Michael E. Greenberg

Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis

Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis.

The regulation and function of c-fos and other immediate early genes in the nervous system

Morgan Sheng and Michael E. Greenberg Department of Microbiology and Molecular Genetics Harvard Medical School Boston. Massachusetts 02115 Introduction The unique morphological and excitable properties of nerve cells endow them with specialized properties that permit the reception, transmission, and storage of information. It has long been recognized that trans- synaptic signals cause rapid responses in neurons. These occur over a time frame ranging from millise- conds (e.g., opening of ligand-gated channels) to sec- onds and minutes (e.g., second messenger-mediated events). Recent studies, however, have revealed that trans.synaptic activation also elicits slower, long-term responses in neural cells that are correlated with, and in some cases shown to be dependent on, the induc- tion of new programs of gene expression (reviewed in Goelet et al., 1986; Black et al., 1987; Comb et al., 1987; Morgan and Curran, 1988). Neuronal gene ex- pression can be modulated by neurotransmitters, mem- brane electrical activity, and neurotrophic growth fac- tors and is likely to play an important role both in the development and in the adaptive plasticity of the ner- vous system. Many of the long-term consequences of trans-synaptic stimulation may therefore be mediated by changes in gene expression. These include altera- tions in neuronal sprouting or synaptic density and changes in the level of expression of neurotransmit- ters, receptors, and ion channel proteins. In inver- tebrates, the activation of new gene expression in neurons has been shown to be critical for the devel- opment of a learning-related long-term facilitation (Montarolo et al., 1986). Substantial progress has been made over the last 5 years in identifying the genes that are responsive to trans-synaptic stimulation and membrane electrical activity in neural cells. These genes fall into two gen- eral classes: genes whose transcription is activated rapidly and transiently within minutes of stimulation (Greenberg et al., 1985; Morgan and Curran, 1986; Bartel et al., 1989; Barzilai et al., 1989), termed the cel- lular immediate early genes (IEGs), and the late re- sponse genes (Merlie et al., 1984; Castellucci et al., 1988; Goldman et al., 1988; Barzilai et al., 1989; Offord and Catterall, 1989; Klarsfeld et al., 1989), whose ex- pression is induced (or repressed) more slowly, over a time frame of hours, via a mechanism that is gener- ally dependent on new protein synthesis. While the distinction between the IECs and the late response genes is not always clear-cut, it has been proposed that IEGs encode regulatory proteins that control the expression of late response genes. The products of the late response genes are then thought to serve more specific effector functions in the neuronal re- sponse. Recent studies, which are the subject of this review, have provided increasing support for this idea. Many IEGs have been shown to encode transcription factors. By directing specific programs of late gene ex- pression, the induction of these proteins could there- fore mediate many of the long-term responses of the neuron to trans-synaptic signals. What Are Immediate Early Genes? The activation of IECs by extracellular stimuli is not specific to neuronal cells. The IEGs were first charac- terized in nonneuronal cells through efforts to iden- tify growth factor-responsive genes that might con- trol the reentry of Go resting cells into cell cycle. This work resulted in the discovery of a class genes whose transcription is activated within minutes after addition of a growth factor. The c-fos and c-myc proto- oncogenes were among the first IECs to be identified (Kelly et 1983; Greenberg and Ziff, 1984) are prototypic members of this family. c-fos transcription- al activation occurs within a few minutes of growth factor stimulation and precedes the activation of c-myc (Greenberg and Ziff, 1984; Kruijer et al., Muller et al., 1984). The induction of these genes is transient; the level of c-fos transcription is once again undetect- able 30 min after growth factor treatment. The obser- vation that growth factors activate the transcription of two genes whose mutation or deregulated expression can lead to cell transformation suggested that Fos and Myc proteins could have important regulatory func- tions during cell proliferation. Indeed, studies using c-fos anti-sense RNA or anti-c-fos antibodies indicate that the activation of c-fos is critical for reentry of qui- escent fibroblasts into the cell cycle (Nishikura and Murray, 1987; Riabowol et al., 1988). In addition to c-fos and c-myc, a large number of other IEGs have been identified by the differential screening of cDNA libraries from growth factor-stimulated cells (Coch- ran et al., 1983; Lau and Nathans, 1985, 1987; Almen- dral et al., 1988; Lim 1987; Kujubu 1987). The total number of IECs is now thought to be close to 100, though relatively few of these genes have been extensively characterized. In general, IEGs share the following characteristics: their expression is low or undetectable in quiescent cells, but is rapidly induced at the transcriptional level within minutes of extracellular stimulation; this tran- scriptional induction is transient and independent of new protein synthesis; the subsequent shut-off of transcription requires new protein synthesis; the mRNAs transcribed from these genes often have a very short half-life (in the case of c-fos, approximately

Huntingtin Acts in the Nucleus to Induce Apoptosis but Death Does Not Correlate with the Formation of Intranuclear Inclusions

The mechanisms by which mutant huntingtin induces neurodegeneration were investigated using a cellular model that recapitulates features of neurodegeneration seen in Huntingtons disease. When transfected into cultured striatal neurons, mutant huntingtin induces neurodegeneration by an apoptotic mechanism. Antiapoptotic compounds or neurotrophic factors protected neurons against mutant huntingtin. Blocking nuclear localization of mutant huntingtin suppressed its ability to form intranuclear inclusions and to induce neurodegeneration. However, the presence of inclusions did not correlate with huntingtin-induced death. The exposure of mutant huntingtin-transfected striatal neurons to conditions that suppress the formation of inclusions resulted in an increase in mutant huntingtin-induced death. These findings suggest that mutant huntingtin acts within the nucleus to induce neurodegeneration. However, intranuclear inclusions may reflect a cellular mechanism to protect against huntingtin-induced cell death.

A brain-specific microRNA regulates dendritic spine development

MicroRNAs are small, non-coding RNAs that control the translation of target messenger RNAs, thereby regulating critical aspects of plant and animal development. In the mammalian nervous system, the spatiotemporal control of mRNA translation has an important role in synaptic development and plasticity. Although a number of microRNAs have been isolated from the mammalian brain, neither the specific microRNAs that regulate synapse function nor their target mRNAs have been identified. Here we show that a brain-specific microRNA, miR-134>, is localized to the synapto-dendritic compartment of rat hippocampal neurons and negatively regulates the size of dendritic spines—postsynaptic sites of excitatory synaptic transmission. This effect is mediated by miR-134 inhibition of the translation of an mRNA encoding a protein kinase, Limk1, that controls spine development. Exposure of neurons to extracellular stimuli such as brain-derived neurotrophic factor relieves miR-134 inhibition of Limk1 translation and in this way may contribute to synaptic development, maturation and/or plasticity.

Widespread transcription at neuronal activity-regulated enhancers

We used genome-wide sequencing methods to study stimulus-dependent enhancer function in mouse cortical neurons. We identified ∼12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Notably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 monomethylated at lysine 4. The level of eRNA expression at neuronal enhancers positively correlates with the level of messenger RNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.

Ca2+ Influx Regulates BDNF Transcription by a CREB Family Transcription Factor-Dependent Mechanism

CREB is a transcription factor implicated in the control of adaptive neuronal responses. Although one function of CREB in neurons is believed to be the regulation of genes whose products control synaptic function, the targets of CREB that mediate synaptic function have not yet been identified. This report describes experiments demonstrating that CREB or a closely related protein mediates Ca2+-dependent regulation of BDNF, a neurotrophin that modulates synaptic activity. In cortical neurons, Ca2+ influx triggers phosphorylation of CREB, which by binding to a critical Ca2+ response element (CRE) within the BDNF gene activates BDNF transcription. Mutation of the BDNF CRE or an adjacent novel regulatory element as well as a blockade of CREB function resulted in a dramatic loss of BDNF transcription. These findings suggest that a CREB family member acts cooperatively with an additional transcription factor(s) to regulate BDNF transcription. We conclude that the BDNF gene is a CREB family target whose protein product functions at synapses to control adaptive neuronal responses.

Coupling of the RAS-MAPK Pathway to Gene Activation by RSK2, a Growth Factor-Regulated CREB Kinase

A signaling pathway has been elucidated whereby growth factors activate the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB), a critical regulator of immediate early gene transcription. Growth factor-stimulated CREB phosphorylation at serine-133 is mediated by the RAS-mitogen-activated protein kinase (MAPK) pathway. MAPK activates CREB kinase, which in turn phosphorylates and activates CREB. Purification, sequencing, and biochemical characterization of CREB kinase revealed that it is identical to a member of the pp90RSK family, RSK2. RSK2 was shown to mediate growth factor induction of CREB serine-133 phosphorylation both in vitro and in vivo. These findings identify a cellular function for RSK2 and define a mechanism whereby growth factor signals mediated by RAS and MAPK are transmitted to the nucleus to activate gene expression.

Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner

Microglia are the resident CNS immune cells and active surveyors of the extracellular environment. While past work has focused on the role of these cells during disease, recent imaging studies reveal dynamic interactions between microglia and synaptic elements in the healthy brain. Despite these intriguing observations, the precise function of microglia at remodeling synapses and the mechanisms that underlie microglia-synapse interactions remain elusive. In the current study, we demonstrate a role for microglia in activity-dependent synaptic pruning in the postnatal retinogeniculate system. We show that microglia engulf presynaptic inputs during peak retinogeniculate pruning and that engulfment is dependent upon neural activity and the microglia-specific phagocytic signaling pathway, complement receptor 3(CR3)/C3. Furthermore, disrupting microglia-specific CR3/C3 signaling resulted in sustained deficits in synaptic connectivity. These results define a role for microglia during postnatal development and identify underlying mechanisms by which microglia engulf and remodel developing synapses.

Transcription-dependent and -independent control of neuronal survival by the PI3K–Akt signaling pathway

The PI3K-Akt signaling pathway plays a critical role in mediating survival signals in a wide range of neuronal cell types. The recent identification of a number of substrates for the serine/threonine kinase Akt suggests that it blocks cell death by both impinging on the cytoplasmic cell death machinery and by regulating the expression of genes involved in cell death and survival. In addition, recent experiments suggest that Akt may also use metabolic pathways to regulate cell survival.

Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB

The mechanism by which the calcium influx signal, triggered by membrane depolarization, is transduced to the nucleus to activate c-fos proto-oncogene transcription has been characterized. A calcium response element (CaRE) that is indistinguishable from a cAMP response element (CRE) mediates transcriptional inducibility by depolarization. Its cognate transcription factor CREB is the target for both calcium and cAMP signals. CREB is rapidly phosphorylated in response to depolarization or cAMP, at a site known to be important for the transcriptional activating function of this protein. The convergent effects of calcium and cAMP on CREB activation are mediated by distinct protein kinase signaling pathways. CREB and its binding site, the Ca/CRE, can thus function as a regulatory element that integrates both calcium and cAMP signals in the control of gene expression.