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1.
Hum Mol Genet ; 24(11): 3058-81, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-25687137

ABSTRACT

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.


Subject(s)
Alzheimer Disease/genetics , Neurons/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Protein Ligases/metabolism , tau Proteins/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Animals , HeLa Cells , Humans , Mice, Inbred C57BL , Mice, Transgenic , Mitochondrial Proteins/metabolism , Mitophagy , Neurons/physiology , Protein Transport , Rats, Wistar , tau Proteins/physiology
2.
Neurobiol Dis ; 62: 489-507, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24411077

ABSTRACT

Functional as well as structural alterations in mitochondria size, shape and distribution are precipitating, early events in progression of Alzheimer's Disease (AD). We reported that a 20-22kDa NH2-tau fragment (aka NH2htau), mapping between 26 and 230 amino acids of the longest human tau isoform, is detected in cellular and animal AD models and is neurotoxic in hippocampal neurons. The NH2htau -but not the physiological full-length protein- interacts with Aß at human AD synapses and cooperates with it in inhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange. Here we show that the NH2htau also adversely affects the interplay between the mitochondria dynamics and their selective autophagic clearance. Fragmentation and perinuclear mislocalization of mitochondria with smaller size and density are early found in dying NH2htau-expressing neurons. The specific effect of NH2htau on quality control of mitochondria is accompanied by (i) net reduction in their mass in correlation with a general Parkin-mediated remodeling of membrane proteome; (ii) their extensive association with LC3 and LAMP1 autophagic markers; (iii) bioenergetic deficits and (iv) in vitro synaptic pathology. These results suggest that NH2htau can compromise the mitochondrial biology thereby contributing to AD synaptic deficits not only by ANT-1 inactivation but also, indirectly, by impairing the quality control mechanism of these organelles.


Subject(s)
Mitochondria/metabolism , Mitochondrial Dynamics/physiology , Neurons/metabolism , Peptide Fragments/metabolism , tau Proteins/metabolism , Alzheimer Disease/metabolism , Cell Line, Tumor , Hippocampus/metabolism , Hippocampus/ultrastructure , Humans , Mitochondria/ultrastructure , Neurons/ultrastructure , Synapses/metabolism
3.
Proc Natl Acad Sci U S A ; 106(27): 11358-63, 2009 Jul 07.
Article in English | MEDLINE | ID: mdl-19549834

ABSTRACT

The present study shows that increased Abeta production in hippocampal neurons, due to a failure of NGF signal, induces an unexpected phosphorylation of tyrosine kinase receptor A (TrkA), followed by activation of the phospholipase C gamma (PLCgamma) pathway and neuronal death. Such phosphorylation seems causally connected with 2 kinases known be involved in amyloidogenesis, Src and CDK5, and associated with alpha and gamma secretase-mediated p75 processing. Pharmacologic inhibition of TrkA phosphorylation and partial silencing of TrkA and/or p75 receptors prevent PLCgamma activation and protect neurons from death. Concomitantly with these events, TrkA, p75, Abeta peptides, and PS1 protein coimmunoprecipitate, suggesting their direct interplay in the subsequent onset of apoptotic death. Together, these findings depict a cellular mechanism whereby the same cellular transducing system may invert its intracellular message from trophic and antiapoptotic to a death signaling, which could also have relevance in the onset of Alzheimer's disease.


Subject(s)
Amyloid beta-Peptides/metabolism , Apoptosis , Receptor, trkA/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , Cell Nucleus/drug effects , Cell Survival/drug effects , Cyclin-Dependent Kinase 5/metabolism , Enzyme Activation/drug effects , Gene Silencing/drug effects , Hippocampus/cytology , Immunoprecipitation , Nerve Growth Factor/pharmacology , Neurons/cytology , Neurons/drug effects , Neurons/enzymology , Phospholipase C gamma/metabolism , Phosphorylation/drug effects , Presenilin-1/chemistry , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Wistar , Signal Transduction/drug effects , Time Factors , src-Family Kinases/metabolism
4.
Mol Neurobiol ; 57(7): 3183-3194, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32504417

ABSTRACT

MicroRNAs have emerged as regulators of brain development and function. Reduction of miR-101 expression has been reported in rodent hippocampus during ageing, in the brain of Alzheimer's disease (AD) patients and in AD animal models. In this study, we investigated the behavioral and molecular consequences of inhibition of endogenous miR-101 in 4-5-month-old C57BL/6J mice, infused with lentiviral particles expressing a miR-101 sponge (pLSyn-miR-101 sponge) in the CA1 field of the hippocampus. The sponge-infected mouse model showed cognitive impairment. The pLSyn-miR-101 sponge-infected mice were unable to discriminate either a novel object location or a novel object as assessed by object place recognition (OPR) and novel object recognition (NOR) tasks, respectively. Moreover, the sponge-infected mice evaluated for contextual memory in inhibitory avoidance task showed shorter retention latency compared to control pLSyn mice. These cognitive impairment features were associated with increased hippocampal expression of relevant miR-101 target genes, amyloid precursor protein (APP), RanBP9 and Rab5 and overproduction of amyloid beta (Aß) 42 levels, the more toxic species of Aß peptide. Notably, phosphorylation-dependent AMP-activated protein kinase (AMPK) hyperactivation is associated with AD pathology and age-dependent memory decline, and we found AMPK hyperphosphorylation in the hippocampus of pLSyn-miR-101 sponge mice. This study demonstrates that mimicking age-associated loss of miR-101 in hippocampal neurons induces cognitive decline and modulation of AD-related genes in mice.


Subject(s)
Alzheimer Disease/genetics , Cognitive Dysfunction/genetics , Hippocampus/metabolism , MicroRNAs/genetics , Neurons/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Avoidance Learning/physiology , Cognitive Dysfunction/metabolism , Disease Models, Animal , Memory/physiology , Mice , MicroRNAs/metabolism , Peptide Fragments/metabolism
5.
Mol Cell Neurosci ; 38(3): 381-92, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18511295

ABSTRACT

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.


Subject(s)
Alzheimer Disease/enzymology , Caspases/metabolism , Disease Models, Animal , Peptide Fragments/metabolism , tau Proteins/chemistry , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amino Acid Motifs/physiology , Animals , Apoptosis/physiology , Caspase Inhibitors , Caspases/genetics , Cell Differentiation/physiology , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Humans , Mice , Mice, Transgenic , Neurotoxins/chemistry , Neurotoxins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , tau Proteins/genetics , tau Proteins/metabolism
6.
Neuropharmacology ; 52(6): 1366-77, 2007 May.
Article in English | MEDLINE | ID: mdl-17397881

ABSTRACT

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.


Subject(s)
Cerebellum/drug effects , Delayed Rectifier Potassium Channels/physiology , Extracellular Signal-Regulated MAP Kinases/physiology , Mitogen-Activated Protein Kinases/physiology , Neuroprotective Agents , Substance P/pharmacology , Animals , Blotting, Western , Calpain/antagonists & inhibitors , Caspases/metabolism , Cerebellum/cytology , Cytoplasmic Granules/physiology , Delayed Rectifier Potassium Channels/drug effects , Electrophysiology , Enzyme Activation/drug effects , Immunohistochemistry , Microscopy, Phase-Contrast , Oncogene Protein v-akt/physiology , Patch-Clamp Techniques , Potassium/physiology , Rats , Rats, Wistar , Receptors, Neurokinin-1/drug effects , Receptors, Neurokinin-1/physiology , Reverse Transcriptase Polymerase Chain Reaction , Substance P/analogs & derivatives , Tachykinins/agonists
7.
Neuropharmacology ; 117: 134-148, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28163105

ABSTRACT

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease. However, the development of an NGF-based therapy is limited by its potent pain activity. We have developed a "painless" derivative form of human NGF (NGF61/100), characterized by identical neurotrophic properties but a reduced nociceptive sensitization activity in vivo. Here we characterized the response of rat dorsal root ganglia neurons (DRG) to the NGF derivative NGF61/100, in comparison to that of control NGF (NGF61), analyzing the expression of noxious pro-nociceptive mediators. NGF61/100 displays a neurotrophic activity on DRG neurons comparable to that of control NGF61, despite a reduced activation of PLCγ, Akt and Erk1/2. NGF61/100 does not differ from NGF61 in its ability to up-regulate Substance P (SP) and Calcitonin Gene Related Peptide (CGRP) expression. However, upon Bradykinin (BK) stimulation, NGF61/100-treated DRG neurons release a much lower amount of SP and CGRP, compared to control NGF61 pre-treated neurons. This effect of painless NGF is explained by the reduced up-regulation of BK receptor 2 (B2R), respect to control NGF61. As a consequence, BK treatment reduced phosphorylation of the transient receptor channel subfamily V member 1 (TRPV1) in NGF61/100-treated cultures and induced a significantly lower intracellular Ca2+ mobilization, responsible for the lower release of noxious mediators. Transcriptomic analysis of DRG neurons treated with NGF61/100 or control NGF allowed identifying a small number of nociceptive-related genes that constitute an "NGF pain fingerprint", whose differential regulation by NGF61/100 provides a strong mechanistic basis for its selective reduced pain sensitizing actions.


Subject(s)
Nerve Growth Factor/adverse effects , Nerve Growth Factor/pharmacology , Pain/chemically induced , Peptide Fragments/adverse effects , Sensory Receptor Cells/cytology , Animals , Bradykinin/pharmacology , Calcitonin Gene-Related Peptide/metabolism , Calcium/metabolism , Ganglia, Spinal/metabolism , Gene Expression Profiling , Humans , Pain/metabolism , Peptide Fragments/pharmacology , Primary Cell Culture , Rats , Receptors, Bradykinin/metabolism , Substance P/metabolism , TRPV Cation Channels/metabolism , Up-Regulation/drug effects
8.
Cancer Res ; 61(10): 4272-7, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11358855

ABSTRACT

157Gd is a potential agent for neutron capture cancer therapy (GdNCT). We directly observed the microdistribution of Gd in cultured human glioblastoma cells exposed to Gd-diethylenetriaminepentaacetic acid (Gd-DTPA). We demonstrated, with three independent techniques, that Gd-DTPA penetrates the plasma membrane, and we observed no deleterious effect on cell survival. A systematic microchemical analysis revealed a higher Gd accumulation in cell nuclei compared with cytoplasm. This is significant for prospective GdNCT because the proximity of Gd to DNA increases the cell-killing potential of the short-range, high-energy electrons emitted during the neutron capture reaction. We also exposed Gd-containing cells to thermal neutrons and demonstrated the GdNC reaction effectiveness in inducing cell death. These results in vitro stimulated in vivo Gd-DTPA uptake studies, currently underway, in human glioblastoma patients.


Subject(s)
Gadolinium/pharmacokinetics , Gadolinium/therapeutic use , Glioblastoma/metabolism , Glioblastoma/radiotherapy , Neutron Capture Therapy , Cell Death/radiation effects , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Gadolinium DTPA/pharmacokinetics , Gadolinium DTPA/toxicity , Humans , Isotopes , Mass Spectrometry , Spectrometry, X-Ray Emission , Tumor Cells, Cultured
9.
Cell Death Discov ; 2: 16050, 2016.
Article in English | MEDLINE | ID: mdl-27551538

ABSTRACT

A change in the delicate equilibrium between apoptosis and survival regulates the neurons fate during the development of nervous system and its homeostasis in adulthood. Signaling pathways promoting or protecting from apoptosis are activated by multiple signals, including those elicited by neurotrophic factors, and depend upon specific transcriptional programs. To decipher the rescue program induced by substance P (SP) in cerebellar granule neurons, we analyzed their whole-genome expression profiles after induction of apoptosis and treatment with SP. Transcriptional pathways associated with the survival effect of SP included genes encoding for proteins that may act as pharmacological targets. Inhibition of one of these, the Myc pro-oncogene by treatment with 10058-F4, reverted in a dose-dependent manner the rescue effect of SP. In addition to elucidate the transcriptional mechanisms at the intersection of neuronal apoptosis and survival, our systems biology-based perspective paves the way towards an innovative pharmacology based on targets downstream of neurotrophic factor receptors.

10.
J Neurosci ; 20(2): 589-99, 2000 Jan 15.
Article in English | MEDLINE | ID: mdl-10632588

ABSTRACT

We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsin-like activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic accumulation of ubiquitinated proteins and decline of proteasome function are reversed by the pan-caspase inhibitor Z-VAD-fmk. The decline in proteasome function is accompanied by, and likely attributable to, a marked and progressive decline of deubiquitinating activities. The finding that the proteasomes are early involved in apoptosis and that ubiquitinated proteins accumulate during this process prospect granule neurons as a model system aimed at correlating these events with neurodegenerative diseases.


Subject(s)
Apoptosis/physiology , Cerebellum/physiology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Multienzyme Complexes/metabolism , Neurons/cytology , Neurons/physiology , Ubiquitins/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Apoptosis/drug effects , Cell Division , Cell Survival/drug effects , Cells, Cultured , Cerebellum/cytology , Culture Media, Serum-Free , Leucine/analogs & derivatives , Leucine/pharmacology , Leupeptins/pharmacology , Neurons/drug effects , Oligopeptides/pharmacology , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational , Rats , Rats, Wistar
11.
Cell Death Differ ; 11(2): 217-30, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14615799

ABSTRACT

Although the role of the microtubule-binding domain of the tau protein in the modulation of microtubule assembly is widely established, other possible functions of this protein have been poorly investigated. We have analyzed the effect of adenovirally mediated expression of two fragments of the N-terminal portion - free of microtubule-binding domain - of the tau protein in cerebellar granule neurons (CGNs). We found that while the expression of the tau (1-230) fragment, as well as of full-length tau, inhibits the onset of apoptosis, the tau (1-44) fragment exerts a powerful toxic action on the same neurons. The antiapoptotic action of tau (1-230) is exerted at the level of Akt-mediated activation of the caspase cascade. On the other hand, the toxic action of the (1-44) fragment is not prevented by inhibitors of CGN apoptosis, but is fully inhibited by NMDA receptor antagonists. These findings point to a novel, physiological role of the N-terminal domain of tau, but also underlay that its possible proteolytic truncation mediated by apoptotic proteases may generate a highly toxic fragment that could contribute to neuronal death.


Subject(s)
Neurons/cytology , Neurons/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , tau Proteins/chemistry , tau Proteins/metabolism , Animals , Apoptosis , Caspase Inhibitors , Caspases/metabolism , Cell Survival , Cells, Cultured , Enzyme Activation , Free Radical Scavengers/antagonists & inhibitors , Gene Expression , Humans , Peptide Fragments/genetics , Phosphoserine/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , tau Proteins/genetics
12.
Eur J Histochem ; 49(1): 87-92, 2005.
Article in English | MEDLINE | ID: mdl-15823799

ABSTRACT

We previously demonstrated that exogenously administered neurokinin A and neurokinin B, but not substance P, increased the sensitivity of cultured cerebellar granule neurons (CGNs) to glutamate. In the present study, the presence of tachykinin neuropeptides in CGNs was tested by confocal-based immunofluorescence. We found that neurokinin A and neurokinin B are present in CGNs but absent in astrocytes while substance P is abundant in astrocytes but absent in CGNs. It is postulated that the different localization of tachykinin neuropeptides in CGNs and astroglial cells has a physiological role in the modulation of excitatory transmission.


Subject(s)
Cerebellum/chemistry , Neurons/chemistry , Neuropeptides/analysis , Tachykinins/analysis , Animals , Antibody Specificity , Astrocytes/chemistry , Astrocytes/cytology , Astrocytes/metabolism , Cells, Cultured , Cerebellum/cytology , Cerebellum/metabolism , Cytoplasmic Granules , Fluorescent Antibody Technique , Immunohistochemistry , Microscopy, Confocal , Neurokinin A/analysis , Neurokinin A/biosynthesis , Neurokinin B/analysis , Neurokinin B/biosynthesis , Neurons/cytology , Neurons/metabolism , Neuropeptides/biosynthesis , Rats , Substance P/analysis , Substance P/biosynthesis , Tachykinins/biosynthesis
13.
Eur J Neurosci ; 4(8): 733-744, 1992.
Article in English | MEDLINE | ID: mdl-12106317

ABSTRACT

The protein complex promoting in vitro terminal differentiation of cerebellar granule cells has been isolated from rabbit serum. We designate the complex the neurite outgrowth and adhesion complex (NOAC). The apparent molecular weight, evaluated by gel filtration, is 80 - 100 kDa. Rat cerebellar granule cells cultured in NOAC exhibit much lower glial cell contamination and survive, in their differentiated state, much longer than in 10% foetal calf serum. While they bind tetanus toxin, express specific antigens such as synapsin I, synaptophisin and A2B5, and release [3H]d-aspartate in a fashion similar to that shown by cells cultured in foetal calf serum, they show a 60% reduction in the total number of kainate binding sites. Excitatory amino acid (EAA)-triggered and depolarization-stimulated calcium influx, measured in the presence of different agonists, is 50 - 80% lower in NOAC-cultured cells. NOAC cells are resistant to excitotoxic stimuli carried by EAAs or by depolarizing treatments with 50 mM KCl or 6 microM veratridine. The marked resistance of NOAC-cultured neurons to EAAs can be attributed to decreased calcium entry through EAA-coupled and voltage-gated calcium channels and possibly to other, as yet unidentified, phenotypic properties of these cells. These findings demonstrate that rabbit serum contains one or more polypeptide(s) endowed with the properties of promoting in vitro survival and differentiation of rat cerebellar granule cells and of conferring an EAA-resistant phenotype.

14.
Neuropharmacology ; 38(9): 1335-42, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10471087

ABSTRACT

In previous studies (Volonté and Merlo, 1996. J. Neurosci. Res. 45, 183-193) basilen blue was shown to be a P2 receptor antagonist which abrogated glutamate-mediated cytotoxicity in cerebellar neurones in primary culture. Our work has now been extended to evaluate the neuroprotective action of the compound in additional neuronal systems, as well as in a different paradigm of cell death. We show that basilen blue prevents L-glutamate-mediated neurotoxicity in rat cerebellar (90-100% inhibition), cortical (60-70%) and hippocampal (50%) neurones. Similarly, glutamate-dependent progressive darkening of cell bodies, loss of phase-brightness and rapid cellular swelling are inhibited. Basilen blue is significantly less toxic and more effective at blocking L-glutamate toxicity in mixed cortical/glial cultures, compared to its structural analogue cibacron blue. Moreover, its neuroprotective effect is correlated with the time of incubation with granule neurones. Other purinoceptor ligands, including 2,2'-pyridylisatogen, but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium, are also effective in preventing glutamate toxicity. Furthermore, basilen blue prevents serum deprivation- and low potassium-induced apoptotic cell death in cerebellar granule neurones. In summary, our data extend and reinforce the possibility of a potential therapeutic use of P2 receptor modulators as neuroprotective agents for the central nervous system.


Subject(s)
Neurons/drug effects , Neuroprotective Agents/pharmacology , Receptors, Purinergic P2/metabolism , Triazines/pharmacology , Animals , Cells, Cultured , Central Nervous System/cytology , Cerebral Cortex/cytology , Enzyme Inhibitors/pharmacology , Glutamic Acid/pharmacology , Hippocampus/cytology , Isatin/analogs & derivatives , Isatin/pharmacology , Neurons/metabolism , Pyridoxal Phosphate/analogs & derivatives , Pyridoxal Phosphate/pharmacology , Rats , Rats, Wistar
15.
J Neuroimmunol ; 123(1-2): 9-17, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11880144

ABSTRACT

We show that treatment of cerebellar granules with interleukin-8 (IL-8), growth-related gene product beta (GRObeta) or AMPA induced activation of PI3-K/Akt and of ERK pathways, the latter being independent of PI3-K and dependent on PTX-sensitive G proteins. We also show that AMPA-mediated neuron survival was abolished both by ERK kinase inhibitor PD98059 and AMPA-Rs blocker CNQX, and that chemokine-mediated survival was blocked by the PI3-K inhibitors LY294002 and wortmannin. We conclude that the neurotrophic effects of AMPA need the contemporary activation of ERKs and stimulation of AMPA-Rs, and that PI3-K/Akt activation is a determinant pathway for the IL-8/GRObeta anti-apoptotic activity.


Subject(s)
Cerebellum/cytology , Chemokines, CXC , Intercellular Signaling Peptides and Proteins , Protein Serine-Threonine Kinases , Receptors, AMPA/physiology , Receptors, Interleukin-8B/physiology , Signal Transduction , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Cell Survival , Chemotactic Factors/pharmacology , Enzyme Activation , Growth Substances/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Rats, Wistar , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/pharmacology
16.
J Neuroimmunol ; 92(1-2): 122-32, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9916887

ABSTRACT

We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROalpha by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROalpha may play a neuromodulatory role on mouse cerebellum.


Subject(s)
Chemokines, CXC , Chemotactic Factors/pharmacology , Growth Inhibitors/pharmacology , Growth Substances/pharmacology , Intercellular Signaling Peptides and Proteins , Interleukin-8/pharmacology , Purkinje Cells/drug effects , Animals , Animals, Newborn , Calcium/metabolism , Chemokine CXCL1 , Cytosol/metabolism , Electric Conductivity , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Fluorescent Dyes , Humans , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/metabolism , Long-Term Potentiation/drug effects , Mice , Mice, Inbred BALB C , Organic Chemicals , Purkinje Cells/metabolism , Purkinje Cells/physiology , Rats , Rats, Wistar , Receptors, Chemokine/metabolism , Receptors, Interleukin/metabolism , Receptors, Interleukin-8B
17.
Neuroscience ; 54(3): 595-604, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8101358

ABSTRACT

Using amino acid transmitter release, inositol phosphate production and 45Ca2+ accumulation as cellular responses to alpha-latrotoxin treatment, we studied the susceptibility of rat primary cerebellar granule cultures to toxin. Binding studies as well as immunofluorescence of toxin-treated cells, allow an evaluation of the expression, properties and distribution of toxin-binding sites. After binding, the toxin develops its releasing action on amino acid transmitters, in a manner reminiscent of the events described for cholinergic and catecholaminergic cell systems. Although few peculiarities emerge, toxin-dependent release of amino acid transmitters from granule cells in culture appears to be a fully exocytotic process.


Subject(s)
Amino Acids/metabolism , Cerebellum/metabolism , Neurotransmitter Agents/metabolism , Spider Venoms/pharmacology , Animals , Calcium/metabolism , Calcium Radioisotopes , Cerebellum/drug effects , Fluorescent Antibody Technique , Inositol Phosphates/metabolism , PC12 Cells/drug effects , PC12 Cells/metabolism
18.
Neuroscience ; 95(1): 163-71, 2000.
Article in English | MEDLINE | ID: mdl-10619472

ABSTRACT

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an "L-cut" lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the "L-cut". Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex. The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.


Subject(s)
Cerebellar Cortex/physiology , DNA Damage , Denervation , Neurons/metabolism , Afferent Pathways/physiology , Animals , Axotomy , Cell Death/physiology , Cells, Cultured , Cerebellar Cortex/cytology , In Situ Nick-End Labeling , Male , Mesencephalon/physiology , Nerve Fibers/physiology , Neurons/physiology , Purkinje Cells/physiology , Rats , Rats, Wistar
19.
Neurochem Int ; 38(3): 189-97, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11099776

ABSTRACT

In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium and 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents.


Subject(s)
Cell Hypoxia/drug effects , Glucose/deficiency , Neuroprotective Agents/pharmacology , Purinergic P2 Receptor Antagonists , Triazines/pharmacology , Adenosine Triphosphate/metabolism , Animals , Cell Hypoxia/physiology , Cell Survival/drug effects , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cerebellum/metabolism , Coloring Agents/pharmacology , Dose-Response Relationship, Drug , Energy Metabolism/drug effects , Glucose/metabolism , NAD/metabolism , NADP/metabolism , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Wistar
20.
Neuroreport ; 11(7): 1375-9, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10841341

ABSTRACT

We have recently shown that, in rat cerebellar granule cells, apoptosis triggered by KCl deprivation is associated with an amyloidogenic shift in the processing of amyloid precursor protein (APP) resulting in an increase of amyloid beta-protein (A beta) secretion. To further investigate this issue we studied the relationship between secretion of APP metabolites (A beta, APPs) and neuronal degeneration. We postulated that the endogenous products of the APP metabolism may modulate neuronal survival by an autocrine loop. Treatment of cerebellar granule cells with various antibodies raised against different epitopes of APPs and A beta oppositely modulates low potassium apoptotic cell death. Antibodies specific for the N-terminal of A beta (4G8, 6E10, R3659) increased neuronal survival by 30% over controls. On the contrary, treatment of cultures undergoing apoptosis with the monoclonal antibody 22C11 directed against the APP N-terminus reduced neuronal survival by 53%, suggesting that endogenous alpha-APPs contribute to neuronal survival. Moreover low KCl culture medium, conditioned by cerebellar granule cells, attenuated the apoptotic process. This anti-apoptotic effect was abolished by removal of APPs from the conditioned medium. Western blotting of APPs removed from the conditioned medium confirmed the presence of alpha-APPs. These data indicate that APP cleavage products oppositely modulate neuronal survival through an autocrine loop and further strengthen an Alzheimer's disease pathogenetic scheme based on altered metabolism of APP.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Apoptosis/physiology , Autocrine Communication/physiology , Neurons/cytology , Amyloid beta-Protein Precursor/immunology , Animals , Antibodies, Monoclonal/pharmacology , Cell Survival/physiology , Cells, Cultured , Cerebellum/cytology , Neutralization Tests , Rats , Rats, Wistar
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