A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantification.

Recombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.

The sense of smell, its signalling pathways, and the dichotomy of cilia and microvilli in olfactory sensory cells.

Smell is often regarded as an ancillary perception in primates, who seem so dominated by their sense of vision. In this paper, we will portray some aspects of the significance of olfaction to human life and speculate on what evolutionary factors contribute to keeping it alive. We then outline the functional architecture of olfactory sensory neurons and their signal transduction pathways, which are the primary detectors that render olfactory perception possible. Throughout the phylogenetic tree, olfactory neurons, at their apical tip, are either decorated with cilia or with microvilli. The significance of this dichotomy is unknown. It is generally assumed that mammalian olfactory neurons are of the ciliary type only. The existence of so-called olfactory microvillar cells in mammals, however, is well documented, but their nature remains unclear and their function orphaned. This paper discusses the possibility, that in the main olfactory epithelium of mammals ciliated and microvillar sensory cells exist concurrently. We review evidence related to this hypothesis and ask, what function olfactory microvillar cells might have and what signalling mechanisms they use.

Neuropeptide Y in the olfactory microvillar cells.

This paper examines a possible role of microvillar cells in coordinating cell death and regeneration of olfactory epithelial neurons. The olfactory neuroepithelium of mammals is a highly dynamic organ. Olfactory neurons periodically degenerate by apoptosis and as a consequence of chemical or physical damage. To compensate for this loss of cells, the olfactory epithelium maintains a lifelong ability to regenerate from a pool of resident multipotent stem cells. To assure functional continuity and histological integrity of the olfactory epithelium over a period of many decades, apoptosis and regeneration require to be precisely coordinated. Among the factors that have been implicated in mediating this regulation is the neuropeptide Y (NPY). Knockout mice that lack functional expression of this neurogenic peptide show defects in embryonic development of the olfactory epithelium and in its ability to regenerate in the adult. Here we show that, in postnatal olfactory epithelia, NPY is exclusively expressed by a specific population of microvillar cells. We previously characterized these cells as a novel type of putative chemosensory cells, which are provided with a phosphatidyl-inositol-mediated signal transduction cascade. Our findings allow for the first time to suggest that microvillar cells are involved in connecting apoptosis to neuronal regeneration by stimulus-induced release of NPY.

Morituri te salutant? Olfactory signal transduction and the role of phosphoinositides.

During the past 150 years, researchers have investigated the cellular, physiological, and molecular mechanisms underlying the sense of smell. Based on these efforts, a conclusive model of olfactory signal transduction in the vertebrate's nose is now available, spanning from G-protein-mediated odorant receptors to ion channels, which are linked by a cyclic adenosine 3',5'-monophosphate-mediated signal transduction cascade. Here we review some historical milestones in the chronology of olfactory research, particularly emphasising the role of cyclic nucleotides and inositol trisphosphate as alternative second messengers in olfactory cells. We will describe the functional anatomy of the nose, outline the cellular composition of the olfactory epithelium, and describe the discovery of the molecular backbone of the olfactory signal transduction cascade. We then summarize our current model, in which cyclic adenosine monophosphate is the sole excitatory second messenger in olfactory sensory neurons. Finally, a possible significance of microvillous olfactory epithelial cells and inositol trisphosphate in olfaction will be discussed.

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.

Phosphatidyl-inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium.

Ciliated sensory neurons, supporting cells and basal stem cells represent major cellular components of the main olfactory epithelium in mammals. Here we describe a novel class of sensory cells in the olfactory neuroepithelium. The cells express phospholipase C beta-2 (PLC beta2), transient receptor potential channels 6 (TRPC6) and inositol 3, 4, 5-trisphosphate receptors type III (InsP3R-III). Unlike ciliated olfactory neurons, they express neither olfactory marker protein nor centrin, adenylyl cyclase or cyclic nucleotide-gated cation channels. Typical components of the cytoskeleton of microvilli, ezrin and actin are found co-localized with PLC beta2 and TRPC6 in apical protrusions of the cells. In Ca2+-imaging experiments, the cells responded to odours. They express neuronal marker proteins and possess an axon-like process, but following bulbectomy the cells do not degenerate. Our results suggest a novel class of microvillous secondary chemosensory cells in the mammalian olfactory system. These cells, which utilize phosphatidyl-inositides in signal transduction, represent about 5% of all olfactory cells. Their abundance indicates that they play an important role in stimulus-dependent functions and/or the regeneration of the olfactory system.

Single amino acid residue influences the distribution pattern of an inwardly rectifying potassium channel in polarized cells.

We used immunohistochemistry to identify the localization of the inwardly rectifying potassium channels K(ir) 2.1 and its mutant K(ir) 2.1 M84K, and P2X(2) receptors heterologously expressed in Xenopus oocytes, opossum kidney (OK) cells and NG108-15 cells. K(ir) 2.1 wild-type channels were unevenly distributed over the surface of the oocytes and the density was higher at the vegetal pole than at the animal pole. In OK cells the protein was detected at the basolateral membrane, and in NG108-15 cells the protein was found in the soma of the cells and in long thick outgrowing neurites. In contrast, mutant K(ir) 2.1 M84K channels were evenly distributed over the membrane of the oocytes, whereas in OK cells the protein was only detected at the tips of the brush border. In NG108-15 cells the protein was found in the soma of the cells and in all growing neurites. The density of P2X(2) was higher at the animal pole of the oocytes and was restricted to the tips of the brush border in OK cells. In NG108-15 cells the protein was restricted to thinner outgrowing structures and the soma of the cells. We conclude that the exchange of a single amino acid residue in the N-terminus of K(ir) 2.1 changes the distribution pattern in all of the cell types studied. Furthermore, we were able to show that another ion channel sharing the same topology with inwardly rectifying potassium channels showed a different distribution pattern in these cell types.