Gustavo C. Munguba

Structural Correlation Between the Nerve Fiber Layer and Retinal Ganglion Cell Loss in Mice with Targeted Disruption of the Brn3b Gene

PURPOSE Mice with a targeted disruption of Brn3b (knockout Brn3b(-/-)) undergo the loss of a majority of retinal ganglion cells (RGCs) before birth. Spectral domain optical coherence tomography (SD-OCT) allows for the noninvasive examination of Brn3b(-/-) cellular loss in vivo. METHODS The central retinas of Brn3b(-/-) and phenotypically wild-type (Brn3b(+/+) and Brn3b(±)) mice were imaged by SD-OCT. The combined nerve fiber layer (NFL) and inner plexiform layer (IPL) were manually segmented and thickness maps were generated. The results were confirmed by histologic and immunofluorescence cell counts of the RGC layer (RGCL) of the same retinas. RESULTS The combined NFL and IPL of the Brn3b(-/-) retinas were significantly thinner, and the histologic cell counts significantly lower, than those of the phenotypically wild-type retinas (paired t-test; P < 0.01 and P < 0.01, respectively). The combined NFL and IPL thickness and the histologic cell count correlated highly (R(2) = 0.9612). Immunofluorescence staining revealed significant RGC-specific loss in Brn3b(-/-) retinas (paired t-test; P < 0.01). The distribution of combined central NFL and IPL loss was not localized or sectorial. CONCLUSIONS The strong correlation between the combined layer thickness and histologic cell counts validates manual OCT segmentation as a method of monitoring cell loss in the RGCL. A retinal thickness map assessed if combined NFL and IPL thickness loss in Brn3b(-/-) eyes was topographically specific. Generalized RGC and combined NFL and IPL loss was observed in the Brn3b(-/-) retinas, in contrast to topographically specific RGC loss observed in glaucomatous DBA2/J eyes.

Nerve Fiber Layer Thinning Lags Retinal Ganglion Cell Density Following Crush Axonopathy

PURPOSE We investigated the progressive nature of neurodegenerative structural changes following injury to retinal ganglion cell (RGC) axons using quantifiable and noninvasive in vivo imaging techniques. METHODS To track degenerative RGC progression in retinas following optic nerve crush (ONC) injury, spectral-domain optical coherence tomography (SD-OCT) was used to quantitate the RGC nerve fiber layer (NFL) density. The RGC soma cell density (RCD) was measured by confocal scanning laser ophthalmoscopy (CSLO). The RCD counts were performed using blood vessels as landmarks to anatomically track defined progressive changes in enhanced yellow fluorescent fusion protein (EYFP)-labeled RGCs. RESULTS Following ONC injury, 68% of the observed decrease in RCD measured by CSLO and 54% of the NFL thickness obtained by SD-OCT imaging (N=4 retinas) occurred within the first week. Between days 7 and 14, an additional 22% decrease in RCD was concurrent with a 31% decrease in overall NFL thickness. Finally, between days 14 and 21, an additional 10% decrease in RCD measured in vivo by CSLO and 15% decrease in NFL thickness by SD-OCT was observed. CONCLUSIONS Our data suggest that in vivo CSLO imaging of EYFP-RGC expression and SD-OCT measured NFL thickness are fast and reliable methods that longitudinally track neurodegenerative progression following ONC injury. Neurodegenerative changes in NFL thickness measured by SD-OCT imaging have the same overall trajectory as those observed by CSLO for RCD; however, changes in NFL thickness initially lag behind in vivo RGC soma counts with a slower decline in overall measurable change.

Effects of Glaucoma on Chrna6 Expression in the Retina

Purpose: Recent advances in technology now provide tools capable of tracking genome-wide expression changes occurring in progressive pathological processes. The present experiments were carried out to determine if acetylcholine receptor α 6 subunit (Chrna6) is a reliable retinal ganglion cell (RGC) marker in adult mouse eyes and if Chrna6 expression can be used to track progressive loss of RGCs, such as is observed in the DBA/2J glaucoma model. Methods: Data sets derived from the BXD strains were used to extract gene expression signatures for RGCs. Pooled retinas from DBA/2J or C57BL/6J cases at 1–3 months, 12 months, and 16–17 months were prepared for gene-array and RT-PCR analysis. Globes were fixed in paraformaldehyde and sectioned for immunofluorescence with antibodies against Chrna6. Results: Chrna6 has a cellular expression signature for RGCs with high correlation to Thy1 (r = 0.65), a recognized RGC marker. Immunofluorescence experiments confirm that in the young and adult mouse retina, Chrna6 is preferentially expressed by RGCs. We further show that C3H/HeJ retinas, which lack photoreceptors, also express Chrna6 in the RGC layer. Gene expression array analyses, confirmed by RT-PCR, show progressive loss of Chrna6 expression in retinas of the DBA/2J glaucomatous mouse retinas. Conclusions: Quantitative trait locus analysis provides support for Chrna6 as a RGC marker. Chrna6 expression decreases with death of RGCs in glaucomatous DBA/2J mice and after optic nerve crush injury, further supporting Chrna6 as a reliable RGC marker. High expression of RGC Chrna6 in the absence of photoreceptors is suggestive that Chrna6 expression by RGCs is independent of photoreceptor-derived stimuli.

Abstract P6-11-03: HACE1 loss results in hyperactive Rac signaling conferring resistance to HER2 targeted therapies

HER2-targeted therapies have been instrumental in improving the treatment of HER2+ breast cancer. However, drug resistance usually emerges and remains a big challenge in using anti-HER2 therapies. Previous studies have implicated activation of the Rho GTPase Rac1 by PTEN loss or IGF-1R overexpression as a mechanism of resistance to HER2-targeted therapies. We identified HACE1, an E3 ubiquitin ligase for Rac1, as a tumor suppressor capable of cooperating with HER2 to transform normal mammary epithelial cells. While HACE1 loss alone resulted in enhanced Rac activation, HER2 activation of Rac1 combined with HACE1 loss resulted in even higher levels of activated Rac resulting in the ability to form tumors in immunocompromised mice. In this study, we show that loss of HACE1 confers resistance to the HER2 tyrosine kinase inhibitor (TKI) Lapatinib due to sustained Rac signaling irrespective of EGFR/HER2 signaling. While Lapatinib inhibition alone is capable of attenuating proliferation of HER2 overexpressing mammary epithelial cells, HACE1 loss continues to drive proliferation of in vitro tumor formation (clonogenicity) as well in vivo tumor growth. We demonstrate that Lapatinib sensitivity can be restored using a Rac inhibitor in HACE1 knockdown cells. Moreover, while the Rac inhibitor alone was capable of attenuating the effects of HER2 overexpression and HACE1 loss, simultaneous inhibition of both HER2 and Rac signaling was superior to either monotherapy alone. These results support Rac activation as a mechanism of resistance to HER2-targeted therapies and highlight the use of a Rac inhibitor to treat HER2+ refractory breast cancers. Citation Format: Erik T Goka, Dana S Senderoff, Gustavo Munguba, Marc E Lippman. HACE1 loss results in hyperactive Rac signaling conferring resistance to HER2 targeted therapies [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-11-03.