MicroRNAs (miRNAs) are crucial regulators of myelination in the peripheral nervous system (PNS). However, the miRNAs species involved and the underlying mechanisms are largely unknown. We found that let-7 miRNAs are highly abundant during PNS myelination and that their levels are inversely correlated to the expression of lin28 homolog B (Lin28B), an antagonist of let-7 accumulation. Sustained expression of Lin28B and consequently reduced levels of let-7 miRNAs results in a failure of Schwann cell myelination in transgenic mouse models and in cell culture. Subsequent analyses revealed that let-7 miRNAs promote expression of the myelination-driving master transcription factor Krox20 (also known as Egr2) through suppression of myelination inhibitory Notch signalling. We conclude that the Lin28B/let-7 axis acts as a critical driver of PNS myelination, in particular by regulating myelination onset, identifying this pathway also as a potential therapeutic target in demyelinating diseases.

Nonalcoholic fatty liver disease, a condition in which excess fat accumulates in the liver, is strongly associated with the metabolic syndrome, including obesity and other related conditions. This disease has the potential to progress from steatosis to steatohepatitis, fibrosis, and cirrhosis. The recent increase in the prevalence of the metabolic syndrome is largely driven by changes in diet and activity levels. Individual variation in the response to this obesogenic environment, however, is attributable in part to genetic variation between individuals, but very few mammalian genetic loci have been identified with effects on fat accumulation in the liver. To study the genetic basis for variation in liver fat content in response to dietary fat, liver fat proportion was determined using quantitative magnetic resonance imaging in 478 mice from 16 LG/J X SM/J recombinant inbred strains fed either a high-fat (42% kcal from fat) or low-fat (15% kcal from fat) diet. An analysis of variance confirmed that there is a genetic basis for variation in liver fat content within the population with significant effects of sex and diet. Three quantitative trail loci that contribute to liver fat content also were mapped.

Myelination is a biosynthetically demanding process in which mTORC1, the gatekeeper of anabolism, occupies a privileged regulatory position. We have shown previously that loss of mTORC1 function in Schwann cells (SCs) hampers myelination. Here, we genetically disrupted key inhibitory components upstream of mTORC1, TSC1 or PTEN, in mouse SC development, adult homeostasis, and nerve injury. Surprisingly, the resulting mTORC1 hyperactivity led to markedly delayed onset of both developmental myelination and remyelination after injury. However, if mTORC1 was hyperactivated after myelination onset, radial hypermyelination was observed. At early developmental stages, physiologically high PI3K-Akt-mTORC1 signaling suppresses expression of Krox20 (Egr2), the master regulator of PNS myelination. This effect is mediated by S6K and contributes to control mechanisms that keep SCs in a not-fully differentiated state to ensure proper timing of myelination initiation. An ensuing decline in mTORC1 activity is crucial to allow myelination to start, while remaining mTORC1 activity drives myelin growth.

Some mutations affecting dynamin 2 (DNM2) can cause dominantly inherited Charcot-Marie-Tooth (CMT) neuropathy. Here, we describe the analysis of mice carrying the DNM2 K562E mutation which has been associated with dominant-intermediate CMT type B (CMTDIB). Contrary to our expectations, heterozygous DNM2 K562E mutant mice did not develop definitive signs of an axonal or demyelinating neuropathy. Rather, we found a primary myopathy-like phenotype in these mice. A likely interpretation of these results is that the lack of a neuropathy in this mouse model has allowed the unmasking of a primary myopathy due to the DNM2 K562E mutation which might be overshadowed by the neuropathy in humans. Consequently, we hypothesize that a primary myopathy may also contribute to the disease mechanism in some CMTDIB patients. We propose that these findings should be considered in the evaluation of patients, the determination of the underlying disease processes and the development of tailored potential treatment strategies.

Diabetic retinopathy (DR) is an increasingly frequent cause of blindness across populations; however, the events that initiate pathophysiology of DR remain elusive. Strong preclinical and clinical evidence suggests that abnormalities in retinal lipid metabolism caused by diabetes may account for the origin of this disease. A major arm of lipid metabolism, de novo biosynthesis, is driven by elevation in available glucose, a common thread binding all forms of vision loss in diabetes. Therefore, we hypothesized that aberrant retinal lipid biogenesis is an important promoter of early DR. In murine models, we observed elevations of diabetes-associated retinal de novo lipogenesis ∼70% over control levels. This shift was primarily because of activation of fatty acid synthase (FAS), a rate-limiting enzyme in the biogenic pathway. Activation of FAS was driven by canonical glucose-mediated disinhibition of acetyl-CoA carboxylase, a major upstream regulatory enzyme. Mutant mice expressing gain-of-function FAS demonstrated increased vulnerability to DR, whereas those with FAS deletion in rod photoreceptors maintained preserved visual responses upon induction of diabetes. Excess retinal de novo lipogenesis-either because of diabetes or because of FAS gain of function-was associated with modestly increased levels of palmitate-containing phosphatidylcholine species in synaptic membranes, a finding with as yet uncertain significance. These findings implicate glucose-dependent increases in photoreceptor de novo lipogenesis in the early pathogenesis of DR, although the mechanism of deleterious action of this pathway remains unclear.

Allele-specific expression (ASE) is a phenomenon in which one allele is preferentially expressed over the other. Genetic and epigenetic factors cause ASE by altering the final composition of a gene's product, leading to expression imbalances that can have functional consequences on phenotypes. Environmental signals also impact allele-specific expression, but how they contribute to this cross talk remains understudied. Here, we explored how genotype, parent-of-origin, tissue, sex, and dietary fat simultaneously influence ASE biases. Male and female mice from a F1 reciprocal cross of the LG/J and SM/J strains were fed a high or low fat diet. We harnessed strain-specific variants to distinguish between two ASE classes: parent-of-origin-dependent (unequal expression based on parental origin) and sequence-dependent (unequal expression based on nucleotide identity). We present a comprehensive map of ASE patterns in 2853 genes across three tissues and nine environmental contexts. We found that both ASE classes are highly dependent on tissue and environmental context. They vary across metabolically relevant tissues, between males and females, and in response to dietary fat. We also found 45 genes with inconsistent ASE biases that switched direction across tissues and/or environments. Finally, we integrated ASE and QTL data from published intercrosses of the LG/J and SM/J strains. Our ASE genes are often enriched in QTLs for metabolic and musculoskeletal traits, highlighting how this orthogonal approach can prioritize candidate genes. Together, our results provide novel insights into how genetic, epigenetic, and environmental mechanisms govern allele-specific expression, which is an essential step toward deciphering the genotype-to-phenotype map.

Small GTPases of the Rho and Ras families are important regulators of Schwann cell biology. The Ras-like GTPases RalA and RalB act downstream of Ras in malignant peripheral nerve sheath tumors. However, the physiological role of Ral proteins in Schwann cell development is unknown. Using transgenic mice with ablation of one or both Ral genes, we report that Ral GTPases are crucial for axonal radial sorting. While lack of only one Ral GTPase was dispensable for early peripheral nerve development, ablation of both RalA and RalB resulted in persistent radial sorting defects, associated with hallmarks of deficits in Schwann cell process formation and maintenance. In agreement, ex vivo-cultured Ral-deficient Schwann cells were impaired in process extension and the formation of lamellipodia. Our data indicate further that RalA contributes to Schwann cell process extensions through the exocyst complex, a known effector of Ral GTPases, consistent with an exocyst-mediated function of Ral GTPases in Schwann cells.

Small ubiquitin-like modifier (SUMO)2/3 but not SUMO1 conjugation is activated after transient cerebral ischemia. To investigate its function, we blocked neuronal SUMO2/3 translation through lentiviral microRNA delivery in primary cortical neurons. Viability was unaffected by SUMO2/3 silencing unless neurons were stressed by transient oxygen-glucose deprivation (OGD). Both 15 and 45 minutes of OGD were tolerated by control microRNA-expressing neurons but damaged >60% of neurons expressing SUMO2/3 microRNA. Damaging OGD (75 minutes) increased neuronal loss to 54% (control microRNA) and to 99% (SUMO2/3 microRNA). This suggests that activation of SUMO2/3 conjugation is an endogenous neuroprotective stress response.

The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. We found that Gdap1 knockout mice (Gdap1(-/-)), mimicking genetic alterations of patients suffering from severe forms of Charcot-Marie-Tooth disease, develop an age-related, hypomyelinating peripheral neuropathy. Ablation of Gdap1 expression in Schwann cells recapitulates this phenotype. Additionally, intra-axonal mitochondria of peripheral neurons are larger in Gdap1(-/-) mice and mitochondrial transport is impaired in cultured sensory neurons of Gdap1(-/-) mice compared with controls. These changes in mitochondrial morphology and dynamics also influence mitochondrial biogenesis. We demonstrate that mitochondrial DNA biogenesis and content is increased in the peripheral nervous system but not in the central nervous system of Gdap1(-/-) mice compared with control littermates. In search for a molecular mechanism we turned to the paralogue of GDAP1, GDAP1L1, which is mainly expressed in the unaffected central nervous system. GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane. This translocation is necessary to substitute for loss of GDAP1 expression. Accordingly, more GDAP1L1 was associated with mitochondria in the spinal cord of aged Gdap1(-/-) mice compared with controls. Our findings demonstrate that Charcot-Marie-Tooth disease caused by mutations in GDAP1 leads to mild, persistent oxidative stress in the peripheral nervous system, which can be compensated by GDAP1L1 in the unaffected central nervous system. We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Membrane lipid composition is central to the highly specialized functions of neurological tissues. In the retina, abnormal lipid metabolism causes severe forms of blindness, often through poorly understood neuronal cell death. Here, we demonstrate that deleting the de novo lipogenic enzyme fatty acid synthase (FAS) from the neural retina, but not the vascular retina, results in progressive neurodegeneration and blindness with a temporal pattern resembling rodent models of retinitis pigmentosa. Blindness was not rescued by protection from light-evoked activity; by eating a diet enriched in palmitate, the product of the FAS reaction; or by treatment with the PPARα agonist fenofibrate. Vision loss was due to aberrant synaptic structure, blunted responsiveness to glial-derived neurotrophic factor and ciliary neurotrophic factor, and eventual apoptotic cell loss. This progressive neurodegeneration was associated with decreased membrane cholesterol content, as well as loss of discrete n-3 polyunsaturated fatty acid- and saturated fatty acid-containing phospholipid species within specialized membrane microdomains. Neurotrophic signaling was restored by exogenous cholesterol delivery. These findings implicate de novo lipogenesis in neurotrophin-dependent cell survival by maintaining retinal membrane configuration and lipid composition, and they suggest that ongoing lipogenesis may be required to prevent cell death in many forms of retinopathy.

Patients with diabetes are at higher risk of developing carotid artery stenosis and resultant stroke. Arachidonoyl phospholipids affect plaque inflammation and vulnerability, but whether diabetic patients have unique carotid artery phospholipidomic profiles is unknown. We performed a comprehensive paired analysis of phospholipids in extracranial carotid endarterectomy (CEA) plaques of matched diabetic and nondiabetic patients and analyzed mass spectrometry-derived profiles of three phospholipids, plasmenyl-phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylinositol (PI), in maximally (MAX) and minimally (MIN) diseased CEA segments. We also measured levels of arachidonic acid (AA), produced by pPE hydrolysis, and choline-ethanolamine phosphotransferase 1 (CEPT1), responsible for most pPE de novo biosynthesis. In paired analysis, MIN CEA segments had higher levels than MAX segments of pPE (P < 0.001), PS (P < 0.001), and PI (P < 0.03). MIN diabetic plaques contained higher levels than MAX diabetic plaques of arachidonoyl pPE38:4 and pPE38:5 and CEPT1 was upregulated in diabetic versus nondiabetic plaques. AA levels were relatively greater in MIN versus MAX segments of all CEA segments, and were higher in diabetic than nondiabetic plaques. Our findings suggest that arachidonoyl phospholipids are more likely to be abundant in the extracranial carotid artery plaque of diabetic rather than nondiabetic patients.

The mitochondrial protein MAVS (also known as IPS-1, VISA, and CARDIF) interacts with RIG-I-like receptors (RLRs) to induce type I interferon (IFN-I). NLRX1 is a mitochondrial nucleotide-binding, leucine-rich repeats (NLR)-containing protein that attenuates MAVS-RLR signaling. Using Nlrx1(-/-) cells, we confirmed that NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High-throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1 and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

Acyl-protein thioesterases 1 and 2 (APT1 and APT2) reverse S-acylation, a potential regulator of systemic glucose metabolism in mammals. Palmitoylation proteomics in liver-specific knockout mice shows that APT1 predominates over APT2, primarily depalmitoylating mitochondrial proteins, including proteins linked to glutamine metabolism. miniTurbo-facilitated determination of the protein-protein proximity network of APT1 and APT2 in HepG2 cells reveals APT proximity networks encompassing mitochondrial proteins including the major translocases Tomm20 and Timm44. APT1 also interacts with Slc1a5 (ASCT2), the only glutamine transporter known to localize to mitochondria. High-fat-diet-fed male mice with dual (but not single) hepatic deletion of APT1 and APT2 have insulin resistance, fasting hyperglycemia, increased glutamine-driven gluconeogenesis, and decreased liver mass. These data suggest that APT1 and APT2 regulation of hepatic glucose metabolism and insulin signaling is functionally redundant. Identification of substrates and protein-protein proximity networks for APT1 and APT2 establishes a framework for defining mechanisms underlying metabolic disease.

During development, Schwann cells (SCs) interpret different extracellular cues to regulate their migration, proliferation, and the remarkable morphological changes associated with the sorting, ensheathment, and myelination of axons. Although interactions between extracellular matrix proteins and integrins are critical to some of these processes, the downstream signaling pathways they control are still poorly understood. Integrin-linked kinase (ILK) is a focal adhesion protein that associates with multiple binding partners to link integrins to the actin cytoskeleton and is thought to participate in integrin and growth factor-mediated signaling. Using SC-specific gene ablation, we report essential functions for ILK in radial sorting of axon bundles and in remyelination in the peripheral nervous system. Our in vivo and in vitro experiments show that ILK negatively regulates Rho/Rho kinase signaling to promote SC process extension and to initiate radial sorting. ILK also facilitates axon remyelination, likely by promoting the activation of downstream molecules such as AKT/protein kinase B.

The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Context-dependent genetic effects, including genotype-by-environment and genotype-by-sex interactions, are a potential mechanism by which genetic variation of complex traits is maintained in populations. Pleiotropic genetic effects are also thought to play an important role in evolution, reflecting functional and developmental relationships among traits. We examine context-dependent genetic effects at pleiotropic loci associated with normal variation in multiple metabolic syndrome (MetS) components (obesity, dyslipidemia, and diabetes-related traits). MetS prevalence is increasing in Western societies and, while environmental in origin, presents substantial variation in individual response. We identify 23 pleiotropic MetS quantitative trait loci (QTL) in an F(16) advanced intercross between the LG/J and SM/J inbred mouse strains (Wustl:LG,SM-G16; n = 1002). Half of each family was fed a high-fat diet and half fed a low-fat diet; and additive, dominance, and parent-of-origin imprinting genotypic effects were examined in animals partitioned into sex, diet, and sex-by-diet cohorts. We examine the context-dependency of the underlying additive, dominance, and imprinting genetic effects of the traits associated with these pleiotropic QTL. Further, we examine sequence polymorphisms (SNPs) between LG/J and SM/J as well as differential expression of positional candidate genes in these regions. We show that genetic associations are different in different sex, diet, and sex-by-diet settings. We also show that over- or underdominance and ecological cross-over interactions for single phenotypes may not be common, however multidimensional synthetic phenotypes at loci with pleiotropic effects can produce situations that favor the maintenance of genetic variation in populations. Our findings have important implications for evolution and the notion of personalized medicine.

The nuclear receptor PPARalpha is activated by drugs to treat human disorders of lipid metabolism. Its endogenous ligand is unknown. PPARalpha-dependent gene expression is impaired with inactivation of fatty acid synthase (FAS), suggesting that FAS is involved in generation of a PPARalpha ligand. Here we demonstrate the FAS-dependent presence of a phospholipid bound to PPARalpha isolated from mouse liver. Binding was increased under conditions that induce FAS activity and displaced by systemic injection of a PPARalpha agonist. Mass spectrometry identified the species as 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-GPC). Knockdown of Cept1, required for phosphatidylcholine synthesis, suppressed PPARalpha-dependent gene expression. Interaction of 16:0/18:1-GPC with the PPARalpha ligand-binding domain and coactivator peptide motifs was comparable to PPARalpha agonists, but interactions with PPARdelta were weak and none were detected with PPARgamma. Portal vein infusion of 16:0/18:1-GPC induced PPARalpha-dependent gene expression and decreased hepatic steatosis. These data suggest that 16:0/18:1-GPC is a physiologically relevant endogenous PPARalpha ligand.

The liver plays a major role in regulating metabolic homeostasis and is vital for nutrient metabolism. Identifying the genetic factors regulating these processes could lead to a greater understanding of how liver function responds to a high-fat diet and how that response may influence susceptibilities to obesity and metabolic syndrome. In this study we examine differences in hepatic gene expression between the LG/J and SM/J inbred mouse strains and how gene expression in these strains is affected by high-fat diet. LG/J and SM/J are known to differ in their responses to a high-fat diet for a variety of obesity- and diabetes-related traits, with the SM/J strain exhibiting a stronger phenotypic response to diet.