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Craig M. Nelson, Patrick O'Hayer, David R. Hyde; Tumor Necrosis Factor Alpha Signaling is Required for Photoreceptor Regeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5991.
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Constant light exposure causes photoreceptor apoptosis in dark-adapted adult zebrafish, which induces Müller glia proliferation to yield transiently amplifying pluripotent neuronal progenitor cells that migrate to the ONL and differentiate into photoreceptors. The signals mediating the initiation of regeneration remain unknown. We hypothesize that dying photoreceptors generate a trans-acting signal that induces regeneration. This analysis attempts to identify this photoreceptor-generated signal that initiates regeneration.
Light-damaged and undamaged adult zebrafish retinas were homogenized and the lysate was injected into healthy eyes. Undamaged and light-damaged protein lysates were analyzed by 2D gel electrophoresis and MALDI-TOF mass spectrometry to identify candidate signal proteins. Intravitreal injection and elctroporation of anti-TNFα morpholinos was used to knockdown expression of TNFα protein. Immunohistochemistry was used to analyze cell death (TUNEL), proliferation (PCNA), and TNFα expression, which was also quantified on immunoblots.
Injection of light-damaged retinal lysates significantly increased the number of dividing Müller glia relative to control lysate-injected eyes. Proteomic techniques revealed increased expression of TNFα pathway proteins at the time of photoreceptor death. TNFα expression was increased by photoreceptor cell death and subsequently localized to the proliferating Müller glia. Knockdown of TNFα prior to initiating constant light treatment resulted in significantly reduced numbers of proliferating Müller glia.
A trans-acting signal is generated by dying photoreceptors that stimulates Müller glia proliferation. Proteins from the TNFα signaling pathway increased in expression when the photoreceptor cells were undergoing cell death and knockdown of TNFα significantly decreased the number of proliferating Müller glia. These data suggest that TNFα signaling is required for Müller glia proliferation in response to photoreceptor death.
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