Ephrin-A5 (AL-1/RAGS) is essential for proper retinal axon guidance and topographic mapping in the mammalian visual system.

Ephrin-A5 (AL-1/RAGS), a ligand for Eph receptor tyrosine kinases, repels retinal axons in vitro and has a graded expression in the superior colliculus (SC), the major midbrain target of retinal ganglion cells. These properties implicate ephrin-A5 in the formation of topographic maps, a fundamental organizational feature of the nervous system. To test this hypothesis, we generated mice lacking ephrin-A5. The majority of ephrin-A5-/- mice develop to adulthood, are morphologically intact, and have normal anterior-posterior patterning of the midbrain. However, within the SC, retinal axons establish and maintain dense arborizations at topographically incorrect sites that correlate with locations of low expression of the related ligand ephrin-A2. In addition, retinal axons transiently overshoot the SC and extend aberrantly into the inferior colliculus (IC). This defect is consistent with the high level of ephrin-A5 expression in the IC and the finding that retinal axon growth on membranes from wild-type IC is inhibited relative to that on membranes from ephrin-A5-/- IC. These findings show that ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.

A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

Biallelic methylation and silencing of mouse Aprt in normal kidney cells.

Heritable gene silencing is an important mechanism of tumor suppressor gene inactivation in a variety of human cancers. In the present study, we show that methylation-associated silencing of the autosomal adenine phosphoribosyltransferase (Aprt) locus occurs in primary mouse kidney cells. Aprt-deficient cells were isolated from mice that were heterozygous for Aprt, i.e., they contained one wild-type Aprt allele and one targeted allele bearing an insertion of the bacterial neo gene. Although silencing of the wild-type allele alone was sufficient for the cells to become completely Aprt-deficient, biallelic methylation of the promoter region was found to occur. Moreover, despite the absence of selective pressure against the targeted allele, phenotypic silencing of the inserted neo gene accompanied silencing of the wild-type Aprt allele. A potential role for allelic homology in these events is discussed.

Topographic-specific axon branching controlled by ephrin-As is the critical event in retinotectal map development.

The retinotectal projection is the predominant model for studying molecular mechanisms controlling development of topographic axonal connections. Our analyses of topographic mapping of retinal ganglion cell (RGC) axons in chick optic tectum indicate that a primary role for guidance molecules is to regulate topographic branching along RGC axons, a process that imposes unique requirements on the molecular control of map development. We show that topographically appropriate connections are established exclusively by branches that form along the axon shaft. Initially, RGC axons overshoot their appropriate termination zone (TZ) along the anterior-posterior (A-P) tectal axis; temporal axons overshoot the greatest distance and nasal axons the least, which correlates with the nonlinear increasing A-P gradient of ephrin-A repellents. In contrast, branches form along the shaft of RGC axons with substantial A-P topographic specificity. Topography is enhanced through the preferential arborization of appropriately positioned branches and elimination of ectopic branches. Using a membrane stripe assay and time-lapse microscopy, we show that branches form de novo along retinal axons. Temporal axons preferentially branch on their topographically appropriate anterior tectal membranes. After the addition of soluble EphA3-Fc, which blocks ephrin-A function, temporal axons branch equally on anterior and posterior tectal membranes, indicating that the level of ephrin-As in posterior tectum is sufficient to inhibit temporal axon branching and generate branching specificity in vitro. Our findings indicate that topographic branch formation and arborization along RGC axons are critical events in retinotectal mapping. Ephrin-As inhibit branching along RGC axons posterior to their correct TZ, but alone cannot account for topographic branching and must cooperate with other molecular activities to generate appropriate mapping along the A-P tectal axis.

Microangiopathic haemolytic anaemia before and after renal transplantation.

A patient who developed chronic renal failure secondary to the haemolytic uraemic syndrome subsequently developed life threatening microangiopathic haemolytic anaemia following renal transplantation. Transplant nephrectomy was necessary to prevent the progression of thrombocytopenia and associated pulmonary haemorrhage.

Cerebral microhemorrhage and brain ß-amyloid in aging and Alzheimer disease.

OBJECTIVES: Incidental cerebral microhemorrhage (MH) is frequently found in older individuals scanned with susceptibility-weighted MRI (SWI) or gradient-recalled echo MRI. MH have been linked with ß-amyloid (Aß) deposition using (11)C-Pittsburgh compound B (PiB) PET in Alzheimer disease (AD) and cerebral amyloid angiopathy (CAA). We hypothesized that Aß deposition in asymptomatic elderly individuals is associated with lobar MH (LMH). METHODS: This was a cross-sectional study of 84 elderly healthy controls (HC), 28 subjects with mild cognitive impairment (MCI), and 26 subjects with probable AD who underwent 3-T SWI and (11)C-PiB PET. (11)C-PiB cortical binding was quantified normalized to cerebellar cortex (standardized uptake value ratio [SUVR]) and scans classified as positive (PiB+) or negative (PiB-) by visual inspection. MH were manually counted and categorized by region and as lobar or nonlobar. RESULTS: LMH were present in 30.8% of AD, 35.7% of MCI, and 19.1% of HC. The prevalence of LMH among PiB+ subjects was similar, regardless of clinical classification (AD 30.8%, MCI 38.9%, HC 41.4%, p > 0.7). HC with LMH had significantly higher mean neocortical SUVR (1.7 ± 0.5) than HC without LMH (1.3 ± 0.3, p ± 0.01). In HC, there was a positive correlation between number of LMH and SUVR, and between LMH and age. In HC, PiB+ (odds ratio [OR] 7.3, 95% confidence interval [CI] 1.6-33.7, p = 0.01) and age (OR 1.2, 95% CI 1.03-1.3, p = 0.02) both independently predicted the occurrence of LMH using logistic regression. CONCLUSION: Asymptomatic Aß deposition in older adults is strongly associated with LMH.

Value of the gamma camera renogram in the differential diagnosis of acute tubular necrosis and rejection in the early post-transplant period. Comparison with biopsy findings.

A group of 128 consecutive patients was identified on whom renal isotope studies had been performed during the first 2 months after renal transplantation and within 7 days of transplant biopsy. The prospective renogram and biopsy reports were reviewed and graded into 4 categories: severe rejection, predominant rejection, predominant acute tubular necrosis (ATN) and pure ATN. Two extreme patterns of renogram were identified: a sharp rise with a fast decline in the first min, attributed to ATN, and a slowly rising curve with no early peak occurring in severe rejection although not specific to this condition. There was a continuous intermediate spectrum. There was no inter-observer variation in gradings at the 2 ends of the spectrum. In the middle part the difference between 2 independent observers never exceeded more than 1 grade. There was good correlation between the biopsy and renogram gradings, with a discrepancy of more than 1 grade in only 5 patients; 2 of these, with severe rejection on the renogram, showed predominant ATN on biopsy, but the final clinical diagnosis was severe rejection (false positive biopsies). Two patients with biopsies showing severe rejection had a sharp initial up-slope in the renograms but a slower down-slope (over 4 min compared with 1 min in true ATN). With better definition of the criteria these renograms would not have been graded as ATN. There was 1 patient in whom no satisfactory explanation for the discrepancy was found (presumed false positive renogram). When properly defined criteria are used to interpret renograms, this simple test is at least as reliable as renal biopsy in differentiating ATN from rejection in the early post-transplant period, especially in the presence of anuria or severe oligurea.

Tandem B1 elements located in a mouse methylation center provide a target for de novo DNA methylation.

A cis-acting methylation center that signals de novo DNA methylation is located upstream of the mouse Aprt gene. In the current study, two approaches were taken to determine if tandem B1 repetitive elements found at the 3' end of the methylation center contribute to the methylation signal. First, bisulfite genomic sequencing demonstrated that CpG sites within the B1 elements were methylated at relative levels of 43% in embryonal stem cells deficient for the maintenance DNA methyltransferase when compared with wild type embryonal stem cells. Second, the ability of the B1 elements to signal de novo methylation upon stable transfection into mouse embryonal carcinoma cells was examined. This approach demonstrated that the B1 elements were methylated de novo to a high level in the embryonal carcinoma cells and that the B1 elements acted synergistically. The results from these experiments provide strong evidence that the tandem B1 repetitive elements provide a significant fraction of the methylation center signal. By extension, they also support the hypothesis that one role for DNA methylation in mammals is to protect the genome from expression and transposition of parasitic elements.

Regulation of peptidergic vesicle mobility by secretagogues.

Neuropeptides are released into the extracellular space from large secretory granules. In order to reach their release sites, these granules are translocated on microtubules and thought to interact with filamentous actin as they approach the cell membrane. We have used a green fluorescent protein-tagged neuropeptide prohormone (prepro-orphanin FQ) to visualize vesicle trafficking dynamics in NS20Y cells and cultures of primary hippocampal neurons. We found that the majority of secretory granules were mobile and accumulated at both the tips of neurites as well as other apparently specialized cellular sites. We also used live-cell imaging to test the notion that peptidergic vesicle mobility was regulated by secretagogues. We show that treatment with forskolin appeared to increase vesicle rates of speed, while depolarization with high K+ had no effect, even though both treatments stimulated neuropeptide secretion. In cultured hippocampal neurons the green fluorescent protein-tagged secretory vesicles were routed to both dendrites and axons, indicating that peptidergic vesicle transport was not polarized. Basal peptidergic vesicle mobility rates in hippocampal neurons were the same as those in NS20Y cells. Taken together, these studies suggest that secretory vesicle mobility is regulated by specific classes of secretagogues and that neuropeptide containing secretory vesicles may be released from dendritic structures.

Hypomethylation of an expanded FMR1 allele is not associated with a global DNA methylation defect.

The vast majority of fragile-X full mutations are heavily methylated throughout the expanded CGG repeat and the surrounding CpG island. Hypermethylation initiates and/or stabilizes transcriptional inactivation of the FMR1 gene, which causes the fragile X-syndrome phenotype characterized, primarily, by mental retardation. The relation between repeat expansion and hypermethylation is not well understood nor is it absolute, as demonstrated by the identification of nonretarded males who carry hypomethylated full mutations. To better characterize the methylation pattern in a patient who carries a hypomethylated full mutation of approximately 60-700 repeats, we have evaluated methylation with the McrBC endonuclease, which allows analysis of numerous sites in the FMR1 CpG island, including those located within the CGG repeat. We report that the expanded-repeat region is completely free of methylation in this full-mutation male. Significantly, this lack of methylation appears to be specific to the expanded FMR1 CGG-repeat region, because various linked and unlinked repetitive-element loci are methylated normally. This finding demonstrates that the lack of methylation in the expanded CGG-repeat region is not associated with a global defect in methylation of highly repeated DNA sequences. We also report that de novo methylation of the expanded CGG-repeat region does not occur when it is moved via microcell-mediated chromosome transfer into a de novo methylation-competent mouse embryonal carcinoma cell line.

Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling.

Topographic maps are a fundamental feature of sensory representations in nervous systems. The formation of one such map, defined by the connection of ganglion cells in the retina to their targets in the superior colliculus of the midbrain, is thought to depend upon an interaction between complementary gradients of retinal EphA receptors and collicular ephrin-A ligands. We have tested this hypothesis by using gene targeting to elevate EphA receptor expression in a subset of mouse ganglion cells, thereby producing two intermingled ganglion cell populations that express distinct EphA receptor gradients. We find that these two populations form separate maps in the colliculus, which can be predicted as a function of the net EphA receptor level that a given ganglion cell expresses relative to its neighbors.