Delio Mercanti

CXC chemokines interleukin-8 (IL-8) and growth-related gene product α (GROα) modulate Purkinje neuron activity in mouse cerebellum

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 GROα 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 GROα may play a neuromodulatory role on mouse cerebellum.

SDF‐1α‐mediated modulation of synaptic transmission in rat cerebellum

The functional expression of the seven‐transmembrane domain G protein‐coupled chemokine receptor CXCR‐4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti‐CXCR‐4 Ab, and by evaluating the calcium responses to the physiological agonist stromal‐derived cell factor‐1α (SDF‐1α) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR‐4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF‐1α, both in cell bodies and in neuronal processes. Whole‐cell patch‐clamped PNs in cerebellar slices responded to SDF‐1α application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF‐1α‐induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR‐4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

NGF and BDNF signaling control amyloidogenic route and Aβ production in hippocampal neurons

Here, we report that interruption of NGF or BDNF signaling in hippocampal neurons rapidly activates the amyloidogenic pathway and causes neuronal apoptotic death. These events are associated with an early intracellular accumulation of PS1 N-terminal catalytic subunits and of APP C-terminal fragments and a progressive accumulation of intra- and extracellular Aβ aggregates partly released into the culture medium. The released pool of Aβ induces an increase of APP and PS1 holoprotein levels, creating a feed-forward toxic loop that might also cause the death of healthy neurons. These events are mimicked by exogenously added Aβ and are prevented by exposure to β- and γ-secretase inhibitors and by antibodies directed against Aβ peptides. The same cultured neurons deprived of serum die, but APP and PS1 overexpression does not occur, Aβ production is undetectable, and cell death is not inhibited by anti-Aβ antibodies, suggesting that hippocampal amyloidogenesis is not a simple consequence of an apoptotic trigger but is due to interruption of neurotrophic signaling.

Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control

Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.

A NH2 tau fragment targets neuronal mitochondria at AD synapses: possible implications for neurodegeneration.

Synapses are ultrastructural sites for memory storage in brain, and synaptic damage is the best pathologic correlate of cognitive decline in Alzheimers disease (AD). Post-translational hyperphosphorylation, enzyme-mediated truncation, conformational modifications, and aggregation of tau protein into neurofibrillary tangles (NFTs) are hallmarks for a heterogeneous group of neurodegenerative disorders, so-called tauopathies. AD is a secondary tauopathy since it is pathologically distinguished by the presence of amyloid-beta (Abeta)-containing senile plaques and the presence of tau-positive NFTs in the neocortex and hippocampus. Here, we report that a 20-22 kDa NH2-truncated tau fragment is largely enriched in human mitochondria from cryopreserved synaptosomes of AD brains and that its amount in terminal fields correlates with the pathological synaptic changes and with the organelle functional impairment. This NH2-truncated tau form is also found in other human, not AD-tauopathies, while its presence in AD patients is linked to Abeta multimeric species and likely to pathology severity. Finally native, patient-derived, Abeta oligomers-enriched extracts likely impair the mitochondrial function by the in vitro production of 20-22 kDa NH2-tau fragments in mature human SY5Y and in rat hippocampal neurons. Thus our findings suggest that the mitochondrial NH2-derived tau peptide distribution may exacerbate the synapse degeneration occurring in tauopathies, including AD, and sustain the in vivo NH-2 tau cleavage inhibitors as an alternative drug discovery strategies for AD therapy.

Effects of Topically Administered Nerve Growth Factor on Axonal Regeneration in Peripheral Nerve Autografts Implanted in the Spinal Cord of Rats

The effect of exogenous nerve growth factor (NGF) on axonal regeneration into autologous peripheral nerve (PN) grafts implanted to the spinal cord (SC) of rats was assessed by retrograde labeling of the parent soma of the regenerating axons with horseradish peroxidase. NGF was delivered at the graft site over periods of 15 and 30 days by using indwelling osmotic minipumps. In control rats, the minipumps were filled with saline. At 15 days after grafting in the NGF-treated rats, the mean number of SC as well as dorsal root ganglion (DRG) neurons that regenerated their axons into the peripheral nerve grafts was increased 55.3 and 26.4 times, respectively, as compared to the control group values. At 30 days, SC and DRG neurons in the NGF-treated group were 10.9 and 3.1 times greater than in the control group. In the NGF-treated group, the regenerating SC neurons were located within a range of 7 to 13 mm from the graft site as compared to 1 to 7 mm in the control group. Finally, the analysis of the soma diameters of the regenerating neurons showed that NGF enhanced and maintained with time the regenerative response from small-sized DRG neurons. Therefore, NGF is thought to promote directly the regenerative potential of SC as well as DRG neurons and to exert an indirect glial cell-mediated effect at the SC-graft interface.

Tyrosine kinase nerve growth factor receptor switches from prosurvival to proapoptotic activity via Abeta-mediated phosphorylation

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 γ (PLCγ) pathway and neuronal death. Such phosphorylation seems causally connected with 2 kinases known be involved in amyloidogenesis, Src and CDK5, and associated with α and γ secretase–mediated p75 processing. Pharmacologic inhibition of TrkA phosphorylation and partial silencing of TrkA and/or p75 receptors prevent PLCγ 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 Alzheimers disease.

APP is Phosphorylated by TrkA and Regulates NGF/TrkA Signaling

The pathogenic model of Alzheimers disease (AD) posits that aggregates of amyloid β, a product of amyloid precursor protein (APP) processing, cause dementia. However, alterations of normal APP functions could contribute to AD pathogenesis, and it is therefore important to understand the role of APP. APP is a member of a gene family that shows functional redundancy as documented by the evidence that single knock-out mice are viable, whereas mice with combined deletions of APP family genes die shortly after birth. A residue in the APP intracellular region, Y682, is indispensable for these essential functions of APP. It is therefore important to identify pathways that regulate phosphorylation of Y682 as well as the role of Y682 in vivo. TrkA is associated with both phosphorylation of APP-Y682 and alteration of APP processing, suggesting that tyrosine phosphorylation of APP links APP processing and neurotrophic signaling to intracellular pathways associated with cellular differentiation and survival. Here we have tested whether the NGF/TrkA signaling pathway is a physiological regulator of APP phosphorylation. We find that NGF induces tyrosine phosphorylation of APP, and that APP interacts with TrkA and this interaction requires Y682. Unpredictably, we also uncover that APP, and specifically Y682, regulates activation of the NGF/TrkA signaling pathway in vivo, the subcellular distribution of TrkA and the sensitivity of neurons to the trophic action of NGF. This evidence suggests that these two membrane proteins functions are strictly interconnected and that the NGF/TrkA signaling pathway is involved in AD pathogenesis and can be used as a therapeutic target.

Are gadolinium contrast agents suitable for gadolinium neutron capture therapy

Abstract Objective: Gadolinium neutron capture therapy (GdNCT) is a potential treatment for malignant tumors based on two steps: (1) injection of a tumor-specific 157Gd compound; (2) tumor irradiation with thermal neutrons. The GdNC reaction can induce cell death provided that Gd is proximate to DNA. Here, we studied the nuclear uptake of Gd by glioblastoma (GBM) tumor cells after treatment with two Gd compounds commonly used for magnetic resonance imaging, to evaluate their potential as GdNCT agents. Methods: Using synchrotron X-ray spectromicroscopy, we analyzed the Gd distribution at the subcellular level in: (1) human cultured GBM cells exposed to Gd-DTPA or Gd-DOTA for 0–72hours; (2) intracerebrally implanted C6 glioma tumors in rats injected with one or two doses of Gd-DOTA, and (3) tumor samples from GBM patients injected with Gd-DTPA. Results: In cell cultures, Gd-DTPA and Gd-DOTA were found in 84% and 56% of the cell nuclei, respectively. In rat tumors, Gd penetrated the nuclei of 47% and 85% of the tumor cells, after single and double injection of Gd-DOTA, respectively. In contrast, in human GBM tumors 6.1% of the cell nuclei contained Gd-DTPA. Discussion: Efficacy of Gd-DTPA and Gd-DOTA as GdNCT agents is predicted to be low, due to the insufficient number of tumor cell nuclei incorporating Gd. Although multiple administration schedules in vivo might induce Gd penetration into more tumor cell nuclei, a search for new Gd compounds with higher nuclear affinity is warranted before planning GdNCT in animal models or clinical trials.

Signaling pathways activated by chemokine receptor CXCR2 and AMPA-type glutamate receptors and involvement in granule cells survival

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.