Jon M. Kornhauser

Positional Cloning of the Mouse Circadian Clock Gene

We used positional cloning to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning approximately 100,000 bp of DNA from which transcript classes of 7.5 and approximately 10 kb arise. Clock encodes a novel member of the bHLH-PAS family of transcription factors. In the Clock mutant allele, an A-->T nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the CLOCK protein. Clock is a unique gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. CLOCK represents the second example of a PAS domain-containing clock protein (besides Drosophila PERIOD), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism.

Calcium regulation of neuronal gene expression

Plasticity is a remarkable feature of the brain, allowing neuronal structure and function to accommodate to patterns of electrical activity. One component of these long-term changes is the activity-driven induction of new gene expression, which is required for both the long-lasting long-term potentiation of synaptic transmission associated with learning and memory, and the activitydependent survival events that help to shape and wire the brain during development. We have characterized molecular mechanisms by which neuronal membrane depolarization and subsequent calcium influx into the cytoplasm lead to the induction of new gene transcription. We have identified three points within this cascade of events where the specificity of genes induced by different types of stimuli can be regulated. By using the induction of the gene that encodes brain-derived neurotrophic factor (BDNF) as a model, we have found that the ability of a calcium influx to induce transcription of this gene is regulated by the route of calcium entry into the cell, by the pattern of phosphorylation induced on the transcription factor cAMP-response element (CRE) binding protein (CREB), and by the complement of active transcription factors recruited to the BDNF promoter. These results refine and expand the working model of activity-induced gene induction in the brain, and help to explain how different types of neuronal stimuli can activate distinct transcriptional responses.

Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus

Photic information entrains a circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus to environmental light/dark cycles. To determine whether light regulates c-fos gene expression in the SCN, we have measured c-fos mRNA levels in the SCN of the golden hamster. We report that, during the subjective night, light causes a rapid increase in levels of c-fos mRNA in the SCN. Light pulses of 5 min duration are sufficient to induce c-fos mRNA, and the highest mRNA levels occur 30 min following the onset of light. The minimum level of illumination required to induce an increase in c-fos mRNA is indistinguishable from the minimum irradiance that produces a phase shift in the hamsters circadian rhythm of activity. In addition, the induction of c-fos mRNA in the SCN by light is itself under circadian regulation. Light induction of c-fos mRNA occurs only during the subjective night, at circadian times when photic phase shifting of activity occurs. Taken together, these data suggest that c-fos may be a molecular component of the photic pathway for entrainment of mammalian circadian rhythms.

Nerve Growth Factor Activates Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways To Stimulate CREB Serine 133 Phosphorylation

ABSTRACT The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

CREB Transcriptional Activity in Neurons Is Regulated by Multiple, Calcium-Specific Phosphorylation Events

The transcription factor CREB mediates diverse responses in the nervous system. It is not known how CREB induces specific patterns of gene expression in response to different extracellular stimuli. We find that Ca(2+) influx into neurons induces CREB phosphorylation at Ser133 and two additional sites, Ser142 and Ser143. While CREB Ser133 phosphorylation is induced by many stimuli, phosphorylation at Ser142 and Ser143 is selectively activated by Ca(2+) influx. The triple phosphorylation of CREB is required for effective Ca(2+) stimulation of CREB-dependent transcription, but the phosphorylation of Ser142 and Ser143, in addition to Ser133, disrupts the interaction of CREB with its cofactor CBP. These results suggest that Ca(2+) influx triggers a specific program of gene expression in neurons by selectively regulating CREB phosphorylation.

A kinase to remember: dual roles for MAP kinase in long-term memory.

The selective down-regulation of the transmembrane apCAM isoform has been proposed to remove inhibitory constraints on neuronal outgrowth by promoting the disassembly of adhesive contacts between sensory neuron processes that normally inhibit growth. However, any role that the internalization of the transmembrane form of apCAM plays in the relief of inhibitory constraints on synaptic reorganization must be reconciled with the continued presence of the GPI-linked form of apCAM. Clearly, the ratio of transmembrane to GPI-linked apCAM may be important. One interesting idea raised by Kandel and colleagues is that the spatial distributions of the transmembrane and GPI-linked isoforms may be distinct, with the transmembrane form localized to the neurite processes that mediate homophilic interactions, and the GPI-linked form localized to the synaptic region that mediates heterophilic (sensory to motor neuron) interactions. If this were the case, the selective removal of the transmembrane form would alter the relative levels of the two apCAM isoforms in favor of the GPI-linked form, thereby promoting outgrowth and association with the postsynaptic neuron.These new findings in Aplysia may have important implications for other organisms including insects and mammals. Although MAP kinase has not yet been shown to be critical for learning and memory in vertebratesMartin et al. 1997xMartin, K.C, Michael, D, Rose, J.C, Barad, M, Casadio, A, Zhu, H, and Kandel, E.R. Neuron. 1997; 18Abstract | Full Text | Full Text PDF | Scopus (411)See all ReferencesMartin et al. 1997 demonstrate that exposure of hippocampal slices to forskolin, an activator of adenylate cyclase, leads to MAP kinase phosphorylation within the nucleus in hippocampal pyramidal neurons. This is consistent with the finding that stimuli that trigger LTP in the hippocampus can induce MAP kinase activity (Thomas et al. 1994xThomas, K.L, Laroche, S, Errington, M.L, Bliss, T.V.P, and Hunt, S.P. Neuron. 1994; 13: 737–745Abstract | Full Text PDF | PubMed | Scopus (150)See all ReferencesThomas et al. 1994), and raises the possibility that PKA is important for the activation of MAP kinase, which could in turn mediate events that are critical for the establishment and maintenance of LTP. Since CAMs have already been demonstrated to play a role in the regulation of synaptic potentiation in Aplysia, Drosophila, and mice, it will be important to determine whether features of MAP kinase regulation of CAM expression observed in Aplysia represent a general mechanism of synaptic facilitation that is conserved through evolution.

Light, Immediate-Early Genes, and Circadian Rhythms

Many diverse behaviors exhibit clear circadian rhythms in their expression. In mammals, these rhythms originate from a neural circadian clock located in the suprachiasmatic nuclei (SCN). Recently, signaling pathways activated by light in the SCN have begun to be identified. A specific set of immediate-early genes is induced by light in the SCN, and their expression is correlated with the resetting of circadian behavioral rhythms. These light-regulated immediate-early genes offer multiple inroads into the biology of the SCN: first, they are functional markers for the activation of SCN neurons by light; second, they can direct us to the upstream light-activated (and clock-regulated) signal transduction pathways which mediate their induction; and finally, they encode transcription factor proteins which may play a role in the molecular mechanism of resetting the circadian clock.

Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, Fos expression and creb phosphorylation in the hamster suprachiasmatic nucleus

Aging is associated with a variety of alterations in circadian rhythms, including changes in the response to environmental stimuli. The underlying causes for these age-related changes in the circadian system remain unknown. Recent studies have demonstrated that light induces the expression of Fos and phosphorylation of the cyclic-AMP response element-binding protein in the rodent suprachiasmatic nuclei, the location of a master circadian pacemaker in mammals, suggesting that these transcription factors may mediate the effects of light on the circadian clock. The purpose of this study was to determine the effects of aging upon light-induced phase-shifting of circadian locomotor activity rhythms, Fos protein expression and cyclic-AMP response element-binding protein phosphorylation in the suprachiasmatic nuclei. Young (three to four months) and old (18-22 months) male golden hamsters free-running in constant darkness were exposed to 5-min monochromatic light pulses of different irradiance levels, at circadian time 19, after which either steady-state phase shifts of locomotor activity rhythms were measured, or else immunocytochemistry for Fos or for phospho-cyclic-AMP response element-binding protein was performed. Old hamsters were approximately 20 times less sensitive to the phase-shifting effects of light on the activity rhythm, and the photic irradiance threshold for Fos-like immunoreactivity induction in the suprachiasmatic nuclei was elevated when compared to young animals. Aging was also associated with a deficit in cyclic-AMP response element-binding protein phosphorylation by light. These data indicate that there are dramatic changes in light-activated molecular responses in the suprachiasmatic nuclei of old hamsters, and suggest that these molecular changes may underlie age-related changes in the effects of light on the circadian clock system.

Magnitude of the CREB-dependent transcriptional response is determined by the strength of the interaction between the kinase-inducible domain of CREB and the KIX domain of CREB-binding protein.

ABSTRACT The activity of the transcription factor CREB is regulated by extracellular stimuli that result in its phosphorylation at a critical serine residue, Ser133. Phosphorylation of Ser133 is believed to promote CREB-dependent transcription by allowing CREB to interact with the transcriptional coactivator CREB-binding protein (CBP). Previous studies have established that the domain encompassing Ser133 on CREB, known as the kinase-inducible domain (KID), interacts specifically with a short domain in CBP termed the KIX domain and that this interaction depends on the phosphorylation of Ser133. In this study, we adapted a recently described Escherichia coli-based two-hybrid system for the examination of phosphorylation-dependent protein-protein interactions, and we used this system to study the kinase-induced interaction between the KID and the KIX domain. We identified residues of the KID and the KIX domain that are critical for their interaction as well as two pairs of oppositely charged residues that apparently interact at the KID-KIX interface. We then isolated a mutant form of the KIX domain that interacts more tightly with wild-type and mutant forms of the KID than does the wild-type KIX domain. We show that in the context of full-length CBP, the corresponding amino acid substitution resulted in an enhanced ability of CBP to stimulate CREB-dependent transcription in mammalian cells. Conversely, an amino acid substitution in the KIX domain that weakens its interaction with the KID resulted in a decreased ability of full-length CBP to stimulate CREB-dependent transcription. These findings demonstrate that the magnitude of CREB-dependent transcription in mammalian cells depends on the strength of the KID-KIX interaction and suggest that the level of transcription induced by coactivator-dependent transcriptional activators can be specified by the strength of the activator-coactivator interaction.

Temporal and spatial changes in GATA transcription factor expression are coincident with development of the chicken optic tectum

The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.