Primate short-wavelength cones share molecular markers with rods.

Macaca, Callithrix jacchus marmoset monkey, Pan troglodytes chimpanzee and human retinas were examined to define if short wavelength (S) cones share molecular markers with L&M cone or rod photoreceptors. S cones showed consistent differences in their immunohistochemical staining and expression levels compared to L&M cones for "rod" Arrestin1 (S-Antigen), "cone" Arrestin4, cone alpha transducin, and Calbindin. Our data verify a similar pattern of expression in these primate retinas and provide clues to the structural divergence of rods and S cones versus L&M cones, suggesting S cone retinal function is "intermediate" between them.

Histologic development of the human fovea from midgestation to maturity.

PURPOSE: To describe the histologic development of the human central retina from fetal week (Fwk) 22 to 13 years. DESIGN: Retrospective observational case series. METHODS: Retinal layers and neuronal substructures were delineated on foveal sections of fixed tissue stained in azure II-methylene blue and on frozen sections immunolabeled for cone, rod, or glial proteins. Postmortem tissue was from 11 eyes at Fwk 20-27; 8 eyes at Fwk 28-37; 6 eyes at postnatal 1 day to 6 weeks; 3 eyes at 9 to 15 months; and 5 eyes at 28 months to 13 years. RESULTS: At Fwk 20-22 the fovea could be identified by the presence of a single layer of cones in the outer nuclear layer. Immunolabeling detected synaptic proteins, cone and rod opsins, and Müller glial processes separating the photoreceptors. The foveal pit appeared at Fwk 25, involving progressive peripheral displacement of ganglion cell, inner plexiform, and inner nuclear layers. The pit became wider and shallower after birth, and appeared mature by 15 months. Between Fwk 25 and Fwk 38, all photoreceptors developed more distinct inner and outer segments, but these were longer on peripheral than foveal cones. After birth the foveal outer nuclear layer became much thicker as cone packing occurred. Cone packing and neuronal migration during pit formation combined to form long central photoreceptor axons, which changed the outer plexiform layer from a thin sheet of synaptic pedicles into the thickest layer in the central retina by 15 months. Foveal inner and outer segment length matched peripheral cones by 15 months and was 4 times longer by 13 years. CONCLUSIONS: These data are necessary to understand the marked changes in human retina from late gestation to early adulthood. They provide qualitative and quantitative morphologic information required to interpret the changes in hyper- and hyporeflexive bands in pediatric spectral-domain optical coherence tomography images at the same ages.

Maturation of the human fovea: correlation of spectral-domain optical coherence tomography findings with histology.

PURPOSE: To correlate human foveal development visualized by spectral-domain optical coherence tomography (SDOCT) with histologic specimens. DESIGN: Retrospective, observational case series. METHODS: Morphology and layer thickness of retinal SDOCT images from 1 eye each of 22 premature infants, 30 term infants, 16 children, and 1 adult without macular disease were compared to light microscopic histology from comparable ages. RESULTS: SDOCT images correlate with major histologic findings at all time points. With both methods, preterm infants demonstrate a shallow foveal pit indenting inner retinal layers (IRL) and short, undeveloped foveal photoreceptors. At term, further IRL displacement forms the pit and peripheral photoreceptors lengthen; the elongation of inner and outer segments (IS and OS, histology) separates the IS band from retinal pigment epithelium. Foveal IS and OS are shorter than peripheral for weeks after birth (both methods). By 13 months, foveal cone cell bodies stack >6 deep, Henle fiber layer (HFL) thickens, and IS/OS length equals peripheral; on SDOCT, foveal outer nuclear layer (which includes HFL) and IS/OS thickens. At 13 to 16 years, the fovea is fully developed with a full complement of SDOCT bands; cone cell bodies >10 deep have thin, elongated, and tightly packed IS/OS. CONCLUSIONS: We define anatomic correlates to SDOCT images from normal prenatal and postnatal human fovea. OCT bands typical of photoreceptors of the adult fovea are absent near birth because of the immaturity of foveal cones, develop by 24 months, and mature into childhood. This validates the source of SDOCT signal and provides a framework to assess foveal development and disease.

Foveal cone density shows a rapid postnatal maturation in the marmoset monkey.

The spatial and temporal pattern of cone packing during marmoset foveal development was explored to understand the variables involved in creating a high acuity area. Retinal ages were between fetal day (Fd) 125 and 6 years. Cone density was determined in wholemounts using a new hexagonal quantification method. Wholemounts were labeled immunocytochemically with rod markers to identify reliably the foveal center. Cones were counted in small windows and density was expressed as cones × 103/mm2 (K). Two weeks before birth (Fd 125-130), cone density had a flat distribution of 20-30 K across the central retina encompassing the fovea. Density began to rise at postnatal day 1 (Pd 1) around, but not in, the foveal center and reached a parafoveal peak of 45-55 K by Pd 10. Between Pd 10 and 33, there was an inversion such that cone density at the foveal center rose rapidly, reaching 283 K by 3 months and 600 K by 5.4 months. Peak foveal density then diminished to 440 K at 6 months and older. Counts done in sections showed the same pattern of low foveal density up to Pd 1, a rapid rise from Pd 30 to 90, followed by a small decrease into adulthood. Increasing foveal cone density was accompanied by 1) a reduction in the amount of Müller cell cytoplasm surrounding each cone, 2) increased stacking of foveal cone nuclei into a mound 6-10 deep, and 3) a progressive narrowing of the rod-free zone surrounding the fovea. Retaining foveal cones in a monolayer precludes final foveal cone densities above 60 K. However, high foveal adult cone density (300 K) can be achieved by having cone nuclei stack into columns and without reducing their nuclear diameter. Marmosets reach adult peak cone density by 3-6 months postnatal, while macaques and humans take much longer. Early weaning and an arboreal environment may require rapid postnatal maturation of the marmoset fovea.

Localizations of visual cycle components in retinal pigment epithelium.

PURPOSE: We used immunocytochemistry and confocal microscopy to determine whether enzymes of the rod visual cycle were uniformly distributed in retinal pigment epithelium (RPE) cells. The localizations of these enzymes were compared to known localizations of retinoid-binding proteins and associated proteins. METHODS: Antibodies to proteins and enzymes associated with the rod visual cycle were used for fluorescence immunocytochemistry with frozen sections of albino mouse and rat retina. Images were obtained with a laser scanning confocal microscope. RESULTS: Components associated with the rod visual cycle were distributed in three distinct patterns in mouse and rat RPE. Three visual cycle enzymes (RDH5, LRAT, and RPE65) were restricted to the somata of RPE cells and were not detected within apical processes. Ezrin, an actin-binding protein, and ERM-binding phosphoprotein50/sodium-hydrogen exchanger regulatory factor1 (EBP50/NHERF1), an ezrin-binding PDZ-domain protein, were largely restricted to RPE apical processes. The fluorescence intensity over Müller cell apical processes was less intense. Cellular retinaldehyde-binding protein (CRALBP), which binds to EBP50/NHERF1, and cellular retinol-binding protein type 1 (CRBP1) were found throughout RPE cells and Müller cells. CONCLUSIONS: Visual cycle enzymes were confined to the somata of RPE cells and did not occur within the long apical processes, either in dark- or light-adapted animals. Other components previously linked to the visual cycle (EBP50/NHERF1 and ezrin) were largely confined to the apical processes, where they could be associated with release of 11-cis-retinal or uptake of all-trans-retinol. CRALBP and CRBP1 were distributed throughout the RPE cell, where they could mediate diffusion of retinoids between apical processes and somata.

Expression of synaptic and phototransduction markers during photoreceptor development in the marmoset monkey Callithrix jacchus.

Marmoset photoreceptor development was studied to determine the expression sequence for synaptic, opsin, and phototransduction proteins. All markers appear first in cones within the incipient foveal center or in rods at the foveal edge. Recoverin appears in cones across 70% of the retina at fetal day (Fd) 88, indicating that it is expressed shortly after photoreceptors are generated. Synaptic markers synaptophysin, SV2, glutamate vesicular transporter 1, and CTBP2 label foveal cones at Fd 88 and cones at the retinal edge around birth. Cones and rods have distinctly different patterns of synaptic protein and opsin expression. Synaptic markers are expressed first in cones, with a considerable delay before they appear in rods at the same eccentricity. Cones express synaptic markers 2-3 weeks before they express opsin, but rods express opsin 2-4 weeks before rod synaptic marker labeling is detected. Medium/long-wavelength-selective (M&L) opsin appears in foveal cones and rod opsin in rods around the fovea at Fd 100. Very few cones expressing short-wavelength-selective (S) opsin are found in the Fd 105 fovea. Across peripheral retina, opsin appears first in rods, followed about 1 week later by M&L cone opsin. S cone opsin appears last, and all opsins reach the retinal edge by 1 week after birth. Cone transducin and rod arrestin are expressed concurrently with opsin, but cone arrestin appears slightly later. Marmoset photoreceptor development differs from that in Macaca and humans. It starts relatively late, at 56% gestation, compared with Macaca at 32% gestation. The marmoset opsin expression sequence is also different from that of either Macaca or human.

Rod photoreceptor differentiation in fetal and infant human retina.

Human rods and cones are arranged in a precise spatial mosaic that is critical for optimal functioning of the visual system. However, the molecular processes that underpin specification of cell types within the mosaic are poorly understood. The progressive differentiation of human rods was tracked from fetal week (Fwk) 9 to postnatal (P) 8 months using immunocytochemical markers of key molecules that represent rod progression from post-mitotic precursors to outer segment-bearing functional photoreceptors. We find two phases associated with rod differentiation. The early phase begins in rods on the foveal edge at Fwk 10.5 when rods are first identified, and the rod-specific proteins NRL and NR2e3 are detected. By Fwk 11-12, these rods label for interphotoreceptor retinoid binding protein, recoverin, and aryl hydrocarbon receptor interacting protein-like 1. The second phase occurs over the next month with the appearance of rod opsin at Fwk 15, closely followed by the outer segment proteins rod GTP-gated sodium channel, rod arrestin, and peripherin. TULP is expressed relatively late at Fwk 18-20 in rods. Each phase proceeds across the retina in a central-peripheral order, such that rods in far peripheral retina are only entering the early phase at the same time that cells in central retina are entering their late phase. During the second half of gestation rods undergo an intracellular reorganization of these proteins, and cellular and OS elongation which continues into infancy. The progression of rod development shown here provides insight into the possible mechanisms underlying human retinal visual dysfunction when there are mutations affecting key rod-related molecules.

Human orbital sympathetic nerve pathways.

PURPOSE: To determine pathways of sympathetic nerves from the orbital apex to the eyelids in human cadaver tissue using immunohistochemistry. METHODS: Human cadaver orbit tissue was sectioned and immunolabeled with a monoclonal antityrosine hydroxylase antibody. RESULTS: In the orbital apex, the nasociliary, frontal, lacrimal, and maxillary branches of the trigeminal nerve demonstrated intense staining upon entering the orbit. Immunoreactive axons from the nasociliary and frontal nerves were observed to join the extraocular motor nerves in the posterior orbit. A plexus of immunolabeled nerves was observed to accompany the ophthalmic artery as it entered the orbital apex. The ophthalmic artery and its branches throughout the orbit demonstrated staining of nerve fibers in the peripheral muscularis. The nasociliary nerve contributed sympathetic branches to the ciliary ganglion. Nerves passing through the ciliary ganglion and a few ganglion cell bodies demonstrated mild to moderate tyrosine hydroxylase reactivity. Axons within the short and long ciliary nerves demonstrated strong tyrosine hydroxylase reactivity and were observed to enter the posterior sclera and the suprachoroidal space. The lacrimal gland demonstrated mild pericapillary staining and occasional stromal nerve fibers reactive to the antityrosine hydroxylase antibody. Müller muscle and the inferior tarsal muscle possessed a strong tyrosine hydroxylase-reactive nerve supply that appeared to originate from the anterior terminal branches of the nasociliary and lacrimal nerves. CONCLUSIONS: Sympathetic nerves enter the orbit via the first and second divisions of the trigeminal nerve and a plexus of nerves surrounding the ophthalmic artery. Extraocular motor nerves receive a sympathetic nerve supply from the sensory nerves in the posterior orbit. Some ciliary ganglion cell bodies demonstrated tyrosine hydroxylase-like reactivity, suggesting a sympathetic modulatory role for the ciliary ganglion. Sympathetics innervate ocular structures via the posterior ciliary nerves. Sympathetic axons travel anteriorly in the orbit via the nasociliary and lacrimal nerves to innervate the sympathetic eyelid muscles. Sympathetic nerves also travel with the frontal branch of the ophthalmic nerve to innervate the forehead skin. The ophthalmic artery and all of its branches contain a perivascular sympathetic nerve supply that may be involved in regulation of blood flow to ocular and orbital structures.

The zinc-binding domain of Nna1 is required to prevent retinal photoreceptor loss and cerebellar ataxia in Purkinje cell degeneration (pcd) mice.

The Purkinje cell degeneration (pcd) mouse undergoes retinal photoreceptor degeneration and Purkinje cell loss. Nna1 is postulated to be the causal gene for pcd. We show that a BAC containing the Nna1 gene rescues retinal photoreceptor loss and Purkinje cell degeneration, confirming that Nna1 loss-of-function is responsible for these phenotypes. Mutation of the zinc-binding domain within the transgene destroyed its ability to rescue neuronal loss in pcd(5J) homozygous mice. In conclusion, Nna1 is required for survival of retinal photoreceptors and other neuron populations that degenerate in pcd mice. A functional zinc-binding domain is crucial for Nna1 to support neuron survival.

Cellular debris and ROS in age-related cortical cataract are caused by inappropriate involution of the surface epithelial cells into the lens cortex.

PURPOSE: To quantify changes in the lens epithelial cells and underlying lens cortex responsible for age-related cortical cataract (ARCC) in the rat. METHODS: Freshly isolated lenses were stained vitally for DNA with Hoechst 33342. Reactive oxygen species (ROS) and mitochondria were visualized and quantified by dihydrorhodamine 123 (DHR). The fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM) of vitally stained lenses. Cortical DNA was verified as such by DNAse I digestion. Cataract reflections were determined from digitalized images of light reflections taken with a low magnification light microscope, or with the LSCM. RESULTS: The anterior surface epithelia of old rat lenses were full of gaps and ragged in appearance with a decrease of over 50% in lens epithelial cell (LEC) density. The surface LECs were frequently seen to have involuted into the cortex at inappropriate sites, forming deposits full of DNA, nuclear and mitochondrial debris, and abundant ROS. These involutions frequently originated near open gaps in the surface epithelia, where they appear to have detached from the capsular membrane. Cortical cataracts in the rat lenses were seen to co-localize with these LEC involutions, as had been seen previously in mice with ARCC. CONCLUSIONS: ARCC in rats co-localized with inappropriate accumulations of nuclei, mitochondria, DNA, and expression of ROS in debris filled foci. These were the result of both involution of surface LECs into areas of cortical ARCC, and by an extension of the normal bow region deep into the anterior and posterior of cataractous lenses. These results were in complete agreement with our previous studies on ARCC in mice.

Development of the human retina in the absence of ganglion cells.

Retinal development was studied in eyes from fetal and neonatal human anencephalic (AnC) and normal age-matched infants to determine the time of retinal ganglion cell (GC) loss and its effect on the development of other retinal neurons. At fetal week (Fwk) 14, GC loss was evident in central retina and by Fwk 19-20 almost all GC were absent, although immunocytochemical labeling for GC markers brain 3, neurofilament M and parvalbumin detected a few GC in the AnC far periphery at older ages. The inner nuclear and inner plexiform (IPL) layers showed variable amounts of thinning but all normal bipolar (BP) and horizontal cell markers were still present. The amacrine (AM) labels calbindin and calretinin were markedly reduced. Lamination for these markers in the IPL was less organized than in normal retinas, with BP and AM markers extending into the degenerated GC layer. Cone and rod photoreceptors had normal morphology and topography in AnC retina and each expressed normal phototransduction and synaptic markers. The prospective fovea was identified in AnC neonatal retina by cone packing and the absence of immunolabeled rod photoreceptors. In one AnC neonatal retina, blood vessels and astrocytes extended across the inner retina in the putative fovea and there was no evidence of a pit. In another AnC neonatal retina, blood vessels and astrocytes formed a foveal avascular zone in the inner retina and a shallow pit was present within this zone. However, both foveas showed evidence for the onset of cone elongation and packing. These findings support the model of Springer and Hendrickson [2005; Vis. Neurosci. 22, 171] in which the foveal avascular zone is critical for pit formation, but suggest that mechanisms inherent to the outer retina may be involved in early stages of foveal cone packing.

Development of the neural retina and its vasculature in the marmoset Callithrix jacchus.

The morphological sequence of retinal development in the New World marmoset monkey Callithrix jacchus is similar to previous reports in Macaca and humans. The incipient fovea is present at fetal day (Fd) 100 as the only part of the retina that contains five distinct layers, including a single layer of cone photoreceptors. A foveal pit begins to form at Fd 135 in the center of the foveal avascular zone which is surrounded by a ring of blood vessels (BV) and astrocytes. At birth (Fd 144) the fovea has a single layer of cones over the pit center where the inner retinal layers are thinned but still separated. After birth the fovea rapidly matures so that foveal cone and pit morphology are similar to adult by 4 months. Five distinct layers and the BV plexus in the nerve fiber layer are present to the retinal edge in neonatal marmosets. Near the optic disc BV are sprouting into outer retinal layers at birth and vascularization of the outer retina is completed by 2 to 3 months. Retinal length increases sharply up to Fd 135, but undergoes a quiescent period around birth during which pit formation begins. Length then increases again up to 4mo, followed by a slow increase into adulthood. The postnatal increase is accompanied by a marked thinning of the peripheral retina. The pars plana appears after birth and its length increases at least until 2 years of age. The major difference between marmoset and Macaca is the relative immaturity of the marmoset fovea at birth, and its rapid development after birth. This makes the marmoset a good candidate for neonatal experimental manipulation of retinal and eye development.

Accumulation of DNA, nuclear and mitochondrial debris, and ROS at sites of age-related cortical cataract in mice.

PURPOSE: Lenses from young and old mice were analyzed by laser scanning confocal microscopy (LSCM) with vital dyes, to determine whether age-related subcapsular and cortical cataracts were linked to the failure of lens fiber cells to degrade nuclei, DNA, and mitochondria properly and whether they result in the overproduction of reactive oxygen species (ROS) at the same sites. RESULTS: As opposed to the clear DNA-free subcapsular and cortical areas of young adult mouse lenses, these areas in cataractous old mouse lenses were found to contain accumulations of nuclei, nuclear fragments, aggregated mitochondria, and amorphous DNA as cortical inclusions (P < 0.001 between young and old lenses). These inclusions correlated spatially with age-related cataracts and with the presence of ROS. The source of such undegraded material was a large expansion of transition nuclei in the bow region and also direct involution of surface lens epithelial cells (LECs) into the underlying cortex, frequently leaving bare patches devoid of nuclei on the surface of the anterior epithelium. METHODS: Live lenses were stained vitally for DNA with Hoechst 33342. ROS and mitochondria were stained and quantified with dihydrorhodamine 123 (DHR). In fixed lenses, DNA was stained with propidium iodide (PI) or 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI). The intensity and position of each probe's fluorescence was determined by LSCM. Cataract localization was ascertained by digitalized microscopy of reflected light. CONCLUSIONS: In aged mice, most subcapsular and cortical cataracts colocalize with accumulations of nuclei, mitochondria, and DNA, These effects are accompanied at the same sites by the production of ROS. The condition is due to the failure of lens fiber cells in the bow region to differentiate properly into the clear fiber state and to the improper involution of cells from the anterior epithelium directly into the underlying cortex, resulting in cataractous opacities.

Essential role of Ca2+-binding protein 4, a Cav1.4 channel regulator, in photoreceptor synaptic function.

CaBP1-8 are neuronal Ca(2+)-binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4(-/-) mice than in control mice. Cabp4(-/-) retinas also had ectopic synapses originating from rod bipolar and horizontal cells tha HJt extended into the outer nuclear layer. Responses of Cabp4(-/-) rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4(-/-) mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Ca(v)1.4 alpha(1)-subunit and shifted the activation of Ca(v)1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca(2+) influx and neurotransmitter release in photoreceptor synaptic terminals.

Cellular retinaldehyde-binding protein interacts with ERM-binding phosphoprotein 50 in retinal pigment epithelium.

PURPOSE: To characterize mechanisms of apical localization of visual cycle components in retinal pigment epithelium (RPE) by the identification of cellular retinaldehyde-binding protein (CRALBP) interaction partners. METHODS: An overlay assay was used to detect interactions of CRALBP with components of RPE microsomes. Interacting proteins were identified with two-dimensional (2D)-PAGE and liquid chromatography tandem mass spectrometry (LC MS/MS). Protein interactions were characterized by affinity chromatography, peptide competition, and expression of protein domains. Protein colocalization in mouse retina was examined using double-label immunocytochemistry and confocal microscopy. RESULTS: CRALBP bound to a 54-kDa protein in RPE microsomes, which was identified as ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50), a PDZ domain protein, also known as sodium/hydrogen exchanger regulatory factory type 1 (NHERF-1). EBP50 and ezrin in solubilized microsomes bound to CRALBP-agarose but not to a control agarose column. CRALBP bound to both recombinant PDZ domains of EBP50 but not to the C-terminal ezrin-binding domain. In outer retina, EBP50 and ezrin were localized to RPE and Müller apical processes. CRALBP was distributed throughout both RPE and Müller cells, including their apical processes. CONCLUSION: RM proteins are multivalent linkers that connect plasma membrane proteins with the cortical actin cytoskeleton. EBP50 interacts with ERM family members through a C-terminal domain and binds targets such as CRALBP through its PDZ domains, thus contributing to an apical localization of target proteins. Our results provide a structural basis for apical localization of a retinoid-processing complex in RPE cells and offer insight into the cell biology of retinoid processing and trafficking in RPE.

Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver.

Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6-8-week-old Lrat-/- mice revealed that the rod outer segments are approximately 35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation.

Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation.

Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in HCE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/-) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/- of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/- of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered cultures at air-liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures.

Polyglutamine-expanded ataxin-7 promotes non-cell-autonomous purkinje cell degeneration and displays proteolytic cleavage in ataxic transgenic mice.

Spinocerebellar ataxia (SCA) type 7 is an inherited neurodegenerative disorder caused by expansion of a polyglutamine tract within the ataxin-7 protein. To determine the molecular basis of polyglutamine neurotoxicity in this and other related disorders, we produced SCA7 transgenic mice that express ataxin-7 with 24 or 92 glutamines in all neurons of the CNS, except for Purkinje cells. Transgenic mice expressing ataxin-7 with 92 glutamines (92Q) developed a dramatic neurological phenotype presenting as a gait ataxia and culminating in premature death. Despite the absence of expression of polyglutamine-expanded ataxin-7 in Purkinje cells, we documented severe Purkinje cell degeneration in 92Q SCA7 transgenic mice. We also detected an N-terminal truncation fragment of ataxin-7 in transgenic mice and in SCA7 patient material with both anti-ataxin-7 and anti-polyglutamine specific antibodies. The appearance of truncated ataxin-7 in nuclear aggregates correlates with the onset of a disease phenotype in the SCA7 mice, suggesting that nuclear localization and proteolytic cleavage may be important features of SCA7 pathogenesis. The non-cell-autonomous nature of the Purkinje cell degeneration in our SCA7 mouse model indicates that polyglutamine-induced dysfunction in adjacent or connecting cell types contributes to the neurodegeneration.