Localization of ATP sources in the mammalian retinaNishika Muddasani, Salvatore Stella Jr, Matthew Voas

UMKC School of Medicine

Introduction•ATP has always been considered the principal energy source that drives biological processes as well as an important signaling molecule. •The exact mechanism of ATP packaging, sorting, and release, however, has been elusive. •The recent discovery of a vesicular nucleotide transporter (VNUT) has established the existence of a nucleotide transporter that can concentrate and store ATP in neuronal vesicles (see Fig.1).•ATP serves an important role as a signaling molecule in the retina, and is the precursor to adenosine (Fig. 2). However, the role of purines in regulating retinal function has not been elucidated.•The aim of this study is to investigate the vesicular localization in the mammalian retina.

Results1. Retinal expression of VNUT

A. Western Blot B. VNUT immunoreactivity

Conclusion•This study reveals VNUT expression is restricted to horizontal cells, amacrine cells, Müller cells, and ganglion cells (see Fig. 4). •ATP released in the inner retina could contribute to activation of P2X7 receptors (death receptors) on ganglion cells and could play a role in glaucoma.• ATP released from horizontal cells could provide a significant source of ATP, a precursor for adenosine. Adenosine is an important aspect of signaling in the outer retina (Ref. 2). Adenosine has also been implicated in the regulation of dark adaptation. This hypothesis requires further investigation (Ref. 3).

References1.Sawada et al., Proc Natl Acad Sci. 105:5683-6, 2008 2.Stella et al., J. Neurophysiol. 87:351-60, 2002.3.Stella et al., J. Neurophysiol. 90:165-74, 2003.

Figure 2. Antibodies and sources

2. VNUT is absent from photoreceptor terminals

4. VNUT is expressed in Müller cells and inner retinal neurons

A. Western blot of retina and brain with two different VNUT antibodies at 1:1000 dilution. Each antibody recognizes two bands in the brain and two bands in the retina. -actin was used as a control. B. Mouse retina immunostained with anti-VNUT antibody (red). All three antibodies used in this study showed the same labeling pattern in the retina. Adjacent to that is mouse retina immunostained with rabbit anti-VNUT (1:1000) preadsorbed to human VNUT protein, incubated overnight at 4oC, and then applied to retinal sections. Scale bars are 10 microns.

VNUT puncta do not co-localize with PSD-95 at photoreceptor terminals A. VNUT (red) in vertical retinal section. B. PSD-95 (green) in a vertical retinal section. C. Merge of PSD-95 and VNUT. D-E: High magnification zoom of a region in the OPL (250X) of the OPL (D, VNUT, and E, VNUT + PSD-95). VNUT puncta and PSD-95 do not co-localize in the OPL. Scale bars are 10 microns.

Figure 1. Loading ATP into vesicles. (Ref. 2)

Fig. 4 VNUT expression profile. Arrows indicate proposed signaling pathway by VNUT expression. (Ref. 2)

AcknowledgementVision Research Foundation of Kansas City to S.L.S., Jr., R.P.B Challenge Grant to the Department of Ophthalmology at UMKC, and a Sarah Morrison Award to N.M.

I. Brn3a + VNUT

H. VNUT

G. Brn3a

IPL

GCL

IPL

GCL

GCL

IPL

A-C: glutamine synthetase (GS) a Muller cell marker (green) and VNUT (red) immunolabeling. VNUT co-localizes with VNUT in the INL, IPL and at the Muller cell endfeet. D-F: CART (Cocaine and Amphetamine-regulated Transcript) propeptide (green) labels a subset of amacrine cells and co-localizes with VNUT (red) positive amacrine cells (see arrow). G-I: Brn3a (green) is a ganglion cell marker that labels the nuclei of retinal ganglion cells. Scale bars are 10 microns.

Mouse retina immunolabeled with A. Calbindin (green) B. VNUT (red). C. the merge of Calbindin (green) and VNUT (red). VNUT co-localizes with the somata, tips and processes of horizontal cells. Scale bars are 10 microns.

Figure 3. Major elements of purine signaling: The two major mechanisms of release are via gap junction hemichannels (HC) and exocytosis (VNUT). (Ref. 3)

OSOS

ONLONL

OPLOPL

INLINL

IPLIPL

GCLGCL

3. VNUT is expressed in the tips, somata, and processes of horizontal cells

A. VNUT B. PSD-95 C. VNUT + PSD-95

D. VNUT (OPL) E. VNUT + PSD-95 (OPL)

OS

ONL

OPL

INL

IPL

GCL

A. Calbindin

B. VNUT

C. Calbindin + VNUT

F. CART + VNUTE. VNUTD. CART

A. GS B. VNUT C. GS + VNUT

OS

ONL

OPLINL

IPL

GCL

GCL

IPLINLOPL

ONL

OS

G. Brn3a

H. VNUT

I. Brn3a + VNUT

MethodsAnimals: Mice [C57BLk6]. All animals were cared for according to institutional guidelines prescribed by the UMKC IACUC Committee, the UMKC division of laboratory animal medicine, and the National Eye Institute. Adult mice 8 to 12 weeks of age, were anesthetized by CO2 asphyxiation.Western blots: Mouse retina and brain tissues were lysed in HEPES buffered saline plus a protease inhibitor cocktail (Thermo/Pierce) and 2 mM EDTA. Western blots were probed with anti-VNUT, and -actin as a control housekeeping protein. Immunohistochemistry: Immunostaining was performed using the indirect immunofluorescence method.Confocal Imaging: All images were acquired sequentially to avoid spectral bleed through on an upright fixed stage confocal microscope (Nikon C2 confocal microscope).