Evidence that minocycline treatment confounds the interpretation of neurofilament as a biomarker.

Neurofilament light (NfL) concentration in cerebrospinal fluid (CSF) and blood serves as an important biomarker in neurology drug development. Changes in NfL are generally assumed to reflect changes in neuronal damage, while little is known about the clearance of NfL from biofluids. We observed an NfL increase of 3.5-fold in plasma and 5.7-fold in CSF in an asymptomatic individual at risk for genetic prion disease following 6 weeks' treatment with oral minocycline for a dermatologic indication. Other biomarkers remained normal, and proteomic analysis of CSF revealed that the spike was exquisitely specific to neurofilaments. NfL dropped nearly to normal levels 5 weeks after minocycline cessation, and the individual remained free of disease 2 years later. Plasma NfL in dermatology patients was not elevated above normal controls. Dramatically high plasma NfL (>500 pg/mL) was variably observed in some hospitalized individuals receiving minocycline. In mice, treatment with minocycline resulted in variable increases of 1.3- to 4.0-fold in plasma NfL, with complete washout 2 weeks after cessation. In neuron-microglia co-cultures, minocycline increased NfL concentration in conditioned media by 3.0-fold without any visually obvious impact on neuronal health. We hypothesize that minocycline does not cause or exacerbate neuronal damage, but instead impacts the clearance of NfL from biofluids, a potential confounder for interpretation of this biomarker.

Microglia and amyloid plaque formation in Alzheimer's disease - Evidence, possible mechanisms, and future challenges.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive decline that severely affects patients and their families. Genetic and environmental risk factors, such as viral infections, synergize to accelerate the aging-associated neurodegeneration. Genetic risk factors for late-onset AD (LOAD), which accounts for most AD cases, are predominantly implicated in microglial and immune cell functions. As such, microglia play a major role in formation of amyloid beta (Aß) plaques, the major pathological hallmark of AD. This review aims to provide an overview of the current knowledge regarding the role of microglia in Aß plaque formation, as well as their impact on morphological and functional diversity of Aß plaques. Based on this discussion, we seek to identify challenges and opportunities in this field with potential therapeutic implications.

An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis.

The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-ß1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell-derived microglia, HSV-2-induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2-treated microglia exerted IKBKE-dependent type I IFN-mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.

ER stress induces caspase-2-tBID-GSDME-dependent cell death in neurons lytically infected with herpes simplex virus type 2.

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV-induced neuronal cell death remain enigmatic. Here, we report that lytic HSV-2 infection of human neuron-like SH-SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV-2-induced GSDME-mediated cell death occurs downstream of replication-induced endoplasmic reticulum stress driven by inositol-requiring kinase 1α (IRE1α), leading to activation of caspase-2, cleavage of the pro-apoptotic protein BH3-interacting domain death agonist (BID), and mitochondria-dependent activation of caspase-3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC-derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress-driven pathway to execute GSDME-mediated cell death and promote inflammation.

TREM2 is down-regulated by HSV1 in microglia and involved in antiviral defense in the brain.

Immunological control of viral infections in the brain exerts immediate protection and also long-term maintenance of brain integrity. Microglia are important for antiviral defense in the brain. Here, we report that herpes simplex virus type 1 (HSV1) infection of human induced pluripotent stem cell (hiPSC)-derived microglia down-regulates expression of genes in the TREM2 pathway. TREM2 was found to be important for virus-induced IFNB induction through the DNA-sensing cGAS-STING pathway in microglia and for phagocytosis of HSV1-infected neurons. Consequently, TREM2 depletion increased susceptibility to HSV1 infection in human microglia-neuron cocultures and in the mouse brain. TREM2 augmented STING signaling and activation of downstream targets TBK1 and IRF3. Thus, TREM2 is important for the antiviral immune response in microglia. Since TREM2 loss-of-function mutations and HSV1 serological status are both linked to Alzheimer's disease, this work poses the question whether genetic or virus-induced alterations of TREM2 activity predispose to post-infection neurological pathologies.

APP-BACE1 Interaction and Intracellular Localization Regulate Aß Production in iPSC-Derived Cortical Neurons.

Alzheimer's disease (AD) is characterized pathologically by amyloid ß (Aß)-containing plaques. Generation of Aß from amyloid precursor protein (APP) by two enzymes, ß- and γ-secretase, has therefore been in the AD research spotlight for decades. Despite this, how the physical interaction of APP with the secretases influences APP processing is not fully understood. Herein, we compared two genetically identical human iPSC-derived neuronal cell types: low Aß-secreting neuroprogenitor cells (NPCs) and high Aß-secreting mature neurons, as models of low versus high Aß production. We investigated levels of substrate, enzymes and products of APP amyloidogenic processing and correlated them with the proximity of APP to ß- and γ-secretase in endo-lysosomal organelles. In mature neurons, increased colocalization of full-length APP with the ß-secretase BACE1 correlated with increased ß-cleavage product sAPPß. Increased flAPP/BACE1 colocalization was mainly found in early endosomes. In the same way, increased colocalization of APP-derived C-terminal fragment (CTF) with presenilin-1 (PSEN1), the catalytic subunit of γ-secretase, was seen in neurons as compared to NPCs. Furthermore, most of the interaction of APP with BACE1 in low Aß-secreting NPCs seemed to derive from CTF, the remaining APP part after BACE1 cleavage, indicating a possible novel product-enzyme inhibition. In conclusion, our results suggest that interaction of APP and APP cleavage products with their secretases can regulate Aß production both positively and negatively. ß- and γ-Secretases are difficult targets for AD treatment due to their ubiquitous nature and wide range of substrates. Therefore, targeting APP-secretase interactions could be a novel treatment strategy for AD. Colocalization of APP species with BACE1 in a novel model of low- versus high-Aß secretion-Two genetically identical human iPSC-derived neuronal cell types: low Aß-secreting neuroprogenitor cells (NPCs) and high Aß secreting mature neurons, were compared. Increased full-length APP (flAPP)/BACE1 colocalization in early endosomes was seen in neurons, while APP-CTF/BACE1 colocalization was much higher than flAPP/BACE1 colocalization in NPCs, although the cellular location was not determined.

Herpes Simplex Virus 1 and 2 Infections during Differentiation of Human Cortical Neurons.

Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) can infect the central nervous system (CNS) with dire consequences; in children and adults, HSV-1 may cause focal encephalitis, while HSV-2 causes meningitis. In neonates, both viruses can cause severe, disseminated CNS infections with high mortality rates. Here, we differentiated human induced pluripotent stem cells (iPSCs) towards cortical neurons for infection with clinical CNS strains of HSV-1 or HSV-2. Progenies from both viruses were produced at equal quantities in iPSCs, neuroprogenitors and cortical neurons. HSV-1 and HSV-2 decreased viability of neuroprogenitors by 36.0% and 57.6% (p < 0.0001), respectively, 48 h post-infection, while cortical neurons were resilient to infection by both viruses. However, in these functional neurons, both HSV-1 and HSV-2 decreased gene expression of two markers of synaptic activity, CAMK2B and ARC, and affected synaptic activity negatively in multielectrode array experiments. However, unaltered secretion levels of the neurodegeneration markers tau and NfL suggested intact axonal integrity. Viral replication of both viruses was found after six days, coinciding with 6-fold and 22-fold increase in gene expression of cellular RNA polymerase II by HSV-1 and HSV-2, respectively. Our results suggest a resilience of human cortical neurons relative to the replication of HSV-1 and HSV-2.

Brain immune cells undergo cGAS/STING-dependent apoptosis during herpes simplex virus type 1 infection to limit type I IFN production.

Protection of the brain from viral infections involves the type I IFN (IFN-I) system, defects in which render humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels lead to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we showed that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, whereas lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices or mice treated with a caspase inhibitor exhibited lower viral load and an improved infection outcome. Collectively, we identify an activation-induced apoptosis program in brain immune cells that downmodulates local immune responses.

HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection.

Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.

Novel Insights into How Overnutrition Disrupts the Hypothalamic Actions of Leptin.

Obesity has become a worldwide health problem, but we still do not understand the molecular mechanisms that contribute to overeating and low expenditure of energy. Leptin has emerged as a major regulator of energy balance through its actions in the hypothalamus. Importantly, obese people exhibit high circulating levels of leptin, yet the hypothalamus no longer responds normally to this hormone to suppress appetite or to increase energy expenditure. Several well-known hypotheses have been proposed to explain impaired central responsiveness to the effects of leptin in obesity, including defective transit across the blood-brain barrier at the arcuate nucleus, hypothalamic endoplasmic reticulum stress, maladaptive sterile inflammation in the hypothalamus, and overexpression of molecules that may inhibit leptin signaling. We also discuss a new explanation that is based on our group's recent discovery of a signaling pathway that we named "NSAPP" after its five main protein components. The NSAPP pathway consists of an oxide transport chain that causes a transient, targeted burst in intracellular hydrogen peroxide (H2O2) to inactivate redox-sensitive members of the protein tyrosine phosphatase gene family. The NSAPP oxide transport chain is required for full activation of canonical leptin signaling in neurons but fails to function normally in states of overnutrition. Remarkably, leptin and insulin both require the NSAPP oxide transport chain, suggesting that a defect in this pathway could explain simultaneous resistance to the appetite-suppressing effects of both hormones in obesity.

The unfolded protein response is a negative regulator of scavenger receptor class B, type I (SR-BI) expression.

Scavenger receptor class B, type I (SR-BI) is the main receptor for high-density lipoprotein (HDL) and an emerging atheroprotective candidate. A central function of SR-BI is the delivery of HDL-derived cholesterol to the liver for subsequent excretion into the bile. Here, we investigated the regulation of SR-BI by the unfolded protein response (UPR), an adaptive mechanism induced by endoplasmic reticulum (ER) stress, which is frequently activated in metabolic disorders. We provide evidence that induction of acute ER stress by well-characterized chemical inducers leads to decreased SR-BI expression in hepatocyte-derived cell lines. This results in a functional reduction of selective lipid uptake from HDL. However, the regulation of SR-BI by ER stress is not a direct consequence of altered cellular cholesterol metabolism. Finally, we show that SR-BI down-regulation by the UPR might be a compensatory mechanism to provide partial adaption to ER stress. The observed down-regulation of SR-BI by ER stress in hepatic cells might contribute to the unfavorable effects of metabolic disorders on cholesterol homeostasis and cardiovascular diseases.

Effect of chronic kidney disease on macrophage cholesterol efflux.

AIMS: Patients with chronic kidney disease (CKD) have a high risk to develop atherosclerosis. The capacity of high-density lipoproteins (HDL) or serum to accept cholesterol from macrophages and the capacity of macrophages to export excess cholesterol are critical for the atheroprotective role of reverse cholesterol transport. HDL cholesterol acceptor capacity was reported to be decreased in middle aged hemodialysis patients, but the role of confounding factors remains unclear. MAIN METHODS: We measured the cholesterol acceptor capacity (CAC) of HDL or serum in 12 pediatric and 17 young adult patients with CKD stages 3-5, 14 young adult hemodialysis patients and 15 adult renal transplant recipients without associated diseases and matched controls using THP-1 macrophages. Moreover we studied the cholesterol export capacity (CEC) of patients' monocyte-derived macrophages (HMDMs) to control serum or HDL. KEY FINDINGS: In adults with CKD stages 3-5 serum CAC was slightly increased, whereas CEC of HMDMs was unaltered in both, adult and pediatric patients. In hemodialysis patients, however, serum CAC was markedly reduced to 85±11% of control (p<0.001), presumably due to low serum apolipoprotein A-I. Interestingly, CEC of HMDMs from dialysis patients was increased. In transplant patients no alterations were found. SIGNIFICANCE: CKD without hemodialysis does not reduce cholesterol export from macrophages. Hemodialysis patients might benefit from therapies aiming to restore serum CAC by increasing apolipoprotein A-I. The enhanced export of cholesterol by HMDMs from dialysis patients may represent an adaptive response.

2-Deoxy-D-glucose treatment changes the Golgi apparatus architecture without blocking synthesis of complex lipids.

The classic Golgi apparatus organization, an arrangement of highly ordered cisternal stacks with tubular-vesicular membrane specializations on both sides, is the functional image of a continuous flow of contents and membranes with input, metabolization, and output in a dynamic steady state. In response to treatment with 2-deoxy-D-glucose (2-DG), which lowers the cellular ATP level by about 70% within minutes, this organization is rapidly replaced by tubular-glomerular membrane convolutes described as Golgi networks and bodies. 2-DG is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis, which has become attractive in the context of therapeutic approaches for several kinds of tumors specifically targeting glycolysis in cancer. With the question of whether the functions of the Golgi apparatus in lipid synthesis would be influenced by the 2-DG-induced Golgi apparatus reorganization, we focused on lipid metabolism within the Golgi bodies. For this, we applied a fluorophore-labeled short-chain ceramide (BODIPY-Cer) in various combinations with 2-DG treatment to HepG2 cell cultures and followed uptake, enrichment and metabolization to higher ordered lipids. The cellular ATP status in each experiment was controlled with a bioluminescence assay, and the response of the Golgi apparatus was tracked by immunostaining of the trans-Golgi network protein TGN46. For electron microscopy, the fluorescent BODIPY-Cer signals were converted into electron-dense precipitates by photooxidation of diaminobenzidine (DAB); DAB precipitates labeled trans-Golgi areas in control cultures but also compartments at the periphery of the Golgi bodies formed in response to 2-DG treatment, thus indicating that concentration of ceramide takes place in spite of the Golgi apparatus reorganization. Lipid analyses by thin-layer chromatography (TLC) performed in parallel showed that BODIPY-Cer is not only concentrated in compartments of the 2-DG-induced Golgi bodies but is partly metabolized to BODIPY-sphingomyelin. Both, uptake and condensation of BODIPY-Cer and its conversion to complex lipids indicate that functions of the Golgi apparatus in the cellular lipid metabolism persist although the classic Golgi apparatus organization is abolished.

Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other.

Differential basolateral-apical distribution of scavenger receptor, class B, type I in cultured cells and the liver.

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral-apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro.

Inhibition of mTOR down-regulates scavenger receptor, class B, type I (SR-BI) expression, reduces endothelial cell migration and impairs nitric oxide production.

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.

High-density lipoprotein endocytosis in endothelial cells.

AIM: To describe the way stations of high-density lipoprotein (HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescence microscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type I mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrin-coated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

Scavenger receptor, Class B, Type I provides an alternative means for beta-VLDL uptake independent of the LDL receptor in tissue culture.

Recent evidence suggests that scavenger receptor, class B, type I (SR-BI) plays a physiological role in VLDL metabolism. SR-BI was reported to mediate beta-VLDL uptake; however, cellular details of this process are not well characterized. In the present study we show that SR-BI delivers cholesterol derived from beta-VLDL to LDL receptor negative SR-BI over-expressing Chinese Hamster Ovarian cells (ldlA7-SRBI). Cell association of beta-VLDL was approximately 3 times higher after SR-BI over-expression, which was competed by beta-VLDL, but only to a lesser extent by HDL and LDL. Almost all of the associated beta-VLDL was located intracellularly, and therefore could not be released by a 50-fold excess of unlabeled beta-VLDL. beta-VLDL was degraded at a rate of 6 ng beta-VLDL/mg cell protein and hour. In contrast to ldlA7 cells, beta-VLDL association was competed by LDL in cells with a functional LDL receptor like CHO and HepG2 cells, indicating a strong impact of the LDL receptor in beta-VLDL uptake. beta-VLDL degradation was similar to ldlA7-SRBI cells. When beta-VLDL uptake was followed using fluorescence microscopy, beta-VLDL showed a different uptake pattern in SR-BI over-expressing cells, ldlA7-SRBI, compared to LDL receptor containing cells, CHO and HepG2.