Cholinergic Atrophy Research Articles

Atrophy of the cholinergic regions advances from early to late mild cognitive impairment.

We investigated the volumetric changes in the components of the cholinergic pathway for patients with early mild cognitive impairment (EMCI) and those with late mild cognitive impairment (LMCI). The effect of patients' apolipoprotein 4 (APOE-ε4) allele status on the structural changes were analyzed. Structural magnetic resonance imaging data were collected. Patients' demographic information, plasma data, and validated global cognitive composite scores were included. Relevant features were extracted for constructing machine learning models to differentiate between EMCI (n = 312) and LMCI (n = 541) and predict patients' neurocognitive function. The data were analyzed primarily through one-way analysis of variance and two-way analysis of covariance. Considerable differences were observed in cholinergic structural changes between patients with EMCI and LMCI. Cholinergic atrophy was more prominent in the LMCI cohort than in the EMCI cohort (P < 0.05 family-wise error corrected). APOE-ε4 differentially affected cholinergic atrophy in the LMCI and EMCI cohorts. For LMCI cohort, APOE-ε4 carriers exhibited increased brain atrophy (left amygdala: P = 0.001; right amygdala: P = 0.006, and right Ch123, P = 0.032). EMCI and LCMI patients showed distinctive associations of gray matter volumes in cholinergic regions with executive (R2 = 0.063 and 0.030 for EMCI and LMCI, respectively) and language (R2 = 0.095 and 0.042 for EMCI and LMCI, respectively) function. Our data confirmed significant cholinergic atrophy differences between early and late stages of mild cognitive impairment. The impact of the APOE-ε4 allele on cholinergic atrophy varied between the LMCI and EMCI groups.

Reduction in Volume of Nucleus Basalis of Meynert Is Specific to Parkinson's Disease and Progressive Supranuclear Palsy but Not to Multiple System Atrophy.

ObjectivesTo study in vivo gray matter (GM) volumes of the nucleus basalis of Meynert (nbM) in different parkinsonian syndromes and assess their relationship with clinical variables.MethodsT1-weighted magnetic resonance images from patients with progressive supranuclear palsy (PSP, N = 43), multiple system atrophy (MSA, N = 23), Parkinson’s disease (PD, N = 26), and healthy controls (HC, N = 29) were included. T1-weighted images were analyzed using a voxel-based morphometry approach implemented in the VBM8 toolbox, and nbM volumes were extracted from the spatially normalized GM images using a cyto-architectonically-defined nbM mask in stereotactic standard space. NbM volumes were compared between groups, while controlling for intracranial volume. Further, within each group correlation analyses between nbM volumes and the Mini Mental Status Examination (MMSE), Hoehn and Yahr stage, PSP Rating Scale, Unified Parkinson’s Disease Rating Scale part III and Frontal Assessment Battery scores were performed.ResultsSignificantly lower nbM volumes in patients with PSP and PD compared to HC or patients with MSA were found. No significant correlations between MMSE and nbM volumes were detected in any of the subgroups. No significant correlations were found between clinical scores and nbM volumes in PSP or other groups.ConclusionnbM volumes were reduced both in PD and PSP but not in MSA. The lack of significant correlations between nbM and cognitive measures suggests that other factors, such as frontal atrophy, may play a more important role than subcortical cholinergic atrophy in PSP patients. These results may indicate that other drug-targets are needed to improve cognitive function in PSP patients.

Alzheimer's Disease: An Update and Insights Into Pathophysiology.

Alzheimer’s disease (AD) is an irreversible brain disorder associated with slow, progressive loss of brain functions mostly in older people. The disease processes start years before the symptoms are manifested at which point most therapies may not be as effective. In the hippocampus, the key proteins involved in the JAK2/STAT3 signaling pathway, such as p-JAK2-Tyr1007 and p-STAT3-Tyr705 were found to be elevated in various models of AD. In addition to neurons, glial cells such as astrocytes also play a crucial role in the progression of AD. Without having a significant effect on tau and amyloid pathologies, the JAK2/STAT3 pathway in reactive astrocytes exhibits a behavioral impact in the experimental models of AD. Cholinergic atrophy in AD has been traced to a trophic failure in the NGF metabolic pathway, which is essential for the survival and maintenance of basal forebrain cholinergic neurons (BFCN). In AD, there is an alteration in the conversion of the proNGF to mature NGF (mNGF), in addition to an increase in degradation of the biologically active mNGF. Thus, the application of exogenous mNGF in experimental studies was shown to improve the recovery of atrophic BFCN. Furthermore, it is now coming to light that the FGF7/FGFR2/PI3K/Akt signaling pathway mediated by microRNA-107 is also involved in AD pathogenesis. Vascular dysfunction has long been associated with cognitive decline and increased risk of AD. Vascular risk factors are associated with higher tau and cerebral beta-amyloid (Aβ) burden, while synergistically acting with Aβ to induce cognitive decline. The apolipoprotein E4 polymorphism is not just one of the vascular risk factors, but also the most prevalent genetic risk factor of AD. More recently, the research focus on AD shifted toward metabolisms of various neurotransmitters, major and minor nutrients, thus giving rise to metabolomics, the most important “omics” tool for the diagnosis and prognosis of neurodegenerative diseases based on an individual’s metabolome. This review will therefore proffer a better understanding of novel signaling pathways associated with neural and glial mechanisms involved in AD, elaborate potential links between vascular dysfunction and AD, and recent developments in “omics”-based biomarkers in AD.

Role of Cholinergic Signaling in Alzheimer's Disease.

Acetylcholine, a neurotransmitter secreted by cholinergic neurons, is involved in signal transduction related to memory and learning ability. Alzheimer’s disease (AD), a progressive and commonly diagnosed neurodegenerative disease, is characterized by memory and cognitive decline and behavioral disorders. The pathogenesis of AD is complex and remains unclear, being affected by various factors. The cholinergic hypothesis is the earliest theory about the pathogenesis of AD. Cholinergic atrophy and cognitive decline are accelerated in age-related neurodegenerative diseases such as AD. In addition, abnormal central cholinergic changes can also induce abnormal phosphorylation of ttau protein, nerve cell inflammation, cell apoptosis, and other pathological phenomena, but the exact mechanism of action is still unclear. Due to the complex and unclear pathogenesis, effective methods to prevent and treat AD are unavailable, and research to explore novel therapeutic drugs is various and active in the world. This review summaries the role of cholinergic signaling and the correlation between the cholinergic signaling pathway with other risk factors in AD and provides the latest research about the efficient therapeutic drugs and treatment of AD.

The Nerve Growth Factor Metabolic Pathway Dysregulation as Cause of Alzheimer's Cholinergic Atrophy.

The cause of the loss of basal forebrain cholinergic neurons (BFCNs) and their terminal synapses in the cerebral cortex and hippocampus in Alzheimer’s disease (AD) has provoked a decades-long controversy. The cholinergic phenotype of this neuronal system, involved in numerous cognitive mechanisms, is tightly dependent on the target-derived nerve growth factor (NGF). Consequently, the loss of BFCNs cholinergic phenotype in AD was initially suspected to be due to an NGF trophic failure. However, in AD there is a normal NGF synthesis and abundance of the NGF precursor (proNGF), therefore the NGF trophic failure hypothesis for the atrophy of BCNs was abandoned. In this review, we discuss the history of NGF-dependency of BFCNs and the atrophy of these neurons in Alzheimer’s disease (AD). Further to it, we propose that trophic factor failure explains the BFCNs atrophy in AD. We discuss evidence of the occurrence of a brain NGF metabolic pathway, the dysregulation of which, in AD explains the severe deficiency of NGF trophic support for the maintenance of BFCNs cholinergic phenotype. Finally, we revise recent evidence that the NGF metabolic dysregulation in AD pathology starts at preclinical stages. We also propose that the alteration of NGF metabolism-related markers in body fluids might assist in the AD preclinical diagnosis.

A new role for matrix metalloproteinase-3 in the NGF metabolic pathway: Proteolysis of mature NGF and sex-specific differences in the continuum of Alzheimer's pathology

Matrix metalloproteinase-3 (MMP-3) has been associated with risk of Alzheimer's disease (AD). In this study we introduce a novel role for MMP-3 in degrading nerve growth factor (NGF) in vivo and examine its mRNA and protein expression across the continuum of AD pathology. We provide evidence that MMP-3 participates in the degradation of mature NGF in vitro and in vivo and that it is secreted from the rat cerebral cortex in an activity-dependent manner. We show that cortical MMP-3 is upregulated in the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis. A similar upregulation was found in AD and MCI brains as well as in cognitively normal individuals with elevated amyloid deposition. We also observed that frontal cortex MMP-3 protein levels are higher in males. MMP-3 protein correlated with more AD neuropathology, markers of NGF metabolism, and lower cognitive scores in males but not in females. These results suggest that MMP-3 upregulation in AD might contribute to NGF dysmetabolism, and therefore to cholinergic atrophy and cognitive deficits, in a sex-specific manner. MMP-3 should be further investigated as a biomarker candidate or as a therapeutic target in AD.

Developmental suppression of forebrain trkA receptors and attentional capacities in aging rats: A longitudinal study

Basal forebrain (BF) cholinergic neurons innervating the cortex regulate cognitive, specifically attentional, processes. Cholinergic atrophy and cognitive decline occur at an accelerated pace in age-related neurodegenerative disorders such as Alzheimer’s disease; however, the mechanism responsible for this phenomenon remains unknown. Here we hypothesized that developmental suppression of nerve growth factor signaling, mediated via tropomyosin-related kinase A (trkA) receptors, would escalate age-related attentional vulnerability. An adeno-associated viral vector expressing trkA shRNA (AAV-trkA) was utilized to knockdown trkA receptors in postnatal rats at an ontogenetic time point when cortical cholinergic inputs mature, and the impact of this manipulation on performance was assessed in animals maintained on an operant attention task throughout adulthood and until old (24 months) age. A within-subject comparison across different time points illustrated a gradual age-related decline in attentional capacities. However, the performance under baseline and distracted conditions did not differ between the AAV-trkA-infused and animals infused with a vector expressing shRNA against the control protein luciferase at any time point. Additional analysis of cholinergic measures conducted at 24 months showed that the capacity of cholinergic terminals to release acetylcholine following a depolarizing stimulus, cortical cholinergic fiber density and BF cholinergic cell size remained comparable between the two groups. Contrary to our predictions, these data indicate that developmental BF trkA disruption does not impact age-related changes in attentional functions. It is possible that life-long engagement in cognitive activity might have potentially rescued the developmental insults on the cholinergic system, thus preserving attentional capacities in advanced age.

Basal forebrain atrophy and cortical amyloid deposition in nondemented elderly subjects

BackgroundBoth neurodegeneration of the cholinergic basal forebrain (BF) and deposition of β-amyloid are early events in the course of Alzheimer's disease (AD). Associations between increased amyloid pathology and cholinergic atrophy have been described in autopsy studies. MethodsWe used structural MRI and AV45-PET amyloid imaging data of 225 cognitively normal or mildly impaired elderly subjects from the Alzheimer's Disease Neuroimaging Initiative to assess in vivo associations between BF atrophy and cortical amyloid deposition. Associations were examined using region-of-interest (ROI) and voxel-based approaches with reference to cytoarchitectonic mappings of the cholinergic BF nuclei. ResultsROI- and voxel-based approaches yielded complementary evidence for an association between BF volume and cortical amyloid deposition in presymptomatic and predementia stages of AD, irrespective of age, gender, and APOE genotype. ConclusionsThe observed correlations between BF atrophy and cortical amyloid load likely reflect associations between cholinergic degeneration and amyloid pathology as reported in neuropathologic examination studies.

Impact of the NGF Maturation and Degradation Pathway on the Cortical Cholinergic System Phenotype

Cortical cholinergic atrophy plays a significant role in the cognitive loss seen with aging and in Alzheimer's disease (AD), but the mechanisms leading to it remain unresolved. Nerve growth factor (NGF) is the neurotrophin responsible for the phenotypic maintenance of basal forebrain cholinergic neurons in the mature and fully differentiated CNS. In consequence, its implication in cholinergic atrophy has been suspected; however, no mechanistic explanation has been provided. We have previously shown that the precursor of NGF (proNGF) is cleaved extracellularly by plasmin to form mature NGF (mNGF) and that mNGF is degraded by matrix metalloproteinase 9. Using cognitive-behavioral tests, Western blotting, and confocal and electron microscopy, this study demonstrates that a pharmacologically induced chronic failure in extracellular NGF maturation leads to a reduction in mNGF levels, proNGF accumulation, cholinergic degeneration, and cognitive impairment in rats. It also shows that inhibiting NGF degradation increases endogenous levels of the mature neurotrophin and increases the density of cortical cholinergic boutons. Together, the data point to a mechanism explaining cholinergic loss in neurodegenerative conditions such as AD and provide a potential therapeutic target for the protection or restoration of this CNS transmitter system in aging and AD.

Cortical peroxynitration of nerve growth factor in aged and cognitively impaired rats

Basal forebrain cholinergic neurons (BFCN), a system involved in learning and memory processes, are highly dependent on a continuous supply of biologically active nerve growth factor (NGF). Age-related cholinergic atrophy and cell loss in normal brains is apparently not complemented by reductions in the levels of NGF as could be expected. In the present work, cortical proNGF/NGF were immunoprecipitated from cortical brain homogenates from young and aged and behaviorally characterized rats and resolved with antinitrotyrosine antibodies to reveal nitration of tyrosine residues in proteins. Cortical proNGF in aged and cognitively impaired rats was found to be a target for peroxynitrite-mediated oxidative damage with correlative impact on decrease in choline acetyltransferase activity. These studies provide evidence for oxidative stress damage of NGF molecules in the cerebral cortex of cognitively impaired aged rats as previously shown in AD human brains.

Correlation of cognitive performance and morphological changes in neocortical pyramidal neurons in aging

It is well established that the cerebral cortex undergoes extensive remodeling in aging. In this study, we used behaviorally characterized rats to correlate age-related morphological changes with cognitive impairment. For this, young and aged animals were tested in the Morris water maze to evaluate their cognitive performance. Following behavioral characterization, the animals were perfused and a combination of intracellular labeling and immunohistochemistry was applied. Using this approach, we characterized the dendritic morphology of cortical pyramidal neurons as well as the pattern of glutamatergic and GABAergic appositions on their cell bodies and dendrites. We focused on the association region of the parietal cortex (LtPA) and the medial prefrontal cortex (mPFC) for their involvement in the Morris water maze task. We found an age-related atrophy of distal basal dendrites that did not differ between aged cognitively unimpaired (AU) and aged cognitively impaired animals (AI). Dendritic spines and glutamatergic appositions generally decreased from young to AU and from AU to AI rats. On the other hand, GABAergic appositions only showed a trend towards a decrease in AU rats. Collectively, the data show that the ratio of excitatory/inhibitory inputs was only altered in AI animals. When cortical cholinergic varicosities were labeled on alternate sections, we found that AI animals also had a significant reduction of cortical cholinergic boutons compared with AU or young animals. In aged animals, the density of cortical cholinergic varicosities correlated with the excitatory/inhibitory ratio. Our data suggest that both cholinergic atrophy and an imbalance towards inhibition may contribute to the observed age-associated behavioral impairment.

Cholinergic Involvement in Alzheimer’s Disease. A Link with NGF Maturation and Degradation

Basal forebrain cholinergic neurons are highly dependent on nerve growth factor (NGF) supply for the maintenance of their cholinergic phenotype as well as their cholinergic synaptic integrity. The precursor form of NGF, proNGF, abounds in the CNS and is highly elevated in Alzheimer's disease. In order to obtain a deeper understanding of the NGF biology in the CNS, we have performed a series of ex vivo and in vivo investigations to elucidate the mechanisms of release, maturation and degradation of this neurotrophin. In this short review, we describe this NGF metabolic pathway, its significance for the maintenance of basal cholinergic neurons, and its dysregulation in Alzheimer's disease. We are proposing that the conversion of proNGF to mature NGF occurs in the extracellular space by the coordinated action of zymogens, convertases, and endogenous regulators, which are released in the extracellular space in an activity-dependent fashion. We further discuss our findings of a diminished conversion of the NGF precursor molecule to its mature form in Alzheimer's disease as well as an augmented degradation of mature NGF. These combined effects on NGF metabolism would explain the well-known cholinergic atrophy found in Alzheimer's disease and would offer new therapeutic opportunities aimed at correcting the NGF dysmetabolism along with Abeta-induced inflammatory responses.

P3-380: Aβ-induced nerve growth factor trophic disconnection in Alzheimer's disease

The causative mechanisms for the atrophy of forebrain cholinergic neurons in Alzheimer's disease (AD) remain undefined. Western blot analysis of members of the NGF maturation/degradation cascade in the CNS and of key inflamatory markers in human brain samples of Alzheimer's patients and controls as well as in rodents after the application of oligomeric A beta 42 and treatment with minocycline. We found evidence for a diminished conversion of the NGF precursor molecule to its mature form and for increased NGF-degradation in Alzheimer's brain samples, resulting in an up-regulation of proNGF levels. We further observed that soluble Aβ-oligomers injections provoked a similar up-regulation of proNGF levels in the rat hippocampus. These experiments revealed an Aβ-induced inflammatory microglia activation, increased inducible nitric oxide synthase (iNOS) which activated the NGF-degrading enzyme MMP-9 and facilitated the CNS accumulation of peroxynitrite-modified proNGF. These combined effects markedly reduced the level and biological activity of mature NGF and provoked the accumulation of proNGF. These changes were prevented with the application of minocycline. Accordingly, the administration of minocycline in a transgenic AD mouse model also corrected the altered levels of MMP-9, iNOS and microglial activation, preserving cognitive behavior and normalizing proNGF levels. These effects of Aβ amyloid CNS burden on NGF metabolism would explain the well-known cholinergic atrophy found in AD and it offers a novel therapeutic target.

Hippocampal hypocellularity in the Ts65Dn mouse originates early in development

Ts65Dn, a well-characterized animal model for Down syndrome, has three copies of the distal end of mouse chromosome 16 and therefore has segmental trisomy for orthologs for nearly half of the genes located on human chromosome 21. Ts65Dn mice have learning and memory impairments, especially in tasks involving the hippocampus. Previous studies have shown that older adult Ts65Dn mice have structural abnormalities in the hippocampus including fewer granule cells in dentate gyrus and more pyramidal cells in the CA3 subfield of cornus ammonis. However, it is not clear whether those changes are secondary to the age-related neurodegeneration of the basal forebrain cholinergic neurons that project to the hippocampus or if they originate earlier during hippocampal development. To address this question, we performed a quantitative study of the hippocampal volume and the numbers of granule cell and pyramidal neurons in young (postnatal day 6, P6) and adult (3-month-old) mice using the optical fractionator method. At P6, Ts65Dn mice had 20% fewer granule cells in dentate gyrus than did euploid littermates. Similarly, compared to euploid, P6 trisomic mice showed an 18% reduction in mitotic cells in the granule cell layer and the hilus, where granule cell precursors divide to generate the internal granule cell layer. Granule cell hypocellularity persists in 3-month-old Ts65Dn mice before the onset of cholinergic atrophy. The hypocellularity seen in the trisomic adult hippocampus originates early in development and may contribute to specific cognitive deficits in these mice.

Progression of age‐associated cognitive impairment correlates with quantitative and qualitative loss of TrkA receptor protein in nucleus basalis and cortex

A direct correlation between disease progression and reduced expression of TrkA receptor in cholinergic neurons has been documented in neurocognitive pathologies including Alzheimer's disease. We investigated whether reduced expression of TrkA protein might also correlate with the level of cognitive impairment in age-associated cognitive impairment. Quantitative and qualitative measurements of TrkA protein levels in the cortex and nucleus basalis of aged rats that had been well-characterized behaviorally as 'unimpaired', 'mildly impaired' or 'fully impaired' demonstrated significant changes in TrkA expression. In the mildly impaired cognitive state phenotypic silencing of TrkA was detected in neurons expressing TrkA at high density but before cholinergic atrophy or loss of TrkA+ neurons was detected. In the fully impaired cognitive state a significant loss in TrkA+ cholinergic neurons together with a more significant phenotypic silencing of TrkA expression then took place. These data suggest that TrkA+ cholinergic cells are associated with cognition, TrkA could be a biomarker of the cognitive state and phenotypic loss of TrkA precedes neuronal loss and probably sensitizes cells to death. We speculate that neurotrophic deficits may be a shared mechanism for cognitive decline in aging and Alzheimer's disease.

Alzheimer's disease: more than a ‘cholinergic disorder' — evidence that cholinergic–monoaminergic interactions contribute to EEG slowing and dementia

The loss of cognitive (particularly mnemonic) abilities constitutes a prominent symptom of Alzheimer's disease (AD). These cognitive symptoms occur in close relation to the slowing of the electroencephalogram (EEG), and it is likely that the inability of cortical circuits to maintain an activated state contributes to the behavioral disorganization in AD. The ‘cholinergic hypothesis’ of AD suggests that many of the cognitive and EEG symptoms are related to the atrophy of basal forebrain cholinergic neurons, which innervate the neocortex and hippocampus, among others. However, data from behavioral and electrophysiological studies in rats suggest that selective reductions in cholinergic transmission result in relatively small mnemonic impairments, and only a partial reduction in EEG activation. Thus, cholinergic atrophy alone may not be sufficient to cause the marked changes in cognition and cortical activity typical of AD. Cholinergic deficits do, however, make neural circuits susceptible to additional neurodegenerative processes. In rats, lowered serotonergic or noradrenergic activity alone often produces only minor impairments in learning/memory tasks and does not block EEG activation. The same monoaminergic deficits, however, result in severe behavioral impairments, and reduce or abolish EEG activation when they occur in a brain already affected by lowered cholinergic activity. There is an abundance of evidence that monoamines are reduced in AD. These degenerative processes, when occurring in a neural environment compromised by cholinergic atrophy, may then contribute to the disturbances in cortical processing and cognition/behavior in AD. A prediction derived from this theory is that an enhancement of monoaminergic functions may have beneficial effects on behavior and cortical activity. Preliminary experiments support this idea: combined cholinergic–monoaminergic stimulation can be more effective in reversing behavioral (Morris water maze) impairments and EEG slowing in rats with multiple neurotransmitter deficiencies than cholinergic enhancement alone. Thus, a stimulation of monoaminergic activity, in conjunction with cholinergic therapies, may provide an effective treatment option for AD.

Nerve growth factor and Alzheimer's disease.

Nerve growth factor (NGF) is a well-characterized protein that exerts pharmacological effects on a group of cholinergic neurons known to atrophy in Alzheimer's disease (AD). Considerable evidence from animal studies suggests that NGF may be useful in reversing, halting, or at least slowing the progression of AD-related cholinergic basal forebrain atrophy, perhaps even attenuating the cognitive deficit associated with the disorder. However, many questions remain concerning the role of NGF in AD. Levels of the low-affinity receptor for NGF appear to be at least stable in AD basal forebrain, and the recent finding of AD-related increases in cortical NGF brings into question whether endogenous NGF levels are related to the observed cholinergic atrophy and whether additional NGF will be useful in treating this disorder. Evidence regarding the localization of NGF within the central nervous system and its presumed role in maintaining basal forebrain cholinergic neurons is summarized, followed by a synopsis of the relevant aspects of AD neuropathology. The available data regarding levels of NGF and its receptor in the AD brain, as well as potential roles for NGF in the pathogenesis and treatment of AD, are also reviewed. NGF and its low affinity receptor are abundantly present within the AD brain, although this does not rule out an NGF-related mechanism in the degeneration of basal forebrain neurons, nor does it eliminate the possibility that exogenous NGF may be successfully used to treat AD. Further studies of the degree and distribution of NGF within the human brain in normal aging and in AD, and of the possible relationship between target NGF levels and the status of basal forebrain neurons in vivo, are necessary before engaging in clinical trials.

Development of effective therapy for Alzheimer's disease based on neurotrophic factors

Neurotrophic factors play a major role in brain development and function. Because administration of these factors can attenuate degeneration of neurons and behavioral deficits in animals, such molecules may become useful in the treatment of AD. At the present time, a solid rationale can be made for clinical trials with NGF, given the pronounced trophic effect of NGF on forebrain cholinergic neurons and the cholinergic atrophy occurring in AD. Future research in the following areas will be crucial toward development of successful neurotrophic factor therapy: (a) New research should focus on identifying new neurotrophic factors, which protect other vulnerable neurons in AD, in particular hippocampal and cortical neurons; (b) Multiple available strategies should be pursued to find ways to deliver neurotrophic proteins across the blood-brain barrier. In addition, a broadly based effort to establish a small-molecule pharmacology of neurotrophic factors is likely to lead to clinically useful drugs; (c) Regulatory guidance is necessary to establish ethically acceptable designs for clinical trials to unequivocally establish efficacy of drugs that attenuate the progression of AD.