Most mammals have two types of cone photoreceptors, which contain either medium wavelength (M) or short wavelength (S) opsin. The number and spatial organization of cone types varies dramatically among species, presumably to fine-tune the retina for different visual environments. In the mouse, S-and M-opsin are expressed in an opposing dorsal-ventral gradient. We previously reported that cone opsin patterning requires thyroid hormone ␤2, a nuclear hormone receptor that regulates transcription in conjunction with its ligand, thyroid hormone (TH). Here we show that exogenous TH inhibits S-opsin expression, but activates M-opsin expression. Binding of endogenous TH to TR␤2 is required to inhibit S-opsin and to activate M-opsin. TH is symmetrically distributed in the retina at birth as S-opsin expression begins, but becomes elevated in the dorsal retina at the time of M-opsin onset (postnatal day 10). Our results show that TH is a critical regulator of both S-opsin and M-opsin, and suggest that a TH gradient may play a role in establishing the gradient of M-opsin. These results also suggest that the ratio and patterning of cone types may be determined by TH availability during retinal development.photoreceptor ͉ retinal development ͉ nuclear hormone receptor T hyroid hormone (TH) is secreted mostly as thyroxine (T4) from the thyroid gland and converted locally to a transcriptionally active form, 3,5,3Ј-triiodothyronine (T3), by iodothyronine deiodinating enzymes (1). T3 receptors bind to DNA regulatory elements, often as heterodimers with retinoid X receptors (RXRs), to activate or repress transcription of target genes (for review, see refs. 2 and 3). T3 regulates diverse developmental processes in multiple regions of the central nervous system including the hippocampus, cerebral cortex, inner ear, and cerebellum (4-8). Numerous developmental processes are TH-dependent, including cell differentiation, migration, and dendritic growth (2, 3, 9-11). Perturbations in maternal or fetal TH in humans can lead to clinical syndromes ranging from severe mental retardation and hearing loss to mild or moderate deficits in motor skills, language, memory, and attention (12)(13)(14).In the retina, the TH ␤2 receptor isoform (TR␤2) is restricted to cone photoreceptors (15, 16), and we previously showed that it is a critical regulator of late-stage cone differentiation. In the first stage of cone differentiation, cones exit the cell cycle and begin to express specific transcription factors, including cone rod homeobox (CRX), TR␤2,. In the second stage of differentiation, cones express opsin and form specific synaptic connections. Most mammalian cones express short wavelength (S-opsin) or medium wavelength (M-opsin) opsin. However, the spatial arrangement of cone types varies considerably among species. In mice, S-opsin expression begins just before birth and is expressed predominantly in ventral cones. By contrast, M-opsin expression begins at the end of the first postnatal week and is expressed predominantly in dorsal cones (21, 22...

RXRgamma is essential (along with TRbeta2) for suppressing S-opsin in all immature cones and in dorsal cones in the mature retina, but it is not necessary for M-opsin regulation. These results demonstrate a critical role for RXRs in regulating cell differentiation in the CNS and highlight a remarkable conservation of opsin regulation from Drosophila to mammals.

The most common form of blindness at birth, Leber's congenital amaurosis (LCA), is inherited in an autosomal recessive fashion. Mutations in six different retina-specific genes, including a recently discovered gene, AIPL1, have been linked to LCA in humans. To understand the molecular basis of LCA caused by aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) mutations, and to elucidate the normal function of AIPL1, we performed a yeast twohybrid screen using AIPL1 as bait. The screen demonstrated that AIPL1 interacts specifically with farnesylated proteins. Mutations in AIPL1 linked to LCA compromise this activity. These findings suggest that the essential function of AIPL1 within photoreceptors requires interactions with farnesylated proteins. Analysis of isoprenylation in cultured human cells shows that AIPL1 enhances the processing of farnesylated proteins. Based on these findings, we propose that AIPL1 interacts with farnesylated proteins and plays an essential role in processing of farnesylated proteins in retina.S ohocki et al. (1) have reported the identification of a gene on chromosome 17 as a candidate for Leber's congenital amaurosis (LCA), a severe form of inherited retinal dystrophy resulting in complete blindness or severely impaired vision at birth. The protein encoded by this gene is 70% similar to the aryl hydrocarbon receptor-interacting protein (AIP), therefore it was named AIP-like 1 (AIPL1). Association of several mutations in AIPL1 with LCA shows that AIPL1 has a function that is essential for the retina. Consistent with this finding, AIPL1 is expressed only in retina (2).Both AIPL1 and AIP contain three tetratricopeptide repeat (TPR) domains (3). This structure is present in a variety of proteins that participate in diverse biological functions, ranging from protein folding to protein translocation. TPR domains are sites of protein-protein interaction; in particular, they are thought to interact with the C termini of binding partners. For example, the TPR1 and TPR2A domains of p60͞Hop, a heat shock protein (Hsp)70͞Hsp90 adapter protein, bind highly conserved short peptides corresponding to the EEVD sequences at the C termini of Hsp70͞90 proteins (4). The presence of TPR domains in AIPL1 suggests that it may also interact with the C termini of specific proteins within the retina.To investigate the role of AIPL1 in retina, we performed a yeast two-hybrid screen to identify proteins that interact with AIPL1. Our results show that AIPL1 specifically binds to farnesylated proteins. Farnesylation, a type of prenylation, is a posttranslational modification that occurs at the C termini of many proteins. Approximately 2% of all proteins are prenylated (5). Prenylation enhances protein-membrane interactions and protein-protein interactions (6). Hypoprenylation of Rabs due to mutations in Rab escort protein-1 (REP-1) has been associated with choroideremia (CHM), a retinal degeneration (7). Protein prenylation is also required for maintenance of retinal cytoarchitecture and photoreceptor stru...

Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCI) to medium containing lower levels of K+ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad‐spectrum caspase inhibitors, tert‐butoxycarbonyl‐Asp·(O‐methyl)·fluoromethyl ketone and benzyloxycarbonyl‐Val‐Ala‐Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N‐acetyl Tyr‐Val‐Ala‐Asp·chloromethyl ketone (YVAD·cmk), an interleukin‐1β converting enzyme‐preferring inhibitor, and N‐acetyl Asp‐Glu‐Val‐Asp·fluoromethyl ketone (DEVD·fmk), a CPP32‐preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin‐1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP‐ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD‐sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+‐deprived granule neurons.