Characterization of RNA binding protein RBPMS as a novel marker for retinal ganglion cells.

The recently discovered RNA binding protein RBPMS appears to be a strong candidate as an RGC marker. To test its specificity and reliability, we performed immunostaining for RBPMS and Brn3a (the latter which labels a large percentage of RGCs), and compared them to retrogradely labeled RGCs. Previous studies demonstrate that retrograde labeling with Fluorogold yields over 99% labeling of RGCs. Based on FG labeling, results indicate that RBPMS was expressed specifically in RGCs, and closely matched Fluorogold's labeling efficiency. This indicates that RBPMS is expressed in over 99% of RGCs, unlike Brn3a--which is expressed in only about 80% of RGCs. Thus, our studies provide strong evidence that RBPMS is a more reliable and specific marker for RGCs, compared to other commonly used markers.

ABSTRACT

AcknowledgementsMajor funding for this work was provided by the National Eye Institute (grant 7R01EY020913-04 ). Thanks also go to the UCSD Division of Biological Sciences and the Undergraduate Research Scholarship program for additional funding—and to Ms. W. Kwok (UCSD) for her generous support through the Eureka! Scholarship to LIS. Original poster layout was provided by Ms. E.K. Nguyen (UCLA). I also thank PV and XZ for their guidance, Mr. A. Kreymerman for help with editing, Dr. T. Stiles for help with printing, and the other members of the Goldberg lab for their continual support and good will.

CONCLUSION

Abridged References

FG labeling colocalizes with immunostaining against RBPMS in over 99% of cells

Immunostaining against RBPMS yields higher levels of RGC labeling than staining against other markers

Immunostaining against RBPMS labels RGCs in a more consistent manner than against current markers

Lior I. Schenk1, Praseeda Venugopalan1,2, Xiong Zhang1, and Jeffrey L. Goldberg1,3

1 University of California, San Diego, La Jolla, CA; 2 University of Miami, Miami, FL; 3 UCSD Shiley Eye Center, La Jolla, CA

BACKGROUNDHow can we identify the cellular impact of retinal disease? One challenge lies in accurately quantifying the loss of cells such as retinal ganglion cells (RGCs). This requires reliable markers that identify all the different subtypes of RGCs. Several studies investigating different RGC markers have been performed--but have yielded varying levels of success, due to inconsistent expression and specificity. As such, there is a need in retinal research for a more efficient RGC marker.

RBPMS yields labeling of RGCs with over 99% specificity,

in a consistent reliable manner, and with higher output than that of current

antibodies. By utilizing this powerful new marker, we can even better visualize each disease’s pathway of action--putting us one step further in the fight for sight.

METHODS

Immunohistochemistry

Retrograde Labeling of RGCs

Fluorogold dye is first applied to the surface of the superior colliculus.

FG taken up by RGC axon terminals is transported retrogradely up the optic nerve to somata in the retina.

Complete FG transport results in labeling of about 98% of RGCs.

1. Kwong JM, Caprioli J, Piri N (2010). RNA binding protein with multiple splicing: a new marker for retinal ganglion cells. Invest Ophthalmol Vis Sci.

2. Nadal-Nicolás FM, Jiménez-López M, Sobrado-Calvo P, Nieto-López L, Cánovas-Martínez I, Salinas-Navarro M, Vidal-Sanz M, Agudo M (2009). Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci.

3. Chiu K, Lau WM, Yeung SC, Chang RC, So KF (2008). Retrograde labeling of retinal ganglion cells by application of fluoro-gold on the surface of superior colliculus. J Vis Exp.

All animals were housed, maintained, and examined with accordance to the IACUC and UCSD’s policies and procedures as set forth by the Animal Research Committee.

Antibodies: RBPMS: uniquely expressed in retinal

ganglion cells, for reasons unknown. RNA binding protein; stains cytoplasm of RGCs.

BRN-3: a family of proteins , each with various roles in RGC development. Transcription factors; stain RGC variably depending on developmental stage (as well as blood vessels due to peroxidase activity).

DAPI: used as a nuclear and chromosome counterstain. Binds strongly to A,T regions of DNA resulting in strong labeling of nuclei.

RESULTS

FG

RBPMS Brn3a

0

<1

06628

5

<1

A B C

D (A,B) More RGCs are present in the RBPMS stain than in the Brn3a stain. (B) Brn3a additionally stains blood vessels, demonstrating a lack of specificity for RGCs alone. (C) A merged image with DAPI staining depicts additional populations of non-RGCs in the amacrine cell layer. (D) A merged image compares RPMS and Brn3a IHC. Arrowheads: Multiple cells were positive for RBPMS but not for Brn3a.

Brn3a RBPMSBrn3aDAPI

RBPMSBrn3aDAPI

RBPMS

RBPMS Brn3a0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

RGC labeling (relative to RBPMS)RBPMSBrn3a

FG

• Over 99% of cells labeled by FG were also positive for RBPMS.

• RBPMS staining colocalizes with FG labeling at a higher rate than Brn3a and FG.

• Though not all cells quantified were positive for FG or for Brn3a, over 99% were positive for RBPMS.

• n=1; more experiments necessary for conclusive data.

(A) RBPMS immunofluorescence overlayed onto Brn3a immunofluorescence and FG retrograde labeling. Arrows: Multiple RGCs (identify confirmed by FG) were positive for RBPMS and FG, but negative for Brn3a—indicating that RBPMS may be more effective than Brn3a as an RGC marker. (B) Quantification performed in 4 retinae (stained for RBPMS, Brn3a, and DAPI) demonstrated higher RBPMS staining overall. On average, and in all zones of the retina, RBPMS achieved higher RGC labeling than Brn3a.

• A portion of the retina was not labeled by FG. This could be due to afferents traveling to areas other than the SC, such as the LGN and the SCN.

• Though Brn3a staining covered the entirety of the retina, relevant areas of examination resulted in higher RBPMS staining overall.

RBPMS labels RGCs in a highly consistent and reliable manner.

RBPMSBrn3a

FG

A B

RBPMS labels more RGCs than other markers do.

Counterstaining against FG confirms RGC specificity.