Targeting Phosphoinositide 3-Kinase – Five Decades of Chemical Space Exploration

Phosphoinositide 3-kinase (PI3K) plays a key role in a plethora of physiologic processes and controls cell growth, metabolism, immunity, cardiovascular and neurological function, and more. The discovery of wort-mannin as the first potent PI3K inhibitor (PI3Ki) in the 1990s provided rapid identification of PI3K-dependent processes, which drove the discovery of the PI3K/protein kinase B (PKB/Akt)/target of rapamycin (mTOR) pathway. Genetic mouse models and first PI3K isoform-specific inhibitors pinpointed putative therapeutic applications. The recognition of PI3K as target for cancer therapy drove subsequently drug development. Here we provide a brief journey through the emerging roles of PI3K to the development of preclinical and clinical PI3Ki candidates.

Tcf1 Sustains the Expression of Multiple Regulators in Promoting Early Natural Killer Cell Development

T cell factor 1 (Tcf1) is known as a critical mediator for natural killer (NK) cell development and terminal maturation. However, its essential targets and precise mechanisms involved in early NK progenitors (NKP) are not well clarified. To investigate the role of Tcf1 in NK cells at distinct developmental phases, we employed three kinds of genetic mouse models, namely, Tcf7fl/flVavCre/+, Tcf7fl/flCD122Cre/+ and Tcf7fl/flNcr1Cre/+ mice, respectively. Similar to Tcf1 germline knockout mice, we found notably diminished cell number and defective development in BM NK cells from all strains. In contrast, Tcf7fl/flNcr1Cre/+ mice exhibited modest defects in splenic NK cells compared with those in the other two strains. By analyzing the published ATAC-seq and ChIP-seq data, we found that Tcf1 directly targeted 110 NK cell-related genes which displayed differential accessibility in the absence of Tcf1. Along with this clue, we further confirmed that a series of essential regulators were expressed aberrantly in distinct BM NK subsets with conditional ablating Tcf1 at NKP stage. Eomes, Ets1, Gata3, Ikzf1, Ikzf2, Nfil3, Runx3, Sh2d1a, Slamf6, Tbx21, Tox, and Zeb2 were downregulated, whereas Spi1 and Gzmb were upregulated in distinct NK subsets due to Tcf1 deficiency. The dysregulation of these genes jointly caused severe defects in NK cells lacking Tcf1. Thus, our study identified essential targets of Tcf1 in NK cells, providing new insights into Tcf1-dependent regulatory programs in step-wise governing NK cell development.

A novel non-genetic murine model of hyperglycemia and hyperlipidemia-associated accelerated atherosclerosis

Objective: Atherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. To establish a new non-genetic model of inducible metabolic risk factors mimicking hyperlipidemia, hyperglycemia, or both and allowing to detect phenotypic differences dependent on the metabolic stressor(s). Methods and Results: Wild type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) or high-cholesterol/high fat-diet (Paigen diet, PD). Combined hyperlipidemic and hyperglycemic (HGHCi) mice, but not hyperlipidemia (HCi) alone, display characteristic features of accelerated atherosclerosis. Atherosclerotic plaques of HGHCi animals were larger, showed a less stable phenotype, contained more macrophages and less smooth muscle cells. These findings were observed both at early (12 weeks) and late (20 weeks) time points on both HFD or PD diet. Differences between the HGHCi and HCi model were confirmed using RNAseq analysis revealing that significantly more genes are dysregulated in mice with combined hyperlipidemia and hyperglycemia as compared to the hyperlipidemia only group. The HGHCi-associated genes were related to pathways regulating inflammation, cellular metabolism and collagen degradation. PD accelerates atherosclerosis in mice and shows plaque formation already after 8 weeks, therefore, representing a fast direct inducible hyperglycemic atherosclerosis model. Conclusion: We established a non-genetic inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses

Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8–P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.

Dopaminergic Dysregulation in Syndromic Autism Spectrum Disorders: Insights From Genetic Mouse Models

Autism spectrum disorder (ASD) is a neurodevelopmental disorder defined by altered social interaction and communication, and repetitive, restricted, inflexible behaviors. Approximately 1.5-2% of the general population meet the diagnostic criteria for ASD and several brain regions including the cortex, amygdala, cerebellum and basal ganglia have been implicated in ASD pathophysiology. The midbrain dopamine system is an important modulator of cellular and synaptic function in multiple ASD-implicated brain regions via anatomically and functionally distinct dopaminergic projections. The dopamine hypothesis of ASD postulates that dysregulation of dopaminergic projection pathways could contribute to the behavioral manifestations of ASD, including altered reward value of social stimuli, changes in sensorimotor processing, and motor stereotypies. In this review, we examine the support for the idea that cell-autonomous changes in dopaminergic function are a core component of ASD pathophysiology. We discuss the human literature supporting the involvement of altered dopamine signaling in ASD including genetic, brain imaging and pharmacologic studies. We then focus on genetic mouse models of syndromic neurodevelopmental disorders in which single gene mutations lead to increased risk for ASD. We highlight studies that have directly examined dopamine neuron number, morphology, physiology, or output in these models. Overall, we find considerable support for the idea that the dopamine system may be dysregulated in syndromic ASDs; however, there does not appear to be a consistent signature and some models show increased dopaminergic function, while others have deficient dopamine signaling. We conclude that dopamine dysregulation is common in syndromic forms of ASD but that the specific changes may be unique to each genetic disorder and may not account for the full spectrum of ASD-related manifestations.

Thyroid hormone receptor-α regulates autophagy, mitochondrial biogenesis, and fatty acid utilization in skeletal muscle

Skeletal muscle (SM) weakness occurs in hypothyroidism and resistance to thyroid hormone alpha (RTHα) syndrome. However, the cell signaling and molecular mechanism(s) underlying muscle weakness under these conditions is not well understood. We thus examined the role of thyroid hormone receptor alpha (TRα), the predominant TR isoform in SM, on autophagy, mitochondrial biogenesis and metabolism to demonstrate the molecular mechanism(s) underlying muscle weakness in these two conditions.Two genetic mouse models, TRα1 PV/+ mice which expresses mutant Thra1PV gene ubiquitously, and SM-TRα1 L400R/+ mice, which expresses TRα1 L400R in a muscle-specific manner, were used in this study. Gastrocnemius muscle from TRα1 PV/+, SM-TRα1 L400R/+, and their control mice was harvested for analyses. We demonstrated that loss of TRα1 signaling in gastrocnemius muscle from both the genetic mouse models led to decreased autophagy as evidenced by accumulation of p62 and decreased expression of lysosomal markers (LAMP1, and LAMP2) and lysosomal proteases (cathepsin B and cathepsin D). The expression of PGC1α, TFAM, and ERRα, key factors contributing to mitochondrial biogenesis as well as mitochondrial proteins were decreased, suggesting that there was reduced mitochondrial biogenesis due to the expression of mutant TRα1. Transcriptomic and metabolomic analyses of SM suggested that lipid catabolism was impaired, and was associated with decreased acylcarnitines and tricarboxylic acid cycle (TCA cycle) intermediates in the SM from the mouse line expressing SM-specific mutant TRα1. Our results provide new insight into TRα1-mediated cell signaling, molecular, and metabolic changes that occur in SM when TR action is impaired.

Saturation mutagenesis defines novel mouse models of severe spine deformity

Embryonic formation and patterning of the vertebrate spinal column requires coordination of many molecular cues. After birth, the integrity of the spine is impacted by developmental abnormalities of the skeletal, muscular, and nervous systems, which may result in deformities such as kyphosis and scoliosis. We sought to identify novel genetic mouse models of severe spine deformity by implementing in vivo skeletal radiography as part of a high-throughput saturation mutagenesis screen. We report selected examples of genetic mouse models following radiographic screening of 54,497 mice from 1,275 pedigrees. An estimated 30.44% of autosomal genes harbored predicted damaging alleles examined twice or more in the homozygous state. Of the 1,275 pedigrees screened, 7.4% presented with severe spine deformity developing in multiple mice, and of these, meiotic mapping implicated ENU alleles in 21% of pedigrees. Our study provides proof-of-concept that saturation mutagenesis is capable of discovering novel mouse models of human disease, including conditions with skeletal, neural, and neuromuscular pathologies. Furthermore, we report a mouse model of skeletal disease, including severe spine deformity, caused by recessive mutation in Scube3. By integrating results with a human clinical exome database, we identified a patient with undiagnosed skeletal disease who harbored recessive mutations in SCUBE3, and we demonstrated that disease-associated mutations are associated with reduced trans-activation of Smad signaling in vitro. All radiographic results and mouse models are made publicly available through the Mutagenetix online database with the goal of advancing understanding of spine development and discovering novel mouse models of human disease.

Vitamin D deficiency exacerbates UV/endorphin and opioid addiction

The current opioid epidemic warrants a better understanding of genetic and environmental factors that contribute to opioid addiction. Here we report an increased prevalence of vitamin D (VitD) deficiency in patients diagnosed with opioid use disorder and an inverse and dose-dependent association of VitD levels with self-reported opioid use. We used multiple pharmacologic approaches and genetic mouse models and found that deficiencies in VitD signaling amplify exogenous opioid responses that are normalized upon restoration of VitD signaling. Similarly, physiologic endogenous opioid analgesia and reward responses triggered by ultraviolet (UV) radiation are repressed by VitD signaling, suggesting that a feedback loop exists whereby VitD deficiency produces increased UV/endorphin-seeking behavior until VitD levels are restored by cutaneous VitD synthesis. This feedback may carry the evolutionary advantage of maximizing VitD synthesis. However, unlike UV exposure, exogenous opioid use is not followed by VitD synthesis (and its opioid suppressive effects), contributing to maladaptive addictive behavior.

An alternative hypothesis for why exposure to static magnetic and electric fields treats type 2 diabetes

Carter et al. report that exposure to static magnetic and electric fields (sBE), for as little as 3 days, reverses glucose intolerance and insulin resistance in diet-induced and genetic mouse models of type 2 diabetes (1). They hypothesize that sBE triggers a systemic redox response to modulate insulin sensitivity and that sBE could therefore be used as a noninvasive treatment for type 2 diabetes. However, these authors were unable to define a mechanism to explain how SBE might alter ROS or to identify the specific proteins that mediate this effect. Given these limitations we propose an alternative hypothesis to explain their findings.