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.

A polymorphism in CALHM1 influences Ca2+ homeostasis, Abeta levels, and Alzheimer's disease risk.

Alzheimer's disease (AD) is a genetically heterogeneous disorder characterized by early hippocampal atrophy and cerebral amyloid-beta (Abeta) peptide deposition. Using TissueInfo to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions, we identified a gene on 10q24.33 that we call CALHM1. We show that CALHM1 encodes a multipass transmembrane glycoprotein that controls cytosolic Ca(2+) concentrations and Abeta levels. CALHM1 homomultimerizes, shares strong sequence similarities with the selectivity filter of the NMDA receptor, and generates a large Ca(2+) conductance across the plasma membrane. Importantly, we determined that the CALHM1 P86L polymorphism (rs2986017) is significantly associated with AD in independent case-control studies of 3404 participants (allele-specific OR = 1.44, p = 2 x 10(-10)). We further found that the P86L polymorphism increases Abeta levels by interfering with CALHM1-mediated Ca(2+) permeability. We propose that CALHM1 encodes an essential component of a previously uncharacterized cerebral Ca(2+) channel that controls Abeta levels and susceptibility to late-onset AD.

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.

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 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes.

Recognition of sweet, bitter and umami tastes requires the non-vesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways. However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel, is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception.

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.

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.

CALHM1 ion channel elicits amyloid-ß clearance by insulin-degrading enzyme in cell lines and in vivo in the mouse brain.

Alzheimer's disease is characterized by amyloid-ß (Aß) peptide accumulation in the brain. CALHM1, a cell-surface Ca(2+) channel expressed in brain neurons, has anti-amyloidogenic properties in cell cultures. Here, we show that CALHM1 controls Aß levels in vivo in the mouse brain through a previously unrecognized mechanism of regulation of Aß clearance. Using pharmacological and genetic approaches in cell lines, we found that CALHM1 ion permeability and extracellular Ca(2+) were required for the Aß-lowering effect of CALHM1. Aß level reduction by CALHM1 could be explained by an increase in extracellular Aß degradation by insulin-degrading enzyme (IDE), extracellular secretion of which was strongly potentiated by CALHM1 activation. Importantly, Calhm1 knockout in mice reduced IDE enzymatic activity in the brain, and increased endogenous Aß concentrations by up to ∼50% in both the whole brain and primary neurons. Thus, CALHM1 controls Aß levels in cell lines and in vivo by facilitating neuronal and Ca(2+)-dependent degradation of extracellular Aß by IDE. This work identifies CALHM1 ion channel as a potential target for promoting amyloid clearance in Alzheimer's disease.

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.

Inhibition of AMP-activated protein kinase signaling alleviates impairments in hippocampal synaptic plasticity induced by amyloid ß.

The AMP-activated protein kinase (AMPK) is a Ser/Thr kinase that is activated in response to low-energy states to coordinate multiple signaling pathways to maintain cellular energy homeostasis. Dysregulation of AMPK signaling has been observed in Alzheimer's disease (AD), which is associated with abnormal neuronal energy metabolism. In the current study we tested the hypothesis that aberrant AMPK signaling underlies AD-associated synaptic plasticity impairments by using pharmacological and genetic approaches. We found that amyloid ß (Aß)-induced inhibition of long-term potentiation (LTP) and enhancement of long-term depression were corrected by the AMPK inhibitor compound C (CC). Similarly, LTP impairments in APP/PS1 transgenic mice that model AD were improved by CC treatment. In addition, Aß-induced LTP failure was prevented in mice with genetic deletion of the AMPK α2-subunit, the predominant AMPK catalytic subunit in the brain. Furthermore, we found that eukaryotic elongation factor 2 (eEF2) and its kinase eEF2K are key downstream effectors that mediate the detrimental effects of hyperactive AMPK in AD pathophysiology. Our findings describe a previously unrecognized role of aberrant AMPK signaling in AD-related synaptic pathophysiology and reveal a potential therapeutic target for AD.

Stimulation of Brain AMP-Activated Protein Kinase Attenuates Inflammation and Acute Lung Injury in Sepsis.

Sepsis and septic shock are enormous public health problems with astronomical financial repercussions on health systems worldwide. The central nervous system (CNS) is closely intertwined in the septic process but the underlying mechanism is still obscure. AMP-activated protein kinase (AMPK) is a ubiquitous energy sensor enzyme and plays a key role in regulation of energy homeostasis and cell survival. In this study, we hypothesized that activation of AMPK in the brain would attenuate inflammatory responses in sepsis, particularly in the lungs. Adult C57BL/6 male mice were treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 20 ng), an AMPK activator, or vehicle (normal saline) by intracerebroventricular (ICV) injection, followed by cecal ligation and puncture (CLP) at 30 min post-ICV. The septic mice treated with AICAR exhibited elevated phosphorylation of AMPKα in the brain along with reduced serum levels of aspartate aminotransferase, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), compared with the vehicle. Similarly, the expressions of TNF-α, IL-1ß, keratinocyte-derived chemokine and macrophage inflammatory protein-2 as well as myeloperoxidase activity in the lungs of AICAR-treated mice were significantly reduced. Moreover, histological findings in the lungs showed improvement of morphologic features and reduction of apoptosis with AICAR treatment. We further found that the beneficial effects of AICAR on septic mice were diminished in AMPKα2 deficient mice, showing that AMPK mediates these effects. In conclusion, our findings reveal a new functional role of activating AMPK in the CNS to attenuate inflammatory responses and acute lung injury in sepsis.

CALHM1 controls the Ca²âº-dependent MEK, ERK, RSK and MSK signaling cascade in neurons.

Calcium homeostasis modulator 1 (CALHM1) is a Ca(2+) channel controlling neuronal excitability and potentially involved in the pathogenesis of Alzheimer's disease (AD). Although strong evidence indicates that CALHM1 is required for neuronal electrical activity, its role in intracellular Ca(2+) signaling remains unknown. In the present study, we show that in hippocampal HT-22 cells, CALHM1 expression led to a robust and relatively selective activation of the Ca(2+)-sensing kinases ERK1/2. CALHM1 also triggered activation of MEK1/2, the upstream ERK1/2-activating kinases, and of RSK1/2/3 and MSK1, two downstream effectors of ERK1/2 signaling. CALHM1-mediated activation of ERK1/2 signaling was controlled by the small GTPase Ras. Pharmacological inhibition of CALHM1 permeability using Ruthenium Red, Zn(2+), and Gd(3+), or expression of the CALHM1 N140A and W114A mutants, which are deficient in mediating Ca(2+) influx, prevented the effect of CALHM1 on the MEK, ERK, RSK and MSK signaling cascade, demonstrating that CALHM1 controlled this pathway via its channel properties. Importantly, expression of CALHM1 bearing the natural P86L polymorphism, which leads to a partial loss of CALHM1 function and is associated with an earlier age at onset in AD patients, showed reduced activation of ERK1/2, RSK1/2/3, and MSK1. In line with these results obtained in transfected cells, primary cerebral neurons isolated from Calhm1 knockout mice showed significant impairments in the activation of MEK, ERK, RSK and MSK signaling. The present study identifies a previously uncharacterized mechanism of control of Ca(2+)-dependent ERK1/2 signaling in neurons, and further establishes CALHM1 as a critical ion channel for neuronal signaling and function.

CALHM1 Deletion in Mice Affects Glossopharyngeal Taste Responses, Food Intake, Body Weight, and Life Span.

Stimulation of Type II taste receptor cells (TRCs) with T1R taste receptors causes sweet or umami taste, whereas T2Rs elicit bitter taste. Type II TRCs contain the calcium channel, calcium homeostasis modulator protein 1 (CALHM1), which releases adenosine triphosphate (ATP) transmitter to taste fibers. We have previously demonstrated with chorda tympani nerve recordings and two-bottle preference (TBP) tests that mice with genetically deleted Calhm1 (knockout [KO]) have severely impaired perception of sweet, bitter, and umami compounds, whereas their sour and salty tasting ability is unaltered. Here, we present data from KO mice of effects on glossopharyngeal (NG) nerve responses, TBP, food intake, body weight, and life span. KO mice have no NG response to sweet and a suppressed response to bitter compared with control (wild-type [WT]) mice. KO mice showed some NG response to umami, suggesting that umami taste involves both CALHM1- and non-CALHM1-modulated signals. NG responses to sour and salty were not significantly different between KO and WT mice. Behavioral data conformed in general with the NG data. Adult KO mice consumed less food, weighed significantly less, and lived almost a year longer than WT mice. Taken together, these data demonstrate that sweet taste majorly influences food intake, body weight, and life span.

Novel resveratrol analogues attenuate renal ischemic injury in rats.

BACKGROUND: Renal ischemia-reperfusion (I/R) is a severe clinical complication with no specific treatment. Resveratrol has been shown as a promising experimental agent in renal I/R due to its effect on cellular energy metabolism, oxidative stress, and inflammation. Recently, we identified two biologically active resveratrol analogues (RSVAs), RSVA405 and RSVA314. We hypothesized that both RSAVs would attenuate I/R-induced renal injury. METHODS: Adult male rats were subjected to renal I/R through bilateral renal pedicle clamping for 60 min, followed by reperfusion. RSVA405 (3 mg/kg Body Weight), RSVA314 (3 mg/kg Body Weight), or vehicle (10% dimethyl sulfoxide and 33% Solutol in phosphate buffered saline) were administered by intraperitoneal injection 1 h before ischemia. Blood and renal tissues were collected 24 h after I/R for evaluation. RESULTS: Administration of RSVA405 and RSVA314 significantly reduced the serum levels of renal dysfunction and injury markers, including creatinine, blood urea nitrogen, aspartate aminotransferase, and lactate dehydrogenase, compared with vehicle. The protective effect of RSVA405 and RSVA314 was also reflected on histologic evaluation. Both RSVAs reduced the number of apoptotic cells by more than 60% as determined by transferase dUTP nick end labeling assay, compared with vehicle. The renal adenosine triphosphate levels of the vehicle group was decreased to 52.4% of control, whereas those of the RSVA405 and RSVA314 groups were restored to 72.3% and 79.6% of control, respectively. Both RSVAs significantly reduced the protein expression of inducible nitric oxide synthase and nitrotyrosine and the messenger RNA levels of tumor necrosis factor-α, interleukin-6, and interleukin-1ß. CONCLUSIONS: RSVA405 and RSVA314 attenuate I/R-induced renal injury through the modulation of energy metabolism, oxidative stress, and inflammation.

How do taste cells lacking synapses mediate neurotransmission? CALHM1, a voltage-gated ATP channel.

CALHM1 was recently demonstrated to be a voltage-gated ATP-permeable ion channel and to serve as a bona fide conduit for ATP release from sweet-, umami-, and bitter-sensing type II taste cells. Calhm1 is expressed in taste buds exclusively in type II cells and its product has structural and functional similarities with connexins and pannexins, two families of channel protein candidates for ATP release by type II cells. Calhm1 knockout in mice leads to loss of perception of sweet, umami, and bitter compounds and to impaired gustatory nerve responses to these tastants. These new studies validate the concept of ATP as the primary neurotransmitter from type II cells to gustatory neurons. Furthermore, they identify voltage-gated ATP release through CALHM1 as an essential molecular mechanism of ATP release in taste buds. We discuss these new findings, as well as unresolved issues in peripheral taste signaling that we hope will stimulate future research.

Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability.

Extracellular Ca(2+) (Ca(2+)(o)) plays important roles in physiology. Changes of Ca(2+)(o) concentration ([Ca(2+)](o)) have been observed to modulate neuronal excitability in various physiological and pathophysiological settings, but the mechanisms by which neurons detect [Ca(2+)](o) are not fully understood. Calcium homeostasis modulator 1 (CALHM1) expression was shown to induce cation currents in cells and elevate cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in response to removal of Ca(2+)(o) and its subsequent addback. However, it is unknown whether CALHM1 is a pore-forming ion channel or modulates endogenous ion channels. Here we identify CALHM1 as the pore-forming subunit of a plasma membrane Ca(2+)-permeable ion channel with distinct ion permeability properties and unique coupled allosteric gating regulation by voltage and [Ca(2+)](o). Furthermore, we show that CALHM1 is expressed in mouse cortical neurons that respond to reducing [Ca(2+)](o) with enhanced conductance and action potential firing and strongly elevated [Ca(2+)](i) upon Ca(2+)(o) removal and its addback. In contrast, these responses are strongly muted in neurons from mice with CALHM1 genetically deleted. These results demonstrate that CALHM1 is an evolutionarily conserved ion channel family that detects membrane voltage and extracellular Ca(2+) levels and plays a role in cortical neuronal excitability and Ca(2+) homeostasis, particularly in response to lowering [Ca(2+)](o) and its restoration to normal levels.

CB2 receptor deficiency increases amyloid pathology and alters tau processing in a transgenic mouse model of Alzheimer's disease.

The endocannabinoid CB2 receptor system has been implicated in the neuropathology of Alzheimer's disease (AD). In order to investigate the impact of the CB2 receptor system on AD pathology, a colony of mice with a deleted CB2 receptor gene, CNR2, was established on a transgenic human mutant APP background for pathological comparison with CB2 receptor-sufficient transgenic mice. J20 APP (PDGFB-APPSwInd) mice were bred over two generations with CNR2(-/-) (Cnr2(tm1Dgen)/J) mice to produce a colony of J20 CNR2(+/+) and J20 CNR2(-/-) mice. Seventeen J20 CNR2(+/+) mice (12 females, 5 males) and 16 J20 CNR2(-/-) mice (11 females, 5 males) were killed at 12 months, and their brains were interrogated for AD-related pathology with both biochemistry and immunocytochemistry (ICC). In addition to amyloid-dependent endpoints such as soluble Aß production and plaque deposition quantified with 6E10 staining, the effect of CB2 receptor deletion on total soluble mouse tau production was assayed by using a recently developed high-sensitivity assay. Results revealed that soluble Aß42 and plaque deposition were significantly increased in J20 CNR2(-/-) mice relative to CNR2(+/+) mice. Microgliosis, quantified with ionized calcium-binding adapter molecule 1 (Iba-1) staining, did not differ between groups, whereas plaque associated microglia was more abundant in J20 CNR2(-/-) mice. Total tau was significantly suppressed in J20 CNR2(-/-) mice relative to J20 CNR2(+/+) mice. The results confirm the constitutive role of the CB2 receptor system both in reducing amyloid plaque pathology in AD and also support tehpotential of cannabinoid therapies targeting CB2 to reduce Aß; however, the results suggest that interventions may have a divergent effect on tau pathology.

Salty taste deficits in CALHM1 knockout mice.

Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt.

Growth arrest-specific 1 binds to and controls the maturation and processing of the amyloid-beta precursor protein.

Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by cerebral deposition of amyloid-ß (Aß), a series of peptides derived from the processing of the amyloid-ß precursor protein (APP). To identify new candidate genes for AD, we recently performed a transcriptome analysis to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions. This strategy identified CALHM1 (calcium homeostasis modulator 1), a gene modulating AD age at onset and Aß metabolism. Here, we focused our attention on another candidate identified using this screen, growth arrest-specific 1 (Gas1), a gene involved in the central nervous system development. We found that Gas1 formed a complex with APP and controlled APP maturation and processing. Gas1 expression inhibited APP full glycosylation and routing to the cell surface by leading to a trafficking blockade of APP between the endoplasmic reticulum and the Golgi. Gas1 expression also resulted in a robust inhibition of APP transport into multivesicular bodies, further demonstrating that Gas1 negatively regulated APP intracellular trafficking. Consequently, Gas1 overexpression led to a reduction in Aß production, and conversely, Gas1 silencing in cells expressing endogenously Gas1 increased Aß levels. These results suggest that Gas1 is a novel APP-interacting protein involved in the control of APP maturation and processing.

CB2 receptor deficiency increases amyloid pathology and alters tau processing in a transgenic mouse model of Alzheimer's disease.

The endocannabinoid CB2 receptor system has been implicated in the neuropathology of Alzheimer's disease (AD). In order to investigate the impact of the CB2 receptor system on AD pathology, a colony of mice with a deleted CB2 receptor gene, CNR2, was established on a transgenic human mutant APP background for pathological comparison with CB2 receptor-sufficient transgenic mice. J20 APP (PDGFB-APPSwInd) mice were bred over two generations with CNR2⁻/⁻ (Cnr2(tm1Dgen)/J) mice to produce a colony of J20 CNR2⁺/⁺ and J20 CNR2⁻/⁻ mice. Seventeen J20 CNR2⁺/⁺ mice (12 females, 5 males) and 16 J20 CNR2⁻/⁻ mice (11 females, 5 males) were killed at 12 months, and their brains were interrogated for AD-related pathology with both biochemistry and immunocytochemistry (ICC). In addition to amyloid-dependent endpoints such as soluble Aß production and plaque deposition quantified with 6E10 staining, the effect of CB2 receptor deletion on total soluble mouse tau production was assayed by using a recently developed high-sensitivity assay. Results revealed that soluble Aß42 and plaque deposition were significantly increased in J20 CNR2⁻/⁻ mice relative to CNR2⁺/⁺ mice. Microgliosis, quantified with ionized calcium-binding adapter molecule 1 (Iba-1) staining, did not differ between groups, whereas plaque associated microglia was more abundant in J20 CNR2⁻/⁻ mice. Total tau was significantly suppressed in J20 CNR2⁻/⁻ mice relative to J20 CNR2⁺/⁺ mice. The results confirm the constitutive role of the CB2 receptor system both in reducing amyloid plaque pathology in AD and also support tehpotential of cannabinoid therapies targeting CB2 to reduce Aß; however, the results suggest that interventions may have a divergent effect on tau pathology.