CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia.

Increased endothelial cell (EC) proliferation is a hallmark of arteriovenous malformations (AVMs) in hereditary hemorrhagic telangiectasia (HHT). The underlying mechanism and disease relevance of this abnormal cell proliferative state of the ECs remain unknown. Here, we report the identification of a CDK6-driven mechanism of cell cycle progression deregulation directly involved in EC proliferation and HHT vascular pathology. Specifically, HHT mouse liver ECs exhibited defects in their cell cycle control characterized by a G1/S checkpoint bypass and acceleration of cell cycle speed. Phosphorylated retinoblastoma (p-RB1)-a marker of G1/S transition through the restriction point-significantly accumulated in ECs of HHT mouse retinal AVMs and HHT patient skin telangiectasias. Mechanistically, ALK1 loss of function increased the expression of key restriction point mediators, and treatment with palbociclib or ribociclib, two CDK4/6 inhibitors, blocked p-RB1 increase and retinal AVMs in HHT mice. Palbociclib also improved vascular pathology in the brain and slowed down endothelial cell cycle speed and EC proliferation. Specific deletion of Cdk6 in ECs was sufficient to protect HHT mice from AVM pathology. Thus, CDK6-mediated endothelial cell cycle acceleration controls EC proliferation in AVMs and is a central determinant of HHT pathogenesis. We propose that clinically approved CDK4/6 inhibitors have repurposing potential in HHT.

ANG2 Blockade Diminishes Proangiogenic Cerebrovascular Defects Associated With Models of Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by arteriovenous malformations and blood vessel enlargements. However, there are no effective drug therapies to combat arteriovenous malformation formation in patients with HHT. Here, we aimed to address whether elevated levels of ANG2 (angiopoietin-2) in the endothelium is a conserved feature in mouse models of the 3 major forms of HHT that could be neutralized to treat brain arteriovenous malformations and associated vascular defects. In addition, we sought to identify the angiogenic molecular signature linked to HHT. METHODS: Cerebrovascular defects, including arteriovenous malformations and increased vessel calibers, were characterized in mouse models of the 3 common forms of HHT using transcriptomic and dye injection labeling methods. RESULTS: Comparative RNA sequencing analyses of isolated brain endothelial cells revealed a common, but unique proangiogenic transcriptional program associated with HHT. This included a consistent upregulation in cerebrovascular expression of ANG2 and downregulation of its receptor Tyr kinase with Ig and EGF homology domains (TIE2/TEK) in HHT mice compared with controls. Furthermore, in vitro experiments revealed TEK signaling activity was hampered in an HHT setting. Pharmacological blockade of ANG2 improved brain vascular pathologies in all HHT models, albeit to varying degrees. Transcriptomic profiling further indicated that ANG2 inhibition normalized the brain vasculature by impacting a subset of genes involved in angiogenesis and cell migration processes. CONCLUSIONS: Elevation of ANG2 in the brain vasculature is a shared trait among the mouse models of the common forms of HHT. Inhibition of ANG2 activity can significantly limit or prevent brain arteriovenous malformation formation and blood vessel enlargement in HHT mice. Thus, ANG2-targeted therapies may represent a compelling approach to treat arteriovenous malformations and vascular pathologies related to all forms of HHT.

Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway.

Abnormal calcium homeostasis, activation of protease calpain, generation of p25 and hyperactivation of cyclin-dependent kinase 5 (Cdk5) have all been implicated in the pathogenesis of neurogenerative diseases including Alzheimer's disease. We have recently shown that extracellular cold-inducible RNA-binding protein (eCIRP) induces Cdk5 activation via p25. However, the precise molecular mechanism by which eCIRP regulates calcium signaling and calpain remains to be addressed. We hypothesized that eCIRP regulates p25 via Ca2+-dependent calpain activation. eCIRP increased calpain activity and decreased the endogenous calpain inhibitor calpastatin in Neuro 2a (N2a) cells. Calpain inhibition with calpeptin attenuated eCIRP-induced calpain activity and p25. eCIRP specifically upregulated cytosolic calpain 1, and calpain 1 silencing attenuated the eCIRP-induced increase in p25. eCIRP stimulation increased cytosolic free Ca2+, especially in hippocampal neuronal HT22 cells, which was attenuated by the eCIRP inhibitor Compound 23 (C23). Endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP3R) inhibition using 2-aminoethoxy-diphenyl-borate or xestospongin-C (X-C), interleukin-6 receptor alpha (IL-6Rα)-neutralization, and phospholipase C (PLC) inhibition with U73122 attenuated eCIRP-induced Ca2+ increase, while Ca2+ influx across the plasma membrane remained unaffected by eCIRP. Finally, C23, IL-6Rα antibody, U73122 and X-C attenuated eCIRP-induced p25 in HT-22 cells. In conclusion, the current study uncovers eCIRP-triggered Ca2+ release from ER stores in an IL-6Rα/PLC/IP3-dependent manner as a novel molecular mechanism underlying eCIRP's induction of Cdk5 activity and potential involvement in neurodegeneration.

Oxidative stress-induced MMP- and γ-secretase-dependent VE-cadherin processing is modulated by the proteasome and BMP9/10.

Classical cadherins, including vascular endothelial (VE)-cadherin, are targeted by matrix metalloproteinases (MMPs) and γ-secretase during adherens junction (AJ) disassembly, a mechanism that might have relevance for endothelial cell (EC) integrity and vascular homeostasis. Here, we show that oxidative stress triggered by H2O2 exposure induced efficient VE-cadherin proteolysis by MMPs and γ-secretase in human umbilical endothelial cells (HUVECs). The cytoplasmic domain of VE-cadherin produced by γ-secretase, VE-Cad/CTF2-a fragment that has eluded identification so far-could readily be detected after H2O2 treatment. VE-Cad/CTF2, released into the cytosol, was tightly regulated by proteasomal degradation and was sequentially produced from an ADAM10/17-generated C-terminal fragment, VE-Cad/CTF1. Interestingly, BMP9 and BMP10, two circulating ligands critically involved in vascular maintenance, significantly reduced VE-Cad/CTF2 levels during H2O2 challenge, as well as mitigated H2O2-mediated actin cytoskeleton disassembly during VE-cadherin processing. Notably, BMP9/10 pretreatments efficiently reduced apoptosis induced by H2O2, favoring endothelial cell recovery. Thus, oxidative stress is a trigger of MMP- and γ-secretase-mediated endoproteolysis of VE-cadherin and AJ disassembly from the cytoskeleton in ECs, a mechanism that is negatively controlled by the EC quiescence factors, BMP9 and BMP10.

Anesthesia promotes acute expression of genes related to Alzheimer's disease and latent tau aggregation in transgenic mouse models of tauopathy.

BACKGROUND: Exposure to anesthesia in the elderly might increase the risk of dementia. Although the mechanism underlying the association is uncertain, anesthesia has been shown to induce acute tau hyperphosphorylation in preclinical models. We sought to investigate the impact of anesthesia on gene expression and on acute and long-term changes in tau biochemistry in transgenic models of tauopathy in order to better understand how anesthesia influences the pathophysiology of dementia. METHODS: We exposed mice with over-expressed human mutant tau (P301L and hyperdopaminergic COMTKO/P301L) to two hours of isoflurane and compared anesthetized mice to controls at several time points. We evaluated tau hyperphosphorylation with quantitative high-sensitivity enzyme-linked immunosorbent assay and performed differential expression and functional transcriptome analyses following bulk mRNA-sequencing. RESULTS: Anesthesia induced acute hyperphosphorylation of tau at epitopes related to Alzheimer's disease (AD) in both P301L-based models. Anesthesia was associated with differential expression of genes in the neurodegenerative pathways (e.g., AD-risk genes ApoE and Trem2) and thermogenesis pathway, which is related to both mammalian hibernation and tau phosphorylation. One and three months after anesthesia, hyperphosphorylated tau aggregates were increased in the anesthetized mice. CONCLUSIONS: Anesthesia may influence the expression of AD-risk genes and induce biochemical changes in tau that promote aggregation even after single exposure. Further preclinical and human studies are necessary to establish the relevance of our transcriptomic and biochemical findings in these preclinical models to the pathogenesis of dementia following anesthesia. TRIAL REGISTRATION: Not applicable.

Extracellular CIRP Activates the IL-6Rα/STAT3/Cdk5 Pathway in Neurons.

Extracellular cold-inducible RNA-binding protein (eCIRP) stimulates microglial inflammation causing neuronal damage during ischemic stroke and is a critical mediator of alcohol-induced cognitive impairment. However, the precise role of eCIRP in mediating neuroinflammation remains unknown. In this study, we report that eCIRP activates neurotoxic cyclin-dependent kinase-5 (Cdk5)/p25 through the induction of IL-6Rα/STAT3 pathway in neurons. Amyloid ß (Aß)-mediated neuronal stress, which is associated with Alzheimer's disease, increased the levels of eCIRP released from BV2 microglial cells. The released eCIRP levels from BV2 cells increased 3.2-fold upon stimulation with conditioned medium from Neuro-2a (N2a) cells containing Aß compared to control N2a supernatant in a time-dependent manner. Stimulation of N2a cells and primary neurons with eCIRP upregulated the neuronal Cdk5 activator p25 expression in a dose- and time-dependent manner. eCIRP directly induced neuronal STAT3 phosphorylation and p25 increase via its novel receptor IL-6Rα. Next, we showed using surface plasmon resonance that eCIRP-derived peptide C23 inhibited the binding of eCIRP to IL-6Rα at 25 µM, with a 40-fold increase in equilibrium dissociation constant (Kd) value (from 8.08 × 10-8 M to 3.43 × 10-6 M), and completely abrogated the binding at 50 µM. Finally, C23 reversed the eCIRP-induced increase in neuronal STAT3 phosphorylation and p25 levels. In conclusion, the current study demonstrates that the upregulation of neuronal IL-6Rα/STAT3/Cdk5 pathway is a key mechanism of eCIRP's role in neuroinflammation and that C23 as a potent inhibitor of this pathway has translational potential in neurodegenerative pathologies controlled by eCIRP.

Intramuscular injection of vectorized-scFvMC1 reduces pathological tau in two different tau transgenic models.

With evidence supporting the prion-like spreading of extracellular tau as a mechanism for the initiation and progression of Alzheimer's disease (AD), immunotherapy has emerged as a potential disease-modifying strategy to target tau. Many studies have proven effective to clear pathological tau species in animal models of AD, and several clinical trials using conventional immunotherapy with anti-tau native antibodies are currently active. We have previously generated a vectorized scFv derived from the conformation-dependent anti-tau antibody MC1, scFvMC1, and demonstrated that its intracranial injection was able to prevent tau pathology in adult tau mice. Here, we show that, in a prevention paradigm and in two different tau transgenic models (JNPL3 and P301S), a one-time intramuscular injection of AAV1-scFvMC1 generated a long-lasting peripheral source of anti-tau scFvMC1 and significantly reduced insoluble and soluble tau species in the brain. Moreover, our data showed that scFvMC1 was internalized by the microglia, in the absence of overt inflammation. This study demonstrates the efficacy of intramuscular delivery of vectorized scFv to target tau, and suggests a new potential application to treat AD and the other tauopathies.

Molecular and Pharmacological Modulation of CALHM1 Promote Neuroprotection against Oxygen and Glucose Deprivation in a Model of Hippocampal Slices.

Calcium homeostasis modulator 1 (CALHM1) is a calcium channel involved in the regulation of cytosolic Ca2+ levels. From a physiological point of view, the open state of CALHM1 depends not only on voltage but also on the extracellular concentration of calcium ([Ca2+]) ions. At low [Ca2+]e or depolarization, the channel is opened, allowing Ca2+ influx; however, high extracellular [Ca2+]e or hyperpolarization promote its resting state. The unique Ca2+ permeation of CALHM1 relates to the molecular events that take place in brain ischemia, such as depolarization and extracellular changes in [Ca2+]e, particularly during the reperfusion phase after the ischemic insult. In this study, we attempted to understand its role in an in vitro model of ischemia, namely oxygen and glucose deprivation, followed by reoxygenation (OGD/Reox). To this end, hippocampal slices from wild-type Calhm1+/+, Calhm1+/-, and Calhm1-/- mice were subjected to OGD/Reox. Our results point out to a neuroprotective effect when CALHM1 is partially or totally absent. Pharmacological manipulation of CALHM1 with CGP37157 reduced cell death in Calhm1+/+ slices but not in that of Calhm1-/- mice after exposure to the OGD/Reox protocol. This ionic protection was also verified by measuring reactive oxygen species production upon OGD/Reox in Calhm1+/+ and Calhm1-/- mice, resulting in a downregulation of ROS production in Calhm1-/- hippocampal slices and increased expression of HIF-1α. Taken together, we can conclude that genetic or pharmacological inhibition of CALHM1 results in a neuroprotective effect against ischemia, due to an attenuation of the neuronal calcium overload and downregulation of oxygen reactive species production.

Correcting Smad1/5/8, mTOR, and VEGFR2 treats pathology in hereditary hemorrhagic telangiectasia models.

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs - including in HHT patient blood outgrowth ECs - and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.

Anti-tau conformational scFv MC1 antibody efficiently reduces pathological tau species in adult JNPL3 mice.

Tau, the main component of the neurofibrillary tangles (NFTs), is an attractive target for immunotherapy in Alzheimer's disease (AD) and other tauopathies. MC1/Alz50 are currently the only antibodies targeting a disease-specific conformational modification of tau. Passive immunization experiments using intra-peritoneal injections have previously shown that MC1 is effective at reducing tau pathology in the forebrain of tau transgenic JNPL3 mice. In order to reach a long-term and sustained brain delivery, and avoid multiple injection protocols, we tested the efficacy of the single-chain variable fragment of MC1 (scFv-MC1) to reduce tau pathology in the same animal model, with focus on brain regional differences. ScFv-MC1 was cloned into an AAV delivery system and was directly injected into the hippocampus of adult JNPL3 mice. Specific promoters were employed to selectively target neurons or astrocytes for scFv-MC1 expression. ScFv-MC1 was able to decrease soluble, oligomeric and insoluble tau species, in our model. The effect was evident in the cortex, hippocampus and hindbrain. The astrocytic machinery appeared more efficient than the neuronal, with significant reduction of pathology in areas distant from the site of injection. To our knowledge, this is the first evidence that an anti-tau conformational scFv antibody, delivered directly into the mouse adult brain, is able to reduce pathological tau, providing further insight into the nature of immunotherapy strategies.

Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex.

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-µm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles.

Blockade and knock-out of CALHM1 channels attenuate ischemic brain damage.

Overactivation of purinergic receptors during cerebral ischemia results in a massive release of neurotransmitters, including adenosine triphosphate (ATP), to the extracellular space which leads to cell death. Some hypothetical pathways of ATP release are large ion channels, such as calcium homeostasis modulator 1 (CALHM1), a membrane ion channel that can permeate ATP. Since this transmitter contributes to postischemic brain damage, we hypothesized that CALHM1 activation may be a relevant target to attenuate stroke injury. Here, we analyzed the contribution of CALHM1 to postanoxic depolarization after ischemia in cultured neurons and in cortical slices. We observed that the onset of postanoxic currents in neurons in those preparations was delayed after its blockade with ruthenium red or silencing of Calhm1 gene by short hairpin RNA, as well as in slices from CALHM1 knockout mice. Subsequently, we used transient middle cerebral artery occlusion and found that ruthenium red, a blocker of CALHM1, or the lack of CALHM1, substantially attenuated the motor symptoms and reduced significantly the infarct volume. These results show that CALHM1 channels mediate postanoxic depolarization in neurons and brain damage after ischemia. Therefore, targeting CALHM1 may have a high therapeutic potential for treating brain damage after ischemia.

Tacrolimus rescues the signaling and gene expression signature of endothelial ALK1 loss-of-function and improves HHT vascular pathology.

Hereditary hemorrhagic telangiectasia (HHT) is a highly debilitating and life-threatening genetic vascular disorder arising from endothelial cell (EC) proliferation and hypervascularization, for which no cure exists. Because HHT is caused by loss-of-function mutations in bone morphogenetic protein 9 (BMP9)-ALK1-Smad1/5/8 signaling, interventions aimed at activating this pathway are of therapeutic value. We interrogated the whole-transcriptome in human umbilical vein ECs (HUVECs) and found that ALK1 signaling inhibition was associated with a specific pro-angiogenic gene expression signature, which included a significant elevation of DLL4 expression. By screening the NIH clinical collections of FDA-approved drugs, we identified tacrolimus (FK-506) as the most potent activator of ALK1 signaling in BMP9-challenged C2C12 reporter cells. In HUVECs, tacrolimus activated Smad1/5/8 and opposed the pro-angiogenic gene expression signature associated with ALK1 loss-of-function, by notably reducing Dll4 expression. In these cells, tacrolimus also inhibited Akt and p38 stimulation by vascular endothelial growth factor, a major driver of angiogenesis. In the BMP9/10-immunodepleted postnatal retina-a mouse model of HHT vascular pathology-tacrolimus activated endothelial Smad1/5/8 and prevented the Dll4 overexpression and hypervascularization associated with this model. Finally, tacrolimus stimulated Smad1/5/8 signaling in C2C12 cells expressing BMP9-unresponsive ALK1 HHT mutants and in HHT patient blood outgrowth ECs. Tacrolimus repurposing has therefore therapeutic potential in HHT.

CALHM1-Mediated ATP Release and Ciliary Beat Frequency Modulation in Nasal Epithelial Cells.

Mechanical stimulation of airway epithelial cells causes apical release of ATP, which increases ciliary beat frequency (CBF) and speeds up mucociliary clearance. The mechanisms responsible for this ATP release are poorly understood. CALHM1, a transmembrane protein with shared structural features to connexins and pannexins, has been implicated in ATP release from taste buds, but it has not been evaluated for a functional role in the airway. In the present study, Calhm1 knockout, Panx1 knockout, and wild-type mouse nasal septal epithelial cells were grown at an air-liquid interface (ALI) and subjected to light mechanical stimulation from an air puff. Apical ATP release was attenuated in Calhm1 knockout cultures following mechanical stimulation at a pressure of 55 mmHg for 50 milliseconds (p < 0.05). Addition of carbenoxolone, a PANX1 channel blocker, completely abolished ATP release in Calhm1 knockout cultures but not in wild type or Panx1 knockout cultures. An increase in CBF was observed in wild-type ALIs following mechanical stimulation, and this increase was significantly lower (p < 0.01) in Calhm1 knockout cultures. These results demonstrate that CALHM1 plays a newly defined role, complementary to PANX1, in ATP release and downstream CBF modulation following a mechanical stimulus in airway epithelial cells.

A modification-specific peptide-based immunization approach using CRM197 carrier protein: Development of a selective vaccine against pyroglutamate Aß peptides.

Strategies aimed at reducing cerebral accumulation of the amyloid-ß (Aß) peptides have therapeutic potential in Alzheimer's disease (AD). Aß immunization has proven to be effective at promoting Aß clearance in animal models but adverse effects have hampered its clinical evaluation. The first anti-Aß immunization clinical trial, which assessed a full-length Aß1-42 vaccine, increased the risk of encephalitis most likely because of autoimmune pro-inflammatory T helper 1 (Th1) response against all forms of Aß. Immunization against less abundant but potentially more pathologically relevant Aß products, such as N-terminally-truncated pyroglutamate-3 Aß (AßpE3), could provide efficacy and improve tolerability in Aß immunotherapy. Here, we describe a selective vaccine against AßpE3, which uses the diphtheria toxin mutant CRM197 as carrier protein for epitope presentation. CRM197 is currently used in licensed vaccines and has demonstrated excellent immunogenicity and safety in humans. In mice, our AßpE3:CRM197 vaccine triggered the production of specific anti-AßpE3 antibodies that did not cross-react with Aß1-42, non-cyclized AßE3, or N-terminally-truncated pyroglutamate-11 Aß (AßpE11). AßpE3:CRM197 antiserum strongly labeled AßpE3 in insoluble protein extracts and decorated cortical amyloid plaques in human AD brains. Anti-AßpE3 antibodies were almost exclusively of the IgG1 isotype, suggesting an anti-inflammatory Th2 response bias to the AßpE3:CRM197 vaccine. To the best of our knowledge, this study shows for the first time that CRM197 has potential as a safe and suitable vaccine carrier for active and selective immunization against specific protein sequence modifications or conformations, such as AßpE3.

A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10.

Hereditary hemorrhagic telangiectasia (HHT) is a potentially life-threatening genetic vascular disorder caused by loss-of-function mutations in the genes encoding activin receptor-like kinase 1 (ALK1), endoglin, Smad4, and bone morphogenetic protein 9 (BMP9). Injections of mouse neonates with BMP9/10 blocking antibodies lead to HHT-like vascular defects in the postnatal retinal angiogenesis model. Mothers and their newborns share the same immunity through the transfer of maternal antibodies during lactation. Here, we investigated whether the transmammary delivery route could improve the ease and consistency of administering anti-BMP9/10 antibodies in the postnatal retinal angiogenesis model. We found that anti-BMP9/10 antibodies, when intraperitoneally injected into lactating dams, are efficiently transferred into the blood circulation of lactationally-exposed neonatal pups. Strikingly, pups receiving anti-BMP9/10 antibodies via lactation displayed consistent and robust vascular pathology in the retina, which included hypervascularization and defects in arteriovenous specification, as well as the presence of multiple and massive arteriovenous malformations. Furthermore, RNA-Seq analyses of neonatal retinas identified an increase in the key pro-angiogenic factor, angiopoietin-2, as the most significant change in gene expression triggered by the transmammary delivery of anti-BMP9/10 antibodies. Transmammary-delivered BMP9/10 immunoblocking in the mouse neonatal retina is therefore a practical, noninvasive, reliable, and robust model of HHT vascular pathology.

ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

Obesity is a major public health problem. An in-depth knowledge of the molecular mechanisms of oro-sensory detection of dietary lipids may help fight it. Humans and rodents can detect fatty acids via lipido-receptors, such as CD36 and GPR120. We studied the implication of the MAPK pathways, in particular, ERK1/2, in the gustatory detection of fatty acids. Linoleic acid, a dietary fatty acid, induced via CD36 the phosphorylation of MEK1/2-ERK1/2-ETS-like transcription factor-1 cascade, which requires Fyn-Src kinase and lipid rafts in human taste bud cells (TBCs). ERK1/2 cascade was activated by Ca2+ signaling via opening of the calcium-homeostasis modulator-1 (CALHM1) channel. Furthermore, fatty acid-evoked Ca2+ signaling and ERK1/2 phosphorylation were decreased in both human TBCs after small interfering RNA knockdown of CALHM1 channel and in TBCs from Calhm1-/- mice. Targeted knockdown of ERK1/2 by small interfering RNA or PD0325901 (MEK1/2 inhibitor) in the tongue and genetic ablation of Erk1 or Calhm1 genes impaired preference for dietary fat in mice. Lingual inhibition of ERK1/2 in healthy volunteers also decreased orogustatory sensitivity for linoleic acid. Our data demonstrate that ERK1/2-MAPK cascade is regulated by the opening of CALHM1 Ca2+ channel in TBCs to modulate orogustatory detection of dietary lipids in mice and humans.-Subramaniam, S., Ozdener, M. H., Abdoul-Azize, S., Saito, K., Malik, B., Maquart, G., Hashimoto, T., Marambaud, P., Aribi, M., Tordoff, M. G., Besnard, P., Khan, N. A. ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo.

Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer's disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology.

CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice.

CALHM1 is a cell surface calcium channel expressed in cerebral neurons. CALHM1 function in the brain remains unknown, but recent results showed that neuronal CALHM1 controls intracellular calcium signaling and cell excitability, two mechanisms required for synaptic function. Here, we describe the generation of Calhm1 knockout (Calhm1(-/-)) mice and investigate CALHM1 role in neuronal and cognitive functions. Structural analysis revealed that Calhm1(-/-) brains had normal regional and cellular architecture, and showed no evidence of neuronal or synaptic loss, indicating that CALHM1 deficiency does not affect brain development or brain integrity in adulthood. However, Calhm1(-/-) mice showed a severe impairment in memory flexibility, assessed in the Morris water maze, and a significant disruption of long-term potentiation without alteration of long-term depression, measured in ex vivo hippocampal slices. Importantly, in primary neurons and hippocampal slices, CALHM1 activation facilitated the phosphorylation of NMDA and AMPA receptors by protein kinase A. Furthermore, neuronal CALHM1 activation potentiated the effect of glutamate on the expression of c-Fos and C/EBPß, two immediate-early gene markers of neuronal activity. Thus, CALHM1 controls synaptic activity in cerebral neurons and is required for the flexible processing of memory in mice. These results shed light on CALHM1 physiology in the mammalian brain.

Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors.

Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with ß-hemoglobinopathies.