Shobi Veleri

The Neuropeptide Pigment-Dispersing Factor Adjusts Period and Phase of Drosophila's Clock

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. PDF is necessary for robust activity rhythms and is thought to couple the circadian oscillations of the clock neurons. However, little is known about the action of PDF on individual clock neurons. Here, we combined the period–luciferase reporter system with immunolabeling of clock proteins in wild-type and Pdf01 mutants to dissect the effects of PDF on specific subgroups of clock neurons. Additionally, PDF levels were elevated to higher than normal levels using specific neural mutants, and a correlation analysis of locomotor activity and clock protein staining served to determine the periods of specific clock cells. We found that PDF has multiple effects on the clock neurons: In some groups of clock neurons, PDF was required for maintaining the oscillations of individual cells, and in others, PDF was required for synchronous cycling of the individual members. Other clock neurons cycled with high amplitude in absence of PDF, but PDF affected their intrinsic clock speed. Sometimes PDF shortened and sometimes PDF lengthened period. Our observations indicate that PDF is crucial for adjusting cycling amplitude, period, and phase of the different players in the circadian clock. Under natural conditions PDF may be required for adapting Drosophilas clock to varying photoperiods. Indeed, we show here that Pdf01 mutants are not able to adapt their activity to long photoperiods in a wild-type manner.

Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila's circadian clock

Organisms use the daily cycles of light and darkness to synchronize their internal circadian clocks with the environment. Because they optimize physiological processes and behavior, properly synchronized circadian clocks are thought to be important for the overall fitness. In Drosophila melanogaster, the circadian clock is synchronized with the natural environment by light-dependent degradation of the clock protein Timeless, mediated by the blue-light photoreceptor Cryptochrome (Cry). Here we report identification of a genetic variant, Veela, which severely disrupts this process, because these genetically altered flies maintain behavioral and molecular rhythmicity under constant-light conditions that usually stop the clock. We show that the Veela strain carries a natural timeless allele (ls-tim), which encodes a less-light-sensitive form of Timeless in combination with a mutant variant of the F-box protein Jetlag. However, neither the ls-tim nor the jetlag genetic variant alone is sufficient to disrupt light input into the central pacemaker. We show a strong interaction between Veela and cryptochrome genetic variants, demonstrating that the Jetlag, Timeless, and Cry proteins function in the same pathway. Veela also reveals a function for the two natural variants of timeless, which differ in their sensitivity to light. In combination with the complex array of retinal and extraretinal photoreceptors known to signal light to the pacemaker, this previously undescribed molecular component of photic sensitivity mediated by the two Timeless proteins reveals that an unexpectedly rich complexity underlies modulation of this process.

Biology and therapy of inherited retinal degenerative disease: insights from mouse models.

Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases.

Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis

Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.

Preservation of Cone Photoreceptors after a Rapid yet Transient Degeneration and Remodeling in Cone-Only Nrl−/− Mouse Retina

Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor neural retina leucine zipper (NRL). The loss of Nrl (Nrl−/−) in mice results in a retina with predominantly S-opsin-containing cones that exhibit molecular and functional characteristics of wild-type cones. Here, we report that Nrl−/− retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by 4 months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic electroretinogram. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl−/− mouse illustrates the long-term viability of cones in the absence of rods and retinal pigment epithelium defects in a rodless retina. We propose that Nrl−/− retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.

Ciliopathy-associated gene Cc2d2a promotes assembly of subdistal appendages on the mother centriole during cilia biogenesis

The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a-/- mouse that recapitulates features of Meckel syndrome including embryonic lethality and multi-organ defects. Cilia are absent in Cc2d2a-/- embryonic node and other somatic tissues; disruption of cilia-dependent Shh signaling appears to underlie exencephaly in mutant embryos. The Cc2d2a-/- mouse embryonic fibroblasts (MEFs) lack cilia though mother centriole and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a-/- MEFs, subdistal appendages are lacking or abnormal by transmission-EM. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis.

Knockdown of Bardet-Biedl Syndrome Gene BBS9/PTHB1 Leads to Cilia Defects

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffers vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals.

NeuroD1 is required for survival of photoreceptors but not pinealocytes: results from targeted gene deletion studies.

NeuroD1 encodes a basic helix‐loop‐helix transcription factor involved in the development of neural and endocrine structures, including the retina and pineal gland. To determine the effect of NeuroD1 knockout in these tissues, a Cre/loxP recombination strategy was used to target a NeuroD1 floxed gene and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, which is selectively expressed in the pineal gland and retina. At 2 months of age, NeuroD1 cKO retinas have a dramatic reduction in rod‐ and cone‐driven electroretinograms and contain shortened and disorganized outer segments; by 4 months, NeuroD1 cKO retinas are devoid of photoreceptors. In contrast, the NeuroD1 cKO pineal gland appears histologically normal. Microarray analysis of 2‐month‐old NeuroD1 cKO retina and pineal gland identified a subset of genes that were affected 2–100‐fold; in addition, a small group of genes exhibit altered differential night/day expression. Included in the down‐regulated genes are Aipl1, which is necessary to prevent retinal degeneration, and Ankrd33, whose protein product is selectively expressed in the outer segments. These findings suggest that NeuroD1 may act through Aipl1 and other genes to maintain photoreceptor homeostasis.

Unique self-sustaining circadian oscillators within the brain of Drosophila melanogaster.

In Drosophila circadian rhythms persist in constant darkness (DD). The small ventral Lateral Neurons (s-LNv) mainly control the behavioral circadian rhythm in consortium with the large ventral Lateral Neurons (l-LNv) and dorsal Lateral Neurons (LNd). It is believed that the molecular oscillations of clock genes are the source of this persistent behavior. Indeed the s-LNv, LNd, Dorsal Neurons (DN)-DN2 and DN3 displayed self-sustained molecular oscillations in DD both at RNA and protein levels, except the DN2 oscillates in anti-phase. In contrast, the l-LNv and DN1 displayed self-sustained oscillations at the RNA level, but protein oscillations quickly dampened. Having self-sustained and dampened molecular oscillators together in the DN groups suggested that they play different roles. However, all DN groups seemed to contribute together to the light–dark (LD) behavioral rhythm. The LD entrainment of LN oscillators is achieved through Rhodopsin (RH) and Cryptochrome (CRY). CRYs expression in all DN groups implicates also its role in LD entrainment of DN, like in DN1. However, mutations in cry and glass that did not inflict LD synchronization of the DN2, DN3 oscillator implicate the existence of a novel photoreceptor at least in DN3.

REEP6 mediates trafficking of a subset of Clathrin-coated vesicles and is critical for rod photoreceptor function and survival

Abstract In retinal photoreceptors, vectorial transport of cargo is critical for transduction of visual signals, and defects in intracellular trafficking can lead to photoreceptor degeneration and vision impairment. Molecular signatures associated with routing of transport vesicles in photoreceptors are poorly understood. We previously reported the identification of a novel rod photoreceptor specific isoform of Receptor Expression Enhancing Protein (REEP) 6, which belongs to a family of proteins involved in intracellular transport of receptors to the plasma membrane. Here we show that loss of REEP6 in mice (Reep6−/−) results in progressive retinal degeneration. Rod photoreceptor dysfunction is observed in Reep6−/− mice as early as one month of age and associated with aberrant accumulation of vacuole-like structures at the apical inner segment and reduction in selected rod phototransduction proteins. We demonstrate that REEP6 is detected in a subset of Clathrin-coated vesicles and interacts with the t-SNARE, Syntaxin3. In concordance with the rod degeneration phenotype in Reep6−/− mice, whole exome sequencing identified homozygous REEP6-E75K mutation in two retinitis pigmentosa families of different ethnicities. Our studies suggest a critical function of REEP6 in trafficking of cargo via a subset of Clathrin-coated vesicles to selected membrane sites in retinal rod photoreceptors.