SummaryMicroglia, the primary resident immune cells of the central nervous system (CNS), exhibit dynamic behavior involving rapid process motility and cellular migration that is thought to underlie key functions of immune surveillance and tissue repair. Although age-related changes in microglial activation have been implicated in the pathogenesis of neurodegenerative diseases of aging, how dynamic behavior in microglia is influenced by aging is not fully understood. In this study, we employed live imaging of retinal microglia in situ to compare microglial morphology and behavioral dynamics in young and aged animals. We found that aged microglia in the resting state have significantly smaller and less branched dendritic arbors, and also slower process motilities, which probably compromise their ability to survey and interact with their environment continuously. We also found that dynamic microglial responses to injury were age-dependent. While young microglia responded to extracellular ATP, an injury-associated signal, by increasing their motility and becoming more ramified, aged microglia exhibited a contrary response, becoming less dynamic and ramified. In response to laser-induced focal tissue injury, aged microglia demonstrated slower acute responses with lower rates of process motility and cellular migration compared with young microglia. Interestingly, the longer term response of disaggregation from the injury site was retarded in aged microglia, indicating that senescent microglial responses, while slower to initiate, are more sustained. Together, these altered features of microglial behavior at rest and following injury reveal an agedependent dysregulation of immune response in the CNS that may illuminate microglial contributions to agerelated neuroinflammatory degeneration.

PurposeMicroglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or “resting” conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates “resting” microglial morphology and behavior.MethodsWe employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia.ResultsRetinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels.ConclusionsOur results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of “resting” microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity.

Ongoing research is warranted because A. vera may represent a new therapeutic class of medications for OSSN treatment.

Purpose To report the identification and characterization of stromal amyloid deposits in patients with keratoconus. Methods The excised corneal buttons from two patients diagnosed clinically with keratoconus underwent histochemical analysis with Masson trichrome, Congo red, Alcian blue and periodic acid-Schiff stains as well as immunohistochemical analysis for the TGFBI protein product (TGFBIp), prealbumin, lysozyme, kappa and lambda light chain expression. Following the collection of DNA from both patients, exons 4 and 11–14 of TGFBI were amplified and sequenced to search for mutations previously associated with dystrophic corneal stromal amyloid deposition. Results Light microscopic examination of the corneal buttons revealed stromal thinning, epithelial basement membrane abnormalities and focal disruption of Bowman’s layer. Multiple stromal deposits were identified that stained red with Masson trichrome, pink with periodic acid-Schiff, and red with Congo red; the Congo red-stained deposits demonstrated birefringence and dichroism with crossed polarized lenses. Immunohistochemical staining demonstrated reactivity of the stromal deposits with antibodies to TGFBIp, but no reactivity with antibodies against prealbumin, lysozyme, or kappa and lambda light chains. Screening of TGFBI exons 4, 11–14 revealed two previously identified SNPs present in the heterozygous state in both individuals, but no other coding region variants. Conclusions Two cases of keratoconus with clinically unsuspected, presumed secondary stromal amyloid deposition are described. Although TGFBIp is identified in the stromal deposits, no previously reported amyloidogenic mutations are identified in TGFBI in either affected individual, indicating a previously undescribed mechanism of stromal amyloid deposition.

Purpose Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder of the corneal endothelium associated with visually significant corneal edema and glaucoma. Statistical genetic analysis of four families with PPCD has demonstrated linkage to a 2.4 cM common support interval on chromosome 20 bordered by the markers D20S182 and D20S139. We sought to identify the genetic basis of PPCD linked to chromosome 20 (PPCD1) by screening the 26 positional candidate genes between these markers in a family previously mapped to the PPCD1 region. Methods The coding regions of the 26 positional candidate genes mapped to the common PPCD1 support interval were amplified and sequenced in affected and unaffected individuals from a family previously linked to the PPCD1 locus. Nine other genes positioned just outside of the common PPCD1 support interval but within the autosomal dominant congenital hereditary endothelial dystrophy (CHED1) interval were also screened. Results Four DNA sequence variants in three of the positional candidate genes demonstrated complete segregation with the affected phenotype: p.Thr109Thr (rs6111803) in OVOL2, p.Arg56Gln (novel variant - RPSnovel) in RPS19P1, and p.Thr85Thr (rs1053834) and p.Pro99Ser (rs1053839) in C20orf79. Each of these four sequence variants demonstrated significant linkage with the affected phenotype in this family (p-value = 2.5 × 10−7 for RPSnovel, rs1053834 and rs1053839; p-value = 8.6 × 10−7 for rs6111803). However, we also identified each of these four sequence variants in ≥ 9% of unaffected control individuals. The haplotype upon which the disease causing mutation is segregating was found to have a population frequency of 4.2% in the CEPH HapMap trios. While a number of other previously described and novel SNPs were identified in the 35 positional candidate genes located within the PPCD1 and CHED1 intervals, none segregated with the affected phenotype. Conclusions We report the absence of a presumed pathogenic coding region mutation in the common PPCD1 support interval. Although minor alleles of four SNPs were identified that segregated with the affected phenotype, the relatively high frequency of each minor allele in the general population indicates that none is a candidate for the causal variant for PPCD. Instead, the causal variant is most likely a coding region deletion or a variant in a non-coding region of the PPCD1 common support interval.

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