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 Kupffer's 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

Analysis of the light--entrainment pathways for the circadian clock of Drosophila melanogaster

In Drosophila, naturally the circadian clock is entrained by environmental light-dark cycles. Photoreceptors like rhodopsins and cryptochrome perceive light signals for light entrainment of the circadian clock. It is known that there is a light-dependent CRY:TIM interaction, leading to the degradation of TIM coupled with molecular re-setting of the clock-gene cyclings. However, functional double mutants (norpAP41;cryb), blocking simultaneously the known rhodopsin mediated transduction cascade and the largely unknown cryptochrome mediated transduction cascade do not confer the circadian clock absolutely blind to light signals. The double mutants� circadian clock still shows residual light entrainment ability, indicating that a novel photoreceptor entrains the circadian clock. On the other hand, in gl60j cryb double mutants the circadian clock is absolutely blind to light signals. The gl60j mutation removes morphological structures like the compound eyes and the ocelli (signaling from them is blocked by norpAP41); gl60j in addition removes the H-B eyelet and a subset of DNs (DN1s). The H-B eyelet projects axons toward the s-LNvs pacemaker neurons. Both in the s-LNvs and in the DN1s the molecular PER and TIM cyclings can still be synchronized by light in norpAP41;cryb double mutant flies. Therefore, we have investigated a possible role of the H-B eyelet in residual light entrainment of the circadian clock. We have shown that the H-B eyelet functions to sense dim white light. Blocking light signaling from it affects the synchronization of molecular TIM cycling in the pacemaker neurons and in a subset of the DNs. However, at higher intensities of light no effect was seen. Therefore, we further investigated a possible role of the DNs, a subset of which also maintained light-synchronized molecular circadian oscillation in norpAP41;cryb double mutant flies. We have shown that the DN2+3s host a self-sustained molecular oscillator, which is entrainable by light dark cycles. Further, it was demonstrated that a novel norpA and cry independent photoreceptor in the dorsal brain could entrain the DN oscillator. As a putative candidate, we have investigated the Rh 7 gene and shown that it is indeed expressed in proximity to the DNs and LNs. Possibly, Rh 7 bypasses the classical visual norpA dependent phototransduction cascade. The data presented in this thesis support the existence of a novel circadian photoreceptor and--pigment and set the stage for analysis of the signaling pathways involved. Finally, isolation of the novel mutant Veela presents a potential missing link in the CRY:TIM signaling and interaction mechanism, which is the central part of the Drosophila light-entrainment pathway

Molecular Chaperone Dysfunction in Neurodegenerative Diseases and Effects of Curcumin

The intra- and extracellular accumulation of misfolded and aggregated amyloid proteins is a common feature in several neurodegenerative diseases, which is thought to play a major role in disease severity and progression. The principal machineries maintaining proteostasis are the ubiquitin proteasomal and lysosomal autophagy systems, where heat shock proteins play a crucial role. Many protein aggregates are degraded by the lysosomes, depending on aggregate size, peptide sequence, and degree of misfolding, while others are selectively tagged for removal by heat shock proteins and degraded by either the proteasome or phagosomes. These systems are compromised in different neurodegenerative diseases. Therefore, developing novel targets and classes of therapeutic drugs, which can reduce aggregates and maintain proteostasis in the brains of neurodegenerative models, is vital. Natural products that can modulate heat shock proteins/proteosomal pathway are considered promising for treating neurodegenerative diseases. Here we discuss the current knowledge on the role of HSPs in protein misfolding diseases and knowledge gained from animal models of Alzheimer’s disease, tauopathies, and Huntington’s diseases. Further, we discuss the emerging treatment regimens for these diseases using natural products, like curcumin, which can augment expression or function of heat shock proteins in the cell

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. Dis Model Mech 2015 Feb; 8(2):109-129

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

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.Fil: Ochocinska, Margaret J.. National Instituto of Child Health & Human Development; Estados UnidosFil: Muñoz, Estela Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Veleri, Shobi. National Institutes of Health; Estados UnidosFil: Weller, Joan L.. National Instituto of Child Health & Human Development; Estados UnidosFil: Coon, Steven L.. National Instituto of Child Health & Human Development; Estados UnidosFil: Pozdeyev, Nikita. University of Emory; Estados UnidosFil: Iuvone, P. Michael. University of Emory; Estados UnidosFil: Goebbels, Sandra. Max Planck Institute of Experimental Medicine; AlemaniaFil: Furukawa, Takahisa. Osaka University; JapónFil: Klein, David C.. National Instituto of Child Health & Human Development; Estados Unido