Diego J. Rodriguez-Gil

Functional expression of the olfactory signaling system in the kidney

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (Golf), are expressed in the kidney. AC3 and Golf colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3−/− mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.

Blood vessels form a migratory scaffold in the rostral migratory stream

In adult mice, new neurons born in the subventricular zone (SVZ), lining the lateral ventricles, migrate tangentially into the olfactory bulb along a well‐delineated path, the rostral migratory stream (RMS). Neuroblasts in the RMS migrate tangentially in chains, without a recognized migratory scaffold. Here we quantitatively examine the distribution of, and relationships between, cells within the RMS, throughout its rostral‐caudal extent. We show that there is a higher density of blood vessels in the RMS than in other brain regions, including areas with equal cell density, and that the orientation of blood vessels parallels the RMS throughout the caudal to rostral path. Of particular interest, migratory neuroblast chains are longitudinally aligned along blood vessels within the RMS, with over 80% of vessel length in rostral areas of the RMS apposed by neuroblasts. Electron micrographs show direct contact between endothelial cells and neuroblasts, although intervening astrocytic processes are often present. Within the RMS, astrocytes arborize extensively, extending long processes that are parallel to blood vessels and the direction of neuroblast migration. Thus, the astrocytic processes establish a longitudinal alignment within the RMS, rather than a more typical stellate shape. This complementary alignment suggests that blood vessels and astrocytes may cooperatively establish a scaffold for migrating neuroblasts, as well as provide and regulate migratory cues. J. Comp. Neurol. 513:94–104, 2009.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110s, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially “compensated” to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Aging in the olfactory system

With advancing age, the ability of humans to detect and discriminate odors declines. In light of the rapid progress in analyzing molecular and structural correlates of developing and adult olfactory systems, the paucity of information available on the aged olfactory system is startling. A rich literature documents the decline of olfactory acuity in aged humans, but the underlying cellular and molecular mechanisms are largely unknown. Using animal models, preliminary work is beginning to uncover differences between young and aged rodents that may help address the deficits seen in humans, but many questions remain unanswered. Recent studies of odorant receptor (OR) expression, synaptic organization, adult neurogenesis, and the contribution of cortical representation during aging suggest possible underlying mechanisms and new research directions.

Chromosomal Location-Dependent Nonstochastic Onset of Odor Receptor Expression

As odorant receptors (ORs) are thought to be critical determinants of olfactory sensory neuron (OSN) axon targeting and organization, we examined the spatiotemporal onset of mice ORs expression from the differentiation of OSNs in the olfactory placode to an aging olfactory epithelium. ORs were first detected in the placode at embryonic day 9 (E9), at the onset of OSN differentiation but before axon extension. By E13, 22 of 23 ORs were expressed. Onset of individual OR expression was diverse; levels and patterns of expression were unique for each OR. Regional distribution of ORs within zones of the olfactory epithelium appeared stable across development; adult-like patterns were observed by E13. Finally, analysis of OR expression and chromosomal location suggests that ORs are not stochastically expressed; they show evidence of coordinated expression. Collectively, these studies demonstrate that ORs are not equally represented in the “olfactome” across an animals lifespan.

Wnt/Frizzled Family Members Mediate Olfactory Sensory Neuron Axon Extension

A comprehensive model has yet to emerge, but it seems likely that numerous mechanisms contribute to the specificity of olfactory sensory neuron (OSN) axon innervation of the olfactory bulb. Elsewhere in the nervous system the Wnt/Fz family has been implicated in patterning of anterior‐posterior axes, cell type specification, cell proliferation, and axon guidance. Because of our work describing cadherin‐catenin family member expression in the primary olfactory pathway, and because mechanisms of Wnt‐Fz interactions can depend in part on catenins, we were encouraged to explore Wnt‐Fz expression and function in OSN axon extension. Here, we show that OSNs express Fz‐1, Fz‐3, and Wnt‐5a, whereas olfactory ensheathing cells (OECs) express Wnt‐4. Fz‐7 is also expressed in the olfactory nerve by cells that delineate large axon fascicles, but are negative for OEC markers. Fz‐1 showed a developmental downregulation. However, in adults it is expressed at different levels across the olfactory epithelium and in restricted glomeruli across the olfactory bulb, suggesting an important role in the formation and maintenance of OSN connections to the olfactory bulb. Reporter TOPGAL mice demonstrated that some OECs located in the inner olfactory nerve layer can respond to Wnt ligands. Of further interest, we show here with in vitro assays that Wnt‐5a increases OSN axon outgrowth and alters growth cone morphology. Our data point to a key role for Wnt/Fz molecules in the development of the mouse olfactory system, providing complementary mechanisms required for OSN axon extension and coalescence. J. Comp. Neurol. 511:301–317, 2008.

Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons

Significance The constant generation of olfactory sensory neurons throughout life makes the system appealing for studies of the mechanisms of axon extension and connectivity. Understanding the mechanisms leading to the genesis of these new neurons is fundamental for the development of therapeutic treatments. We provide here, to our knowledge, the first detailed analysis of the sequential steps leading toward fully differentiated sensory neurons. We show that odorant receptors are not involved in the initial steps of differentiation, but only later in the final process of maturation. Moreover, the proteins that we studied here also have been implicated in normal and pathophysiological events ranging from kidney function to cancer development, making our data valuable across different disciplines. Odorant receptors (OR) are strongly implicated in coalescence of olfactory sensory neuron (OSN) axons and the formation of olfactory bulb (OB) glomeruli. However, when ORs are first expressed relative to basal cell division and OSN axon extension is unknown. We developed an in vivo fate-mapping strategy that enabled us to follow OSN maturation and axon extension beginning at basal cell division. In parallel, we mapped the molecular development of OSNs beginning at basal cell division, including the onset of OR expression. Our data show that ORs are first expressed around 4 d following basal cell division, 24 h after OSN axons have reached the OB. Over the next 6+ days the OSN axons navigate the OB nerve layer and ultimately coalesce in glomeruli. These data provide a previously unidentified perspective on the role of ORs in homophilic OSN axon adhesion and lead us to propose a new model dividing axon extension into two phases. Phase I is OR-independent and accounts for up to 50% of the time during which axons approach the OB and begin navigating the olfactory nerve layer. Phase II is OR-dependent and concludes as OSN axons coalesce in glomeruli.

Renal Cystic Disease Proteins Play Critical Roles in the Organization of the Olfactory Epithelium

It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases – polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) – localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (Golf, AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.

Novel Paracrine Modulation of Notch–DLL4 Signaling by Fibulin-3 Promotes Angiogenesis in High-Grade Gliomas

High-grade gliomas are characterized by exuberant vascularization, diffuse invasion, and significant chemoresistance, resulting in a recurrent phenotype that makes them impossible to eradicate in the long term. Targeting protumoral signals in the glioma microenvironment could have significant impact against tumor cells and the supporting niche that facilitates their growth. Fibulin-3 is a protein secreted by glioma cells, but absent in normal brain, that promotes tumor invasion and survival. We show here that fibulin-3 is a paracrine activator of Notch signaling in endothelial cells and promotes glioma angiogenesis. Fibulin-3 overexpression increased tumor VEGF levels, microvascular density, and vessel permeability, whereas fibulin-3 knockdown reduced vessel density in xenograft models of glioma. Fibulin-3 localization in human glioblastomas showed dense fiber-like condensations around tumor blood vessels, which were absent in normal brain, suggesting a remarkable association of this protein with tumor endothelium. At the cellular level, fibulin-3 enhanced endothelial cell motility and association to glioma cells, reduced endothelial cell sprouting, and increased formation of endothelial tubules in a VEGF-independent and Notch-dependent manner. Fibulin-3 increased ADAM10/17 activity in endothelial cells by inhibiting the metalloprotease inhibitor TIMP3; this resulted in increased Notch cleavage and increased expression of DLL4 independently of VEGF signaling. Inhibition of ADAM10/17 or knockdown of DLL4 reduced the proangiogenic effects of fibulin-3 in culture. Taken together, these results reveal a novel, proangiogenic role of fibulin-3 in gliomas, highlighting the relevance of this protein as an important molecular target in the tumor microenvironment.

Onset of odorant receptors.

Odorant receptors are thought to be critical determinants of olfactory sensory neuron axon targeting and organization. Nonetheless, a systematic characterization of the onset of odorant receptor expression has not yet been done in the main olfactory epithelium. Here, we briefly review our current understanding regarding the onset of odorant receptor expression in the main olfactory epithelium and identify some of those questions which we believe must be of high priority for future study.