Phospholipid transfer protein (PLTP) deficiency accelerates memory dysfunction through altering amyloid precursor protein (APP) processing in a mouse model of Alzheimer's disease.

Phospholipid transfer protein (PLTP) is a widely expressed lipid transfer protein participating in the transport of cholesterol and other lipids in the plasma and peripheral tissues. Recently, elevated amyloid ß (Aß) in young and aged PLTP-deficient brains had been reported. However, the role of PLTP in amyloid precursor protein (APP) processing and Alzheimer's disease (AD) pathology remains elusive. Here we first found that deficiency of PLTP accelerated memory dysfunction in APP/PS1ΔE9 AD model mice at the age of 3 months. Further characterization showed that PLTP deficiency increased soluble Aß peptides, and intracellular accumulation of Aß was illustrated, which might be due to disrupted APP turnover and the enhanced amyloidogenic pathway. Besides, reduced brain-derived neurotrophic factor (BDNF) was found in PLTP-deficient APP/PS1ΔE9 mice, and the BDNF level was negatively correlated with Aß42 content, instead of Aß40 content. In addition, autophagic dysfunction was found in the PLTP-deficient APP/PS1ΔE9 mice. Our data presented a novel model to link phospholipid metabolism to APP processing and also suggested that PLTP played an important role in Aß metabolism and would be useful to further elucidate functions of PLTP in AD susceptibility.

Effects of hypoxic preconditioning on synaptic ultrastructure in mice.

Hypoxic preconditioning (HPC) elicits resistance to more drastic subsequent insults, which potentially provide neuroprotective therapeutic strategy, but the underlying mechanisms remain to be fully elucidated. Here, we examined the effects of HPC on synaptic ultrastructure in olfactory bulb of mice. Mice underwent up to five cycles of repeated HPC treatments, and hypoxic tolerance was assessed with a standard gasp reflex assay. As expected, HPC induced an increase in tolerance time. To assess synaptic responses, Western blots were used to quantify protein levels of representative markers for glia, neuron, and synapse, and transmission electron microscopy was used to examine synaptic ultrastructure and mitochondrial density. HPC did not significantly alter the protein levels of astroglial marker (GFAP), neuron-specific markers (GAP43, Tuj-1, and OMP), synaptic number markers (synaptophysin and SNAP25) or the percentage of excitatory synapses versus inhibitory synapses. However, HPC significantly affected synaptic curvature and the percentage of synapses with presynaptic mitochondria, which showed concomitant change pattern. These findings demonstrate that HPC is associated with changes in synaptic ultrastructure.

Elevation of brain magnesium prevents and reverses cognitive deficits and synaptic loss in Alzheimer's disease mouse model.

Profound synapse loss is one of the major pathological hallmarks associated with Alzheimer's disease (AD) and might underlie memory impairment. Our previous work demonstrated that the magnesium ion is a critical factor in controlling synapse density/plasticity. Here, we investigated whether elevation of brain magnesium by the use of a recently developed compound, magnesium-l-threonate (MgT), can ameliorate the AD-like pathologies and cognitive deficits in the APPswe/PS1dE9 mice, a transgenic (Tg) mouse model of AD. MgT treatment reduced Aß plaque and prevented synapse loss and memory decline in the Tg mice. Strikingly, MgT treatment was effective even when given to the mice at the end stage of their AD-like pathological progression. To explore how elevation of brain magnesium ameliorates the AD-like pathologies in the brains of Tg mice, we studied molecules critical for APP metabolism and signaling pathways implicated in synaptic plasticity/density. In the Tg mice, the NMDAR/CREB/BDNF signaling was downregulated, whereas calpain/calcineurin/Cdk5 neurodegenerative signaling and ß-secretase (BACE1) expression were upregulated. MgT treatment prevented the impairment of these signaling pathways, stabilized BACE1 expression, and reduced soluble APPß and ß-C-terminal fragments in the Tg mice. At the molecular level, elevation of extracellular magnesium prevented the high-Aß-induced reductions in synaptic NMDARs by preventing calcineurin overactivation in hippocampal slices. Correlation studies suggested that the protection of NMDAR signaling might underlie the stabilization of BACE1 expression. Our results suggest that elevation of brain magnesium exerts substantial synaptoprotective effects in a mouse model of AD and may have therapeutic potential for treating AD in humans.

Hypoxia-inducible factor-1α mediates up-regulation of neprilysin by histone deacetylase-1 under hypoxia condition in neuroblastoma cells.

Hypoxia-inducible factor (HIF)-1 is the key transcriptional activator mediating both adaptive and pathological responses to hypoxia. The purpose of this study was to find the role of HIF-1 in regulating neprilysin (NEP) at the early stage of hypoxia and explore the underlying mechanism. In this study, we demonstrated that both NEP mRNA and protein levels in neuroblastoma cells were elevated in early stages of hypoxia. Over-expression of HIF-1α gene increased NEP mRNA/protein levels, as well as enzyme activity while knockdown of HIF-1α decreased them. Meanwhile, HIF-1α was shown to bind to histone deacetylase (HDAC)-1 and reduced the association of HDAC-1 with NEP promoter, thus activating NEP gene transcription in a de-repression way. In summary, our results indicated that hypoxia in the early stages would up-regulate NEP expression, in which interaction of HIF-1α and HDAC-1 may play a role. This study suggested that NEP up-regulation might be an adaptive response to hypoxia, which was mediated by HIF-1α binding to HDAC-1 at the early stage of hypoxia.

Phospholipid transfer protein (PLTP) deficiency impaired blood-brain barrier integrity by increasing cerebrovascular oxidative stress.

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood-brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.

Prophylactic angiotensin type 1 receptor antagonism confers neuroprotection in an aged rat model of postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is a common geriatric complication, although its exact neuropathogenesis remains elusive. Blockers of the renin-angiotensin system (RAS) ameliorate cognitive deficits in inflammatory brain disorders, with its effects on POCD not yet fully elucidated. The aim of the present study was to investigate regulation of the brain RAS and the effect of angiotensin II receptor type 1 (AT1) inhibition on surgery-induced cognitive impairment in a well-established rat POCD model. We observed upregulation of angiotensin II protein expression and AT1 subtype B transcript levels in the hippocampus after laparotomy, suggesting surgical stress activates the hippocampal RAS in aged rats. Chronic pretreatment with 0.1 mg/kg/day candesartan, an AT1 antagonist, significantly attenuated surgery-induced cognitive deficits in the Morris water maze task without altering blood pressure. Candesartan also decreased hippocampal blood-brain barrier (BBB) permeability. Concomitant with these functional benefits, we observed significant inhibition of hippocampal neuroinflammation, evidenced by decreased glial reactivity and phosphorylation of the NF-κB p65 subunit, as well as marked reductions in interleukin-1ß, tumor necrosis factor-α, and cyclooxygenase-2. Our results are the first to show that activation of the brain RAS after surgery contributes to POCD in aged rats. Chronic treatment with low doses of candesartan may elicit blood pressure-independent neuroprotective effects in POCD by improving BBB function and promoting resolution of neuroinflammation.

Blood-brain barrier dysfunction in mice induced by lipopolysaccharide is attenuated by dapsone.

Blood-brain barrier (BBB) dysfunction is a key event in the development of many central nervous system (CNS) diseases, such as septic encephalopathy and stroke. 4,4'-Diaminodiphenylsulfone (DDS, Dapsone) has displayed neuroprotective effect, but whether DDS has protective role on BBB integrity is not clear. This study was designed to examine the effect of DDS on lipopolysaccharide (LPS)-induced BBB disruption and oxidative stress in brain vessels. Using in vivo multiphoton imaging, we found that DDS administration significantly restored BBB integrity compromised by LPS. DDS also increased the expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Level of reactive oxygen species (ROS) was reduced by DDS treatment, which may due to decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and NOX2 expression. Our results showed that LPS-induced BBB dysfunction could be attenuated by DDS, indicated that DDS has a therapeutic potential for treating CNS infection and other BBB related diseases.

The profiling and identification of the metabolites of (+)-catechin and study on their distribution in rats by HPLC-DAD-ESI-IT-TOF-MS(n) technique.

(+)-Catechin, a potential beneficial compound to human health, is widely distributed in plants and foods. A high-performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry method was applied to profile and identify the metabolites of (+)-catechin in rats and to study the distribution of these metabolites in rat organs for the first time. In total, 51 phase II metabolites (44 new) and three phase I metabolites were tentatively identified, comprising 16 (+)-catechin conjugates, 14 diarylpropan-2-ol metabolites, 6 phenyl valerolactone metabolites and 18 aromatic acid metabolites. Further, 19 phase II metabolites were new compounds. The in vivo metabolic reactions of (+)-catechin in rats were found to be ring-cleavage, sulfation, glucuronidation, methylation, dehydroxylation and dehydrogenation. The numbers of detected metabolites in urine, plasma, small intestine, kidney, liver, lung, heart, brain and spleen were 53, 23, 27, 9, 7, 5, 3, 2 and 1, respectively. This indicated that small intestine, kidney and liver were the major organs for the distribution of (+)-catechin metabolites. In addition, eight metabolites were found to possess bioactivities according to literature. These results are very helpful for better comprehension of the in vivo metabolism of (+)-catechin and its pharmacological actions, and also can give strong indications on the effective forms of (+)-catechin in vivo.

Activation of the canonical nuclear factor-κB pathway is involved in isoflurane-induced hippocampal interleukin-1ß elevation and the resultant cognitive deficits in aged rats.

Although much recent evidence has demonstrated that neuroinflammation contributes to volatile anesthetic-induced cognitive deficits, there are few existing mechanistic explanations for this inflammatory process. This study was conducted to investigate the effects of the volatile anesthetic isoflurane on canonical nuclear factor (NF)-κB signaling, and to explore its association with hippocampal interleukin (IL)-1ß levels and anesthetic-related cognitive changes in aged rats. After a 4-h exposure to 1.5% isoflurane in 20-month-old rats, increases in IκB kinase and IκB phosphorylation, as well as a reduction in the NF-κB inhibitory protein (IκBα), were observed in the hippocampi of isoflurane-exposed rats compared with control rats. These events were accompanied by an increase in NF-κB p65 nuclear translocation at 6h after isoflurane exposure and hippocampal IL-1ß elevation from 1 to 6h after isoflurane exposure. Nevertheless, no significant neuroglia activation was observed. Pharmacological inhibition of NF-κB activation by pyrrolidine dithiocarbamate markedly suppressed the IL-1ß increase and NF-κB signaling, and also mitigated the severity of cognitive deficits in the Morris water maze task. Overall, our results demonstrate that isoflurane-induced cognitive deficits may stem from upregulation of hippocampal IL-1ß, partially via activation of the canonical NF-κB pathway, in aged rats.

NAD+ rescues aging-induced blood-brain barrier damage via the CX43-PARP1 axis.

Blood-brain barrier (BBB) function deteriorates during aging, contributing to cognitive impairment and neurodegeneration. It is unclear what drives BBB leakage in aging and how it can be prevented. Using single-nucleus transcriptomics, we identified decreased connexin 43 (CX43) expression in cadherin-5+ (Cdh5+) cerebral vascular cells in naturally aging mice and confirmed it in human brain samples. Global or Cdh5+ cell-specific CX43 deletion in mice exacerbated BBB dysfunction during aging. The CX43-dependent effect was not due to its canonical gap junction function but was associated with reduced NAD+ levels and mitochondrial dysfunction through NAD+-dependent sirtuin 3 (SIRT3). CX43 interacts with and negatively regulates poly(ADP-ribose) polymerase 1 (PARP1). Pharmacologic inhibition of PARP1 by olaparib or nicotinamide mononucleotide (NMN) supplementation rescued NAD+ levels and alleviated aging-associated BBB leakage. These findings establish the endothelial CX43-PARP1-NAD+ pathway's role in vascular aging and identify a potential therapeutic strategy to combat aging-associated BBB leakage with neuroprotective implications.

Prominent activation of the intraparietal and somatosensory areas during angle discrimination by intra-active touch.

Intra-active touch (IAT) is a process that involves a body part doing the touching (active touch [AT]) and another body part being touched (passive touch [PT]) simultaneously. The brain representation related to IAT is still unclear. A total of 23 subjects carried out angle discrimination under PT, AT and IAT conditions with functional magnetic resonance imaging. All of the tasks were strictly dependent on cutaneous feedback from the finger(s). As the subjects were able to perceive the angle stimuli from the right (touching) and left (touched) sides during the IAT condition, we expected there would be greater brain activation with the IAT condition than for the AT or PT condition. Therefore, we hypothesized that the region within and/or around the intraparietal sulcus (IPS) and the part of the primary somatosensory cortex (SI) that is associated with high-level tactile spatial processing would be more active during the IAT task than during the AT and PT tasks. Compared with the areas activated by the motor somatosensory control task, the most prominent activation areas evoked by the three-angle discrimination tasks were in the SI and secondary somatosensory cortex areas in the bilateral parietal operculum, IPS, lateral occipital complex, insula and cerebellum. Finally, we directly compared IAT with AT and PT, and the results suggest that the contralateral part of IPS and part of the SI are more active under IAT conditions than under either AT or PT conditions. These results suggest that both hemispheres contribute to angle discrimination during IAT.

Prospective memory tasks: a more sensitive method for screening cognitive impairment in ALS?

BACKGROUND: Cognitive change is prevalent in patients with amyotrophic lateral sclerosis (ALS), but still lack a widely accepted and sensitive screening method. In this study, we try to find a sensitive screening battery for detecting subtle cognitive deficits in patients with ALS. METHODS: Eighty consecutive ALS patients and 57 matched normal controls underwent the Mini-Mental Status Examination (MMSE), the verbal fluency test (VFT), the Stroop Color Word Interference Test (CWT), and the prospective memory (PM) tests, including event-based (EBPM) and time-based (TBPM). RESULTS: The patients did not differ from the controls in the MMSE, the VFT and the CWT. By contrast, statistically significant differences were found in the PM tests (EBPM: P=0.043; TBPM: P<0.001). More interestingly, TBPM was more sensitive than EBPM in the early-phase patients. CONCLUSIONS: Prefrontal lobar dysfunction does exist among ALS patients and may spread from the medial to the lateral region. The PM tests seem more sensitive in ALS patients with frontotemporal dysfunction than are the classical cognitive measures.

Presynaptic defects underlying impaired learning and memory function in lipoprotein lipase-deficient mice.

Lipoprotein lipase (LPL) is predominantly expressed in adipose and muscle where it plays a crucial role in the metabolism of triglyceride-rich plasma lipoproteins. LPL is also expressed in the brain with highest levels found in the pyramidal cells of the hippocampus, suggesting a possible role for LPL in the regulation of cognitive function. However, very little is currently known about the specific role of LPL in the brain. We have generated a mouse model of LPL deficiency which was rescued from neonatal lethality by somatic gene transfer. These mice show no exogenous and endogenous LPL expression in the brain. To study the role of LPL in learning and memory, the performance of LPL-deficient mice was tested in two cognitive tests. In a water maze test, LPL-deficient mice exhibited increased latency to escape platform and increased mistake frequency. Decreased latency to platform in the step-down inhibitory avoidance test was observed, consistent with impaired learning and memory in these mice. Transmission electron microscopy revealed a significant decrease in the number of presynaptic vesicles in the hippocampus of LPL-deficient mice. The levels of the presynaptic marker synaptophysin were also reduced in the hippocampus, whereas postsynaptic marker postsynaptic density protein 95 levels remained unchanged in LPL-deficient mice. Theses findings indicate that LPL plays an important role in learning and memory function possibly by influencing presynaptic function.

A family of membrane proteins associated with presenilin expression and gamma-secretase function.

Presenilin 1 (PS1) forms the gamma-secretase complex with at least three components: nicastrin, APH-1, and PEN-2. This complex mediates intramembrane cleavage of amyloid precursor protein (APP) to generate beta-amyloid protein (Abeta) as well as other type 1 transmembrane proteins. Although PS1 mutations linked to familial Alzheimer's disease influence these cleavages, their biological consequences have not been fully understood. In this study, we used mRNA differential display analysis to identify a gene, denoted adoplin-1/ORMDL-1, which displays significantly reduced expression in association with PS1 mutations. Adoplin-1 and two highly homologous genes (adoplin-2, -3) constitute a gene family that encodes transmembrane proteins. The mRNA and protein levels of adoplins (particularly adoplin-1, -2) were markedly elevated in PS-deficient fibroblasts, compared to wild-type cells. Moreover, knockdown of the three adoplins by RNA interference affected maturation of nicastrin and its association with PS1. Adoplin knockdown additionally resulted in elevated levels of APP C-terminal fragments and decreased Abeta production, suggestive of reduced gamma-secretase activity. Our data collectively indicate that adoplins are unique molecules with PS-related expression and functions that may play important role(s) in the maturation and activity of the gamma-secretase complex.

Stimulation Modeling on Three-Dimensional Anisotropic Diffusion of MRI Tracer in the Brain Interstitial Space.

Purpose: To build a mathematical model based magnetic resonance (MR) method to simulate drug anisotropic distribution in vivo in the interstitial space (ISS) of the brain. Materials and Methods: An injection of signal intensity-related gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), which is an exogenous drug, was administered, and its diffusion was traced in the ISS of the brain using MRI. Dynamic MRI scans were performed to monitor and record the changes in signal intensity in each pixel of the region of interest. The transport parameters were calculated using the modified equation to simulate three-dimensional anisotropic diffusion, which was resolved using a Laplace transform and a linear regressive model. Results: After Gd-DTPA was introduced into the caudate nucleus, its distribution was demonstrated in real time. As the Gd-DTPA gradually cleared, the associated hyperintensity attenuated over time. The average diffusion coefficient (D) and the clearance rate constant (k) were (1.305 ± 0.364) × 10-4 mm2/s and (1.40 ± 0.206) × 10-5 s-1, respectively. Discussion: The combination of trace-based MRI and modified diffusion mathematical models can visualize and measure the three-dimensional anisotropic distribution of drugs in the ISS of the brain.

The Neuroscience Research Institute at Peking University: a place for the solution of pain and drug abuse.

Neuroscience research in China has undergone rapid expansion since 1980. The Neuroscience Research Institute of Peking University, one of the most active neuroscience research groups in China, was founded in 1987. Currently, the institute is overseeing four research areas, i.e., (1) pain and analgesia, (2) drug abuse and acupuncture treatment for drug addiction, (3) the mechanism of neurological degenerative disorders, and (4) the role of neuroglia in central nervous system injury. The institute is simultaneously investigating both theoretical and clinical studies. Acupuncture remains the core of research, while pain and drug abuse form the two disciplines.

DNA context and promoter activity affect gene expression in lentiviral vectors.

Several varieties oflentiviral delivery systems have been used to generate stable cell lines and transgenic animals. Some enhancing elements have been identified to promote stable transgene expression. In this study, we describe that the promoter activity is affected by the lentiviral genomic context. We have examined the promoter activities using green fluorescence protein (GFP) as a reporter in different cell lines to demonstrate that the cytomegalovirus (CMV) promoter may not always be the best choice to overexpress transgenes in all cell types. Our data showed that the polypeptide chain elongation factor 1 alpha (EF1alpha) promoter is relatively active in all three model cell lines (293T, HOS, and Hela) while the CMV promoter is less effective in Hela cells.

[Roles of cyclooxygenase 2/2', 3'-cyclic nucleotide3' phosphohydrolase in the oligodendrocyte apoptosis in heroin-induced spongiform leucoencephalopathy].

OBJECTIVE: To investigate the regulation of cyclooxygenase (Cox)-2/2', 3'-cyclic nucleotide3' phosphohydrolase (CNPase) on the oligodendrocyte apoptosis in the pathogenesis of the heroin-induced spongiform leucoencephalopathy (HSLE). METHODS: Samples of frontal lobe, cerebellum, and corpus callosum were obtained from the brains during autopsy of 4 HSLE patients and 5 patients who died of diseases other than cerebral diseases (controls) and underwent light microscopy and electron microscopy. Immunocytochemistry was carried out to detect the expression of myelin basic protein (MBP), caspase-3, COX-2, and CNPase protein. Apoptosis was examined by TUNEL staining. RESULTS: Widespread demyelination was seen in the white matter of the frontal lobe, cerebellum, and corpus callosum of the HSLE cases, most severely in cerebellum. In he HSLE group, the levels of caspase-3 and COX-2 expression were significantly higher, and the level of CNPase was significantly lower than those of the control group (all P < 0.05). CONCLUSION: Widespread demyelination in the white matter is a prevailing pathological change of HSLE. Oligodendrocyte apoptosis is one of the causes of HSLE. The upregulation of COX-2 and downregulation of CNPase may contribute to the pathogenesis.

Dapsone protects brain microvascular integrity from high-fat diet induced LDL oxidation.

Atherosclerosis was considered to induce many vascular-related complications, such as acute myocardial infarction and stroke. Abnormal lipid metabolism and its peroxidation inducing blood-brain barrier (BBB) leakage were associated with the pre-clinical stage of stroke. Dapsone (DDS), an anti-inflammation and anti-oxidation drug, has been found to have protective effects on vascular. However, whether DDS has a protective role on brain microvessels during lipid oxidation had yet to be elucidated. We investigated brain microvascular integrity in a high-fat diet (HFD) mouse model. We designed this study to explore whether DDS had protective effects on brain microvessels under lipid oxidation and tried to explain the underlying mechanism. In our live optical study, we found that DDS significantly attenuated brain microvascular leakage through reducing serum oxidized low-density lipoprotein (oxLDL) in HFD mice (p < 0.001), and DDS significantly inhibited LDL oxidation in vitro (p < 0.001). Our study showed that DDS protected tight junction proteins: ZO-1 (p < 0.001), occludin (p < 0.01), claudin-5 (p < 0.05) of microvascular endothelial cells in vivo and in vitro. DDS reversed LAMP1 aggregation in cytoplasm, and decreased the destruction of tight junction protein: ZO-1 in vitro. We first revealed that DDS had a protective role on cerebral microvessels through preventing tight junction ZO-1 from abnormal degradation by autophagy and reducing lysosome accumulation. Our findings suggested the significance of DDS in protecting brain microvessels under lipid metabolic disorders, which revealed a novel potential therapeutic strategy in brain microvascular-related diseases.

c-jun N-terminal kinase hyperphosphorylates R406W tau at the PHF-1 site during mitosis.

Tauopathies such as Alzheimer disease (AD) probably involve a type of phosphorylation imbalance causing the accumulation of abnormally hyperphosphorylated tau in neurons and/or glias. Investigation of R406W tau mutation may provide insight into such abnormal tau hyperphosphorylation, since this mutation causes AD-like dementia and tauopathy in humans and because it has the unique ability to reduce tau phosphorylation in vitro and in cultured cells. Here we show that R406W mutation primarily disrupts tau phosphorylation at Ser404, a priming phosphorylation site of glycogen synthase kinase-3beta (GSK-3beta), thereby reducing subsequent GSK-3beta-mediated phosphorylation at the PHF-1 site (mostly Ser396). In contrast, c-jun N-terminal kinase (JNK) as activated in the mitotic phase directly hyperphosphorylates R406W tau at the PHF-1 site. This was confirmed by PHF-1 hyperphosphorylation of R406W tau in mitotic cells, its association with cytoplasmic JNK activation, and its inhibition by a JNK inhibitor, SP600125. These data unveil the unknown mechanisms of physiological tau phosphorylation at the PHF-1 site and suggest that cytoplasmic JNK activation may play an important role in the abnormal tau hyperphosphorylation associated with R406W tau mutation and in AD.