The energy metabolism of the retina might comply with daily changes in energy demand and is impaired in diabetic retinopathy - one of the most common causes of blindness in Europe and the USA. The aim of this study was to investigate putative adaptation of energy metabolism in healthy and diabetic retina. Hence expression analysis of metabolic pathway genes was performed using quantitative polymerase chain reaction, semi-quantitative western blot and immunohistochemistry. Transcriptional profiling of key enzymes of energy metabolism identified transcripts of mitochondrial fatty acid β-oxidation enzymes, i.e. carnitine palmitoyltransferase-1α (Cpt-1α) and medium chain acyl-CoA dehydrogenase (Acadm) to display daily rhythms with peak values during daytime in preparations of the whole retina and microdissected photoreceptors. The cycling of both enzymes persisted in constant darkness, was dampened in mice deficient for dopamine D4 (D4) receptors and was altered in db/db mice - a model of diabetic retinopathy. The data of the present study are consistent with circadian clock-dependent and dopaminergic regulation of fatty acid oxidation in retina and its putative disturbance in diabetic retina.
The anti-alcoholism medication, disulfiram (Antabuse), decreases cocaine use in humans regardless of concurrent alcohol consumption and facilitates cocaine sensitization in rats, but the functional targets are unknown. Disulfiram inhibits dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons. The goal of this study was to test the effects of chronic genetic or pharmacological DBH inhibition on behavioral responses to cocaine using DBH knockout (Dbh -/-) mice, disulfiram, and the selective DBH inhibitor, nepicastat. Locomotor activity was measured in control (Dbh +/-) and Dbh -/- mice during a 5 day regimen of saline+saline, disulfiram+saline, nepicastat+saline, saline+cocaine, disulfiram+cocaine, or nepicastat+cocaine. After a 10 day withdrawal period, all groups were administered cocaine, and locomotor activity and stereotypy were measured. Drug-naïve Dbh -/- mice were hypersensitive to cocaine-induced locomotion and resembled cocaine-sensitized Dbh +/- mice. Chronic disulfiram administration facilitated cocaine-induced locomotion in some mice and induced stereotypy in others during the development of sensitization, while cocaine-induced stereotypy was evident in all nepicastat-treated mice. Cocaine-induced stereotypy was profoundly increased in the disulfiram+cocaine, nepicastat+cocaine, and nepicastat+saline groups upon cocaine challenge after withdrawal in Dbh +/- mice. Disulfiram or nepicastat treatment had no effect on behavioral responses to cocaine in Dbh -/- mice. These results demonstrate that chronic DBH inhibition facilitates behavioral responses to cocaine, although different methods of inhibition (genetic vs. non-selective inhibitor vs. selective inhibitor) enhance qualitatively different cocaine-induced behaviors.
Purpose: The neuromodulator dopamine (DA) has been implicated in the prevention of excessive ocular elongation and myopia in various animal models. This study used retina-specific DA knockout mice to investigate the role of retinal DA in refractive development and susceptibility to experimental myopia.
Methods: Measurements of refractive error, corneal curvature, and ocular biometrics were obtained as a function of age for both untreated and form-deprived (FD) groups of retina-specific tyrosine hydroxylase knockout (rTHKO) and control (Ctrl) mice. Retinas from each group were analyzed by HPLC for levels of DA and its primary metabolite (DOPAC).
Results: Under normal visual conditions, rTHKO mice showed significantly myopic refractions (F(1,188) = 7.602, P < 0.001) and steeper corneas (main effect of genotype F(1,180) = 5.1, P < 0.01) at 4 and 6 weeks of age compared with Ctrl mice. Retina-specific THKO mice also had thinner corneas (main effect of genotype F(1,181) = 37.17, P < 0.001), thinner retinas (F(6,181) = 6.07, P < 0.001), and shorter axial lengths (F(6,181) = 3.78, P < 0.01) than Ctrl mice. Retina-specific THKO retinas contained less than 15% of DA and DOPAC compared with Ctrl retinas, and the remaining DA had a significantly higher turnover, as indicated by DOPAC/DA ratios (Student's t-test, P < 0.05). Retina-specific THKO mice showed similar, yet more variable, responses to 6 weeks of FD compared with Ctrl mice.
Conclusions: Diminished retinal DA induced spontaneous myopia in mice raised under laboratory conditions without form deprivation. The relative myopic shift in rTHKO mice may be explained by steeper corneas, an unexpected finding. The chronic loss of DA did not significantly alter the FD myopia response in rTHKO mice.
PURPOSE: The hypothesis that outdoor exposure might protect against myopia has generated much interest, although available data find only modest clinical efficacy. We tested the effect of outdoor rearing on form-deprivation myopia in chicks, a myopia model markedly inhibited by high-intensity indoor laboratory lighting.
METHODS: Unilaterally goggled cohorts of White Leghorn chicks were maintained in a species- appropriate, outdoor rural setting during daylight hours to the extent permitted by weather. Control chicks were reared indoors with incandescent lighting. Besides ocular refraction and ultrasound, we determined dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content in retina and vitreous and measured mRNA expression levels of selected clock and circadian rhythm-related genes in the retina/RPE.
RESULTS: Myopia developed in the goggled eyes of all cohorts. Whereas outdoor rearing lessened myopia by 44% at 4 days, a protective effect was no longer evident at 11 days. Outdoor rearing had no consistent effect on retinal or vitreous content of dopamine or DOPAC. Conforming to prior data on form-deprivation myopia, retina and vitreous levels of DOPAC were reduced in goggled eyes. Compared with contralateral eyes, the retinal expression of clock and circadian rhythm-related genes was modestly altered in myopic eyes of chicks reared indoors or outdoors.
CONCLUSIONS. Outdoor rearing of chicks induces only a partial decrease of goggle-induced myopia that is not maintained, without evidence that retinal dopamine metabolism accounts for the partial myopia inhibition under these outdoor conditions. Although modest, alterations in retinal gene expression suggest that studying circadian signals might be informative for understanding refractive mechanisms.
The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson's disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylationdependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP + ), whereas PIKE-L knockdown elevates α-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or α-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that α-synuclein neutralizes PIKEL's neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.
Myopia, or nearsightedness, is the most common form of refractive abnormality and is characterized by excessive ocular elongation in relation to ocular power. Retinal neurotransmitter signaling, including dopamine, is implicated in myopic ocular growth, but the visual pathways that initiate and sustain myopia remain unclear. Melanopsin-expressing retinal ganglion cells (mRGCs), which detect light, are important for visual function, and have connections with retinal dopamine cells. Here, we investigated how mRGCs influence normal and myopic refractive development using two mutant mouse models: Opn4−/− mice that lack functional melanopsin photopigments and intrinsic mRGC responses but still receive other photoreceptor-mediated input to these cells; and Opn4DTA/DTA mice that lack intrinsic and photoreceptor-mediated mRGC responses due to mRGC cell death. In mice with intact vision or form-deprivation, we measured refractive error, ocular properties including axial length and corneal curvature, and the levels of retinal dopamine and its primary metabolite, L-3,4-dihydroxyphenylalanine (DOPAC). Myopia was measured as a myopic shift, or the difference in refractive error between the form-deprived and contralateral eyes. We found that Opn4−/− mice had altered normal refractive development compared to Opn4+/+ wildtype mice, starting ∼4D more myopic but developing ∼2D greater hyperopia by 16 weeks of age. Consistent with hyperopia at older ages, 16 week-old Opn4−/− mice also had shorter eyes compared to Opn4+/+ mice (3.34 vs 3.42 mm). Opn4DTA/DTA mice, however, were more hyperopic than both Opn4+/+ and Opn4−/− mice across development ending with even shorter axial lengths. Despite these differences, both Opn4−/− and Opn4DTA/DTA mice had ∼2D greater myopic shifts in response to form-deprivation compared to Opn4+/+ mice. Furthermore, when vision was intact, dopamine and DOPAC levels were similar between Opn4−/− and Opn4+/+ mice, but higher in Opn4DTA/DTA mice, which differed with age. However, form-deprivation reduced retinal dopamine and DOAPC by ∼20% in Opn4−/− compared to Opn4+/+ mice but did not affect retinal dopamine and DOPAC in Opn4DTA/DTA mice. Lastly, systemically treating Opn4−/− mice with the dopamine precursor L-DOPA reduced their form-deprivation myopia by half compared to non-treated mice. Collectively our findings show that disruption of retinal melanopsin signaling alters the rate and magnitude of normal refractive development, yields greater susceptibility to form-deprivation myopia, and changes dopamine signaling. Our results suggest that mRGCs participate in the eye's response to myopigenic stimuli, acting partly through dopaminergic mechanisms, and provide a potential therapeutic target underling myopia progression. We conclude that proper mRGC function is necessary for correct refractive development and protection from myopia progression.
The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expandi ng the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.
Neonatal aphakia is associated with retardation of the axial elongation of the neonatal eye. In contrast, form deprivation increases axial elongation, an effect that has been associated with decreased retinal dopamine metabolism. The present investigation was conducted to test the hypothesis that neonatal aphakia induces an effect on the levels of retinal dopamine opposite to form deprivation. Lensectomy and vitrectomy were performed on the right eyes of rhesus monkeys at approximately 1 week of age; their left eyes were unmanipulated. Axial length was measured by A-scan ultrasonography. Prior to surgery, mean axial length of the right and left eyes was identical. Following lens removal, both eyes continued to elongate, however the aphakic eyes elongated at a slower rate resulting in a significant shorter axial length compared to that of the unmanipulated eye. Removal of the crystalline lens had no effect on steady-state dopamine levels in either central or peripheral retina. However, levels of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly elevated in central retina, but not in the peripheral retina of aphakic eyes. Our results support the hypothesis that dopamine is a component of the retinal signaling pathways that are involved in the regulation of eye growth and emmetropization.
Retinal photoreceptors are important in visual signaling for normal eye growth in animals. We used Gnat2 cplf3/cplf3 (Gnat2 −/− ) mice, a genetic mouse model of cone dysfunction to investigate the influence of cone signaling in ocular refractive development and myopia susceptibility in mice. Refractive development under normal visual conditions was measured for Gnat2 −/− and age-matched Gnat2 +/+ mice, every 2 weeks from 4 to 14 weeks of age. Weekly measurements were performed on a separate cohort of mice that underwent monocular form-deprivation (FD) in the right eye from 4 weeks of age using head-mounted diffusers.
Refraction, corneal curvature, and ocular biometrics were obtained using photorefraction, keratometry and optical coherence tomography, respectively. Retinas from FD mice were harvested, and analyzed for dopamine (DA) and 3,4-dihydroxyphenylacetate (DOPAC) using high-performance liquid chromatography. Under normal visual conditions, Gnat2 +/+ and Gnat2 −/− mice showed similar refractive error, axial length, and corneal radii across development (p > 0.05), indicating no significant effects of the Gnat2 mutation on normal ocular refractive development in mice.
Three weeks of FD produced a significantly greater myopic shift in Gnat2 −/− mice compared to Gnat2 +/+ controls (−5.40 ± 1.33 D vs −2.28 ± 0.28 D, p = 0.042). Neither the Gnat2 mutation nor FD altered retinal levels of DA or DOPAC. Our results indicate that cone pathways needed for high acuity vision in primates are not as critical for normal refractive development in mice, and that both rods and cones contribute to visual signalling pathways needed to respond to FD in mammalian eyes.
Purpose: Proper visual transmission depends on the retinal ON and OFF pathways. We used Vs x1-/- mice with a retinal OFF visual pathway defect to determine the role of OFF pathway signaling in refractive development (RD) of the eye.