Intestinal FGF15 regulates bile acid and cholesterol metabolism but not glucose and energy balance.

Fibroblast growth factor 15/19 (FGF15/19, mouse/human ortholog) is expressed in the ileal enterocytes of the small intestine and released postprandially in response to bile acid absorption. Previous reports of FGF15-/- mice have limited our understanding of gut-specific FGF15's role in metabolism. Therefore, we studied the role of endogenous gut-derived FGF15 in bile acid, cholesterol, glucose, and energy balance. We found that circulating levels of FGF19 were reduced in individuals with obesity and comorbidities, such as type 2 diabetes and metabolic dysfunction-associated fatty liver disease. Gene expression analysis of ileal FGF15-positive cells revealed differential expression during the obesogenic state. We fed standard chow or a high-fat metabolic dysfunction-associated steatohepatitis-inducing diet to control and intestine-derived FGF15-knockout (FGF15INT-KO) mice. Control and FGF15INT-KO mice gained similar body weight and adiposity and did not show genotype-specific differences in glucose, mixed meal, pyruvate, and glycerol tolerance. FGF15INT-KO mice had increased systemic bile acid levels but decreased cholesterol levels, pointing to a primary role for gut-derived FGF15 in regulating bile acid and cholesterol metabolism when exposed to obesogenic diet. These studies show that intestinal FGF15 plays a specific role in bile acid and cholesterol metabolism regulation but is not essential for energy and glucose balance.

Suppression of food intake by Glp1r/Lepr-coexpressing neurons prevents obesity in mouse models.

The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A potentially unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-Seq of enriched mouse hypothalamic LepRb cells, identifying several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations displayed strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons, which expressed more Lepr than other LepRb cell populations. Ablating Lepr from LepRbGlp1r cells provoked hyperphagic obesity without impairing energy expenditure. Similarly, improvements in energy balance caused by Lepr reactivation in GABA neurons of otherwise Lepr-null mice required Lepr expression in GABAergic Glp1r-expressing neurons. Furthermore, restoration of Glp1r expression in LepRbGlp1r neurons in otherwise Glp1r-null mice enabled food intake suppression by the GLP1R agonist, liraglutide. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the suppression of food intake by leptin and GLP1R agonists.

Neither GLP-1 receptors nor GFRAL neurons are required for aversive or anorectic response to DON (vomitoxin).

Deoxynivalenol (DON), a type B trichothecene mycotoxin contaminating grains, promotes nausea, emesis and anorexia. With DON exposure, circulating levels of intestinally derived satiation hormones, including glucagon-like peptide 1 (GLP-1) are elevated. To directly test whether GLP-1 signaling mediates the effects of DON, we examined the response of GLP-1 or GLP-1R-deficient mice to DON injection. We found comparable anorectic and conditioned taste avoidance learning responses in GLP-1/GLP-1R deficient mice compared to control littermates, suggesting that GLP-1 is not necessary for the effects of DON on food intake and visceral illness. We then used our previously published data from translating ribosome affinity purification with RNA sequencing (TRAP-seq) analysis of area postrema neurons that express the receptor for the circulating cytokine growth differentiation factor (GDF15), growth differentiation factor a-like (GFRAL). Interestingly, this analysis showed that a cell surface receptor for DON, calcium sensing receptor (CaSR), is heavily enriched in GFRAL neurons. Given that GDF15 potently reduces food intake and can cause visceral illness by signaling through GFRAL neurons, we hypothesized that DON may also signal by activating CaSR on GFRAL neurons. Indeed, circulating GDF15 levels are elevated after DON administration but both GFRAL knockout and GFRAL neuron-ablated mice exhibited similar anorectic and conditioned taste avoidance responses compared to WT littermates. Thus, GLP-1 signaling and GFRAL signaling and neurons are not required for DON-induced visceral illness or anorexia.

The gut peptide Reg3g links the small intestine microbiome to the regulation of energy balance, glucose levels, and gut function.

Changing composition of the gut microbiome is an important component of the gut adaptation to various environments, which have been implicated in various metabolic diseases including obesity and type 2 diabetes, but the mechanisms by which the microbiota influence host physiology remain contentious. Here we find that both diets high in the fermentable fiber inulin and vertical sleeve gastrectomy increase intestinal expression and circulating levels of the anti-microbial peptide Reg3g. Moreover, a number of beneficial effects of these manipulations on gut function, energy balance, and glucose regulation are absent in Reg3g knockout mice. Peripheral administration of various preparations of Reg3g improves glucose tolerance, and this effect is dependent on the putative receptor Extl3 in the pancreas. These data suggest Reg3g acts both within the lumen and as a gut hormone to link the intestinal microbiome to various aspects of host physiology that may be leveraged for novel treatment strategies.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice.

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.

NTS Prlh overcomes orexigenic stimuli and ameliorates dietary and genetic forms of obesity.

Calcitonin receptor (Calcr)-expressing neurons of the nucleus tractus solitarius (NTS; CalcrNTS cells) contribute to the long-term control of food intake and body weight. Here, we show that Prlh-expressing NTS (PrlhNTS) neurons represent a subset of CalcrNTS cells and that Prlh expression in these cells restrains body weight gain in the face of high fat diet challenge in mice. To understand the relationship of PrlhNTS cells to hypothalamic feeding circuits, we determined the ability of PrlhNTS-mediated signals to overcome enforced activation of AgRP neurons. We found that PrlhNTS neuron activation and Prlh overexpression in PrlhNTS cells abrogates AgRP neuron-driven hyperphagia and ameliorates the obesity of mice deficient in melanocortin signaling or leptin. Thus, enhancing Prlh-mediated neurotransmission from the NTS dampens hypothalamically-driven hyperphagia and obesity, demonstrating that NTS-mediated signals can override the effects of orexigenic hypothalamic signals on long-term energy balance.

ARCGHR Neurons Regulate Muscle Glucose Uptake.

The growth hormone receptor (GHR) is expressed in brain regions that are known to participate in the regulation of energy homeostasis and glucose metabolism. We generated a novel transgenic mouse line (GHRcre) to characterize GHR-expressing neurons specifically in the arcuate nucleus of the hypothalamus (ARC). Here, we demonstrate that ARCGHR+ neurons are co-localized with agouti-related peptide (AgRP), growth hormone releasing hormone (GHRH), and somatostatin neurons, which are activated by GH stimulation. Using the designer receptors exclusively activated by designer drugs (DREADD) technique to control the ARCGHR+ neuronal activity, we demonstrate that the activation of ARCGHR+ neurons elevates a respiratory exchange ratio (RER) under both fed and fasted conditions. However, while the activation of ARCGHR+ promotes feeding, under fasting conditions, the activation of ARCGHR+ neurons promotes glucose over fat utilization in the body. This effect was accompanied by significant improvements in glucose tolerance, and was specific to GHR+ versus GHRH+ neurons. The activation of ARCGHR+ neurons increased glucose turnover and whole-body glycolysis, as revealed by hyperinsulinemic-euglycemic clamp studies. Remarkably, the increased insulin sensitivity upon the activation of ARCGHR+ neurons was tissue-specific, as the insulin-stimulated glucose uptake was specifically elevated in the skeletal muscle, in parallel with the increased expression of muscle glycolytic genes. Overall, our results identify the GHR-expressing neuronal population in the ARC as a major regulator of glycolysis and muscle insulin sensitivity in vivo.

Cross-species analysis defines the conservation of anatomically segregated VMH neuron populations.

The ventromedial hypothalamic nucleus (VMH) controls diverse behaviors and physiologic functions, suggesting the existence of multiple VMH neural subtypes with distinct functions. Combing translating ribosome affinity purification with RNA-sequencing (TRAP-seq) data with single-nucleus RNA-sequencing (snRNA-seq) data, we identified 24 mouse VMH neuron clusters. Further analysis, including snRNA-seq data from macaque tissue, defined a more tractable VMH parceling scheme consisting of six major genetically and anatomically differentiated VMH neuron classes with good cross-species conservation. In addition to two major ventrolateral classes, we identified three distinct classes of dorsomedial VMH neurons. Consistent with previously suggested unique roles for leptin receptor (Lepr)-expressing VMH neurons, Lepr expression marked a single dorsomedial class. We also identified a class of glutamatergic VMH neurons that resides in the tuberal region, anterolateral to the neuroanatomical core of the VMH. This atlas of conserved VMH neuron populations provides an unbiased starting point for the analysis of VMH circuitry and function.

tTARGIT AAVs mediate the sensitive and flexible manipulation of intersectional neuronal populations in mice.

While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) adeno-associated viruses (AAVs). tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) 'Driver' AAV and a tetracycline response element-driven, Cre-dependent 'Payload' AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.

The brain contains hundreds of types of neurons, which differ in size, shape and behavior. But neuroscientists often wish to study individual neuronal types in isolation. They are able to do this with the aid of a toolkit made up of two parts: viral vectors and genetically modified mice. Viral vectors are viruses that have been modified so that they are no longer harmful and can instead be used to introduce genetic material into cells on demand. To create a viral vector, the virus' own genetic material is replaced with a 'cargo' gene, such as the gene for a fluorescent protein. The virus is then introduced into a new host such as a mouse. Importantly, the virus only produces the protein encoded by its 'cargo' gene if it is inside a cell that also contains one of two specific enzymes. These enzymes are called Cre and Flp. This is where the second part of the toolkit comes in. Mice can be genetically engineered to produce either Cre or Flp exclusively in specific cell types. By introducing a viral vector into mice that produce either Cre or Flp only in one particular type of neuron, researchers can limit the activity of the cargo gene to that neuronal type. But sometimes even this approach is not selective enough. Researchers may wish to limit the activity of the cargo gene to a subpopulation of cells that produce Cre or Flp. Or they may wish to target only Cre- or Flp-producing cells in a small area of the brain, while leaving cells in neighboring areas unaffected. Sabatini et al. have now overcome this limitation by developing and testing a new set of viral vectors that are active only in neurons that produce both Cre and Flp. The vectors are called tTARGIT AAVs and allow researchers to target cells more precisely than was possible with the previous version of the toolkit. Sabatini et al. show tTARGIT AAVs in action by using them to identify a group of neurons that control how much energy mice use and how much food they eat. As well as applying the vectors to their own research on obesity, Sabatini et al. have also made them freely available for other researchers to use in their own projects.

GFRAL-expressing neurons suppress food intake via aversive pathways.

The TGFß cytokine family member, GDF-15, reduces food intake and body weight and represents a potential treatment for obesity. Because the brainstem-restricted expression pattern of its receptor, GDNF Family Receptor α-like (GFRAL), presents an exciting opportunity to understand mechanisms of action for area postrema neurons in food intake; we generated GfralCre and conditional GfralCreERT mice to visualize and manipulate GFRAL neurons. We found infection or pathophysiologic states (rather than meal ingestion) stimulate GFRAL neurons. TRAP-Seq analysis of GFRAL neurons revealed their expression of a wide range of neurotransmitters and neuropeptides. Artificially activating GfralCre -expressing neurons inhibited feeding, decreased gastric emptying, and promoted a conditioned taste aversion (CTA). GFRAL neurons most strongly innervate the parabrachial nucleus (PBN), where they target CGRP-expressing (CGRPPBN) neurons. Silencing CGRPPBN neurons abrogated the aversive and anorexic effects of GDF-15. These findings suggest that GFRAL neurons link non-meal-associated pathophysiologic signals to suppress nutrient uptake and absorption.

Deletion of the Brain-Specific α and δ Isoforms of Adapter Protein SH2B1 Protects Mice From Obesity.

Mice lacking SH2B1 and humans with variants of SH2B1 display severe obesity and insulin resistance. SH2B1 is an adapter protein that is recruited to the receptors of multiple hormones and neurotrophic factors. Of the four known alternatively spliced SH2B1 isoforms, SH2B1ß and SH2B1γ exhibit ubiquitous expression, whereas SH2B1α and SH2B1δ are essentially restricted to the brain. To understand the roles for SH2B1α and SH2B1δ in energy balance and glucose metabolism, we generated mice lacking these brain-specific isoforms (αδ knockout [αδKO] mice). αδKO mice exhibit decreased food intake, protection from weight gain on standard and high-fat diets, and an adiposity-dependent improvement in glucose homeostasis. SH2B1 has been suggested to impact energy balance via the modulation of leptin action. However, αδKO mice exhibit leptin sensitivity that is similar to that of wild-type mice by multiple measures. Thus, decreasing the abundance of SH2B1α and/or SH2B1δ relative to the other SH2B1 isoforms likely shifts energy balance toward a lean phenotype via a primarily leptin-independent mechanism. Our findings suggest that the different alternatively spliced isoforms of SH2B1 perform different functions in vivo.

Ventromedial hypothalamic nucleus neuronal subset regulates blood glucose independently of insulin.

To identify neurons that specifically increase blood glucose from among the diversely functioning cell types in the ventromedial hypothalamic nucleus (VMN), we studied the cholecystokinin receptor B-expressing (CCKBR-expressing) VMN targets of glucose-elevating parabrachial nucleus neurons. Activation of these VMNCCKBR neurons increased blood glucose. Furthermore, although silencing the broader VMN decreased energy expenditure and promoted weight gain without altering blood glucose levels, silencing VMNCCKBR neurons decreased hIepatic glucose production, insulin-independently decreasing blood glucose without altering energy balance. Silencing VMNCCKBR neurons also impaired the counterregulatory response to insulin-induced hypoglycemia and glucoprivation and replicated hypoglycemia-associated autonomic failure. Hence, VMNCCKBR cells represent a specialized subset of VMN cells that function to elevate glucose. These cells not only mediate the allostatic response to hypoglycemia but also modulate the homeostatic setpoint for blood glucose in an insulin-independent manner, consistent with a role for the brain in the insulin-independent control of glucose homeostasis.

Distinct ipRGC subpopulations mediate light's acute and circadian effects on body temperature and sleep.

The light environment greatly impacts human alertness, mood, and cognition by both acute regulation of physiology and indirect alignment of circadian rhythms. These processes require the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), but the relevant downstream brain areas involved remain elusive. ipRGCs project widely in the brain, including to the central circadian pacemaker, the suprachiasmatic nucleus (SCN). Here we show that body temperature and sleep responses to acute light exposure are absent after genetic ablation of all ipRGCs except a subpopulation that projects to the SCN. Furthermore, by chemogenetic activation of the ipRGCs that avoid the SCN, we show that these cells are sufficient for acute changes in body temperature. Our results challenge the idea that the SCN is a major relay for the acute effects of light on non-image forming behaviors and identify the sensory cells that initiate light's profound effects on body temperature and sleep.

Transcriptional and physiological roles for STAT proteins in leptin action.

OBJECTIVES: Leptin acts via its receptor LepRb on specialized neurons in the brain to modulate food intake, energy expenditure, and body weight. LepRb activates signal transducers and activators of transcription (STATs, including STAT1, STAT3, and STAT5) to control gene expression. METHODS: Because STAT3 is crucial for physiologic leptin action, we used TRAP-seq to examine gene expression in LepRb neurons of mice ablated for Stat3 in LepRb neurons (Stat3LepRbKO mice), revealing the STAT3-dependent transcriptional targets of leptin. To understand roles for STAT proteins in leptin action, we also ablated STAT1 or STAT5 from LepRb neurons and expressed a constitutively-active STAT3 (CASTAT3) in LepRb neurons. RESULTS: While we also found increased Stat1 expression and STAT1-mediated transcription of leptin-regulated genes in Stat3LepRbKO mice, ablating Stat1 in LepRb neurons failed to alter energy balance (even on the Stat3LepRbKO background); ablating Stat5 in LepRb neurons also failed to alter energy balance. Importantly, expression of a constitutively-active STAT3 (CASTAT3) in LepRb neurons decreased food intake and body weight and improved metabolic parameters in leptin-deficient (ob/ob) mice, as well as in wild-type animals. CONCLUSIONS: Thus, STAT3 represents the unique STAT protein required for leptin action and STAT3 suffices to mediate important components of leptin action in the absence of other LepRb signals.

Specific subpopulations of hypothalamic leptin receptor-expressing neurons mediate the effects of early developmental leptin receptor deletion on energy balance.

OBJECTIVE: To date, early developmental ablation of leptin receptor (LepRb) expression from circumscribed populations of hypothalamic neurons (e.g., arcuate nucleus (ARC) Pomc- or Agrp-expressing cells) has only minimally affected energy balance. In contrast, removal of LepRb from at least two large populations (expressing vGat or Nos1) spanning multiple hypothalamic regions produced profound obesity and metabolic dysfunction. Thus, we tested the notion that the total number of leptin-responsive hypothalamic neurons (rather than specific subsets of cells with a particular molecular or anatomical signature) subjected to early LepRb deletion might determine energy balance. METHODS: We generated new mouse lines deleted for LepRb in ARC GhrhCre neurons or in Htr2cCre neurons (representing roughly half of all hypothalamic LepRb neurons, distributed across many nuclei). We compared the phenotypes of these mice to previously-reported models lacking LepRb in Pomc, Agrp, vGat or Nos1 cells. RESULTS: The early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance. CONCLUSIONS: Although early developmental deletion of LepRb from known populations of ARC neurons fails to substantially alter body weight, the minimal phenotype of mice lacking LepRb in Htr2c cells suggests that the phenotype that results from early developmental LepRb deficiency depends not simply upon the total number of leptin-responsive hypothalamic LepRb cells. Rather, specific populations of LepRb neurons must play particularly important roles in body energy homeostasis; these as yet unidentified LepRb cells likely reside in the DMH.

Defining the Transcriptional Targets of Leptin Reveals a Role for Atf3 in Leptin Action.

Leptin acts via its receptor (LepRb) to modulate gene expression in hypothalamic LepRb-expressing neurons, thereby controlling energy balance and glucose homeostasis. Despite the importance of the control of gene expression in hypothalamic LepRb neurons for leptin action, the transcriptional targets of LepRb signaling have remained undefined because LepRb cells contribute a small fraction to the aggregate transcriptome of the brain regions in which they reside. We thus employed translating ribosome affinity purification followed by RNA sequencing to isolate and analyze mRNA from the hypothalamic LepRb neurons of wild-type or leptin-deficient (Lepob/ob) mice treated with vehicle or exogenous leptin. Although the expression of most of the genes encoding the neuropeptides commonly considered to represent the main targets of leptin action were altered only following chronic leptin deprivation, our analysis revealed other transcripts that were coordinately regulated by leptin under multiple treatment conditions. Among these, acute leptin treatment increased expression of the transcription factor Atf3 in LepRb neurons. Furthermore, ablation of Atf3 from LepRb neurons (Atf3LepRbKO mice) decreased leptin efficacy and promoted positive energy balance in mice. Thus, this analysis revealed the gene targets of leptin action, including Atf3, which represents a cellular mediator of leptin action.

C-terminal phosphorylation regulates the kinetics of a subset of melanopsin-mediated behaviors in mice.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and mediate several non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex (PLR). ipRGCs act as autonomous photoreceptors via the intrinsic melanopsin-based phototransduction pathway and as a relay for rod/cone input via synaptically driven responses. Under low light intensities, where only synaptically driven rod/cone input activates ipRGCs, the duration of the ipRGC response will be determined by the termination kinetics of the rod/cone circuits. Little is known, however, about the termination kinetics of the intrinsic melanopsin-based phototransduction pathway and its contribution to several melanopsin-mediated behaviors. Here, we show that C-terminal phosphorylation of melanopsin determines the recovery kinetics of the intrinsic melanopsin-based photoresponse in ipRGCs, the duration of the PLR, and the speed of reentrainment. In contrast, circadian phase alignment and direct effects of light on activity (masking) are not influenced by C-terminal phosphorylation of melanopsin. Electrophysiological measurements demonstrate that expression of a virally encoded melanopsin lacking all C-terminal phosphorylation sites (C terminus phosphonull) leads to a prolonged intrinsic light response. In addition, mice expressing the C terminus phosphonull in ipRGCs reentrain faster to a delayed light/dark cycle compared with mice expressing virally encoded WT melanopsin; however, the phase angle of entrainment and masking were indistinguishable. Importantly, a sustained PLR in the phosphonull animals is only observed at brighter light intensities that activate melanopsin phototransduction, but not at dimmer light intensities that activate only the rod/cone pathway. Taken together, our results highlight how the kinetics of the melanopsin photoresponse differentially regulate distinct light-mediated behaviors.

A visual circuit uses complementary mechanisms to support transient and sustained pupil constriction.

Rapid and stable control of pupil size in response to light is critical for vision, but the neural coding mechanisms remain unclear. Here, we investigated the neural basis of pupil control by monitoring pupil size across time while manipulating each photoreceptor input or neurotransmitter output of intrinsically photosensitive retinal ganglion cells (ipRGCs), a critical relay in the control of pupil size. We show that transient and sustained pupil responses are mediated by distinct photoreceptors and neurotransmitters. Transient responses utilize input from rod photoreceptors and output by the classical neurotransmitter glutamate, but adapt within minutes. In contrast, sustained responses are dominated by non-conventional signaling mechanisms: melanopsin phototransduction in ipRGCs and output by the neuropeptide PACAP, which provide stable pupil maintenance across the day. These results highlight a temporal switch in the coding mechanisms of a neural circuit to support proper behavioral dynamics.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells.

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2(-/-) mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2(-/-) were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2(-/-) mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light.

Phenotypic and functional characterization of Bst+/- mouse retina.

The belly spot and tail (Bst(+/-)) mouse phenotype is caused by mutations of the ribosomal protein L24 (Rpl24). Among various phenotypes in Bst(+/-) mice, the most interesting are its retinal abnormalities, consisting of delayed closure of choroid fissures, decreased ganglion cells and subretinal vascularization. We further characterized the Bst(+/-) mouse and investigated the underlying molecular mechanisms to assess the feasibility of using this strain as a model for stem cell therapy of retinal degenerative diseases due to retinal ganglion cell (RGC) loss. We found that, although RGCs are significantly reduced in retinal ganglion cell layer in Bst(+/-) mouse, melanopsin(+) RGCs, also called ipRGCs, appear to be unchanged. Pupillary light reflex was completely absent in Bst(+/-) mice but they had a normal circadian rhythm. In order to examine the pathological abnormalities in Bst(+/-) mice, we performed electron microscopy in RGC and found that mitochondria morphology was deformed, having irregular borders and lacking cristae. The complex activities of the mitochondrial electron transport chain were significantly decreased. Finally, for subretinal vascularization, we also found that angiogenesis is delayed in Bst(+/-) associated with delayed hyaloid regression. Characterization of Bst(+/-) retina suggests that the Bst(+/-) mouse strain could be a useful murine model. It might be used to explore further the pathogenesis and strategy of treatment of retinal degenerative diseases by employing stem cell technology.