AMPK activation regulates neuronal structure in developing hippocampal neurons.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a cellular and whole organism energy sensor to regulate ATP-consuming (anabolic) and ATP-generating (catabolic) pathways. The heterotrimeric AMPK complex consists of a catalytic α-subunit, regulatory ß-subunit, and an AMP/ATP-binding γ-subunit. Several alternate isoforms exist for each subunit (α1, α2, ß1, ß2, γ1, γ2 and γ3). However, little is known of the expression pattern or function of the individual catalytic complexes in regulating neuronal structure. In this study, we examined the role of AMPK subunits in differentiating hippocampal neurons. We found that during development, the expression of AMPK subunits increase and that activation of AMPK by energetic stress inhibits neuronal development at multiple stages, not only during axon outgrowth, but also during dendrite growth and arborization. The presence of a single functional AMPK catalytic complex was sufficient to mediate these inhibitory effects of energetic stress. Activation of AMPK mediates these effects by suppressing both the mTOR and Akt signaling pathways. These findings demonstrate that the energy-sensing AMPK pathway regulates neuronal structure in distinct regions of developing neurons at multiple stages of development.

Normal mitral cell dendritic development in the setting of Mecp2 mutation.

Rett syndrome (RTT) is an autism spectrum disorder caused by mutation in the gene encoding methyl CpG binding protein 2 (MECP2). Evidence to date suggests that these disorders display defects in synaptic organization and plasticity. A hallmark of the pathology in RTT has been identified as decreased dendritic arborization, which has been interpreted to represent abnormal dendritic formation and pruning during development. Our previous studies revealed that olfactory axons display defective pathfinding and targeting in the setting of Mecp2 mutation. In the present work, we use Mecp2 mutant mouse models and the olfactory system to investigate dendritic development. Here, we demonstrate that mitral cell dendritic development proceeds normally in mutant mice, resulting in typical dendritic morphology at early postnatal ages. We also failed to detect abnormalities in dendritic inputs at symptomatic stages when glomeruli from mutant mice appear smaller in area than the wild type (WT) (6 weeks postnatally). Collectively, these findings suggest that the initial defects in glomeruli impairment seen with Mecp2 mutation do not result from abnormal dendritic development. Our results using the olfactory system indicate that dendritic abnormalities are not an early feature in the abnormalities incurred by Mecp2 mutation.

AMP-activated protein kinase in the brain.

Since its discovery as an important regulator of fuel utilization in the periphery, AMP-activated protein kinase (AMPK) has become a contender for many important cell-intrinsic and organismal roles regarding energy balance in the central nervous system. The challenge will be to delineate the mechanisms by which neuronal AMPK can respond to cellular energy requirements as well as whole body energy demands. Thus, under physiological conditions in the brain, hypothalamic AMPK responds to changes in energy balance/food intake, whereas under pathological conditions, AMPK responds globally in the brain to energy challenge. Modulation of fatty acid metabolism affects energy balance in a context-specific manner and may provide an insight into other mechanisms for selective activation or inhibition of AMPK activity for therapeutic applications.

Developmental changes in the expression of ATP7A during a critical period in postnatal neurodevelopment.

ATP7A is a P-type ATPase that transports copper from cytosol into the secretory pathway for loading onto cuproproteins or efflux. Mutations in Atp7a cause Menkes disease, a copper-deficiency disorder fatal in the postnatal period due to severe neurodegeneration. Early postnatal copper injections are known to diminish degenerative changes in some human patients and mice bearing mutations in Atp7a. In situ hybridization studies previously demonstrated that ATP7A transcripts are expressed widely in the brain. ATP7A-specific antibody was used to study the neurodevelopmental expression and localization of ATP7A protein in the mouse brain. Based on immunoblot analyses, ATP7A expression is most abundant in the early postnatal period, reaching peak levels at P4 in neocortex and cerebellum. In the developing and adult brain, ATP7A levels are greatest in the choroid plexus/ependymal cells of the lateral and third ventricles. ATP7A expression decreases in most neuronal subpopulations from birth to adulthood. In contrast, ATP7A expression increases in CA2 hippocampal pyramidal and cerebellar Purkinje neurons. ATP7A is expressed in a subset of astrocytes, microglia, oligodendrocytes, tanycytes and endothelial cells. ATP7A is largely localized to the trans-Golgi network, adopting the cell-specific and developmentally-regulated morphology of this organelle. The presence of ATP7A in the axons of postnatal, but not adult, optic nerve suggests stage-specific roles for this enzyme. In sum, the precisely-regulated neurodevelopmental expression of ATP7A correlates well with the limited therapeutic window for effective treatment of Menkes disease.

Regulation of olfactory neurogenesis by amidated neuropeptides.

The existence of stem cells in the CNS raises issues concerning the ability of nervous tissues to regenerate in the adult mammal and provides new perspectives on the treatment of degenerative disease and traumatic injury of the nervous system. These cells have a relatively limited range of locations within the nervous system and include cells of the rostral migratory stream, hippocampus, retina, and olfactory epithelium. The olfactory epithelium has been studied as a model of adult neuronal regeneration, with neuronal precursor/basal cells serving as the olfactory "stem cells." The identification of factors that promote neuronal proliferation or regeneration within the olfactory epithelium can provide clues to the process of adult mammalian nervous system repair and treatment. Multiple factors have been examined that appear to influence the proliferation and subsequent maturation of basal cells. These factors include nerve growth factor, fibroblast growth factor-2, epidermal growth factor, and insulin/insulin-like growth factor-1. Recently, two amidated neuropeptides, neuropeptide Y (NPY) and pituitary adenylate cyclase-activating polypeptide (PACAP38), identified in the olfactory epithelium have been shown to promote dramatically neuronal proliferation. The effects of NPY and PACAP suggest that amidated neuropeptides may serve a broad developmental and regenerative role in the mammalian olfactory epithelium.

Kalirin, a GDP/GTP exchange factor of the Dbl family, is localized to nerve, muscle, and endocrine tissue during embryonic rat development.

Kalirin, a homologue of trio and UNC-73, has been previously demonstrated to cause cytoskeletal rearrangements, enhanced outgrowth of neuritic processes, and altered secretion. In the adult rat, kalirin is specifically localized to the central nervous system, with the main adult isoform, kalirin-7, concentrated in neuronal postsynaptic densities. In this study we examined the expression of kalirin in rat tissue from embryonic Day 10 (E10) through E18, using an antibody that detects all known kalirin isoforms. Kalirin expression in the embryo was more widespread than in the adult, with localization of kalirin protein to both neuronal and non-neuronal tissue, such as muscle, lung, intestinal epithelium, and pancreas. In neurons, kalirin was localized both in cell bodies and axon processes; in muscle tissue, kalirin was highly localized to migrating myogenic cells and at muscle attachment sites. Western blotting analysis indicated that kalirin-7, the major adult isoform, was a minor component of embryonic kalirin; the main isoform expressed in the embryo was kalirin-9. This is the first identification of kalirin expression in embryonic tissue and the first demonstration of non-neuronal expression of kalirin. (J Histochem Cytochem 49:833-844, 2001)

Pituitary adenylyl cyclase-activating peptides and alpha-amidation in olfactory neurogenesis and neuronal survival in vitro.

We investigated the role of amidated neuropeptides, and specifically pituitary adenylyl cyclase-activating polypeptide (PACAP), in olfactory neurogenesis and olfactory receptor neuronal survival. Using both immunohistochemistry and in situ hybridization, we find that both peptidylglycine alpha-amidating monooxygenase (PAM), the enzyme responsible for amidation and therefore activation of all amidated neuropeptides, and amidated PACAP are expressed in developing and adult olfactory epithelium. Amidated PACAP is highly expressed in proliferative basal cells and in immature olfactory neurons. The PACAP-specific receptor PAC(1) receptor is also expressed in this population, establishing that these cells can be PACAP responsive. Experiments were conducted to determine whether amidated neuropeptides, such as PACAP38, might function in olfactory neurogenesis and neuronal survival. Addition of PACAP38 to olfactory cultures increased the number of neurons to >250% of control and stimulated neuronal proliferation and survival. In primary olfactory cultures, pharmacologically decreased PAM activity, as well as neutralization of PACAP38, caused neuron-specific loss that was reversed by PACAP38. Mottled (Brindled) mice, which lack a functional ATP7A copper transporter and serve as a model for Menkes disease, provided an in vivo partial loss-of-function PAM knock-out. These mice had decreased amidated PACAP production and concomitant decreased numbers of olfactory receptor neurons. These data establish amidated peptides and specifically PACAP as having important roles in proliferation in the olfactory system and suggest that a similar function exists in vivo.

Neuropeptide Y functions as a neuroproliferative factor.

Neuropeptide Y (NPY) has a number of functions in mammalian physiology. Here we identify a role for NPY in promoting proliferation of postnatal neuronal precursor cells. NPY is synthesized in the postnatal olfactory epithelium by sustentacular cells, previously proposed to function only in structural support. Mice with a targeted deletion of NPY contain half as many dividing olfactory neuronal precursor cells as do controls. Furthermore, NPY-deficient mice develop significantly fewer olfactory neurons by adulthood. NPY acts on multipotent neuronal precursor or basal cells to activate rapidly and transiently the extracellular signal-regulated kinase (ERK)1/2 subgroup of mitogen-activated protein kinases. The NPY Y1 receptor subtype appears to mediate this effect. The ability of NPY to induce neuronal precursor proliferation is mediated by protein kinase C (PKC), indicating an upstream PKC-dependent activation of ERK1/2. These results indicate that NPY may regulate neuronal precursor proliferation in the adult mammal.

Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors.

With the escalation of obesity-related disease, there is great interest in defining the mechanisms that control appetite and body weight. We have identified a link between anabolic energy metabolism and appetite control. Both systemic and intracerebroventricular treatment of mice with fatty acid synthase (FAS) inhibitors (cerulenin and a synthetic compound C75) led to inhibition of feeding and dramatic weight loss. C75 inhibited expression of the prophagic signal neuropeptide Y in the hypothalamus and acted in a leptin-independent manner that appears to be mediated by malonyl-coenzyme A. Thus, FAS may represent an important link in feeding regulation and may be a potential therapeutic target.

Expression of semaphorins in developing and regenerating olfactory epithelium.

Semaphorins provide signals that guide growing axons to their appropriate destinations. The secreted semaphorin, Sema3A, mediates repulsive effects on axons from various neuronal populations in embryonic rats. The authors localized Sema3A mRNA expression in the primary olfactory pathway during development, in adult rats, and in adult rats that were subjected to a unilateral olfactory bulbectomy. Developing rats at ages from embryonic day 14 (E14) to E19 expressed Sema3A in the olfactory receptor neurons (ORNs) of the olfactory epithelium and in chondrogenic structures surrounding the nasal cavity. In vitro, ORN axons at E14 avoided substrate-bound Sema3A. Low levels of Sema3A expression persisted in the normal adult epithelium both in ORNs scattered throughout the epithelium and in small clusters. Three days after a unilateral olfactory bulbectomy, Sema3A transcript levels increased in regenerating neurons. High levels of Sema3A transcript were found at 1 week postbulbectomy, persisted for 2 weeks, and diminished by 3 weeks. Several other murine semaphorins (Sema4A, Sema4B, and Sema4C) were expressed differentially in the primary olfactory pathway both during development and regeneration. These findings suggest that Sema3A and perhaps other semaphorins play a role in directing ORNs out of the epithelium and to the olfactory bulb, their target structure, during both development and regeneration.

Isolation and in vitro differentiation of conditionally immortalized murine olfactory receptor neurons.

Two major challenges exist in our understanding of the olfactory system. One concerns the enormous combinatorial code underlying odorant discrimination by odorant receptors. The other relates to neurogenesis and neuronal development in the olfactory epithelium. To address these issues, continuous cell cultures containing olfactory receptor neurons (ORNs) were obtained from olfactory epithelia of H-2K(b)-tsA58 transgenic mice. ORNs were detected and characterized by immunocytochemistry, RT-PCR, and Western blot for the markers Galpha(olf), adenylyl cyclase III, the olfactory cyclic nucleotide-gated channel subunits, and olfactory marker protein. In culture, epidermal growth factor and nerve growth factor stimulated proliferation, and brain-derived neurotrophic factor and neurotrophin-3 induced cellular maturation. Clonal cell lines were isolated by fluorescence-activated cell sorting with anti-neural cell adhesion molecule antibodies, and of 144 single cells plated, 39 clones were expanded, propagated, and stored in liquid nitrogen. All attempts at recovery of clonal lines from frozen stocks have been successful. The most thoroughly characterized clone, 3NA12, expressed ORN markers and responded to stimulation by single odorants. Each odorant activated approximately 1% of cells in a clonal line, and this suggests that many different odorant receptors may be expressed by these clonal cells. Therefore, these cell lines and the method by which they have been obtained represent a significant advance in the generation of olfactory cell cultures and provide a system to investigate odorant coding and olfactory neurogenesis.

Reconstructing smell.

Odorant signal transduction and neurogenesis are fundamental properties of the olfactory epithelium. Many preparations have been used to elucidate some of the mechanisms underlying these properties. In this article, we briefly review these research areas and describe some of the techniques used to obtain the data. We focus specifically on the cell-culture paradigm and the data obtained from various immortal cell lines in their attempts to reconstruct the olfactory epithelium in vitro.

Localization of the olfactory cyclic nucleotide-gated channel subunit 1 in normal, embryonic and regenerating olfactory epithelium.

The spatial and temporal expression of subunit 1 of the olfactory cyclic nucleotide-gated channel was investigated using affinity-purified anti-fusion protein antibodies. Immunoreactivity was most prominent in the ciliary layer of the olfactory epithelium, but high protein expression was also seen along the entire length of olfactory receptor neuronal axons to the level of the glomeruli. Electron microscopy showed that the long, thin distal compartments of olfactory cilia labeled more prominently than their thicker proximal segments. This was true as soon as these distal parts began to develop. Using light microscopy, developmental expression of olfactory cyclic nucleotide-gated channel subunit 1 could be detected in discrete populations of olfactory receptor neurons by embryonic day 14. Other signaling molecules are expressed either later (Golf) or only at the level of the epithelial surface and not in axons (adenylyl cyclase type III). Following unilateral lesions of the olfactory bulb, olfactory cyclic nucleotide-gated channel subunit 1 immunoreactivity was present early and throughout developing olfactory receptor neurons; adenylyl cyclase type III immunoreactivity, in contrast, was detectable only later, and again present only in the cilial layer. These results support the hypothesis that this subunit of the olfactory cyclic nucleotide-gated channel may be involved in olfactory axon guidance, in addition to its well-described role in olfactory signal transduction.

Odorants induce the phosphorylation of the cAMP response element binding protein in olfactory receptor neurons.

Although odorants are known to activate olfactory receptor neurons through cAMP, the long-term effects of odorant detection are not known. Our recent findings indicate that there is also a delayed and sustained cAMP response, with kinetics sufficient to mediate long-term cellular responses. This cAMP response is mediated by cGMP through activation of adenylyl cyclase by protein kinase G (PKG). Therefore, we investigated the ability of odorants to regulate gene expression in rat olfactory epithelium. The cAMP-responsive binding protein (CREB) is a well-characterized transcription factor regulated by cAMP. We examined CREB activity in rat olfactory epithelium and olfactory receptor neurons (ORNs) after stimulation with odorants. Odorants increased levels of phosphorylated CREB in olfactory epithelium in vivo, and this increase was localized to ORNs in vitro. Incubation with 8-bromo-cGMP or sodium nitroprusside, a guanylyl cyclase activator, also increased phosphorylated CREB. In vitro, cAMP-dependent protein kinase phosphorylated CREB. In contrast, PKG failed to phosphorylate CREB directly in vitro. Our results demonstrate that the delayed odorant-induced cAMP signal activates CREB, which in turn may modulate gene expression in ORNs. In addition, cGMP indirectly affects CREB activation. This effect of cGMP on CREB activity through cAMP provides another mechanism for the modulation of CREB.

Olfactory receptor neurons exist as distinct subclasses of immature and mature cells in primary culture.

The processes of neuronal differentiation and survival are key questions in neurobiology. The olfactory system possesses unique regenerative capacity, as its neurons are continually replaced throughout adulthood from a maintained population of precursor cells. Primary cultures of olfactory epithelium enriched in olfactory neurons would provide a useful model to study the processes of neurogenesis, differentiation and senescence. To determine whether immature olfactory neurons could be isolated in primary culture and to investigate the mechanisms underlying these processes, culture conditions which selectively favored the presence of immature olfactory neurons were optimized. Using low plating densities, a population of cells was identified which, by reverse transcription-polymerase chain reaction, demonstrated messages for olfactory neuronal markers, including Golf, olfactory cyclic nucleotide-gated channel and olfactory marker protein, as well as the p75 low-affinity nerve growth factor receptor. Immunocytochemical analysis showed that these putative immature olfactory neurons possessed immunoreactivity to G(olf), neuron-specific tubulin, neural cell adhesion molecule, synaptophysin and neurofilament. These neurons were defined as olfactory receptor neuron-1 cells. Under these conditions, a separate class of rarely occurring cells with different morphology demonstrated immunoreactivity to mature markers, such as adenylyl cyclase III and olfactory marker protein. Electrophysiologically, these cells displayed properties consistent with those of acutely dissociated olfactory receptor neurons. Another class of rarer cells which represented less than 2% of cells in culture demonstrated immunoreactivity to glial fibrillary acidic protein. These cultures can serve as a model for in vitro analysis of olfactory receptor neuronal development and maintenance, and provide a potential substrate for the development of cell lines.

The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits.

Cyclic nucleotide-gated (CNG) channels play central roles in visual and olfactory signal transduction. In the retina, rod photoreceptors express the subunits CNCalpha1 and CNCbeta1a. In cone photoreceptors, only CNCalpha2 expression has been demonstrated so far. Rat olfactory sensory neurons (OSNs) express two homologous subunits, here designated CNCalpha3 and CNCalpha4. This paper describes the characterization of CNCbeta1b, a third subunit expressed in OSNs and establishes it as a component of the native channel. CNCbeta1b is an alternate splice form of the rod photoreceptor CNCbeta1a subunit. Analysis of mRNA and protein expression together suggest co-expression of all three subunits in sensory cilia of OSNs. From single-channel analyses of native rat olfactory channels and of channels expressed heterologously from all possible combinations of the CNCalpha3, -alpha4, and -beta1b subunits, we conclude that the native CNG channel in OSNs is composed of all three subunits. Thus, CNG channels in both rod photoreceptors and olfactory sensory neurons result from coassembly of specific alpha subunits with various forms of an alternatively spliced beta subunit.

Synaptic transmission and hippocampal long-term potentiation in olfactory cyclic nucleotide-gated channel type 1 null mouse.

Field potential recording was used to investigate properties of synaptic transmission and long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in both hippocampal slices of mutant mice in which the alpha-subunit of the olfactory cyclic nucleotide-gated channel (alpha3/OCNC)1 was rendered null and also in slices prepared from their wild-type (Wt) littermates. Several measures of basal synaptic transmission were unaltered in the OCNC1 knockout (KO), including maximum field excitatory postsynaptic potential (fEPSP) slope, maximum fEPSP and fiber volley amplitude, and the function relating fiber volley amplitude to fEPSP slope and paired-pulse facilitation. When a high-frequency stimulation protocol was used to induce LTP, similar responses were seen in both groups [KO: 1 min, 299 +/- 50% (mean +/- SE), 60 min, 123 +/- 10%; Wt: 1 min, 287 +/- 63%; 60 min, 132 +/- 19%). However, on theta-burst stimulation, the initial amplitude of LTP was smaller (1 min after induction, 147 +/- 16% of baseline) and the response decayed faster in the OCNC1 KO (60 min, 127 +/- 18%) than in Wt (1 min, 200 +/- 14%; 60 min, 169 +/- 19%). Analysis of waveforms evoked by LTP-inducing tetanic stimuli revealed a similar difference between groups. The development of potentiation throughout the tetanic stimulus was similar in OCNC1 KO and Wt mice when high-frequency stimulation was used, but OCNC1 KO mice showed a significant decrease when compared with Wt mice receiving theta-burst stimulation. These results suggest that activation of cyclic nucleotide-gated channels may contribute to the induction of LTP by weaker, more physiological stimuli, possibly via Ca2+ influx.

Calcium-sensitive particulate guanylyl cyclase as a modulator of cAMP in olfactory receptor neurons.

The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events.

Two novel odorant receptor families expressed in spermatids undergo 5'-splicing.

We report the identification of two novel families of odorant receptor (OdR)-like proteins, termed spermatid chemoreceptors (SCRs), in rat spermatids of the testis. The full-length genomic clones encode seven transmembrane domain receptors that share 35-40% identity with certain OdRs and are among the most divergent members of the OdR superfamily based on phylogenetic analysis. RNase protection assays and in situ hybridization studies confirmed the expression of SCRs in spermatids, the post-meiotic, differentiating cell population in the testis. SCR transcripts were undetectable in the prepubertal testis but were readily identified in spermatids of sexually maturing and mature testis. Rapid amplification of cDNA end-polymerase chain reaction and genomic clone sequencing led to the discovery that SCRs are spliced upstream of their presumptive starting methionines. 5'-Splicing of OdRs may regulate the expression of functional chemoreceptors.

Expression of neuron-specific beta-III tubulin during olfactory neurogenesis in the embryonic and adult rat.

The olfactory neuroepithelium retains the unique capacity to produce a new set of mature neurons every three to four weeks from a precursor population situated at the base of the epithelium. It is not known however, whether developing olfactory neurons in the adult rat follow the same program that is initiated embryonically. By tracking the expression of beta-III tubulin (by immunoreactivity to TuJ-1, an isoform-specific antibody) throughout embryogenesis, we have demonstrated a commitment to the olfactory neuron lineage in a subset of cells in the embryonic olfactory placode and followed their development into adulthood. We have also shown that this developmental pattern of beta-III tubulin expression is recapitulated in neurons undergoing a synchronized neurogenic response to either physical or chemical lesion in the adult neuroepithelium. The embryonic expression pattern reported here is similar to, but earlier than that reported for other markers of developing neurons, such as growth-associated protein-43 and neural cell adhesion molecule. The results of these studies suggest the retention of a conserved neurogenic program from embryonic to adult life in the olfactory neuron and, in addition, support the use of a readily accessible system such as the regenerating olfactory neuroepithelium as an alternative means of studying genes which may be crucial to normal neuronal development.