James I. Morgan

Stimulus-transcription coupling in neurons: role of cellular immediate-early genes

Excitation of neurons results in a series of finely orchestrated responses that occur over a time frame ranging from fractions of a second to hours or days. In the short term, stimulation evokes an array of biochemical and biophysical events that represent the execution of the neurophysiological phenotype of a particular cell. These processes, which contribute to the overall behavior of a neural circuit, do not require de novo protein synthesis. In contrast, stimulation is also linked to long-term phenotypic changes that require alterations in gene expression. Thus, one or more mechanisms must exist that couple cell-surface stimuli to the transcriptional regulatory apparatus of the neuron. In this article James Morgan and Tom Curran detail a stimulus-transcription coupling cascade, involving the products of the proto-oncogenes, c-fos and c-jun, that operates in many cell types including neurons.

TARGETED DISRUPTION OF NMDA RECEPTOR 1 GENE ABOLISHES NMDA RESPONSE AND RESULTS IN NEONATAL DEATH

In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.

Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements

Transcription control regions of eukaryotic genes contain multiple sequence elements proposed to function independently to regulate transcription. We developed transgenic mice carrying fos-lacZ fusion genes with clustered point mutations in each of several distinct regulatory sequences: the sis-inducible element, the serum response element, the fos AP-1 site, and the calcium/cAMP response element. Analysis of Fos-lacZ expression in the CNS and in cultured cells demonstrated that all of the regulatory elements tested were required in concert for tissue- and stimulus-specific regulation of the c-fos promoter. This implies that the regulation of c-fos expression requires the concerted action of multiple control elements that direct the assembly of an interdependent transcription complex.

Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns

In primary dissociated cultures of mouse cerebellum a number of Purkinje cell-specific marker proteins and characteristic ionic currents appear at the appropriate developmental time. During the first week after plating, Purkinje cell dendrites elongate, but as electrical activity emerges the dendrites stop growing and branch. If endogenous electrical activity is inhibited by chronic tetrodotoxin or high magnesium treatment, dendrites continue to elongate, as if they were still immature. At the time that branching begins, intracellular calcium levels become sensitive to tetrodotoxin, suggesting that this cation may be involved in dendrite growth. Even apparently mature Purkinje cells alter their dendritic growth in response to changes in activity, suggesting long-term plasticity.

Proto-oncogene transcription factors and epilepsy

Chemically and electrically induced seizures elicit the rapid transcriptional activation in neurons of a class of genes referred to as cellular immediate-early genes. Since the products of these genes include transcription factors and cytokines, they are proposed to be involved in coupling neuronal excitation to a complex, and poorly understood, programme of cellular responses that involves the regulation of gene expression. Products of two cellular immediate-early genes, c-fos and c-jun, are components of the transcription factor AP-1. In this review, Jim Morgan and Tom Curran discuss how these gene products have begun to reveal some of the molecular details of stimulus-transcription coupling in the nervous system following seizures. In addition, these genes have provided novel reagents and concepts for investigating the biochemical and cellular sequelae of seizure in the CNS, and point towards new avenues of research and potential therapeutic targets in epilepsy.

Fos-IacZ transgenic mice: Mapping sites of gene induction in the central nervous system

Abstract A transgenic mouse line containing a fos-lacZ fusion gene was derived in which β-galactosidase activity identified cell populations expressing fos either constitutively or after stimulation. Seizures and light pulses induced nuclear lacZ activity in defined populations of neurons in vivo, and an array of neurotransmitters, including glutamate, induced the transgene in primary brain cultures. In unstimulated mice, the major sites of fos-lacZ expression were skin, hair follicle, and bone. fos-lacZ mice provide a new avenue for activity mapping studies based on gene expression.

Immediate-early genes: ten years on

T HE REQUEST to write this essay came at an appropriate time for personal reflection. Approximately a decade ago, a chance encounter In the corridars of the Roche Institute of Molecular Biology (RIMB) Instigated a collaborative research programme between a Scottish molecular oncologist and an English neuroscientist on the Induction of gene expression In neurones, Recently, Hoffman-La Roche Inc, annaunced the rclocatlon of the RIMB, glvlng us occasion to think back on the comequences of this transnethnic collaboration. Cellular Immediate-early Genes (c1EG.s) were first descrlbcd as a class of genes Induced within mlnutcs after treatment of strum-deprived flbroblasts wlth growth factors, These observations provldcd the backdrop that cntnlyscd an lntcractlon bctwccn two expatrlot Britons, one lntcrcstcd In the biology of the Induclblc pr~to.onco&~~~c, c+s, and the other in stlmulus~transcrlptlon coupling In ncurones: a calm laboratlvc interaction that has lasted to the present day. The tongcvlty of thls collaboration Is all the more remarkable glvcn the vast cultural gulf defined by Hadrian’s Wall. Thus, despite the fact that lnltlal studies of clEGs had focusscd upon the potentlal role of these genes In cctl.cyclc control, we began to Invest&ate thelr cxpresslon In postmltatlc neuronal cells. The Outcome of these early studies formed the basis of the first revlrw In thls area In Tf!#. Thr early phase of clEG research focussed largely upon c-fbs, and In a hlstorlcal sense, could be referred to as the Fos-pcrlod. Thls phase was driven both by the research Interests of the Investigators that worked initially In the field and, contrary to the notoriously thrifty nature of the Scottish race, the free avartabllity of reagents. One of the most notable achievements of the Fos.pcrlod Is the now wldc. spread USC of /bs exprcsslon as a form of surrogate nruronal-actlvlty mappIn@. Indeed, we developed a fos.lncZ transgcnlc mouse, which enables the use of p=gatactosldase hlstochcmistry to assess fis.ex. pression patterns rapidly and unambiguously irl vA

The cellular prion protein (PrP) selectively binds to Bcl-2 in the yeast two-hybrid system

, The focus on c+s made a substantial contrlbutlon also to our understandlng of how transcriptlou-factor complexes assemble and Interact wlth DNA. For example, the work on Fos and Jun, and thdr llnk to the transcription factor activator protein 1 (API), helped establish the theory that regulation of gene cxpr@sslOll was effected vla homeand heterodlmerlc protein complexes. However, the narrow focus upon c-fos also had the cffcct of limiting perspective on the clEG response, and attributing to It general chatactetlstlcs hased largely upon the behaviour of c-fis In cell culture. As enunciated In the TfNS review’, the early view of the clEG iesponsc was one in which stlmull actlvatcd the transcrlption of clBGs, encoding transc:rlp. tlon factors that regulated other gents that were lnvolvcd In adaptive and plasticity responses. in addition, the response was belleved to be rapid, tran. slent and Stereotypic; that Is, all clEGs were Induced co-ordinately by all stimuli. In the post-Fos period, this basic model has been embellished and expanded. The early emphasis in cIEG research was upon members of the baslc.zlpper (for example, Fos and Jun), zinc-finger (for example, Egrs1) and steroid* receptor [for example, nerve grawth factor= Induclbled (NGFI=B)] superfamliies of transcrlptton factors. However, It had been known for some time that genes that encode non-nuclear pro&Ins belong to rhe cIEG class. Indeed, these genes attracted Increased Interest followlng the reaiiaatlon that it 1s cxtrcmcly dlfflolh to Identify specific target genes for lndlvidual clEG transcrtptlon factors. This pm scnts a serious limltatlan for studies that are almed at clucldatlng the blolaglcai proccsscs that arc controlled by clBGs. In contrast, the general or speclflc function of some of the non-ttanscrlptton factor clEGs is clear. For example, some cIEGs encode enzymes such as, cyclooxygenases, haemoxylenases and proteln phosphatases. Others encode slgnal transduction molecules such as G proteins and cytaklnes. In general, these types of clEG products appear to bc Involved in slgnai transduction processes. Therefore, we suggest that they serve to alter lntraeellular slynnlllng In stlmulated cells and to transmit the clEG response to nelghbourlng tissues via soluble factors. Wlth the advent of detalled studies of clEG expression irr v&o, other general tenets of the clEG response have to be challenged or modified. Axiomatically, the cIEG response is considered to be both rapld dnd transient. But is thls always the case? A number of sttuations exist In viva where induction Is neither rapld nor transient. For example, following transectlon of the? sclatlc nerve, cxpresslan of c-jv# is not upregulated for 12-24 h. Furthermore, once Induced In axotamlzed motor or sensory neurons, its exptesslon can perslst for weeks or months. Similarly, In the hlppocampus of kalnic. acld*treated rodents expression of both fos anti jrct~ appears with a lag of days and remains elevated far an extended period’. Furthermore, continuous ex. prcsslon of fis is observed in several tissues (far example, skin, halr, follicles and bone) In adult and developing anlmels. Taken together, these studies establish that there are at least three modes of expression of clEG. The first is the classical rapid, transient expression seen In culture and 1r1 vlw. The second is characterlaed by a delayed but persistent upregulatlon and the third represents continuous tissue-specific expression, The non-ro

Control of segment-like patterns of gene expression in the mouse cerebellum.

#tosccati in the field assume frequently that ail clEGs are induced by ail stimuli. However, in the delayed phase of expression of clECi, individual clBGs might be expressed In the absence of even closely related family members. For example, In the sciatic-nerve transection experiments described above, Jun is expressed in the complete absence of Fos in axotomked motor neurones. In eon&as& in

Calcium as a modulator of the immediate-early gene cascade in neurons

Bcl-2 can rescue neurons from death and might, therefore, exert its action by associating with neuron-specific proteins. Using LexA-Bcl-2 as bait, we find that the cellular prion protein (PrP) interacts with Bcl-2, but not Bax, in the yeast two-hybrid system. Since the PrP gene has been implicated in neurodegenerative disorders, this preliminary observation suggests a potential pathogenic mechanism for these conditions.