Enteric α-synuclein impairs intestinal epithelial barrier through caspase-1-inflammasome signaling in Parkinson's disease before brain pathology.

Bowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson's disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1ß, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1ß release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP-1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology.

ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aß-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aß oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

A dual mechanism linking NGF/proNGF imbalance and early inflammation to Alzheimer's disease neurodegeneration in the AD11 anti-NGF mouse model.

The neurotrophin Nerve Growth Factor (NGF) is essential for the maintenance and differentiation of basal forebrain cholinergic neurons. Since basal forebrain cholinergic neurons represent one major neuronal population affected and progressively degenerating in Alzheimer's disease (AD), interest has grown for NGF as a potential therapeutic agent in neurodegenerative disorders linked to aging, particularly for AD. However, no evidence was available, to link, in a cause-effect manner, deficits in NGF signalling to the broader activation in the Alzheimer's cascade, besides cholinergic deficits. The phenotypic analysis of the AD11 anti-NGF transgenic mouse, obtained by the "neuroantibodies" phenotypic protein knock out strategy, allowed demonstrating a direct causal link between NGF deprivation and AD pathology. Since then, extensive mechanistic studies on the AD11 model provided a new twist to the concept that alterations in NGF transport and signalling play a crucial role in sporadic Alzheimer's neurodegeneration, leading to the hypothesis of "Neurotrophic imbalance" as an upstream driver for sporadic AD. The results obtained with the AD11 anti-NGF mice highlight the fact that the particular mode of NGF neutralization, with an NGF antibody expressed in the brain, selectively interfering with mature NGF versus unprocessed proNGF, plays a major role in the mechanism of neurodegeneration, and could lead to new insights into the mechanisms of human sporadic AD. Here, we will review (1) the renewed neurotrophic imbalance hypothesis for AD and (2) the mechanisms underlying the neurodegenerative phenotype of AD11 anti-NGF mice.

Endogenous Aß causes cell death via early tau hyperphosphorylation.

Alzheimer's disease (AD) is characterized by Aß overproduction and tau hyperphosphorylation. We report that an early, transient and site-specific AD-like tau hyperphosphorylation at Ser262 and Thr231 epitopes is temporally and causally related with an activation of the endogenous amyloidogenic pathway that we previously reported in hippocampal neurons undergoing cell death upon NGF withdrawal [Matrone, C., Ciotti, M.T., Mercanti, D., Marolda, R., Calissano, P., 2008b. NGF and BDNF signaling control amyloidogenic route and Ab production in hippocampal neurons. Proc. Natl. Acad. Sci. 105, 13138-13143]. Such tau hyperphosphorylation, as well as apoptotic death, is (i) blocked by 4G8 and 6E10 Aß antibodies or by specific ß and/or γ-secretases inhibitors; (ii) temporally precedes tau cleavage mediated by a delayed (6-12h after NGF withdrawal) activation of caspase-3 and calpain-I; (iii) under control of Akt-GSK3ß-mediated signaling. Finally, we show that such site-specific tau hyperphosphorylation causes tau detachment from microtubules and an impairment of mitochondrial trafficking. These results depict, for the first time, a rapid interplay between endogenous Aß and tau post-translational modifications which act co-ordinately to compromise neuronal functions in the same neuronal system, under physiological conditions as seen in AD brain.

Identification of a caspase-derived N-terminal tau fragment in cellular and animal Alzheimer's disease models.

Biochemical modifications of tau proteins have been proposed to be among the earliest neurobiological changes in Alzheimer's disease (AD) and correlate better with cognitive symptoms than do beta-amyloid plaques. We have recently reported that adenovirus-mediated overexpression of the NH2 26-230aa tau fragment evokes a potent NMDA-mediated neurotoxic effect in primary neuronal cultures. In order to assess whether such N-terminal tau fragment(s) are indeed produced during apoptosis or neurodegeneration in vivo, we attempted to ascertain their presence in cell and animal models using an anti-tau antibody directed against the N-terminal sequence of human protein located downstream of the caspase(s)-cleavage site DRKD(25)-QGGYTMHQDQ. We provide biochemical evidence that a caspase(s)-cleaved NH2-terminal tau fragment of 20-22 kDa, consistent with the size of the NH2 26-230aa neurotoxic fragment of tau, is generated in vitro in differentiated human SH-SY5Y cells undergoing apoptosis by BDNF withdrawal or following treatment with staurosporine. In addition this NH2-terminally cleaved tau fragment, whose expression correlates with a significant up-regulation of caspase(s) activity, is also specifically detected in vivo in the hippocampus of 15 month-old AD11 transgenic mice, a model in which a progressive AD-like neurodegeneration is induced by the expression of transgenic anti-NGF antibodies. The results support the idea that aberrant activation of caspase(s), following apoptotic stimuli or neurodegeneration insults, may produce one or more toxic NH2 tau fragments, that further contribute to propagate and increase cellular dysfunctions in AD.

Mismatch between BDNF mRNA and protein expression in the developing visual cortex: the role of visual experience.

Brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) expression in the rat visual cortex of young and postnatal day 90 (P90) animals is developmentally regulated and influenced by visual experience. In the present paper we compared the expression of BDNF mRNA to the actual changes of BDNF protein occurring during postnatal development and verified whether BDNF protein distribution is controlled by visual activity. To achieve this aim we analysed BDNF mRNA and/or BDNF protein cellular distribution in the rat visual cortex at different postnatal ages by using immunohistochemistry and highly sensitive in situ hybridization. We found that before eye opening (P13), in all cortical layers a large number of visual cortical neurons contain BDNF mRNA with no detectable amount of BDNF protein. At later ages (P23 and P90), the number of BDNF-immunostained cells increases; most neurons are double labelled for BDNF mRNA and protein, and a small group of neurons is labelled only for BDNF protein. The cellular increase of BDNF immunolabelling is blocked in animals deprived of visual experience from birth (dark rearing), with a large population of neurons containing BDNF mRNA but not BDNF protein. This is similar to what is observed before eye opening. Exposure of dark-reared rats to a brief period (2 h) of light restores a good match between BDNF mRNA and BDNF protein cellular expression. We propose that visual experience controls the neuronal content of BDNF mRNA and BDNF protein in developing visual cortex.

Alzheimer-like neurodegeneration in aged antinerve growth factor transgenic mice.

Neurotrophin nerve growth factor (NGF) has been suggested to be involved in age-related neurodegenerative diseases, but no transgenic model is currently available to study this concept. We have obtained transgenic mice expressing a neutralizing anti-NGF recombinant antibody, in which the levels of antibodies are three orders of magnitude higher in adult than in newborn mice [F.R., S. C. , A.C., E. Di Daniel, J. Franzot, S. Gonfloni, G. Rossi, N. B. & A. C. (2000) J. Neurosci., 20, 2589-2601]. In this paper, we analyze the phenotype of aged anti-NGF transgenic mice and demonstrate that these mice acquire an age-dependent neurodegenerative pathology including amyloid plaques, insoluble and hyperphosphorylated tau, and neurofibrillary tangles in cortical and hippocampal neurons. Aged anti-NGF mice also display extensive neuronal loss throughout the cortex, cholinergic deficit in the basal forebrain, and behavioral deficits. The overall picture is strikingly reminiscent of human Alzheimer's disease. Aged anti-NGF mice represent, to our knowledge, the most comprehensive animal model for this severe neurodegenerative disease. Also, these results demonstrate that, in mice, a deficit in the signaling and/or transport of NGF leads to neurodegeneration.

Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy.

The disruption of the nerve growth factor (NGF) gene in transgenic mice leads to a lethal phenotype (Crowley et al., 1994) and hinders the study of NGF functions in the adult. In this study the phenotypic knockout of NGF in adult mice was achieved by expressing transgenic anti-NGF antibodies, under the control of the human cytomegalovirus promoter. In adult mice, antibody levels are 2000-fold higher than in newborns. Classical NGF targets, including sympathetic and sensory neurons, are severely affected. In the CNS, basal forebrain and hippocampal cholinergic neurons are not affected in the early postnatal period, whereas they are greatly reduced in the adult (55 and 62% reduction, respectively). Adult mice show a reduced ability in spatial learning behavioral tasks. Adult, but not neonatal, transgenic mice further show a new phenotype at the level of peripheral tissues, such as apoptosis in the spleen and dystrophy of skeletal muscles. The analysis of this novel comprehensive transgenic model settles the controversial issue regarding the NGF dependence of cholinergic neurons in adult animals and reveals new NGF functions in adult non-neuronal tissues. The results demonstrate that the decreased availability of NGF in the adult causes phenotypic effects via processes that are at least partially distinct from early developmental effects of NGF deprivation.

Muscular dystrophy in adult and aged anti-NGF transgenic mice resembles an inclusion body myopathy.

The role of nerve growth factor (NGF) and its receptors in the physiology of skeletal muscles has not been extensively studied in animal models. We describe the production of transgenic lines of mice expressing a neutralizing antibody against NGF (alphaD11) and the morphological and histochemical analysis of skeletal muscles from adult and aged anti-NGF mice. This study reveals that the chronic deprivation of NGF results in a decreased size of myofibers of dorsal and hindlimb muscles in adult but not in postnatal day (P)2 mice. In myofibers from adult anti-NGF mice, the presence of central nuclei, vacuolization of the cytoplasm, and inflammatory cell infiltration was observed. The immunohistochemical analysis of these muscular fibers revealed an upregulation of p75 expression, a decrease in adenosine triphosphatase (ATP)ase activity, and a subsarcolemmal Congo Red-positive staining. Immunostaining with an antibody against amyloid precursor protein showed an increased labeling of the cytoplasm of myofibers from adult and aged anti-NGF mice. These features are reminiscent of human myopathies, such as inclusion body myositis. We conclude that NGF deficits might be relevant for a class of human myopathies.

Functional blockade of tyrosine kinase A in the rat basal forebrain by a novel antagonistic anti-receptor monoclonal antibody.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

Differential regulation of brain-derived neurotrophic factor messenger RNA cellular expression in the adult rat visual cortex.

In this study, we report a comparative analysis of the distribution of brain-derived neurotrophic factor messenger RNA in the binocular primary visual cortex of rats analysed at the end of the critical period for monocular deprivation (postnatal day 35) and during adulthood (postnatal day 90). High-resolution non-isotopic in situ hybridization coupled with Nissl staining allowed to determine the relative number of neurons expressing brain-derived neurotrophic factor messenger RNA. In postnatal day 90 rats, the relative number of neurons positive for brain-derived neurotrophic factor messenger RNA significantly decreases in layer II/III with respect to postnatal day 35 animals, being constant in all the other cortical layers. Moreover, we demonstrate that dark rearing for 22 days, starting from postnatal day 90, determines: (i) a decrease of the overall level of brain-derived neurotrophic factor messenger RNA with a consequent reduction of labelling intensity in all cells throughout cortical layers II-VI; (ii) an increase of cell numbers expressing brain-derived neurotrophic factor messenger RNA in layers IV and V; and (iii) a decreased intensity of staining for brain-derived neurotrophic factor messenger RNA in dendrites after dark rearing. A re-exposure to light for 2 h after the period of darkness almost restores the number of brain-derived neurotrophic factor RNA-positive neurons. We conclude that the maturation of brain-derived neurotrophic factor messenger RNA in neurons of layer II/III goes beyond postnatal days 35-40, which can be considered the end of the critical period [Fagiolini M. et al. (1994) Vis. Res., 34, 709-720]. Moreover, we show that the cellular expression of brain-derived neurotrophic factor messenger RNA is regulated by light in adult rats as well as during development.

Dark rearing blocks the developmental down-regulation of brain-derived neurotrophic factor messenger RNA expression in layers IV and V of the rat visual cortex.

In this study, we describe the distribution of brain-derived neurotrophic factor messenger RNA in the binocular primary visual cortex of the rat during postnatal development, starting at postnatal day (P) 13. High-resolution non-isotopic in situ hybridization combined with Nissl staining were used to quantify the number of cells expressing brain-derived neurotrophic factor messenger RNA. At P13, most of the cells express brain-derived neurotrophic factor messenger RNA. After eye opening (P14-P15), the relative number of brain-derived neurotrophic factor messenger RNA-positive cells decreases by a factor of two in layer IV, i.e. that receiving the visual input, and in layer V. To verify the hypothesis that light could trigger this decrease, pups were kept in complete darkness from birth. At P22, pups reared in the dark were killed and the visual cortex processed for in situ hybridization and northern blotting. The results obtained in dark-reared animals prove that light deprivation can: (i) decrease the general levels of brain-derived neurotrophic factor messenger RNA, and (ii) increase the relative number of brain-derived neurotrophic factor messenger RNA-positive cells in layers IV and V with respect to control rats. Exposure to light for five days after the period of darkness restored the number of brain-derived neurotrophic factor messenger RNA-positive cells. We conclude that the expression of brain-derived neurotrophic factor messenger RNA in the rat primary visual cortex is regulated during development and that this process is under the control of visual input.

Expression of the melatonin receptor in Xenopus laevis: a comparative study between protein and mRNA distribution.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis performed on total RNA from different tissues of Xenopus laevis showed that the melatonin receptor gene cloned from dermal melanophores is expressed in the whole brain, skin, and retina, and that apart from the ovary, there is no expression in tissues having origin outside the central nervous system. Comparative studies using in vitro autoradiography and in situ hybridization demonstrated that the melatonin receptor is expressed with discrete allocation in Xenopus brain. Though the distribution pattern of the specific messenger RNA conforms well with that of the corresponding receptor protein, it is not always coincident.

Reduction of regional cerebral blood flow by melatonin in young rats.

Melatonin (10 ng) was subcutaneously administered to 14-day-old Sprague-Dawley rats. Regional blood flow (rCBF) was measured in 22 anatomically defined structures 20 min later using iodo[14C]antipyrine and quantitative autoradiography. rCBF was markedly reduced in the cerebral areas supplied by circle of Willis and the basilar arteries. Melatonin also significantly decreased blood flow to choroid plexuses. These findings suggest that circulating melatonin may contribute to regulation of cerebral blood flow and brain fluid balance.

Distribution and characterization of the melatonin receptors in the hypothalamus and pituitary gland of three domestic ungulates.

With some exceptions, in most of the mammals the pituitary pars tuberalis and the hypothalamic suprachiasmatic nuclei are reportedly the main targets for the pineal hormone melatonin. However, it is not known if the conspicuous diversity in the distribution pattern of melatonin binding sites in these areas depicts differences in reproductive behavior observed in the seasonally breeding species in the temperate zones. We explored the distribution and the characteristics of melatonin binding sites in the hypothalamus and pituitary of three species (bovine, horse, and donkey) different in terms of seasonal reproductive competence. The topographical localization, investigated by in vitro autoradiography, revealed 2-[125I]iodomelatonin binding sites only in the pituitary gland in all three species, primarily in the pars tuberalis (PT), but also in the pars distalis (PD) and pars intermedia (PI). Kinetic, inhibition, and saturation studies, performed by means of in vitro binding, revealed presence of a single class high affinity binding sites. The Kd values, melatonin, and 2-iodomelatonin Ki values were in the low picomolar range. Coincubation with GTP gamma S inhibited 2-[125I]iodomelatonin binding, demonstrating that these putative receptors are linked to a G protein in their signal-transduction pathway. The hypothalamus was devoid of specific binding. In conclusion, the results suggest that in these species, the hypophysis may be a principal target for the melatonin action on the reproductive system.

Characterization of endothelinA receptors in cerebral and peripheral arteries of the rat.

We have characterized and quantified endothelin receptors in rat brain (anterior cerebral) and peripheral (aorta, carotid, and caudal) arteries, with the use of [125I]endothelin and quantitative autoradiography. Endothelin binding was saturable, of high affinity, and totally displaced by the selective endothelin ETA antagonist BQ 123. A single class of ETA receptors is located in the medial layer of peripheral and cerebral arteries, and its quantification by autoradiography allows study of their regulation and function.

A carnivore species (Canis familiaris) expresses circadian melatonin rhythm in the peripheral blood and melatonin receptors in the brain.

Dogs kept under controlled photoperiodic conditions of 12 h light and 12 h dark expressed a clear diurnal melatonin rhythm in the peripheral blood, with a swift peak restricted to the late part of the scotophase. The highest density of high-affinity, G-protein-linked 2-[125I]iodomelatonin binding sites was found in the pars tuberalis of the pituitary gland. Binding sites were found also in the pars distalis, and light microscopy/high-resolution autoradiography showed that binding was located exclusively over the chromophobe and basophilic cells forming the adenopituitary zona tuberalis, well developed in this species, and extending into the gland as a continuation of pars tuberalis. Cords of basophilic cells located in the pars distalis proper also expressed high receptor density. The eosinophils in the adenohypophysis and the neural lobe were devoid of binding. Heavily labeled were the external laminar and the mitral cell layers of the olfactory bulbs, but no binding was detected in the filae nervi olfactorii or tractus olfactorius. The hypothalamic suprachiasmatic nuclei were discernible clearly. Quantitative autoradiography inhibition experiments revealed that the apparent melatonin inhibitory constant (IC50) in all those areas was around 0.1 nmol/l, which is a physiologically appropriate value considering the peripheral blood melatonin levels. Co-incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) led to a consequential decrease in the binding density. The specific binding observed in other areas (hippocampus, frontal, parietal, occipital cortex and cerebellum) was rather weak, diffuse and could not be attributed to a particular layer; the apparent IC50 for melatonin was about 1 mumol/l, and co-incubation with GTP gamma S did not modify the binding density. Collectively, these data show that the dog possess all the prerequisites for an efficient network adapted to photoperiodic time measurements. A circadian melatonin signal in the peripheral blood and an apparently functional readout receptor system located in key positions within the brain are both present in this species.

Characterization of melatonin receptors and signal transduction system in rat arteries forming the circle of Willis.

The aims of this study were to characterize the melatonin receptors in rat brain arteries forming the circle of Willis. Saturation studies performed using in vitro autoradiography and [125I]iodomelatonin revealed the presence of two binding sites: one with a Kd of 13 pM, and the second characterized by a Kd of 832 pM. Coincubation with a nonhydrolyzable guanine nucleotide analog [guanosine-5'-O-(3-thiotriphosphate)] inhibited 2-[125I]iodomelatonin binding in a concentration-dependent manner, whereas adenine nucleotide adenosine-5'-O-(3-thiotriphosphate) was ineffective. In saturation studies performed in the presence of guanosine-5'-O-(3-thiotriphosphate), the high affinity site was no longer detectable, and the affinity of the receptor was decreased to the high picomolar range. Melatonin, at nanomolar concentrations, was able to inhibit forskolin-stimulated cAMP production in rat circle of Willis arteries. Preincubation with pertussis toxin counteracted the effect of melatonin. Our results demonstrate that melatonin receptors in rat cerebral arteries are linked to their second messenger through a pertussis toxin-sensitive G-protein, similar to what has been described for melatonin receptors in different areas of vertebrate brain.

Distribution and characterization of melatonin receptors in the brain of the Japanese quail, Coturnix japonica.

2-[125I]iodomelatonin was used to study the distribution and properties of the melatonin receptor in the Japanese quail brain. High receptor density was detected in the major targets of direct retinal input (optic tectum, nucleus of the optic basal rout, ventrolateral geniculate nucleus), as well as areas representing terminals in the visual pathways (nucleus rotundus, ectostriatum, thalamo-hyperstriatal pathway). Binding was also found in the piriform cortex, the hypophyseal pars tuberalis, the oculomotorius nucleus and the associated Edinger-Westphal nucleus, and in the nuclei of the third, fourth and sixth cranial nerves. A comparison of the receptor pharmacological profile to that of the mammalian brain demonstrated pharmacological identity of the two binding sites. In the saturation experiments, GPT gamma S decreased the binding affinity, numerical Kd values increasing from approximately 35 pM to approximately 150 pM.