ATP6AP2 over-expression causes morphological alterations in the hippocampus and in hippocampus-related behaviour.

The (pro)renin receptor [(P)RR], also known as ATP6AP2 [ATPase 6 accessory protein 2], is highly expressed in the brain. ATP6AP2 plays a role in early brain development, adult hippocampal neurogenesis and in cognitive functions. Lack of ATP6AP2 has deleterious effects, and mutations of ATP6AP2 in humans are associated with, e.g. X-linked intellectual disability. However, little is known about the effects of over-expression of ATP6AP2 in the adult brain. We hypothesized that mice over-expressing ATP6AP2 in the brain might exhibit altered neuroanatomical features and behavioural responses. To this end, we investigated heterozygous transgenic female mice and confirmed increased levels of ATP6AP2 in the brain. Our data show that over-expression of ATP6AP2 does not affect adult hippocampal neurogenesis, exercise-induced cell proliferation, or dendritic spine densities in the hippocampus. Only a reduced ventricular volume on the gross morphological level was found. However, ATP6AP2 over-expressing mice displayed altered exploratory behaviour with respect to the hole-board and novel object recognition tests. Moreover, primary adult hippocampal neural stem cells over-expressing ATP6AP2 exhibit a faster cell cycle progression and increased cell proliferation. Together, in contrast to the known deleterious effects of ATP6AP2 depletion, a moderate over-expression results in moderate behavioural changes and affects cell proliferation rate in vitro.

Implications of p75NTR for dentate gyrus morphology and hippocampus-related behavior revisited.

The pan-neurotrophin receptor p75NTR is expressed in the adult brain in a discrete pattern. Although numerous studies have addressed its implications for hippocampal functions, the generated sets of data are surprisingly conflicting. We have therefore set out to re-investigate the impact of a deletion of the full-length p75NTR receptor on several parameters of the dentate gyrus (DG), including neurogenesis and hippocampus-related behavior by using p75NTR(ExIII) knockout mice. Moreover, we investigated further parameters of the DG (cholinergic innervation, dendritic spines). In addition, we analyzed on the morphological level the impact of aging by comparing adult and aged p75NTR(ExIII) mice and their age-matched littermates. Adult (4-6 months old), but not aged (20 months old), p75NTR(ExIII) knockout mice display an enhanced volume of the DG. However, adult neurogenesis within the adult DG was unaffected in both adult and aged p75NTR(ExIII) knockout mice. We could further demonstrate that the change in the volume of the DG was accompanied by an increased cholinergic innervation and increased spine densities of granule cells in adult, but not aged p75NTR deficient mice. These morphological changes in the adult p75NTR deficient mice were accompanied by specific alterations in their behavior, including altered behavior in the Morris water maze test, indicating impairments in spatial memory retention.

Effects of the angiotensin-(1-7) receptor Mas on cell proliferation and on the population of doublecortin positive cells within the dentate gyrus and the piriform cortex.

Aside from the well-known biologically active angiotensin II, other biologically active angiotensins have been discovered, including angiotensin IV and angiotensin-(1-7). Some years ago, we and others discovered that the Mas proto-oncogene encodes a G protein-coupled receptor being essential for angiotensin-(1-7) signaling. Mas is not only expressed in the periphery but also within the brain, e.g. in the dentate gyrus (DG) and the piriform cortex (PC). Since the DG is capable of adult neurogenesis, we examined the impact of a deletion of Mas upon adult neurogenesis. Deletion of Mas did not alter cell proliferation in the adult DG (as monitored with phosphohistone H3) and did not alter cell death (as monitored with activated Caspase 3). However, Mas deficiency resulted in an increase in the number of doublecortin (DCX) positive cells, indicating that lack of Mas increases the number of this cell population. Concerning the PC, it is discussed whether adult neurogenesis occurs under physiological conditions in this area. We could demonstrate that Mas deficiency has an impact on cell division and on the population of DCX-positive cells within the PC. Since Mas is not expressed before birth within the brain, our data may suggest that adult hippocampal neurogenesis and neurogenesis occurring during prenatal development share several common mechanisms, but are, at least in part, differentially regulated. Moreover, since deficiency for Mas increases the numbers of DCX-positive young neurons, blockage of Mas might be beneficial in stimulating neurogenesis in adults.

The (pro)renin receptor / ATP6ap2 is expressed in the murine hippocampus by adult and newly generated neurons.

PURPOSE: The (pro)renin receptor ((P)RR) is receptor that has been shown to be involved in developmental processes. Adult neurogenesis shares many similarities with fetal and embryonic neuronal development, but is restricted to some brain areas, including the hippocampus. We therefore investigated the expression of the (P)RR within the adult hippocampal formation and investigated whether (P)PR is expressed by adult and newly generated neurons in the dentate gyrus. METHODS: (P)PR protein expressing cells in the hippocampus were analyzed using immunohistochemistry. Double-labeling with markers for adult neurogenesis was used to investigate whether newly formed cells also express (P)PR. RESULTS: (P)PR is expressed by neuronal cells with the hippocampus. (P)RR protein is expressed during different stages of adult neurogenesis within the dentate gyrus (DG). (P)RR is not expressed by Sox2 positive neuronal stem cells, but by doublecortin positive cells located both in the subgranular zone and the granular layer of the DG. CONCLUSIONS: The results indicate that (P)PR is mainly expressed by adult neurons in the hippocampus as well as in late stages of adult neurogenesis within the hippocampus. However, to clarify the involvement of this receptor in adult hippocampal neurogenesis and neuronal cell differentiation in detail, functional analyses needed to be performed.

Immunohistochemical localization of the angiotensin-(1-7) receptor Mas in the murine forebrain.

Apart from the well-known biologically active angiotensin II, other biologically active angiotensins have been discovered, including angiotensin IV and angiotensin-(1-7). Some years ago, we and others discovered that the Mas proto-oncogene encodes a receptor that is essential for angiotensin-(1-7) signaling. Angiotensin-(1-7) is not only expressed in the periphery but also within the brain. Based on that, we examined the distribution of Mas within the murine brain, using an antibody directed against the 3(rd) cytoplasmic loop of the receptor protein. Strongest Mas protein expression was detected in the dentate gyrus of the hippocampus and within the piriform cortex. However, Mas protein expression is not restricted to these areas, since Mas immunopositive neurons were also seen in different parts of the cortex, hippocampus, amygdala, basal ganglia, thalamus and hypothalamus. Based on the expression of Mas protein in the cortex and the limbic system, angiotensin-(1-7) signaling may play a role in synaptic plasticity, learning, memory and emotion, as has been described for angiotensin II and IV.

Roles of exogenous and endogenous FGF-2 in animal models of depression.

PURPOSE: Several members of the fibroblast growth factor (FGF) family have been shown to be dysregulated in individuals with major depression, and treatment with antidepressants has been reported to increase FGF-2 mRNA levels in the forebrain. METHODS: We have used the tail suspension test (TST), and olfactory bulbectomy (OBX), and FGF-2 deficient mice to investigate putative roles of FGF-2 as an antidepressant and mediator of antidepressive drug actions. RESULTS: FGF-2 applied intraventricularly generated antidepressant-like effects in the TST. FGF-2, similar to the antidepressant amitriptyline, attenuated neuron demise in the piriform cortex and posterolateral cortical nucleus of the amygdala following OBX. Moreover, OBX induced reduction in hippocampal neurogenesis could be ameliorated by subsequent treatment with either amitriptyline or FGF-2. Furthermore, FGF-2 was effective in reversing depressive-like behavior induced by OBX, monitored in the locomotor activity and the passive avoidance test. In bulbectomized FGF-2 deficient mice, treatment with amitriptyline protected neurons, but failed to reverse behavioral alterations. CONCLUSIONS: Together, these results suggest that FGF-2 constitutes both a potential target for antidepressive treatments and an important growth factor in the cytokine network underlying the actions of antidepressive drugs. The results further suggest a requirement of endogenous FGF-2 for mediating behavioral, but not neuroprotective actions of amitriptyline.

Expression of trkB and trkC receptors and their ligands brain-derived neurotrophic factor and neurotrophin-3 in the murine amygdala.

The neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their cognate receptors, trkB and trkC, have a variety of physiological brain functions, ranging from cell survival to mechanisms involved in learning and memory and long-term potentiation (LTP). LTP can be induced in the cortex and hippocampus, as well as within the amygdala. However, the role of neurotrophins in amygdalar LTP is largely unknown. Expression patterns of BDNF and NT-3 and their cognate receptors in the adult mouse amygdala have not been analyzed in detail. We have therefore examined the expression of trkB, trkC, BDNF, and NT-3 mRNA and protein in different amygdalar nuclei as well as in the hippocampal areas CA1-CA3 and the dentate gyrus. The distribution pattern of trkB, trkC, BDNF, and NT-3 mRNA in the murine hippocampus is comparable to that seen in rats. Within most amygdalar nuclei, a moderate BDNF mRNA expression was found; however, BDNF mRNA was virtually absent from the central nucleus. No expression of NT-3 mRNA was found within the amygdala, but trkC mRNA-expressing cells were widely distributed within this brain region. trkB mRNA was strongly expressed in the amygdala. Because trkB is expressed in a full-length and a truncated form (the latter form is also expressed by nonneuronal cells), we also investigated the distribution of full-length trkB mRNA-expressing cells and could demonstrate that this version of trkB receptors is also widely expressed in the amygdala. These results can serve as a basis for studies elucidating the physiological roles of these receptors in the amygdala.

TrkB but not trkC receptors are necessary for postnatal maintenance of hippocampal spines.

Dendritic spines are major sites of excitatory synaptic transmission and changes in their densities have been linked to alterations in learning and memory. The neurotrophins brain-derived neurotrophic factor and neurotrophin-3 and their receptors, trkB and trkC, are thought to be involved in learning, memory and long-term potentiation (LTP). LTP is known to induce trkB and trkC gene expression as well as spinogenesis in the hippocampus. In the aging hippocampus, declines in trkB and trkC mRNA levels may underlie, at least in part, impairments in spatial memory and reductions in spine densities. To determine the significance of trkB and trkC for the maintenance of dendritic spines, we have analyzed Golgi-impregnated hippocampi of adult and aged mice heterozygous for trkB, trkC, or both along with respective wildtype littermates. Deletion of one allele of trkB, but not trkC, significantly reduces spine densities of CA1 pyramidal neurons in both adult and aged mice, as compared to age-matched controls. This indicates that trkB, but not trkC, receptors are necessary for the maintenance of hippocampal spines during postnatal life.

Immunohistological markers for staging neurogenesis in adult hippocampus.

Neurogenesis in the adult dentate gyrus (DG) of the hippocampus occurs constitutively throughout postnatal life, and the rate of neurogenesis within the DG can be altered under various physiological and pathophysiological conditions. Adult neurogenesis includes the process in which the division of a precursor cell takes place and the multi-step process (proliferation, differentiation, migration, targeting, and synaptic integration) that ends with the formation of a postmitotic functionally integrated new neuron. During specific time-frames of adult neurogenesis, various markers are expressed that correlate with the differentiation steps along the pathway from early progenitor cells to newly generated postmitotic neurons within the DG. Markers that are currently widely used for the investigation of adult hippocampal neurogenesis are: glial fibrillary acidic protein, nestin, Pax6, NeuroD, PSA-NCAM, doublecortin, TUC-4, Tuj-1, and calretinin. The discovery and development of specific markers that allow the time-course and fate of neurons to be followed during adult neurogenesis in a detailed and precise fashion are not only helpful for gaining further insights into the genesis of new neurons in the hippocampus, but also might be applicable to the development of strategies for therapeutic interventions.

Antidepressant-mediated reversal of abnormal behavior and neurodegeneration in mice following olfactory bulbectomy.

Olfactory bulbectomy is an established animal model of depression that has been mostly investigated in rats. As in human major depression, bulbectomy induces behavioral alterations that can be ameliorated by chronic antidepressant treatment. Furthermore, bulbectomy in rats is known to induce neurodegeneration in some brain areas. The aim of the present study was to evaluate patterns of behavioral alterations and neurodegeneration, and their drug-induced reversibility, in bulbectomized mice. Our results reveal that in mice bulbectomy increases locomotor activity and induces deficits in the passive avoidance test, comparable to the effects seen in rats. Bulbectomy also induced neuronal degeneration, visualized by incorporation of Fluoro-Jade B, in the piriform cortex and the posterolateral cortical amygdaloid nucleus (PLCo). Chronic treatment with the antidepressant amitriptyline protected neurons in both areas and efficiently reversed abnormal locomotor activity induced by bulbectomy. Treatment with citalopram was effective in reversing the behavioral deficits induced by bulbectomy, but did not protect against neurodegeneration. Our data indicate significant overlap between mice and rats in terms of behavioral alterations and neurodegeneration following olfactory bulbectomy. However, there are differences in drug-mediated reversibility of neurodegeneration suggesting that neurodegeneration and protection may be "second-order" features in this animal model of depression. This may also be relevant in the context of studies using genetically altered mice.

Neurotrophin receptor heterozygosity causes deficits in catecholaminergic innervation of amygdala and hippocampus in aged mice.

We have recently shown that aged mice with haploinsufficiencies for the neurotrophin receptors trkB, trkC or both, trkB and trkC, display reduced cell numbers in the substantia nigra and in the dentate gyrus, but not in the amygdala. Moreover, both hippocampus and amygdala contain increased numbers of degenerated axonal fragments. Consistent with this observation and the expression of trkB and trkC by midbrain dopaminergic neurons, we show now that heterozygous deletion of the trkB or/and trkC receptor genes significantly reduces catecholaminergic, tyrosine hydroxylase (TH-) positive fiber densities in the hippocampus and amygdala mainly in aged (21-23 month old) mice. In the amygdala the phenotype was restricted to the lateral and basolateral nucleus of the amygdala. In adult (6 month old) mice, reductions in catecholaminergic fiber densities were only found in the hippocampal area CA3 and the dentate gyrus of heterozygous trkB and trkB/C mice. Our observations suggest that signaling through trkB and trkC neurotrophin receptors is important for the maintenance of the catecholaminergic innervation of two limbic key regions, the hippocampus and amygdala.

The CNS renin-angiotensin system.

The renin-angiotensin system (RAS) is one of the best-studied enzyme-neuropeptide systems in the brain and can serve as a model for the action of peptides on neuronal function in general. It is now well established that the brain has its own intrinsic RAS with all its components present in the central nervous system. The RAS generates a family of bioactive angiotensin peptides with variable biological and neurobiological activities. These include angiotensin-(1-8) [Ang II], angiotensin-(3-8) [Ang IV], and angiotensin-(1-7) [Ang-(1-7)]. These neuroactive forms of angiotensin act through specific receptors. Only Ang II acts through two different high-specific receptors, termed AT1 and AT2. Neuronal AT1 receptors mediate the stimulatory actions of Ang II on blood pressure, water and salt intake, and the secretion of vasopressin. In contrast, neuronal AT2 receptors have been implicated in the stimulation of apoptosis and as being antagonistic to AT1 receptors. Among the many potential effects mediated by stimulation of AT2 are neuronal regeneration after injury and the inhibition of pathological growth. Ang-(1-7) mediates its antihypertensive effects by stimulating the synthesis and release of vasodilator prostaglandins and nitric oxide and by potentiating the hypotensive effects of bradykinin. New data concerning the roles of Ang IV and Ang-(1-7) in cognition also support the existence of complex site-specific interactions between multiple angiotensins and multiple receptors in the mediation of important central functions of the RAS. Thus, the RAS of the brain is involved not only in the regulation of blood pressure, but also in the modulation of multiple additional functions in the brain, including processes of sensory information, learning, and memory, and the regulation of emotional responses.

FGF-2 deficiency does not alter vulnerability of the dopaminergic nigrostriatal system towards MPTP intoxication in mice.

Fibroblast growth factor 2 (FGF-2) was the first growth factor discovered that exerted prominent protective and regenerative effects in an animal model of Parkinson's disease, the MPTP-lesioned dopaminergic nigrostriatal system. To address the putative physiological relevance of endogenous FGF-2 for midbrain dopaminergic neurons, we have analysed densities of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum and amygdala of adult FGF-2-deficient mice. We found that densities of TH-immunoreactive (ir) cells in the SN as well as densities of TH-ir fibers in the striatum and amygdala were unaltered as compared with wild-type littermates. There is evidence to suggest that growth factor deficits do not become apparent unless a system is challenged in a lesioning paradigm. We therefore tested the ability of the nigrostriatal system with respect to its ability to cope with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) intoxication. Treatment with 20 mg/kg MPTP on three consecutive days reduced dopamine levels in the striatum by about 80%. Densities of TH-positive neurons in the SN were reduced by 71%. However, both parameters did not significantly differ between FGF-2(-/-) mice and wild-type littermates. Our results therefore suggest that FGF-2, despite its prominent pharmacological potency as a neurotrophic factor for the dopaminergic nigrostriatal system, is not crucial for maintaining its structural integrity and ability to cope with MPTP intoxication.

Age-related alterations in hippocampal spines and deficiencies in spatial memory in mice.

Alterations in neuronal morphology occur in the brain during normal aging, but vary depending on neuronal cell types and brain regions. Such alterations have been related to memory and cognitive impairment. Changes in hippocampal spine densities are thought to represent a morphological correlate of altered brain functions associated with hippocampal-dependent learning and memory. We therefore have analyzed the impact of aging on different hippocampal-dependent learning tasks and on changes in dendritic spines of CA1 hippocampal and dentate gyrus neurons by analyzing adult (6-7 months) and aged (21-22 months) C57/Bl6 mice. We found a significant decrease in spine numbers of basal CA1 dendrites and decreases in spine length of apical dendrites of CA1 and dentate gyrus neurons. Furthermore, aged mice exhibited significant deficits in hippocampus-dependent learning tasks, such as the probe trial of the Morris water maze and T maze learning. Given the fact that there is no neuronal loss in the hippocampus in aged mice (von Bohlen und Halbach and Unsicker [2002] Eur. J. Neurosci. 16:2434-2440), we suggest that the memory and cognitive decline in the context of aging may be accompanied by rather subtle anatomical changes, such as numbers and morphology of dendritic spines.

The renin-angiotensin system in the mammalian central nervous system.

The brain renin-angiotensin system enables the formation of different biological active forms of angiotensins within the brain. All enzymes and peptides necessary for the biosynthesis of these angiotensins have been recognized within the central nervous system. Since there are considerable mismatches concerning the localization of the different enzymes, this system is not fully understood. Moreover, since alternative pathways of the angiotensin biosynthesis exists, localization and generation, especially of the short forms of biologically active angiotensins, are largely enigmatic. The brain renin-angiotensin system mediates several classic physiological effects including body water balance, maintenance of blood pressure, sexual behaviors, and regulation of pituitary gland hormones. Beside these classic functions, the brain renin-angiotensin system has more subtle functions involving complex mechanisms such as learning and memory. The mechanisms of action seem to differ depending on the utilized different bioactive angiotensin fragments, which are formed by the action of a variety of enzymes. This phenomenon appears to represent an important mechanism for neuromodulation. Moreover, there is evidence to suggest that the renin-angiotensin system is involved in neurological disorders, as e.g. Alzheimer's or Parkinson's disease.

Localization of DJ-1 protein in the murine brain.

Mutations in the DJ-1 gene have been identified to cause Parkinson's disease. In humans, nonmutated DJ-1 is expressed in specific brain areas but seems to be expressed by astrocytes rather than by neurons. In contrast, DJ-1 mRNA is mainly found in neurons in the mouse brain. We have investigated the distribution of DJ-1 protein in the mouse brain and found that DJ-1 protein is predominantly expressed by neurons but can also be detected in astrocytes. Consistent with a global role of DJ-1 in the brain, we found immunoreactivity, for example, in cortical areas, hippocampus, basolateral amygdala, the reticular nucleus of the thalamus, zona incerta, and locus coeruleus. Within the substantia nigra, however, DJ-1 is localized in both neuronal and nonneuronal cells, suggesting a distinct role in this area.

Modeling neurodegenerative diseases in vivo review.

Parkinson's disease (PD) is one of the major neurodegenerative disorders. The etiology of this disease is likely due to combinations of environmental and genetic factors. Symptomatic hallmarks of PD are tremor, bradykinesia, rigidity and postural instability. On the morphological and anatomical level, PD is characterized by massive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to a severe loss of striatal dopaminergic fibers and to a massive reduction of dopamine levels in the striatum. In addition, PD is characterized by the appearance of Lewy bodies within the surviving dopaminergic neurons. Animal models of PD allow getting insight into the mechanisms of several symptoms of PD thereby providing indispensable tools for basic and applied research. The biochemical and cellular changes that occur following administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in rodents or monkeys are remarkably similar to those seen in idiopathic PD. In this review, the main characteristics of experimental models of PD induced by the neurotoxic compound MPTP are reviewed.

Genes, proteins, and neurotoxins involved in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder. The etiology of PD is likely due to combinations of environmental and genetic factors. In addition to the loss of neurons, including dopaminergic neurons in the substantia nigra pars compacta, a further morphologic hallmark of PD is the presence of Lewy bodies and Lewy neurites. The formation of these proteinaceous inclusions involves interaction of several proteins, including alpha-synuclein, synphilin-1, parkin and UCH-L1. Animal models allow to get insight into the mechanisms of several symptoms of PD, allow investigating new therapeutic strategies and, in addition, provide an indispensable tool for basic research. In animals PD does not arise spontaneously, thus, characteristic and specific functional changes have to be mimicked by application of neurotoxic agents or by genetic manipulations. In this review we will focus on genes and gene loci involved in PD, the functions of proteins involved in the formation of cytoplasmatic inclusions, their interactions, and their possible role in PD. In addition, we will review the current animal models of PD.

Genetic deletion of angiotensin AT2 receptor leads to increased cell numbers in different brain structures of mice.

Angiotensin II (Ang II) is a potent vasoactive peptide and displays growth factor-like properties. Different high-affinity Ang II receptor subtypes (AT1A, AT1B and AT2) have been cloned. They are expressed in various brain structures. Additionally, it has been assumed that Mas could interact directly or indirectly with the renin-angiotensin system. The AT1 receptor mediates pressor and mitogenic effects of Ang II, whereas physiological function and signaling mechanisms of the AT2 receptor remain poorly understood. Recent reports have shown that Ang II could mediate apoptosis through AT2 receptors. Since the AT1A, AT2 and Mas knockout mice provide new tools for uncovering potential actions of Ang II, the cell number in different brain structures of male adult wild-type mice and mice deficient for AT1A, AT2 or Mas was evaluated to get more insight into the role of Ang II in central nervous system development. In nearly all investigated brain structures (cortex, hippocampus, amygdala, thalamus), the cell number was significantly higher in AT2-deficient mice in comparison to wild-type mice. To the contrary, in AT1A-deficient mice the cell number was significantly less than in controls in the lateral geniculate and the medial amygdaloid nucleus. However, cell numbers were not changed in Mas-knockout mice compared to their wild-types. These results show the contrary effects of both angiotensin receptors on cell growth and represent the first demonstration of their action on neuronal cell development evidenced in the adult mouse brain.

Identification of angiotensin IV binding sites in the mouse brain by a fluorescent binding study.

In the brain the renin-angiotensin system has been implicated in the regulation of the cardiovascular system as well as of regulation of reproductive hormones, water balance and behavior. Angiotensin II (Ang II) binds to two distinct receptors called AT1 and AT2. Recently a binding site has been discovered which selectively binds a fragment of Ang II. This biologically active Ang II 3-8 fragment is referred to as angiotensin IV (Ang IV) and has been reported to participate in memory acquisition and recall as well as in the regulation of blood flow, neurite outgrowth, angiogenesis and kidney function. The distribution of binding sites for Ang IV within the mouse brain is unknown. Using a nonradioactive binding assay with a fluorescence-coupled Ang IV, we were able to demonstrate specific Ang IV binding sites in the forebrain of mice, in particular in the cortex, the hippocampus, the amygdala, the thalamus and the hypothalamus.