An impairment in sniffing contributes to the olfactory impairment in Parkinson's disease

Although the presence of an olfactory impairment in Parkinson's disease (PD) has been recognized for 25 years, its cause remains unclear. Here we suggest a contributing factor to this impairment, namely, that PD impairs active sniffing of odorants. We tested 10 men and 10 women with clinically typical PD, and 20 age- and gender-matched healthy controls, in four olfactory tasks: (i) the University of Pennsylvania smell identification test; (ii and iii) detection threshold tests for the odorants vanillin and propionic acid; and (iv) a two-alternative forced-choice detection paradigm during which sniff parameters (airflow peak rate, mean rate, volume, and duration) were recorded with a pneomatotachograph-coupled spirometer. An additional experiment tested the effect of intentionally increasing sniff vigor on olfactory performance in 20 additional patients. PD patients were significantly impaired in olfactory identification (P < 0.0001) and detection (P < 0.007). As predicted, PD patients were also significantly impaired at sniffing, demonstrating significantly reduced sniff airflow rate (P < 0.01) and volume (P < 0.002). Furthermore, a patient's ability to sniff predicted his or her performance on olfactory tasks, i.e., the more poorly patients sniffed, the worse their performance on olfaction tests (P < 0.009). Finally, increasing sniff vigor improved olfactory performance in those patients whose baseline performance had been poorest (P < 0.05). These findings implicate a sniffing impairment as a component of the olfactory impairment in PD and further depict sniffing as an important component of human olfaction.

X-ray structure of the human hyperplastic discs protein: An ortholog of the C-terminal domain of poly(A)-binding protein

The poly(A)-binding protein (PABP) recognizes the 3′ mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein–protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four α-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

The generation, migration, and differentiation of olfactory neurons in the adult primate brain

In adult rodents, neural progenitor cells in the subependymal (SZ) zone of the lateral cerebral ventricle generate neuroblasts that migrate in chains via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into interneurons. However, the existence of this neurogenic migratory system in other mammals has remained unknown. Here, we report the presence of a homologue of the rodent SZ/RMS in the adult macaque monkey, a nonhuman Old World primate with a relatively smaller OB. Our results—obtained by using combined immunohistochemical detection of a marker for DNA replication (5-bromodeoxyuridine) and several cell type-specific markers—indicate that dividing cells in the adult monkey SZ generate neuroblasts that undergo restricted chain migration over an extended distance of more than 2 cm to the OB and differentiate into granule interneurons. These findings in a nonhuman primate extend and support the use of the SZ/RMS as a model system for studying neural regenerative mechanisms in the human brain.

A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb

Neuronal synchronization in the olfactory bulb has been proposed to arise from a diffuse action of glutamate released from mitral cells (MC, olfactory bulb relay neurons). According to this hypothesis, glutamate spills over from dendrodendritic synapses formed between MC and granule cells (GC, olfactory bulb interneurons) to activate neighboring MC. The excitation of MC is balanced by a strong inhibition from GC. Here we show that MC excitation is caused by glutamate released from bulbar interneurons located in the GC layer. These reciprocal synapses depend on an unusual, 2-amino-5-phosphonovaleric acid-resistant, N-methyl-d-aspartate receptor. This type of feedback excitation onto relay neurons may strengthen the original sensory input signal and further extend the function of the dendritic microcircuit within the main olfactory bulb.

The area code hypothesis revisited: Olfactory receptors and other related transmembrane receptors may function as the last digits in a cell surface code for assembling embryos

Recent evidence emerging from several laboratories, integrated with new data obtained by searching the genome databases, suggests that the area code hypothesis provides a good heuristic model for explaining the remarkable specificity of cell migration and tissue assembly that occurs throughout embryogenesis. The area code hypothesis proposes that cells assemble organisms, including their brains and nervous systems, with the aid of a molecular-addressing code that functions much like the country, area, regional, and local portions of the telephone dialing system. The complexity of the information required to code cells for the construction of entire organisms is so enormous that we assume that the code must make combinatorial use of members of large multigene families. Such a system would reuse the same receptors as molecular digits in various regions of the embryo, thus greatly reducing the total number of genes required. We present the hypothesis that members of the very large families of olfactory receptors and vomeronasal receptors fulfill the criteria proposed for area code molecules and could serve as the last digits in such a code. We discuss our evidence indicating that receptors of these families are expressed in many parts of developing embryos and suggest that they play a key functional role in cell recognition and targeting not only in the olfactory system but also throughout the brain and numerous other organs as they are assembled.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Organization and expression of canine olfactory receptor genes.

Four members of the canine olfactory receptor gene family were characterized. The predicted proteins shared 40-64% identity with previously identified olfactory receptors. The four subfamilies identified in Southern hybridization experiments had as few as 2 and as many as 20 members. All four genes were expressed exclusively in olfactory epithelium. Expression of multiple members of the larger subfamilies was detected, suggesting that most if not all of the cross-hybridizing bands in genomic Southern blots represented actively transcribed olfactory receptor genes. Analysis of large DNA fragments using Southern blots of pulsed-field gels indicated that subfamily members were clustered together, and that two of the subfamilies were closely linked in the dog genome. Analysis of the four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.

Colony-forming progenitors from mouse olfactory epithelium: evidence for feedback regulation of neuron production.

The mammalian olfactory epithelium (OE) supports continual neurogenesis throughout life, suggesting that a neuronal stem cell exists in this system. In tissue culture, however, the capacity of the OE for neurogenesis ceases after a few days. In an attempt to identify conditions that support the survival of neuronal stem cells, a population of neuronal progenitors was isolated from embryonic mouse OE and cultured in defined serum-free medium. The vast majority of cells rapidly gave rise to neurons, which died shortly thereafter. However, when purified progenitors were co-cultured with cells derived from the stroma underlying the OE, a small subpopulation (0.07-0.1%) gave rise to proliferative colonies. A morphologically identifiable subset of these colonies generated new neurons as late as 7 days in vitro. Interestingly, development of these neuronal colonies was specifically inhibited when purified progenitors were plated onto stromal feeder cells in the presence of a large excess of differentiated OE neurons. These results indicate that a rare cell type, with the potential to undergo prolonged neurogenesis, can be isolated from mammalian OE and that stroma-derived factors are important in supporting neurogenesis by this cell. The data further suggest that differentiated neurons provide a signal that feeds back to inhibit production of new neurons by their own progenitors.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons.

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs.

In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels.

Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing sarcoma.

Olfactory neuroblastoma (ONB) is a malignant tumor of the nasal mucosa whose histogenesis is unclear. A relationship to neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is based on morphologic similarities and the expression of similar neural antigens. However, the clinical presentation of ONB differs from that of NB, and MYCN amplification characteristic of NB is not observed. We have therefore examined the relationship of this malignancy to other classes of neural tumors. In previous studies, two ONB cell lines demonstrated cytogenetic features and patterns of protooncogene expression suggestive of a relationship to the Ewing sarcoma family of childhood peripheral primitive neuroectodermal tumors (pPNETs). The pPNETs show t(11;22)(q24;q12) or t(21;22)(q22;q12) chromosomal translocations fusing the EWS gene from 22q12 with either the FL11 gene on 11q24 or the ERG gene on 21q22. We therefore analyzed ONBs for the presence of pPNET-associated gene fusions. Both cell lines showed rearrangement of the EWS gene, and fluorescence in situ hybridization (FISH) of each case demonstrated fusion of EWS and FL11 genomic sequences. Moreover, both lines expressed EWS/FL11 fusion transcripts with in-frame junctions between exon 7 of EWS and exon 6 of FL11 as described for pPNETs. We identified similar gene fusions in four of six primary ONB cases. None of the cases expressed tyrosine hydroxylase, a catecholamine biosynthetic enzyme widely expressed in NB. Our studies indicate that ONB is not a NB but is a member of the pPNET family.

Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-dependent PDE family designated as PDE1C. This enzyme shows high affinity for cAMP and cGMP, having a Km for cAMP much lower than that of any other neuronal Ca2+/calmodulin-dependent PDE. The mRNA encoding this enzyme is highly enriched in olfactory epithelium and is not detected in six other tissues tested. However, RNase protection analyses indicate that other alternative splice variants related to this enzyme are expressed in several other tissues. Within the olfactory epithelium, this enzyme appears to be expressed exclusively in the sensory neurons. The high affinity for cAMP of this Ca2+/calmodulin-dependent PDE and the fact that its mRNA is highly concentrated in olfactory sensory neurons suggest an important role for it in a Ca(2+)-regulated olfactory signal termination.