, and we found that the presentation of pups induces FosB protein in the POA. This observation, taken together with our finding that the POA and the FosB gene are critical for nurturing behavior, strongly suggests that FosB acts in the POA to mediate the nurturing response

2. Neurotrophins promote the survival of neurons and their withdrawal can trigger cell death by apoptosis

We have obtained evidence that inhibition of the Ras/ERK pathway and activation of two related kinase cascades that involve the ERK relatives p38 MAP kinase and the Jun kinase (JNK) are critical for nerve growth factor (NGF) withdrawal-induced apoptosis. Recently, we have identified another signaling pathway by which extracellular stimuli suppress apoptosis. Survival factors, such as Insulin like Growth Factor-1 (IGF-I) or neurotrophins, including NGF, bind to their cell-surface receptors and trigger the activation of several different categories of enzymes, including the phosphatidyl inositol 3' kinase (PI3K) and Ca²⁺/calmodulin dependent kinase, that then phosphorylate and activate a serine/threonine kinase termed Akt. We have shown that Akt plays a central role in promoting the survival of a wide range of cell types. We have characterized a substrate of Akt, the transcriptional regulator FKHRL, a member of the forkhead family of transcription factors, and have defined FKHRL1's role as a mediator of growth withdrawal-mediated apoptosis. Akt phosphorylates FKHRL1, thereby supressing FKHRL1 function and promoting survival. We have found that Akt regulates the activity of FKHRL1 by phosphorylating this transcriptional regulator at three sites. Akt phosphorylation of FKHRL1 leads to FKHRL1 association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm away from its target genes within the nucleus. Survival factor withdrawal leads to FKHRL1 dephosphorylation , nuclear translocation and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

We have found that activated Akt also promotes cell survival by phosphorylating a specific member of the BCL2 family, BAD, at Ser-136. The BCL2 family of proteins function as heterodimers which depending on the specific composition of the dimers can promote either survival or apoptosis. The phosphorylation of BAD at Ser-136 has been postulated to cause a shift in the type of BCL2 family dimers formed towards dimers that promote cell survival. In the absence of IGF-1, or other survival factors, it is hypothesized that BAD is not phosphorylated at either of its two regulatory sites, Ser-112 or Ser-136. Dephosphorylated BAD is believed to interact with other BCl2 family members to form complexes that trigger apoptosis, possibly through the formation of channels within the mitochondrial membrane that lead to the dissipation of the mitochondrial membrane potential, the release of cytochrome c, and the activation of a family of proteases termed caspases that orchestrate the execution phase of apoptosis. In a recent study we have conclusively identified Akt as a survival factor-regulated kinase that phosphorylates BAD at Ser-136 and inactivates its apoptotic function. We found that Akt and BAD interact with one another and that when Akt is activated and BAD is phosphorylated BAD's interaction with BCL2 family members is disrupted. Phosphorylated BAD moves from the mitochondrial membrane to the cytoplasm where it interacts with 14-3-3 proteins. 14-3-3 proteins may then serve as a nexus of interaction of a variety of signal transduction events, some of which could be relevant to cell survival.

3. A long range objective of our research is to understand the mechanisms by which neuronal activity regulates gene expression

To achieve this goal we have characterized the signal transduction pathways by which Ca²⁺ influx through L-type voltage sensitive Ca²⁺ channels (L-VSCCs) stimulates the transcription of the c-fos proto-oncogene and brain derived neurotrophic factor (BDNF). We discovered that c-fos transcription is activated rapidly and transiently in a variety of mammalian cell types in response to a diverse set of extra-cellular stimuli. Our analysis of the signaling pathways by which Ca²⁺ influx through L-VSCCs triggers c-fos transcription has established a paradigm for studying Ca²⁺ signaling in neurons and has served as a basis for our more recent analysis of the cellular mechanisms that control BDNF transcription in neurons.

We began by identifying the critical regulatory elements within the c-fos gene that mediate the Ca²⁺ response. One of the Ca²⁺ responsive elements (CaREs) is located 60 nucleotides 5= of the site of initiation of c-fos mRNA synthesis and binds the transcription factor CREB. Membrane depolarization of neurons under conditions that selectively stimulate L-VSCCs leads within minutes to the phosphorylation of this cyclic AMP response element-binding protein (CREB) at a critical amino acid residue, Ser-133. Pharmacologic and genetic studies have established that Ca²⁺ influx triggers the activation of several distinct kinase cascades that culminate in the phosphorylation of CREB at Ser-133. These pathways include the Ras/Erk/Rsk pathway, the CaMKK/CaMKIV pathway, and the Ca²⁺sensitive adenylate cyclase/PK-A pathway. The phosphorylation of CREB Ser-133 promotes CREB=s interaction with a transcriptional co-factor termed CREB binding protein (CBP). Once CBP is brought to the promoter it promotes the formation of an active Pol II transcription complex that transcribes the c-fos mRNA.

We and others have found that the BDNF gene transcription is specifically activated by Ca²⁺ influx into neurons and, like c-fos transcription, BDNF transcription is controlled by CREB. BDNF is encoded by a 40 kB gene that gives rise to eight distinct mRNAs whose synthesis is driven by four separate promoters. Our analysis during the previous funding period revealed that BDNF promoter III is the most Ca²⁺ responsive of the four BDNF promoters. A CREB binding site located at -35 relative to the site of initiation of BDNF promoter III transcription is critical for Ca²⁺ induction of BDNF transcription. In addition, we have identified a second CaRE within the BDNF promoter that appears to cooperate with the CREB binding site in conferring Ca²⁺ responsiveness. We believe that this 5= element binds a novel Ca²⁺ regulated transcription factor that may confer neuronal specificity to the BDNF Ca²⁺ response.

Despite considerable progress toward defining the mechanisms by which Ca²⁺ influx into neurons regulates c-fos and BDNF transcription, many questions still remain to be addressed. In addition to the phosphorylation of CREB Ser-133, there appear to be other phosphorylation-dependent events that are critical for Ca²⁺ induction of CREB-dependent gene transcription. Furthermore, there are transcription factors in addition to CREB that are capable of conferring a Ca²⁺ response in neurons. Among these transcription factors are members of the MEF2 family and a potentially novel transcription factor that binds to the 5= regulatory element of the BDNF gene. We are currently addressing several unanswered questions regarding Ca²⁺ regulation of gene expression. Specifically we are 1) characterizing phosphorylation modifications of the CREB/CBP complex that, in addition to the phosphorylation of CREB Ser-133, play a critical role in mediating the Ca²⁺ response, 2) purifying, cloning, and characterizing the transcription factor(s) that bind the 5= CaRE within the BDNF promoter, and 3) characterizing the mechanism(s) by which Ca²⁺ regulates MEF2 activity and establishing the importance of MEF2 proteins as mediators of Ca²⁺ effects on gene expression.

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