Francesco Longo

The AGILE silicon tracker: an innovative γ-ray instrument for space

Abstract AGILE (Light Imager for Gamma-ray Astrophysics) is the first small scientific mission of ASI, the Italian Space Agency. It is a light ( 100 kg for the scientific instrument) satellite for the detection of γ-ray sources in the energy range 30 MeV – 50 GeV within a large field of view ( 1 4 of the sky). It is planned to be operational in the years 2003–2006, a period in which no other gamma-ray mission in the same energy range is foreseen. AGILE is made of a silicon tungsten tracker, a CsI(Tl) minicalorimeter (1.5 X 0 ), an anticoincidence system of segmented plastic scintillators and a X-ray imaging detector sensitive in the 10– 40 keV range. The tracker consists of 14 planes, each of them made of two layers of 16 single-sided, AC coupled, 410 μm thick, 9.5×9.5 cm 2 silicon detectors with a readout pitch of 242 μm and a floating strip. The readout ASIC is the TAA1, an analog-digital, low noise, self-triggering ASIC used in a very low power configuration ( μW / channel ) with full analog readout. The trigger of the satellite is given by the tracker. The total number of readout channels is around 43xa0000. We present a detailed description of the tracker, its trigger and readout logic, its assembly procedures and the prototype performance in several testbeam periods at the CERN PS.

The AGILE silicon tracker: testbeam results of the prototype silicon detector

Abstract AGILE (Light Imager for Gamma-ray Astrophysics) is a small scientific satellite for the detection of cosmic γ-ray sources in the energy range 30 MeV – 50 GeV with a very large field of view (1/4 of the sky). It is planned to be operational in the years 2003–2006, a period in which no other γ-ray mission in the same energy range is foreseen. The heart of the AGILE scientific instrument is a silicon–tungsten tracker made of 14 planes of single sided silicon detectors for a total of 43xa0000 readout channels. Each detector has a dimension of 9.5×9.5 cm 2 and a thickness of 410 μm . We present here a detailed description of the performance of the detector prototype during a testbeam period at the CERN PS in May 2000. The Tracker performance is described in terms of position resolution and signal-to-noise ratio for on and off-axis incident charged particles. The measured 40 μm resolution for a large range of incident angles will provide an excellent angular resolution for cosmic γ-ray imaging.

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinsons disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex.

Genetic and pharmacological evidence that G2019S LRRK2 confers a hyperkinetic phenotype, resistant to motor decline associated with aging

The leucine-rich repeat kinase 2 mutation G2019S in the kinase-domain is the most common genetic cause of Parkinsons disease. To investigate the impact of the G2019S mutation on motor activity in vivo, a longitudinal phenotyping approach was developed in knock-in (KI) mice bearing this kinase-enhancing mutation. Two cohorts of G2019S KI mice and wild-type littermates (WT) were subjected to behavioral tests, specific for akinesia, bradykinesia and overall gait ability, at different ages (3, 6, 10, 15 and 19 months). The motor performance of G2019S KI mice remained stable up to the age of 19 months and did not show the typical age-related decline in immobility time and stepping activity of WT. Several lines of evidence suggest that enhanced LRRK2 kinase activity is the main contributor to the observed hyperkinetic phenotype of G2019S KI mice: i) KI mice carrying a LRRK2 kinase-dead mutation (D1994S KD) showed a similar progressive motor decline as WT; ii) two LRRK2 kinase inhibitors, H-1152 and Nov-LRRK2-11, acutely reversed the hyperkinetic phenotype of G2019S KI mice, while being ineffective in WT or D1994S KD animals. LRRK2 target engagement in vivo was further substantiated by reduction of LRRK2 phosphorylation at Ser935 in the striatum and cortex at efficacious doses of Nov-LRRK2-11, and in the striatum at efficacious doses of H-1152. In summary, expression of the G2019S mutation in the mouse LRRK2 gene confers a hyperkinetic phenotype that is resistant to age-related motor decline, likely via enhancement of LRRK2 kinase activity. This study provides an in vivo model to investigate the effects of LRRK2 inhibitors on motor function.

The next generation of high-energy gamma-ray detectors for satellites: The AGILE silicon tracker

AGILE (Light Imaging Detector for Gamma Astronomy) is a satellite for the detection of gamma-ray sources in the energy range 30 MeV–50 GeV within a large field of view (∼1/5 of the sky) and it is planned to fly in the years 2003–2006, a period in which no other mission entirely dedicated to photon detection above 30 MeV is planned. AGILE is made of a Tungsten-Silicon Tracker, a CsI Minicalorimeter, an anticoincidence system and a X-ray detector sensitive in the 10–40 keV range. The Tracker consists of 14 planes, each one made of 2 layers of 16 single-sided, AC coupled, 410 μm thick, 9.5×9.5u2009cm2 Silicon detectors with a readout pitch of 242 μm and a floating strip. The AGILE trigger is generated by the Silicon strips which are readout by the TAAI, a low noise, self triggering ASIC used in a very low power configuration (∼400 μW/channel) with analog readout. The number of Tracker readout channels is 43000. We present a description of the Tracker and the performance of the detector (position resolution, clus...

D-Aspartate oxidase influences glutamatergic system homeostasis in mammalian brain

We have investigated the relevance of d-aspartate oxidase, the only enzyme known to selectively degrade d-aspartate (d-Asp), in modulating glutamatergic system homeostasis. Interestingly, the lack of the Ddo gene, by raising d-Asp content, induces a substantial increase in extracellular glutamate (Glu) levels in Ddo-mutant brains. Consistent with an exaggerated and persistent N-methyl-d-aspartate receptor (NMDAR) stimulation, we documented in Ddo knockouts severe age-dependent structural and functional alterations mirrored by expression of active caspases 3 and 7 along with appearance ofxa0dystrophic microglia and reactive astrocytes. In addition, prolonged elevation of d-Asp triggered in mutants alterations of NMDAR-dependent synaptic plasticity associated to reduction of hippocampalxa0GluN1 and GluN2B subunits selectively located at synaptic sites and to increase in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-to-N-methyl-d-aspartate ratio. These effects, all of which converged on a progressive hyporesponsiveness at NMDAR sites, functionally resulted in a greater vulnerability to phencyclidine-induced prepulse inhibition deficits in mutants. In conclusion, our results indicate that d-aspartate oxidase, by strictly regulating d-Asp levels, impacts on the homeostasis of glutamatergic system, thus preventing accelerated neurodegenerative processes.

Age-dependent dopamine transporter dysfunction and Serine129 phospho-α-synuclein overload in G2019S LRRK2 mice

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson’s disease. Here, we investigated whether the G2019S LRRK2 mutation causes morphological and/or functional changes at nigro-striatal dopamine neurons. Density of striatal dopaminergic terminals, nigral cell counts, tyrosine hydroxylase protein levels as well as exocytotic dopamine release measured in striatal synaptosomes, or striatal extracellular dopamine levels monitored by in vivo microdialysis were similar between ≥12-month-old G2019S knock-in mice and wild-type controls. In vivo striatal dopamine release was insensitive to the LRRK2 inhibitor Nov-LRRK2-11, and was elevated by the membrane dopamine transporter blocker GBR-12783. However, G2019S knock-in mice showed a blunted neurochemical and motor activation response to GBR-12783 compared to wild-type controls. Western blot and dopamine uptake analysis revealed an increase in dopamine transporter levels and activity in the striatum of 12-month-old G2019S KI mice. This phenotype correlated with a reduction in vesicular monoamine transporter 2 levels and an enhancement of vesicular dopamine uptake, which was consistent with greater resistance to reserpine-induced hypolocomotion. These changes were not observed in 3-month-old mice. Finally, Western blot analysis revealed no genotype difference in striatal levels of endogenous α-synuclein or α-synuclein bound to DOPAL (a toxic metabolite of dopamine). However, Serine129-phosphorylated α-synuclein levels were higher in 12-month-old G2019S knock-in mice. Immunohistochemistry confirmed this finding, also showing no genotype difference in 3-month-old mice. We conclude that the G2019S mutation causes progressive dysfunctions of dopamine transporters, along with Serine129-phosphorylated α-synuclein overload, at striatal dopaminergic terminals, which are not associated with dopamine homeostasis dysregulation or neuron loss but might contribute to intrinsic dopaminergic terminal vulnerability. We propose G2019S knock-in mice as a presymptomatic Parkinson’s disease model, useful to investigate the pathogenic interaction among genetics, aging, and internal or environmental factors leading to the disease.

Genetic and pharmacological evidence that endogenous nociceptin/orphanin FQ contributes to dopamine cell loss in Parkinson's disease

To investigate whether the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) contributes to the death of dopamine neurons in Parkinsons disease, we undertook a genetic and a pharmacological approach using NOP receptor knockout (NOP(-/-)) mice, and the selective and potent small molecule NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). Stereological unbiased methods were used to estimate the total number of dopamine neurons in the substantia nigra of i) NOP(-/-) mice acutely treated with the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), ii) naïve mice subacutely treated with MPTP, alone or in combination with SB-612111, iii) rats injected with a recombinant adeno-associated viral (AAV) vector overexpressing human mutant p.A53T α-synuclein, treated with vehicle or SB-612111. NOP(-/-) mice showed a 50% greater amount of nigral dopamine neurons spared in response to acute MPTP compared to controls, which was associated with a milder motor impairment. SB-612111, given 4 days after MPTP treatment to mimic the clinical condition, prevented the loss of nigral dopamine neurons and striatal dopaminergic terminals caused by subacute MPTP. SB-612111, administered a week after the AAV injections in a clinically-driven protocol, also increased by 50% both the number of spared nigral dopamine neurons and striatal dopamine terminals, and prevented accompanying motor deficits induced by α-synuclein. We conclude that endogenous N/OFQ contributes to dopamine neuron loss in pathogenic and etiologic models of Parkinsons disease through NOP receptor-mediated mechanisms. NOP receptor antagonists might prove effective as disease-modifying agents in Parkinsons disease, through the rescue of degenerating nigral dopamine neurons and/or the protection of the healthy ones.

Super-agile—The X-ray detector for the gamma-ray mission agile

The gamma-ray observatory AGILE, the first ASI Small Mission, is planned to operate in 2002–2005. We present here Super-AGILE, an X-ray detector added on top of the gamma-ray tracker. Super-AGILE will have a large field of view, providing hard X-ray imaging and moderate spectroscopy together with the gamma-ray detector. Super-AGILE is composed by Si-microstrip detectors, equipped with a low-noise electronics allowing a sensitive range of 10–40 keV, and coupled with a set of mutually orthogonal one-dimensional coded masks. A bi-dimensional source location capability is obtained by dividing the total 1444 cm2 geometric area of the detectors in two orthogonal directions.

Lipid nanocarriers containing a levodopa prodrug with potential antiparkinsonian activity.

This paper describes the production, characterization and in vivo activity of lipid nanocarriers (LN) containing a levodopa prodrug (LD-PD) with therapeutic potential in Parkinsons disease. LD is the mainstay of the pharmacotherapy of Parkinsons disease. However, after a good initial response, motor fluctuations, dyskinesia and loss of efficacy, develop over time, partly due to oscillations in plasma and brain levels of the drug. LD-PD was produced with the aim of prolonging the pharmacological activity of LD. To improve solubility, and simultaneously provide a long lasting release and therapeutic efficacy, the prodrug was formulated in tristearin/lecithin LN. The obtained formulation was homogeneous in particle size and remained stable for up to 2months from preparation. For the three different tested LD concentrations, namely 1.25, 2.5 and 5.0mg/ml, the morphological characterization revealed no substantial differences between unloaded and LD-PD loaded LN. The calorimetric test showed an interaction between the lipid phase and the loaded prodrug. In vitro studies using the dialysis method and enzymatic degradation procedure showed that the LD-PD loaded LN provided a controlled prodrug release. Finally, two behavioural tests specific to akinesia (bar test) or akinesia/bradykinesia (drag test) performed in 6-hydroxydopamine hemilesioned mice (a model of Parkinsons disease) demonstrated that the LD-PD loaded LN attenuated parkinsonian disabilities, showing a slightly reduced maximal efficacy but a longer lasting action (up to 24h) than an equal dose of LD. We conclude that LD-PD loaded LN may represent a future LD formulation useful in Parkinsons disease therapy.