Bruch's Membrane Calcification in Pseudoxanthoma Elasticum: Comparing Histopathology and Clinical Imaging.

Purpose: To investigate the histology of Bruch's membrane (BM) calcification in pseudoxanthoma elasticum (PXE) and correlate this to clinical retinal imaging. Design: Experimental study with clinicopathological correlation. Subjects and Controls: Six postmortem eyes from 4 PXE patients and 1 comparison eye from an anonymous donor without PXE. One of the eyes had a multimodal clinical image set for comparison. Methods: Calcification was labeled with OsteSense 680RD, a fluorescent dye specific for hydroxyapatite, and visualized with confocal microscopy. Scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy (SEM-EDX) and time-of-flight secondary ion mass spectrometry (TOF-SIMs) were used to analyze the elemental and ionic composition of different anatomical locations. Findings on cadaver tissues were compared with clinical imaging of 1 PXE patient. Main Outcome Measures: The characteristics and topographical distribution of hydroxyapatite in BM in eyes with PXE were compared with the clinical manifestations of the disease. Results: Analyses of whole-mount and sectioned PXE eyes revealed an extensive, confluent OsteoSense labeling in the central and midperipheral BM, transitioning to a speckled labeling in the midperiphery. These areas corresponded to hyperreflective and isoreflective zones on clinical imaging. Scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy and TOF-SIMs analyses identified these calcifications as hydroxyapatite in BM of PXE eyes. The confluent fluorescent appearance originates from heavily calcified fibrous structures of both the collagen and the elastic layers of BM. Calcification was also detected in an aged comparison eye, but this was markedly different from PXE eyes and presented as small snowflake-like deposits in the posterior pole. Conclusions: Pseudoxanthoma elasticum eyes show extensive hydroxyapatite deposition in the inner and outer collagenous and elastic BM layers in the macula with a gradual change toward the midperiphery, which seems to correlate with the clinical phenotype. The snowflake-like calcification in BM of an aged comparison eye differed markedly from the extensive calcification in PXE. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

Expression of glucose transporter-2 in murine retina: Evidence for glucose transport from horizontal cells to photoreceptor synapses.

The retina has the highest relative energy consumption of any tissue, depending on a steady supply of glucose from the bloodstream. Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases, including diabetes and diabetic retinopathy. Here, we used immunofluorescence to show that glucose transporter-2 (GLUT2) is expressed in horizontal cells of the mouse neuroretina in proximity to inner retinal capillaries. To study the function of GLUT2 in the murine retina, we used organotypic retinal explants, cultivated under entirely controlled, serum-free conditions and exposed them to streptozotocin, a cytotoxic drug transported exclusively by GLUT2. Contrary to our expectations, streptozotocin did not measurably affect horizontal cell viability, while it ablated rod and cone photoreceptors in a concentration-dependent manner. Staining for poly-ADP-ribose (PAR) indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA damage. The negative effect of streptozotocin on the viability of rod photoreceptors was counteracted by co-administration of either the inhibitor of connexin-formed hemi-channels meclofenamic acid or the blocker of clathrin-mediated endocytosis dynasore. Remarkably, cone photoreceptors were not protected from streptozotocin-induced degeneration by neither of the two drugs. Overall, these data suggest the existence of a GLUT2-dependent glucose transport shuttle, from horizontal cells into photoreceptor synapses. Moreover, our study points at different glucose uptake mechanisms in rod and cone photoreceptors.

Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina.

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1ß, il-33 and tgf-ß were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.

Characterization of Calcium Phosphate Spherical Particles in the Subretinal Pigment Epithelium-Basal Lamina Space in Aged Human Eyes.

Purpose: Micrometer-sized spherules formed of hydroxyapatite or whitlockite were identified within extracellular deposits that accumulate in the space between the basal lamina (BL) of retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch's membrane (sub-RPE-BL space). This investigation aimed to characterize the morphologic features, structure, and distribution of these spherules in aged human eyes with and without clinical indications of age-related macular degeneration (AMD). Design: Experimental study. Participants: Five human eyes with varying degrees of sub-RPE-BL deposits were obtained from the University College London Institute of Ophthalmology and Moorfield's Eye Hospital Tissue Repository or the Advancing Sight Network. Two eyes were reported as having clinical indications of AMD (age, 76-87 years), whereas 3 were considered healthy (age, 69-91 years). Methods: Cadaveric eyes with sub-RPE-BL deposits were embedded in paraffin wax and sectioned to a thickness of 4-10 µm. Spherules were identified and characterized using high-resolution scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy, and time-of-flight secondary ion mass spectroscopy. Main Outcome Measures: High-resolution scanning electron micrographs of spherules, the size-frequency distribution of spherules including average diameter, and the distribution of particles across the central-peripheral axis. Elemental maps and time-of-flight secondary ion mass spectra also were obtained. Results: The precipitation of spherules is ubiquitous across the central, mid-peripheral, and far-peripheral axis in aged human eyes. No significant difference was found in the frequency of spherules along this axis. However, statistical analysis indicated that spherules exhibited significantly different sizes in these regions. In-depth analysis revealed that spherules in the sub-RPE-BL space of eyes with clinical signs of AMD were significantly larger (median diameter, 1.64 µm) than those in healthy aged eyes (median diameter, 1.16 µm). Finally, spherules showed great variation in surface topography and internal structure. Conclusions: The precipitation of spherules in the sub-RPE-BL space is ubiquitous across the central-peripheral axis in aged human eyes. However, a marked difference exists in the size and frequency of spherules in eyes with clinical signs of AMD compared to those without, suggesting that the size and frequency of spherules may be associated with AMD.

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.

Modeling the activation of the alternative complement pathway and its effects on hemolysis in health and disease.

The complement system is a powerful mechanism of innate immunity poised to eliminate foreign cells and pathogens. It is an intricate network of >35 proteins, which, once activated, leads to the tagging of the surface to be eliminated, produces potent chemoattractants to recruit immune cells, and inserts cytotoxic pores into nearby lipid surfaces. Although it can be triggered via different pathways, its net output is largely based on the direct or indirect activation of the alternative pathway. Complement dysregulation or deficiencies may cause severe pathologies, such as paroxysmal nocturnal hemoglobinuria (PNH), where a lack of complement control proteins leads to hemolysis and life-threatening anemia. The complexity of the system poses a challenge for the interpretation of experimental data and the design of effective pharmacological therapies. To address this issue, we developed a mathematical model of the alternative complement pathway building on previous modelling efforts. The model links complement activation to the hemolytic activity of the terminal alternative pathway, providing an accurate description of pathway activity as observed in vitro and in vivo, in health and disease. Through adjustment of the parameters describing experimental conditions, the model was capable of reproducing the results of an array of standard assays used in complement research. To demonstrate its clinical applicability, we compared model predictions with clinical observations of the recovery of hematological biomarkers in PNH patients treated with the complement inhibiting anti-C5 antibody eculizumab. In conclusion, the model can enhance the understanding of complement biology and its role in disease pathogenesis, help identifying promising targets for pharmacological intervention, and predict the outcome of complement-targeting pharmacological interventions.

MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked.

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

microRNA regulatory circuits in a mouse model of inherited retinal degeneration.

miRNA dysregulation is a hallmark of many neurodegenerative disorders, including those involving the retina. Up-regulation of miR-1/133 and miR-142, and down-regulation of miR-183/96/182 has been described in the RHO-P347S mouse retina, a model for a common form of inherited blindness. High-throughput LC-MS/MS was employed to analyse the protein expression of predicted targets for these miRNAs in RHO-P347S mouse retinas; 133 potential target genes were identified. Pathway over-representation analysis suggests G-protein signaling/visual transduction, and synaptic transmission for miR-1, and transmembrane transport, cell-adhesion, signal transduction and apoptosis for miR-183/96/182 as regulated functions in retina. Validation of miRNA-target mRNA interactions for miR-1, miR-96/182 and miR-96 targeting Ctbp2, Rac1 and Slc6a9, respectively, was demonstrated in vitro. In vivo interaction of miR-183/96/182 and Rac1 mRNA in retina was confirmed using miR-CATCH. Additional miRNAs (including miR-103-3p, miR-9-5p) were both predicted to target Rac1 mRNA and enriched by Rac1-miR-CATCH. Other Rac1-miR-CATCH-enriched miRNAs (including miR-125a/b-5p, miR-378a-3p) were not predicted to target Rac1. Furthermore, levels of ~25% of the retinal Rac1 interactors were determined by LC-MS/MS; expression of Rap1gds1 and Cav1 was elevated. Our data suggest significant utilisation of miRNA-based regulation in retina. Possibly more than 30 miRNAs interact with Rac1 in retina, targeting both UTRs and coding regions.

The unconventional secretion of ARMS2.

Age-related maculopathy susceptibility 2 (ARMS2) is a small (11 kDa), primate-specific protein found in the extracellular matrix of the choroid layer in the eye. Variants in the corresponding genetic locus are highly associated with age-related macular degeneration, a leading cause of blindness in the elderly. So far, the physiological function of ARMS2 has remained enigmatic. It has been demonstrated that ARMS2 is a genuine secreted protein devoid of an N-terminal leader sequence, yet the mechanism how it exits the cells and enters the choroidal matrix is not understood. Here, we show that ARMS2 efficiently recruits lectin chaperones from the cytosol and colocalizes with calnexin-positive and protein disulfide isomerase-negative vesicle-like structures. Site-directed mutagenesis revealed critical elements for this interaction. Mutant forms proving unable to interact with the calnexin/calreticulin system failed secretion. On the other hand, blocking the endoplasmic reticulum to Golgi transport with brefeldin A had no effect on ARMS2 secretion. As we found ARMS2 colocalizing with GRASP65, a marker for unconventional protein secretion, autophagic factors are likely to be key in its export. Interleukin-1ß (IL-1ß) is the most established example of secretory autophagy. Co-expression experiments, however, suggest that the transport of ARMS2 is different from that of IL-1ß. In conclusion, in this work we show that ARMS2 is externalized via an unconventional pathway bypassing Golgi. Its intracellular separation from the classical secretion pathway suggests that the maturation of the protein requires a specific biochemical niche and/or may be needed to impede the premature formation of unwanted protein-protein interactions.

MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of ∼ 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.

Gene Structure of the 10q26 Locus: A Clue to Cracking the ARMS2/HTRA1 Riddle?

Age-related macular degeneration (AMD) is a sight-threatening disorder of the central retina. Being the leading cause of visual impairment in senior citizens, it represents a major public health issue in developed countries. Genetic studies of AMD identified two major susceptibility loci on chromosomes 1 and 10. The high-risk allele of the 10q26 locus encompasses three genes, PLEKHA1, ARMS2, and HTRA1 with high linkage disequilibrium and the individual contribution of the encoded proteins to disease etiology remains controversial. While PLEKHA1 and HTRA1 are highly conserved proteins, ARMS2 is only present in primates and can be detected by using RT-PCR. On the other hand, there is no unequivocal evidence for the existence of the encoded protein. However, it has been reported that risk haplotypes only affect the expression of ARMS2 (but not of HTRA1), making ARMS2 the best candidate for being the genuine AMD gene within this locus. Yet, homozygous carriers of a common haplotype carry a premature stop codon in the ARMS2 gene (R38X) and therefore lack ARMS2, but this variant is not associated with AMD. In this work we aimed at characterizing the diversity of transcripts originating from this locus, in order to find new hints on how to resolve this perplexing paradox. We found chimeric transcripts originating from the PLEKHA1 gene but ending in ARMS2. This finding may give a new explanation as to how variants in this locus contribute to AMD.

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye.

Accumulation of protein- and lipid-containing deposits external to the retinal pigment epithelium (RPE) is common in the aging eye, and has long been viewed as the hallmark of age-related macular degeneration (AMD). The cause for the accumulation and retention of molecules in the sub-RPE space, however, remains an enigma. Here, we present fluorescence microscopy and X-ray diffraction evidence for the formation of small (0.5-20 µm in diameter), hollow, hydroxyapatite (HAP) spherules in Bruch's membrane in human eyes. These spherules are distinct in form, placement, and staining from the well-known calcification of the elastin layer of the aging Bruch's membrane. Secondary ion mass spectrometry (SIMS) imaging confirmed the presence of calcium phosphate in the spherules and identified cholesterol enrichment in their core. Using HAP-selective fluorescent dyes, we show that all types of sub-RPE deposits in the macula, as well as in the periphery, contain numerous HAP spherules. Immunohistochemical labeling for proteins characteristic of sub-RPE deposits, such as complement factor H, vitronectin, and amyloid beta, revealed that HAP spherules were coated with these proteins. HAP spherules were also found outside the sub-RPE deposits, ready to bind proteins at the RPE/choroid interface. Based on these results, we propose a novel mechanism for the growth, and possibly even the formation, of sub-RPE deposits, namely, that the deposit growth and formation begin with the deposition of insoluble HAP shells around naturally occurring, cholesterol-containing extracellular lipid droplets at the RPE/choroid interface; proteins and lipids then attach to these shells, initiating or supporting the growth of sub-RPE deposits.

Complement factor D in age-related macular degeneration.

PURPOSE: To examine the role of complement factor D (CFD) in age-related macular degeneration (AMD) by analysis of genetic association, copy number variation, and plasma CFD concentrations. METHODS: Single nucleotide polymorphisms (SNPs) in the CFD gene were genotyped and the results analyzed by binary logistic regression. CFD gene copy number was analyzed by gene copy number assay. Plasma CFD was measured by an enzyme-linked immunosorbent assay. RESULTS: Genetic association was found between CFD gene SNP rs3826945 and AMD (odds ratio 1.44; P = 0.028) in a small discovery case-control series (462 cases and 325 controls) and replicated in a combined cohorts meta-analysis of 4765 cases and 2693 controls, with an odds ratio of 1.11 (P = 0.032), with the association almost confined to females. Copy number variation in the CFD gene was identified in 13 out of 640 samples examined but there was no difference in frequency between AMD cases (1.3%) and controls (2.7%). Plasma CFD concentration was measured in 751 AMD cases and 474 controls and found to be elevated in AMD cases (P = 0.00025). The odds ratio for those in the highest versus lowest quartile for plasma CFD was 1.81. The difference in plasma CFD was again almost confined to females. CONCLUSIONS: CFD regulates activation of the alternative complement pathway, which is implicated in AMD pathogenesis. The authors found evidence for genetic association between a CFD gene SNP and AMD and a significant increase in plasma CFD concentration in AMD cases compared with controls, consistent with a role for CFD in AMD pathogenesis.

ARMS2 is a constituent of the extracellular matrix providing a link between familial and sporadic age-related macular degenerations.

PURPOSE: SNPs in chromosomal region 10q26 harboring PLEKHA1, ARMS2, and Htra1 showed the strongest association with age-related macular degeneration. Recent evidence suggests that in patients homozygous for the risk allele, the lack of synthesis of the poorly characterized ARMS2 is causative of this disorder. The present study was undertaken to gain an understanding of the genuine (patho)physiological role of this protein. METHODS: ARMS2-interacting proteins were identified by using a yeast two-hybrid system and validated by coprecipitation. Immunofluorescence was applied to reveal the localization of ARMS2 in transfected cells and in human eyes. Western blot analyses were performed on extra- and intracellular fractions of ARMS2-expressing cells to demonstrate the secretion of ARMS2. RESULTS: Contrary to previous reports, this study showed that ARMS2 is a secreted protein that binds several matrix proteins. Notably, ARMS2 directly interacts with fibulin-6 (hemicentin-1). Mutations in the fibulin-6 gene have been demonstrated to cause familial AMD. ARMS2 also interacts with further extracellular proteins, several of which have been implicated in macular dystrophies. Although ARMS2 apparently lacks any classic targeting sequence, it is translocated to the endoplasmic reticulum in cultured cells before secretion. ARMS2 is mostly confined to choroid pillars in human eyes, representing a part of extracellular matrix and corresponding to the principal sites of drusen formation. CONCLUSIONS: The pivotal role of the extracellular matrix in the progression of AMD is underlined by the abnormal deposition of extracellular debris in the macula, observed frequently in affected individuals. The results have shown that ARMS2 may be necessary for proper matrix function.

Cloning and characterization of rat importin 9: implication for its neuronal function.

We describe the structure of the rat importin 9 gene, together with its transcripts and the encoded protein with its putative functional domains. The importin 9 gene contains 24 exons in a genomic region spanning >52,000 bp. It is transcribed into two mRNAs, generated by means of alternative polyadenylation site usage arranged in tandem. Both transcripts possess the same noncanonical polyadenylation signal (AGUAAA) in rat, this hexamer being conserved in all vertebrates examined. Additionally, intron 8 is bordered by AT-AC dinucleotides. Importin 9 is expressed throughout adult rat tissues, but the 114-kDa Importin 9 protein was detected only in the brain. The localization of the Importin 9 protein was examined by immunohistochemistry in both adult rat tissues and primary hippocampal cell cultures. The strongest labeling was detected in vivo in areas populated by neurons in high density and also in the dendritic processes emanating from these cells. This protein was clearly concentrated in the nuclei of these cells, although their cytoplasms too were heavily labeled. Strong cytoplasmic and very strong nuclear staining was found in a vast majority of the cells with neuronal morphology in vitro. Cultured cells with glial morphology generally exhibited a weaker cytoplasmic labeling. In these cells, the signal decorated the nuclear envelope without nuclear staining and gradually dwindled toward the cell periphery. These results hint at the cell- or tissue-type specific functions of this type of importin protein.

Multiple calmodulin mRNAs are selectively transported to functionally different neuronal and glial compartments in the rat hippocampus. An electron microscopic in situ hybridization study.

The ultrastructural distribution of the calmodulin (CaM) mRNAs transcribed from the three CaM genes was studied in the CA1 region of the adult rat hippocampus by means of electron microscopic in situ hybridization. Digoxigenin-labeled CaM gene-specific riboprobes were detected with nanogold-anti-digoxigenin antibody conjugate. The CaM mRNAs were differentially distributed in both the neuronal and glial cell compartments. The greatest difference in neuronal distribution of the CaM mRNAs was found in the dendrites, where the mRNAs transcribed from the CaM I and III genes were much more abundant than the CaM II mRNA. The neuronal perikarya were heavy labeled for all the CaM mRNAs. Interestingly, the myelinated axons and axon terminals also contained small amounts of nanogold particles for all the CaM mRNAs, which diminished with increasing distance from the soma. Most of the synaptic profiles, however, contained labeling only in the postsynaptic region. The CaM mRNAs were differentially distributed in the glial cells. While the glial cell somata were only lightly labeled, surprisingly concentrated labeling was present in the perisynaptic and perivascular astrocytic processes. In general, the CaM II mRNA was the least represented in the glial processes. Only a very low CaM gene expression was observed in the endothelial and resting microglial cells. These results provide ultrastructural evidence for differential targeting of the multiple CaM mRNA transcripts to the intracellular compartments and suggest their microdomain-specific regulation.

Calmodulin, and various ways to regulate its activity.

Calmodulin (CaM), the ubiquitous calcium sensor protein, is involved in almost all intracellular events. In higher vertebrates, a single protein is encoded by multiple, co-expressed genes, and the number of discrete CaM transcripts produced by a single cell is further increased by intense alternative polyadenylation signal usage. It appears most likely that the individual transcripts possess unique intracellular fates, so that this apparent redundancy multiplies the number of challenges which the cell is able to respond to. The promoter regions of the different CaM genes have been analyzed. Several putative transcription factor binding sites have been identified; however, the elements responsible for their generally strong co-expression, and even those providing different spatial and temporal control, remain to be elucidated. Moreover, a powerful posttranscriptional control mechanism is responsible for the establishment of local intracellular CaM mRNA pools. This is mainly achieved by the selective targeting of mRNAs to various cellular domains, although regulation via mRNA stability cannot be ruled out. Finally, tailoring of the CaM protein itself offers the fastest way whereby the properties of this Ca2+-receptor protein can be changed. Indeed, several posttranslational modifications of CaM were described earlier, but their functions are not yet understood. Here, we briefly review the regulatory levels from the gene transcription to the covalent modifications of the synthesized protein.

Intracellular targeting of calmodulin mRNAs in primary hippocampal cells.

We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling.