The aging brain, neuroinflammatory signaling and sleep-wake regulation.

During the development of the dentate gyrus (DG), both at embryonic and postnatal stages, radial glial cells (RGCs) and neural progenitors proliferate and generate mature granule neurons, the principal neuron of the DG. In an unique way, in the adult DG, a subpopulation of progenitors with a radial morphology are retained in a quiescent state as adult radial glial-like cells (RGLs) in the subgranular zone (SGZ) of the DG and continue to produce new granule neurons throughout adult life. This raises questions about when and how adult RGLs are generated in the DG, which are essential questions to understand how neurogenic niches are generated and maintained in the adult brain. HMG-box transcription factors of Sox family genes could be at the core of those processes, as many of them have essential regulatory functions in both developmental and adult neurogenesis. In this study, we have focused on SoxD transcription factors (Sox5 and Sox6) in DG neurogenesis, as our laboratory has previously shown that they play a critical role in regulating cell cycle progression in progenitor cells and that they are expressed in the SGZ, both during DG development and in adulthood. We describe now that during DG development both Sox5 and Sox6 are persistently expressed in RGCs/RGLs and that their expression gradually turns off along the progression of those cells towards the neuronal lineage. By conditional deletion of Sox5 and Sox6 from early central nervous system development, we have determined that Sox5 is required for RGCs/RGLs to enter the quiescent state during postnatal development. Thus, deleting Sox5 expression during development results first in an increase in hippocampal neurogenesis in young adults and then, in mature adults, leads to an exhaustion of RGLs pool. Furthermore, we have found that BMP signaling target, Id2, could be mediating Sox5-regulated quiescence acquisition during DG development. Furthermore, we have found that Sox6 alone is less required than Sox5 for the development of DG, at least during first three postnatal weeks. Interestingly, selective creERT/tamoxifen-induced deletion of SoxD genes in the adult DG, showed that both Sox5 and Sox6 are required for RGLs to transit from quiescent into active proliferative states, and consequently they are needed for adult neurogenesis. Taken together, our results prove that the transition from developmental RGCs into adult RGLs during DG development is regulated by Sox5. These results set up the basis to further explore Sox5 direct targets to understand the molecular mechanism that involve how adult neurogenesis is specifically generated at certain brain areas and how could we modulate the neurogenic process in pathological and ageing brains.

Evidence for the generation of young neurons out of precursor cells in the adult brain, i.e. adult neurogenesis, exists for at least two brain regions. New nerve cells are generated in the subventricular zone of the olfactory bulb and in the subgranular zone of hippocampal dentate gyrus. Young neurons of the subgranular zone migrate along the rostral migratory stream to the olfactory bulb, where they functionally integrate and contribute to the discrimination of odors. In the hippocampus the function of newly formed granule cells is still a matter of debate, yet it is thought that adult neurogenesis functionally contributes to hippocampal functions. Over the last twenty years of extensive research it became clear that adult hippocampal neurogenesis (AHN) in laboratory rodents can be up-and down regulated by different internal and external stimuli. Physical exercise in a running wheel being among the factors that have been most investigated. Since voluntary exercise not only increases adult neurogenesis in the hippocampus but also beneficially affects learning and memory in laboratory mice and rats, a widespread assumption holds a direct relationship between AHN and cognitive brain health also in higher order species, including humans. However, translating findings in laboratory rodents to the human condition faces difficulties. Enormous differences in basal rates of adult neurogenesis have been reported between mammalian species. The low level of AHN in primates and the complete lack of adult neurogenesis in bat species indicate species-specific differences in adult neurogenesis not only on a regulatory but also on a functional level. For a better understanding of species-specific differences in the regulation of AHN, we investigated basal rates of adult neurogenesis in laboratory mice and closely related wild mouse strains as well as the reaction of AHN to motivationally different running conditions. Testing different wild- and laboratory mice in the same environment allowed the identification of species-specific differences as well as possible domestication effects. Basal rates of adult hippocampal neurogenesis in equally-aged and genetically identical laboratory C57BL/6 mice show individual differences possibly reflecting epigenetic factors. However, the initial level of adult neurogenesis does not influence the response to wheel-exercise. Voluntary physical exercise in laboratory mice always increases AHN but this positive effect cannot be additively stimulated by enhanced running and is even lost as soon as the mice are forced to run. Rewarding the mice for their performance leads to an increase in wheel activity but does not translate into a corresponding additive increase in adult neurogenesis. Likewise, a more naturalistic situation, in which laboratory mice must run to obtain their daily food does not lead to an increase in cell proliferation and entails only a small increase in the number of young neurons, far below the one in voluntary running mice. Wild wood mice (Apodemus sylvaticus) and wild-derived western house mice (Mus musculus domesticus), both close relatives of the common laboratory mouse strains, were tested in the same running situations as laboratory C57BL/6 mice. Besides species-differences in basal neurogenesis rate, we find adult neurogenesis in wild mice remaining relatively constant in response to external influences. None of the factors that normally affect AHN in laboratory animals, such as stress, environmental changes or physical exercise, have an effect on adult neurogenesis in these animals. In wood mice, neither voluntary wheel running nor stress or an impoverished cage environment affect the number of newly generated neurons. House mice also show a stable adult neurogenesis, which shows no significant change after voluntary running or running for food. Adult neurogenesis in the dentate gyrus is thus regulated differently in laboratory and wild mice. However, even in laboratory mice it is not as plastic as initially suggested: laboratory mice, which are tested in a more naturalistic and complex running situation, show rather weak plasticity of AHN, resembling wild mice. Hence, it seems that the regulatory difference in adult neurogenesis between laboratory- and wild mice is, that laboratory animals react to a single stimulus in absence of other inputs. We believe that the constant exposure to different stimuli potentially affecting AHN has led to a natural selection that stabilizes adult neurogenesis in the wild. In contrast, during domestication - including inbreeding - much of the homeostatic capacity in regulating adult neurogenesis might have been lost. Taken together, our data imply that genetic (species-specific differences as well as within-species variation) play an important role in determining basal rates of adult neurogenesis, while motivational-contextual factors modulate the response of AHN to physical exercise, albeit chiefly in domesticated laboratory strains . As such differences appear already between phylogenetically closely related species, extrapolating findings in laboratory mice to distantly related taxonomic groups, such as humans, obviously requires much caution.

Compared to the adult mammalian brain, the brain of the adult zebrafish Danio rerio exhibits a very high proliferative and regenerative potential. The adult mammalian brain in contrast has a very limited neurogenic capacity mainly restricted to two zones, the subventricular zone of the lateral telencephalic ventricles and the subgranular zone of the dentate gyrus of the hippocampus. In contrast, the zebrafish brain harbours 16 proliferation zones distributed all over the brain. The zebrafish has thus become a model for the study of adult neurogenesis and regeneration of nervous tissue. I characterized the expression of the two transcription factors p53 and p73 in the adult zebrafish brain. Both p53 and p73 were shown to play crucial roles in mammalian adult neurogenesis: p53 suppresses the self-renewal of adult neural stem cells and is involved in apoptotic death of neurons following damage. p73 is relevant for the survival of neurons, self-renewal and maintenance of neural stem cells as well as differentiation of precursor cells. It was thus of interest whether these genes have similar roles in the adult zebrafish brain. I established a detailed map of the expression pattern of p53 and p73 mRNA and p53 protein in the adult zebrafish brain. p53 and p73 mRNA expression overlaps in many regions including neurogenic zones. The p53 protein is expressed in most of these regions indicating that the mRNA expression reflects the protein expression. The p53 protein is expressed in mature neurons, Type I cells (non-dividing radial glial cells) and Type IIIa and Type IIIb cells (neuroblasts) in the adult zebrafish telencephalon. In cells of the oligodendrocyte lineage and in Type II cells (dividing radial glial cells) an expression of the p53 protein is not detectable. After stab injury of the adult zebrafish telencephalon both p53 and p73 genes are up-regulated. p53 is up-regulated in Type I cells. In contrast to the uninjured brain, p53 is expressed in cells of the oligodendrocyte lineage following injury. Furthermore, target genes of p53 are up-regulated and apoptosis is induced after stab injury. These results suggest a role for p53 in constitutive and regenerative neurogenesis. However, tp53M214K mutant zebrafish do not show any phenotype. The structurally related p73 is expressed in a very similar pattern as p53 in the uninjured and injured zebrafish brain. Therefore, redundancy between p53 and p73 may occlude the manifestation of a phenotype in the p53 mutant. Taken together, the analysis of expression of both p53 and p73 in the adult zebrafish brain suggests a role of these genes during constitutive and regenerative neurogenesis. The future elucidation of the precise function of the two genes in these processes requires, however, double mutant analysis.

A long-standing problem in the field of adult neurogenesis has been the need to identify newborn neurons and their precursors within a much larger population of preexisting mature neurons and glia. If these nascent cells could be identified, it would be possible to visualize and enumerate such cells in vivo, to access them for electrophysiological and molecular studies, to identify their connections in the neuronal networks, and to alter their activity and function. Several strategies have been developed to solve this problem of finding the proverbial needle in a haystack. Methods such as labeling with thymidine analogs, phenotypic analysis based on the expression of developmental markers, and retro- and lentiviral labeling have each had an important role in advancing our understanding of the proliferation and maturation of newborn neurons in the adult brain. As with all methods, these techniques have advantages and limits that demarcate their appropriate application. In this review, we focus on genetic approaches to studying adult mammalian neurogenesis, describing reporter lines of transgenic mice and summarizing recent advances that employ these emerging technologies. The general strategy of these genetic approaches is to drive the expression of “live” markers such as green fluorescent protein (GFP) in a defined population of neurons, neuronal progenitors, or stem cells. Cytoplasmic expression of fluorescent proteins (FPs) allows the full morphology of labeled cells to be visualized, whereas nuclear expression of such proteins facilitates cell enumeration. FP expression also allows labeled cells to be identified and accessed in live animals and in acute...

Special issue editorial: Glial plasticity in health and disease.

BackgroundAging significantly elevates the risk of developing neurodegenerative diseases. Neuroinflammation is a universal hallmark of neurodegeneration as well as normal brain aging. Which branches of age-related neuroinflammation, and how they precondition the brain toward pathological progression, remain ill-understood. The presence of elevated type I interferon (IFN-I) has been documented in the aged brain, but its role in promoting degenerative processes, such as the loss of neurons in vulnerable regions, has not been studied in depth.MethodsTo comprehend the scope of IFN-I activity in the aging brain, we surveyed IFN-I-responsive reporter mice at multiple ages. We also examined 5- and 24-month-old mice harboring selective ablation of Ifnar1 in microglia to observe the effects of manipulating this pathway during the aging process using bulk RNA sequencing and histological parameters.ResultsWe detected age-dependent IFN-I signal escalation in multiple brain cell types from various regions, especially in microglia. Selective ablation of Ifnar1 from microglia in aged mice significantly reduced overall brain IFN-I signature, dampened microglial reactivity, lessened neuronal loss, restored expression of key neuronal genes and pathways, and diminished the accumulation of lipofuscin, a core hallmark of cellular aging in the brain.ConclusionsOverall, our study demonstrates pervasive IFN-I activity during normal mouse brain aging and reveals a pathogenic, pro-degenerative role played by microglial IFN-I signaling in perpetuating neuroinflammation, neuronal dysfunction, and molecular aggregation. These findings extend the understanding of a principal axis of age-related inflammation in the brain, one likely shared with multiple neurological disorders, and provide a rationale to modulate aberrant immune activation to mitigate neurodegenerative process at all stages.

From physical motion to cognition, every physiological process of the human body is precisely controlled by our brain. To maintain its vital activity, our brain cells demand elevated levels of energy, which are tightly regulated to satisfy a critical homeostatic cellular balance. Thus, anomalies in brain homeostasis provoke devastating neurodegenerative diseases (NDs), which are among the most common cause of death in the Western world. To date, a cure for NDs does not exist. This is due to the current lack of knowledge on the understanding of the molecular mechanisms involved in normal brain ageing and the key players that trigger neuropathology. Human genetics have confirmed risk alleles associated with protein aggregation, mitochondrial biology and protein-quality control. A common root of NDs and senescence is protein misfolding and brain deposits of ordered protein structures referred to as amyloid. Parkinson’s Disease (PD) is the second most common neurodegenerative disease affecting the central and peripheral nervous system. PD pathology is defined by the abnormal accumulation of the protein alpha-synuclein in neurons, nerve fibers and glial cells, characteristic of alpha-synucleinopathies. Key neuropathological hallmarks of PD are Lewy bodies (LBs) and Lewy neurites (LNs), which are alpha-synuclein-positive brain inclusions. Interestingly, the biological processes by which LBs and LNs are formed as well as their role in neurodegeneration await to be defined. Overall, our knowledge on the ultrastructure of the human brain is extremely limited. Understanding human brain ultrastructure is central to describe its function, and therefore, identify physical alterations of senescence vs disease. This is essential to design novel therapies and translational animal and cellular models for NDs and ageing. Electron microscopy (EM) visualizes cellular and tissue ultrastructure at high-resolution by two- (2D) and three-dimensional (3D) imaging. These studies have helped to unravel the nature of the structural components that compose human brain bodies, gaining insights into their possible origins and biological roles. However, a modern description of the ultrastructure of human brain aggregates by applying modern 3D EM technologies is largely lacking. The first part of my thesis is focused on the ultrastructural characterization of human brain aggregates in PD and senile non-demented donors by means of 3D correlative light and electron microscopy (CLEM) approaches. In Chapter 2, we describe the detailed EM characterization of Lewy pathology alongside a multi-scale CLEM pipeline specifically developed for this study. Furthermore, in Chapter 3, we analysed the ultrastructural composition and cellular origins of the age-related brain body defined as Corpora amylacea (CA). Overall, these studies revealed an astonishing heterogeneity in Lewy pathology: previously described filament-containing LBs are identified, however, the vast majority of Lewy pathology, including LBs and LNs, possessed a predominant organellar nature composed of membrane stacks, disrupted cell organelles and dysmorphic cytoskeletal elements. In the case of CA, they are visualized as electron-dense granular aggregates composed of packed membranes and morphologically preserved cell organelles. Further, we provide strong indications that CA originate intracellularly in astrocytic feet forming the glymphatic system of the brain, pointing to a physiological role on sequestering toxic metabolites to prevent tissue inflammation via clearance through the cerebral spinal fluid. The second part of this thesis is focused on cryo-electron tomography (cryo-ET) and the software developments implemented for 3D image processing and subtomogram averaging (STA). Cryo-ET has the enormous potential of in situ visualization of hydrated-frozen biological samples within their native context at high-resolution and in 3D. However, several bottlenecks are identified in the cryo-ET pipeline, such as sample preparation, data acquisition, tilt series alignment and STA, thus far preventing high-throughput cryo-ET. Usually specimen preparation and data acquisition are sample-dependent; however, tilt series alignment is a deterministic step that should be straightforward, but it is currently an important drawback in cryo-ET since alignment is not automated and errors require several rounds of manual intervention. Importantly, alignment errors result in a substantial decrease of the final resolution in the 3D reconstructed tomograms. Thus, in Part II, Chapter 2, we present an automatic tilt series alignment approach for high-resolution cryo-ET data sets. We described our algorithm and the factors to be considered when aiming for high-resolution STA. In Part II, Chapter 3, we implement computational tools and strategies for STA of membrane-bound protein macro-complexes. Furthermore, we present several user-friendly and flexible protocols for STA that can be applied to a wide spectrum of cryo-ET data sets. The results comprised in this thesis highly improve our understanding of the structural basis and origins of human brain bodies in senescence and neurodegeneration. Alongside the biological results, a multi-scale pipeline that combines microscopy and advanced image processing approaches are established for nanoscale visualization of the human brain.

Tuberous sclerosis complex (TSC) is a relatively rare genetic disease characterized by the formation of benign tumors or hamartomas in multiple organs. The tumors are noninvasive and rarely transform to metastatic lesions. TSC is an autosomal dominant disorder that results from mutations in the TSC1 or TSC2 genes. Neurologically, individuals with TSC have severe complications, including refractory seizures, autism, mental retardation, learning difficulties, and changes in behavior. Tubers in the cerebral cortex, subependymal nodules (SENs) along the lateral walls of the lateral ventricles, and subependymal giant cell (GC) astrocytomas are characteristic brain lesions in patients with TSC. Astrocytic-like cells immunopositive for both glial and neuronal markers, dysplastic neurons (DNs), and GCs immunopositive for nestin and vimentin, as well as for proliferation markers such as proliferating nuclear cell antigen (PCNA) and Ki-67, are histological hallmarks of the disease. DNs and GCs retain their ability to re-enter the cell cycle and are immunopositive for markers of neural progenitor and stem cells. Neurogenesis occurs in the adult brain of mammals, particularly in the hippocampus and subventricular zone (SVZ). In the SVZ, newly generated neuronal cells migrate along the ventricle and a SVZ origin for brain tumors in the adult brain have been reported. These brain tumors express markers of neural progenitor and stem cells. The study of analogies and differences between SENs in TSC, neurogenesis in the SVZ, and tumors in the adult brain would reveal clues on the development and origin of SENs and brain tumors. Keywords: Epilepsy, Cancer, Drug, Disease, Neural stem cells, Rapamycin, Therapy, Tumor DOI:10.5055/jndr.2013.0012

The cerebral cortex is one of the most important anatomic foundations of cognitive and memory functions; both functions are affected by aging, particularly when this process is associated with demential such as the Alzheimer type (1,2). Nevertheless, available knowledge about the cytoarchitecture and quantitative structural changes in normal brain aging, particularly in the cortex are still scarce and even contradictory (3,4). The heterogeneity of the studies, as well as the fact that the effects of aging differ in cerebral zones, animal models, and individuals, are the main causes of these discrepancies (2,5).

The term “adult neurogenesis” is used to describe the observation that, in the adult mammalian brain, new neurons are born from stem cells residing in discrete locations and these new neurons migrate, differentiate, and mature into newly integrated, functioning cells. By virtue of this definition, adult neurogenesis is a process, not an event, and as such, can be dissected and examined in evermore discrete components. In general, researchers seek a complete understanding of not only the details of these separate components but also the purpose and function of this process as a whole. Once the tools became available to monitor and measure adult neurogenesis, the interest in this process grew enormously, not the least because the birth and integration of new neurons in the adult brain constitute the most extreme cases of neuroplasticity in the adult brain. While the phenomenon is interesting enough to investigate and understand in the normal, healthy brain, the fact that this process is also disrupted in many disease states adds substantially to the numbers of those studying adult neurogenesis. As a result, a new way of looking at brain therapy has emerged that incorporates the potential of generating new neurons in the context of aging and disease into the search for a strategy for “self-repair.” The idea for this book originated from a meeting on adult neurogenesis in the adult brain held at the Banbury Conference Center at Cold Spring Harbor Laboratory in February 2006. In the secluded and intimate setting of this event, the

The fact that continuous proliferation of stem cells and progenitors, as well as the production of neurons, occurs in the adult CNS raises several basic questions concerning the number of neurons required in a particular system: Can we observe a continued growth of brain regions that sustain neurogenesis? Or does an elimination mechanism exist that keeps the number of cells constant? If so, are the old ones replaced or are the new neurons competing for limited network access? What signals would support their survival and integration and what factors are responsible for their elimination? This chapter addresses these and other questions regarding regulatory mechanisms affecting adult neurogenesis by controlling cell survival. ARE NEUROGENIC BRAIN REGIONS EXPANDING DESPITE SPACE LIMITATIONS? This question was initially addressed several decades ago, following the first evidence that adult mammalian neurogenesis exists. Total neuronal cell counts of the olfactory bulb (OB) and dentate gyrus (DG) at different ages revealed that in both regions, a continued growth of the granule cell layer occurs throughout adult life. From 1 month of age, when the developmental production of granule cells can be considered complete, until 1 year of age, the number of DG granule cells doubles in the rat (Bayer 1982; Bayer et al. 1982). A rise in total volume and increased cell density due to reduced cell diameter both contribute to this phenomenon. In the rat OB, a linear growth of the granule cell layer was observed with age (Kaplan et al. 1985), with the number of olfactory...

Age and activity might be considered the two antagonistic key regulators of adult neurogenesis. Whereas adult neurogenesis declines with age, different kinds of “activities” positively regulate adult neurogenesis. An interaction between these two mechanisms exists. Aging influences aging, and activity affects aging processes. Aging is a principal determinant of life and as such cuts across all biological, psychological, and sociological research. The very essence of aging is difficult to conceptualize, because it is a uniquely omnipresent variable. Aging can thus only be addressed in an transdisciplinary approach, especially if the consequences of aging on complex brain functions are to be studied. In the context of neurogenesis research, “aging” has so far been largely equaled with the biology of long timescales. Implicit in this understanding is that age-dependent changes essentially reflect a unidirectional development in that everything builds on what has occurred before. In this sense, aging can also be seen as continued or lifelong development. This idea has limitations but is instructive with regard to adult neurogenesis because adult neurogenesis is neuronal development under the conditions of the adult brain. The age-related alterations of adult neurogenesis themselves have quantitative and qualitative components. So far, most research has focused on the quantitative aspects. But there can be little doubt that qualitative changes do not simply follow quantitative changes, for example, in cell or synapse numbers but emerge on a systems level and above, when an organism ages. The observation that adult neurogenesis is regulated by activity relates to this idea. From...

Rho small GTPases are members of the Ras superfamily of monomeric 20 ~ 30 kDa GTP-binding proteins. These proteins function as molecular switches that regulate various cellular processes such as migration, adhesion and proliferation. Cycling between GDP-bound inactive and GTP-bound active forms is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and GDP-dissociation inhibitors (GDIs). Among 20 different mammalian Rho GTPases identified to date, RhoA, Rac1 and Cdc42 have been most extensively investigated; regulation of migration, adhesion and proliferation by these proteins have been well documented in a variety of cell types, including neurons. In neurons, RhoA, Rac1 and Cdc42 are crucial for axon guidance, dendrite formation and spine morphogenesis, where molecular machineries required for cell migration and adhesion play essential roles. Recently, accumulating experimental data indicate the participation of Rho GTPases in neuronal cell migration. To establish the cortical lamination and synapse network formation, highly specialized modes of neuron migration are essential, which include 1) radial migration of excitatory pyramidal neurons along radial glial fibers, 2) tangential migration of GABAergic cortical (inhibitory) interneurons along emerging axon tracts and 3) chain migration of interneurons ensheathed in a glial network, which is observed only in olfactory bulb-directed adult neurogenesis. While roles of Rho GTPases in the radial migration have been well reviewed, knowledge of their functions in tangential migration and chain migration are fragmentary to date. In this review, we focus on the roles of Rho small GTPases and their related molecules in tangential migration, together with the possible application of the electroporation method to analyses for this mode of migration in embryonic and postnatal mouse brain.

Normal brain aging is commonly associated with neural activity alteration, β-amyloid (Aβ) deposition, and tau aggregation, driving a progressive cognitive decline in normal elderly individuals. Positron emission tomography (PET) with radiotracers targeting these age-related changes has been increasingly employed to clarify the sequence of their occurrence and the evolution of clinically cognitive deficits. Herein, we reviewed recent literature on PET-based imaging of normal human brain aging in terms of neural activity, Aβ, and tau. Neural hypoactivity reflected by decreased glucose utilization with PET imaging has been predominately reported in the frontal, cingulate, and temporal lobes of the normal aging brain. Aβ PET imaging uncovers the pathophysiological association of Aβ deposition with cognitive aging, as well as the potential mechanisms. Tau-associated cognitive changes in normal aging are likely independent of but facilitated by Aβ as indicated by tau and Aβ PET imaging. Future longitudinal studies using multi-radiotracer PET imaging combined with other neuroimaging modalities, such as magnetic resonance imaging (MRI) morphometry, functional MRI, and magnetoencephalography, are essential to elucidate the neuropathological underpinnings and interactions in normal brain aging.

Adult neurogenesis (ANGE) is a process of generating new neurons in the brains of adult mammals, including humans. It takes place, e.g., in the subgranular zone of the dentate gyrus in the hippocampal formation. The function of the new neurons is not fully explained; however, they are considered to play an important role in learning and memory processes. There is also evidence that ANGE can mediate the response of hippocampal formation to stress, preventing the onset of depression. Besides, newly-generated neurons seem to play an important role in therapeutic action of antidepressants (AD). Results from animal models and human studies, confirming and questioning the hypothesis of a key connection between depression and ANGE, are presented. It is not clear whether the suppression of the production of new neurons influences the pathogenesis of depression and it seems that some other factors are more important. However, it is likely that the level of ANGE is important in treatment of at least some forms of depression. Several experiments, using animal models, have shown that AD, mood stabilizers or other depression therapies increase the level of ANGE. Also, blocking the generation of new neurons abolishes their therapeutic effect. Nevertheless, some recent publications question the significance of ANGE in AD action. The discrepancies described herein, concerning the significance of ANGE in aetiology and treatment of depression, may reflect the complexity of the depressive disorder. This complexity is manifested by the different response (or no response) to various AD and other depression therapies in human patients.