Metabolic characteristics of transmembrane prolyl 4-hydroxylase (P4H-TM) deficient mice.

Transmembrane prolyl 4-hydroxylase (P4H-TM) is an enigmatic enzyme whose cellular function and primary substrate remain to be identified. Its loss-of-function mutations cause a severe neurological HIDEA syndrome with hypotonia, intellectual disability, dysautonomia and hypoventilation. Previously, P4H-TM deficiency in mice was associated with reduced atherogenesis and lower serum triglyceride levels. Here, we characterized the glucose and lipid metabolism of P4h-tm-/- mice in physiological and tissue analyses. P4h-tm-/- mice showed variations in 24-h oscillations of energy expenditure, VO2 and VCO2 and locomotor activity compared to wild-type (WT) mice. Their rearing activity was reduced, and they showed significant muscle weakness and compromised coordination. Sedated P4h-tm-/- mice had better glucose tolerance, lower fasting insulin levels, higher fasting lactate levels and lower fasting free fatty acid levels compared to WT. These alterations were not present in conscious P4h-tm-/- mice. Fasted P4h-tm-/- mice presented with faster hepatic glycogenolysis. The respiratory rate of conscious P4h-tm-/- mice was significantly lower compared to the WT, the decrease being further exacerbated by sedation and associated with acidosis and a reduced ventilatory response to both hypoxia and hypercapnia. P4H-TM deficiency in mice is associated with alterations in whole-body energy metabolism, day-night rhythm of activity, glucose homeostasis and neuromuscular and respiratory functions. Although the underlying mechanism(s) are not yet fully understood, the phenotype appears to have neurological origins, controlled by brain and central nervous system circuits. The phenotype of P4h-tm-/- mice recapitulates some of the symptoms of HIDEA patients, making this mouse model a valuable tool to study and develop tailored therapies.

Sex-Specific Early Retinal Dysfunction in Mutant TDP-43 Transgenic Mice.

BACKGROUND: Increasing evidence has highlighted retinal impairments in neurodegenerative diseases. Dominant mutations in TAR DNA-binding protein 43 (TDP-43) cause amyotrophic lateral sclerosis (ALS), and the accumulation of TDP-43 in the cytoplasm is a pathological hallmark of ALS, frontotemporal dementia (FTD), and many other neurodegenerative diseases. OBJECTIVE: While homozygous transgenic mice expressing the disease-causing human TDP-43 M337V mutant (TDP-43M337V mice) experience premature death, hemizygous TDP-43M337V mice do not suffer sudden death, but they exhibit age-dependent motor-coordinative and cognitive deficits. This study aims to leverage the hemizygous TDP-43M337V mice as a valuable ALS/FTD disease model for the assessment also of retinal changes during the disease progression. METHODS: We evaluated the retinal function of young TDP-43M337V mice by full field electroretinogram (ERG) recordings. RESULTS: At 3-4 months of age, well before the onset of brain dysfunction at 8 months, the ERG responses were notably impaired in the retinas of young female TDP-43M337V mice in contrast to their male counterparts and age-matched non-transgenic mice. Mitochondria have been implicated as critical targets of TDP-43. Further investigation revealed that significant changes in the key regulators of mitochondrial dynamics and bioenergetics were only observed in the retinas of young female TDP-43M337V mice, while these alterations were not present in the brains of either gender. CONCLUSIONS: Together our findings suggest a sex-specific vulnerability within the retina in the early disease stage, and highlight the importance of retinal changes and mitochondrial markers as potential early diagnostic indicators for ALS, FTD, and other TDP-43 related neurodegenerative conditions.

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Neural and Müller glial adaptation of the retina to photoreceptor degeneration.

The majority of inherited retinal degenerative diseases and dry age-related macular degeneration are characterized by decay of the outer retina and photoreceptors, which leads to progressive loss of vision. The inner retina, including second- and third-order retinal neurons, also shows aberrant structural changes at all stages of degeneration. Müller glia, the major glial cells maintain retinal homeostasis, activating and rearranging immediately in response to photoreceptor stress. These phenomena are collectively known as retinal remodeling and are anatomically well described, but their impact on visual function is less well characterized. Retinal remodeling has traditionally been considered a detrimental chain of events that decreases visual function. However, emerging evidence from functional assays suggests that remodeling could also be a part of a survival mechanism wherein the inner retina responds plastically to outer retinal degeneration. The visual system´s first synapses between the photoreceptors and bipolar cells undergo rewiring and functionally compensate to maintain normal signal output to the brain. Distinct classes of retinal ganglion cells remain even after the massive loss of photoreceptors. Müller glia possess the regenerative potential for retinal recovery and possibly exert adaptive transcriptional changes in response to neuronal loss. These types of homeostatic changes could potentially explain the well-maintained visual function observed in patients with inherited retinal degenerative diseases who display prominent anatomic retinal pathology. This review will focus on our current understanding of retinal neuronal and Müller glial adaptation for the potential preservation of retinal activity during photoreceptor degeneration. Targeting retinal self-compensatory responses could help generate universal strategies to delay sensory disease progression.

Retinoid therapy restores eye-specific cortical responses in adult mice with retinal degeneration.

Despite the recent emergence of multiple cellular and molecular strategies to restore vision in retinal disorders, it remains unclear to what extent central visual circuits can recover when retinal defects are corrected in adulthood. We addressed this question in an Lrat-/- mouse model of Leber congenital amaurosis (LCA) in which retinal light sensitivity and optomotor responses are partially restored by 9-cis-retinyl acetate administration in adulthood. Following treatment, two-photon calcium imaging revealed increases in the number and response amplitude of visually responsive neurons in the primary visual cortex (V1). In particular, retinoid treatment enhanced responses from the ipsilateral eye, restoring the normal balance of eye-specific responses in V1. Additionally, the treatment rescued the modulation of cortical responses by arousal. These findings illustrate the significant plasticity of the adult central visual system and underscore the therapeutic potential of retinoid administration for adults with retinal diseases.

Visual System Hyperexcitability and Compromised V1 Receptive Field Properties in Early-Stage Retinitis Pigmentosa in Mice.

Inherited retinal degenerative diseases are a prominent cause of blindness. Although mutations causing death of photoreceptors are mostly known, the pathophysiology downstream in the inner retina and along the visual pathway is incompletely characterized in the earliest disease stages. Here, we investigated retinal, midbrain and cortical visual function using electroretinography (ERG), the optomotor response (OMR), visual evoked potentials (VEPs), respectively, and single unit electrophysiology at the primary visual cortex (V1) in light-adapted juvenile (approximately one-month-old) and young adult (three-month-old) RhoP23H/WT mice, representative of early-stage retinitis pigmentosa (RP). Photopic ERG revealed up to ∼30% hypersensitivity to light in RhoP23H/WT mice, as measured by the light intensity required to generate half-maximal b-wave (I50 parameter). RhoP23H/WT mice also showed increased OMRs toward low spatial frequency (SF) drifting gratings, indicative of visual overexcitation at the midbrain level. At the V1 level, VEPs and single-cell recordings revealed prominent hyperexcitability in the juvenile RhoP23H/WT mice. Mean VEP amplitudes for light ON stimuli were nearly doubled in one-month-old RhoP23H/WT mice compared with controls, and more than doubled for light OFF. Single-cell recordings showed a significantly increased spontaneous V1 neuron firing in the RhoP23H/WT mice, and persistent contrast and temporal sensitivities. In contrast, direction selectivity was severely compromised. Our data suggest that during early RP, the visual pathway becomes hyperexcited. This could have both compensatory and deleterious consequences for visual behavior. Further studies on the mechanisms of hyperexcitability are warranted as this could lead to therapeutic interventions for RP.

In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration.

Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.

Inhibition of ceramide accumulation in AdipoR1-/- mice increases photoreceptor survival and improves vision.

Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1. We discovered an accumulation of ceramides in the AdipoR1-/- retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1-/- mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1-related retinopathy.

VCP/p97 inhibitor CB-5083 modulates muscle pathology in a mouse model of VCP inclusion body myopathy.

BACKGROUND: Pathogenic gain of function variants in Valosin-containing protein (VCP) cause a unique disease characterized by inclusion body myopathy with early-onset Paget disease of bone and frontotemporal dementia (also known as Multisystem proteinopathy (MSP)). Previous studies in drosophila models of VCP disease indicate treatment with VCP inhibitors mitigates disease pathology. Earlier-generation VCP inhibitors display off-target effects and relatively low therapeutic potency. New generation of VCP inhibitors needs to be evaluated in a mouse model of VCP disease. In this study, we tested the safety and efficacy of a novel and potent VCP inhibitor, CB-5083 using VCP patient-derived myoblast cells and an animal model of VCP disease. METHODS: First, we analyzed the effect of CB-5083 in patient-derived myoblasts on the typical disease autophagy and TDP-43 profile by Western blot. Next, we determined the maximum tolerated dosage of CB-5083 in mice and treated the 2-month-old VCPR155H/R155H mice for 5 months with 15 mg/kg CB-5083. We analyzed motor function monthly by Rotarod; and we assessed the end-point blood toxicology, and the muscle and brain pathology, including autophagy and TDP-43 profile, using Western blot and immunohistochemistry. We also treated 12-month-old VCPR155H/+ mice for 6 months and performed similar analysis. Finally, we assessed the potential side effects of CB-5083 on retinal function, using electroretinography in chronically treated VCPR155H/155H mice. RESULTS: In vitro analyses using patient-derived myoblasts confirmed that CB-5083 can modulate expression of the proteins in the autophagy pathways. We found that chronic CB-5083 treatment is well tolerated in the homozygous mice harboring patient-specific VCP variant, R155H, and can ameliorate the muscle pathology characteristic of the disease. VCP-associated pathology biomarkers, such as elevated TDP-43 and p62 levels, were significantly reduced. Finally, to address the potential adverse effect of CB-5083 on visual function observed in a previous oncology clinical trial, we analyzed retinal function in mice treated with moderate doses of CB-5083 for 5 months and documented the absence of permanent ocular toxicity. CONCLUSIONS: Altogether, these findings suggest that long-term use of CB-5083 by moderate doses is safe and can improve VCP disease-associated muscle pathology. Our results provide translationally relevant evidence that VCP inhibitors could be beneficial in the treatment of VCP disease.

Regulation of Adrenergic, Serotonin, and Dopamine Receptors to Inhibit Diabetic Retinopathy: Monotherapies versus Combination Therapies.

We compared monotherapies and combinations of therapies that regulate G-protein-coupled receptors (GPCRs) with respect to their abilities to inhibit early stages of diabetic retinopathy (DR) in streptozotocin-diabetic mice. Metoprolol (MTP; 0.04-1.0 mg/kg b.wt./day), bromocriptine (BRM; 0.01-0.1 mg/kg b.wt./day), doxazosin (DOX; 0.01-1.0 mg/kg b.wt./day), or tamsulosin (TAM; 0.05-0.25 mg/kg b.wt./day) were injected individually daily for 2 months in dose-response studies to assess their effects on the diabetes-induced increases in retinal superoxide and leukocyte-mediated cytotoxicity against vascular endothelial cells, both of which abnormalities have been implicated in the development of DR. Each of the individual drugs inhibited the diabetes-induced increase in retinal superoxide at the higher concentrations tested, but the inhibition was lost at lower doses. To determine whether combination therapies had superior effects over individual drugs, we intentionally selected for each drug a low dose that had little or no effect on the diabetes-induced retinal superoxide for use separately or in combinations in 8-month studies of retinal function, vascular permeability, and capillary degeneration in diabetes. At the low doses used, combinations of the drugs generally were more effective than individual drugs, but the low-dose MTP alone totally inhibited diabetes-induced reduction in a vision task, BRM or DOX alone totally inhibited the vascular permeability defect, and DOX alone totally inhibited diabetes-induced degeneration of retinal capillaries. Although low-dose MTP, BRM, DOX, or TAM individually had beneficial effects on some endpoints, combination of the therapies better inhibited the spectrum of DR lesions evaluated. SIGNIFICANCE STATEMENT: The pathogenesis of early stages of diabetic retinopathy remains incompletely understood, but multiple different cell types are believed to be involved in the pathogenic process. We have compared the effects of monotherapies to those of combinations of drugs that regulate GPCR signaling pathways with respect to their relative abilities to inhibit the development of early diabetic retinopathy.

A p97/Valosin-Containing Protein Inhibitor Drug CB-5083 Has a Potent but Reversible Off-Target Effect on Phosphodiesterase-6.

CB-5083 is an inhibitor of p97/valosin-containing protein (VCP), for which phase I trials for cancer were terminated because of adverse effects on vision, such as photophobia and dyschromatopsia. Lower dose CB-5083 could combat inclusion body myopathy with early-onset Paget disease and frontotemporal dementia or multisystem proteinopathy caused by gain-of-function mutations in VCP. We hypothesized that the visual impairment in the cancer trial was due to CB-5083's inhibition of phosphodiesterase (PDE)-6, which mediates signal transduction in photoreceptors. To test our hypothesis, we used in vivo and ex vivo electroretinography (ERG) in mice and a PDE6 activity assay of bovine rod outer segment (ROS) extracts. Additionally, histology and optical coherence tomography were used to assess CB-5083's long-term ocular toxicity. A single administration of CB-5083 led to robust ERG signal deterioration, specifically in photoresponse kinetics. Similar recordings with known PDE inhibitors sildenafil, tadalafil, vardenafil, and zaprinast showed that only vardenafil had as strong an effect on the ERG signal in vivo as did CB-5083. In the biochemical assay, CB-5083 inhibited PDE6 activity with a potency higher than sildenafil but lower than that of vardenafil. Ex vivo ERG revealed a PDE6 inhibition constant of 80 nM for CB-5083, which is 7-fold smaller than that for sildenafil. Finally, we showed that the inhibitory effect of CB-5083 on visual function is reversible, and its chronic administration does not cause permanent retinal anomalies in aged VCP-disease model mice. Our results warrant re-evaluation of CB-5083 as a clinical therapeutic agent. We recommend preclinical ERG recordings as a routine drug safety screen. SIGNIFICANCE STATEMENT: This report supports the use of a valosin-containing protein (VCP) inhibitor drug, CB-5083, for the treatment of neuromuscular VCP disease despite CB-5083's initial clinical failure for cancer treatment due to side effects on vision. The data show that CB-5083 displays a dose-dependent but reversible inhibitory action on phosphodiesterase-6, an essential enzyme in retinal photoreceptor function, but no long-term consequences on retinal function or structure.

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease.

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.

Retinoids in the visual cycle: role of the retinal G protein-coupled receptor.

Driven by the energy of a photon, the visual pigments in rod and cone photoreceptor cells isomerize 11-cis-retinal to the all-trans configuration. This photochemical reaction initiates the signal transduction pathway that eventually leads to the transmission of a visual signal to the brain and leaves the opsins insensitive to further light stimulation. For the eye to restore light sensitivity, opsins require recharging with 11-cis-retinal. This trans-cis back conversion is achieved through a series of enzymatic reactions composing the retinoid (visual) cycle. Although it is evident that the classical retinoid cycle is critical for vision, the existence of an adjunct pathway for 11-cis-retinal regeneration has been debated for many years. Retinal pigment epithelium (RPE)-retinal G protein-coupled receptor (RGR) has been identified previously as a mammalian retinaldehyde photoisomerase homologous to retinochrome found in invertebrates. Using pharmacological, genetic, and biochemical approaches, researchers have now established the physiological relevance of the RGR in 11-cis-retinal regeneration. The photoisomerase activity of RGR in the RPE and Müller glia explains how the eye can remain responsive in daylight. In this review, we will focus on retinoid metabolism in the eye and visual chromophore regeneration mediated by RGR.

Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.

Publisher Correction: Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Homeostatic plasticity in the retina is associated with maintenance of night vision during retinal degenerative disease.

Neuronal plasticity of the inner retina has been observed in response to photoreceptor degeneration. Typically, this phenomenon has been considered maladaptive and may preclude vision restoration in the blind. However, several recent studies utilizing triggered photoreceptor ablation have shown adaptive responses in bipolar cells expected to support normal vision. Whether such homeostatic plasticity occurs during progressive photoreceptor degenerative disease to help maintain normal visual behavior is unknown. We addressed this issue in an established mouse model of Retinitis Pigmentosa caused by the P23H mutation in rhodopsin. We show robust modulation of the retinal transcriptomic network, reminiscent of the neurodevelopmental state, and potentiation of rod - rod bipolar cell signaling following rod photoreceptor degeneration. Additionally, we found highly sensitive night vision in P23H mice even when more than half of the rod photoreceptors were lost. These results suggest retinal adaptation leading to persistent visual function during photoreceptor degenerative disease.

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium.

Photoisomerization of the 11-cis-retinal chromophore of rod and cone visual pigments to an all-trans-configuration is the initiating event for vision in vertebrates. The regeneration of 11-cis-retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11-cis-retinal also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein-coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11-cis-retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Müller glia, whereas mouse RGR is expressed in RPE but not in Müller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.

Loss of Cln5 leads to altered Gad1 expression and deficits in interneuron development in mice.

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5-/- embryos of various ages and cells harvested from Cln5-/- brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5-/- mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5-/- embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.

A Mixture of U.S. Food and Drug Administration-Approved Monoaminergic Drugs Protects the Retina From Light Damage in Diverse Models of Night Blindness.

Purpose: The purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors. Methods: Acute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (ß1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity. Results: The Gnat1-/- mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2-/- mice were very resistant. The Arr1-/- and Grk1-/- mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1-/- mice. The therapeutic drug doses increased in parallel with light-damage severity. Conclusions: Our results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases.