Gaia Rocchitta

Estrogen, neuroinflammation and neuroprotection in Parkinson’s disease: Glia dictates resistance versus vulnerability to neurodegeneration

Post-menopausal estrogen deficiency is recognized to play a pivotal role in the pathogenesis of a number of age-related diseases in women, such as osteoporosis, coronary heart disease and Alzheimers disease. There are also sexual differences in the progression of diseases associated with the nigrostriatal dopaminergic system, such as Parkinsons disease, a chronic progressive degenerative disorder characterized by the selective degeneration of mesencephalic dopaminergic neurons in the substancia nigra pars compacta. The mechanism(s) responsible for dopaminergic neuron degeneration in Parkinsons disease are still unknown, but oxidative stress and neuroinflammation are believed to play a key role in nigrostriatal dopaminergic neuron demise. Estrogen neuroprotective effects have been widely reported in a number of neuronal cell systems including the nigrostriatal dopaminergic neurons, via both genomic and non-genomic effects, however, little is known on estrogen modulation of astrocyte and microglia function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinsons disease. We here highlight estrogen modulation of glial neuroinflammatory reaction in the protection of mesencephalic dopaminergic neurons and emphasize the cardinal role of glia-neuron crosstalk in directing neuroprotection vs neurodegeneration. In particular, the specific role of astroglia and its pro-/anti-inflammatory mechanisms in estrogen neuroprotection are presented. This study shows that astrocyte and microglia response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injury vary according to the estrogenic status with direct consequences for dopaminergic neuron survival, recovery and repair. These findings provide a new insight into the protective action of estrogen that may possibly contribute to the development of novel therapeutic treatment strategies for Parkinsons disease.

Glucocorticoid receptor deficiency increases vulnerability of the nigrostriatal dopaminergic system: critical role of glial nitric oxide.

Glucocorticoids (GCs) exert via glucocorticoid receptors (GRs) potent anti‐inflammatory and immunosuppressive effects. Emerging evidence indicates that an inflammatory process is involved in dopaminergic nigro‐striatal neuronal loss in Parkinsons disease. We here report that the GR deficiency of transgenic (Tg) mice expressing GR antisense RNA from early embryonic life has a dramatic impact in “programming” the vulnerability of dopaminergic neurons to 1‐ methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). The GR deficiency of Tg mice exacerbates MPTP‐induced toxicity to dopaminergic neurons, as revealed by both severe loss of tyrosine hydroxylase positive nigral neurons and sharp decreases in striatal levels of dopamine and its metabolites. In addition, the late increase in dopamine oxidative metabolism and ascorbic acid oxidative status in GR‐deficient mice was far greater than in wild‐type (Wt) mice. Inducible nitric oxide synthase (iNOS) was sharply increased in activated astrocytes, macrophages/microglia of GR‐deficient as compared with Wt mice. Moreover, GR‐deficient microglia produced three‐ to fourfold higher nitrite levels than Wt mice; these increases preceded the loss of dopaminergic function and were resistant to GR the inhibitory effect of GC, pointing to peroxynitrites as candidate neurotoxic effectors. The iNOS inhibitor N6‐(1‐ iminoethyl)‐L‐lysine normalized vulnerability of Tg mice, thus establishing a novel link between genetic impairment of GR function and vulnerability to MPTP.

Real-Time Monitoring of Brain Tissue Oxygen Using a Miniaturized Biotelemetric Device Implanted in Freely Moving Rats

A miniaturized biotelemetric device for the amperometric detection of brain tissue oxygen is presented. The new system, derived from a previous design, has been coupled with a carbon microsensor for the real-time detection of dissolved O(2) in the striatum of freely moving rats. The implantable device consists of a single-supply sensor driver, a current-to-voltage converter, a microcontroller, and a miniaturized data transmitter. The oxygen current is converted to a digital value by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC). The digital data is sent to a personal computer using a six-byte packet protocol by means of a miniaturized 434 MHz amplitude modulation (AM) transmitter. The receiver unit is connected to a personal computer (PC) via a universal serial bus. Custom developed software allows the PC to store and plot received data. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption, and good linear response in the nanoampere current range. The in vivo results confirmed previously published observations on oxygen dynamics in the striatum of freely moving rats. The system serves as a rapid and reliable model for studying the effects of different drugs on brain oxygen and brain blood flow and it is suited to work with direct-reduction sensors or O(2)-consuming biosensors.

Simultaneous telemetric monitoring of brain glucose and lactate and motion in freely moving rats

A new telemetry system for simultaneous detection of extracellular brain glucose and lactate and motion is presented. The device consists of dual-channel, single-supply miniature potentiostat-I/V converter, a microcontroller unit, a signal transmitter, and a miniaturized microvibration sensor. Although based on simple and inexpensive components, the biotelemetry device has been used for accurate transduction of the anodic oxidation currents generated on the surface of implanted glucose and lactate biosensors and animal microvibrations. The device was characterized and validated in vitro before in vivo experiments. The biosensors were implanted in the striatum of freely moving animals and the biotelemetric device was fixed to the animals head. Physiological and pharmacological stimulations were given in order to induce striatal neural activation and to modify the motor behavior in awake, untethered animals.

Hormones Are Key Actors in Gene X Environment Interactions Programming the Vulnerability to Parkinson's Disease: Glia as a Common Final Pathway

Alterations in developmental programming of neuroendocrine and immune system function may critically modulate vulnerability to various diseases. In particular, genetic factors, including gender, may interact with early life events such as exposure to hormones, endotoxins, or neurotoxins, thereby influencing disease predisposition and/or severity, but little is known about the role of the astroglial cell compartment and its mediators in this phenomenon. Indeed, in the context of innate inflammatory mechanisms, a dysfunction of the astroglial cell compartment is believed to contribute to the selective degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta in Parkinsons disease (PD) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of PD. Hence, in response to brain injury the roles of astrocytes and microglia are very dynamic and cell type‐dependent, in that they may exert the known proinflammatory (harmful) effects, but in certain circumstances they can turn into highly protective cells and exert anti‐inflammatory (beneficial) functions, thereby facilitating neuronal recovery and repair. Here, we summarize our work suggesting a chief role of hormonal programming of glial response to inflammation and oxidative stress in MPTP‐induced loss of DA neuron functionality and demonstrate that endogenous glucocorticoids and the female hormone estrogen (E2) inhibit the aberrant neuroinflammatory cascade, protect astrocytes and microglia from programmed cell death, and stimulate recovery of DA neuron functionality, thereby triggering the repair process. The overall results highlight glia as a final common pathway directing neuroprotection versus neurodegeneration. Such recognition of endogenous glial protective pathways may provide a new insight and may contribute to the development of novel therapeutic treatment strategies for PD and possibly other neurodegenerative disorders.

Biotelemetric Monitoring of Brain Neurochemistry in Conscious Rats Using Microsensors and Biosensors

In this study we present the real-time monitoring of three key brain neurochemical species in conscious rats using implantable amperometric electrodes interfaced to a biotelemetric device. The new system, derived from a previous design, was coupled with carbon-based microsensors and a platinum-based biosensor for the detection of ascorbic acid (AA), O2 and glucose in the striatum of untethered, freely-moving rats. The miniaturized device consisted of a single-supply sensor driver, a current-to-voltage converter, a microcontroller and a miniaturized data transmitter. The redox currents were digitized to digital values by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC), and sent to a personal computer by means of a miniaturized AM transmitter. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption and good linear response in the nanoampere current range. The in-vivo results confirmed previously published observations on striatal AA, oxygen and glucose dynamics recorded in tethered rats. This approach, based on simple and inexpensive components, could be used as a rapid and reliable model for studying the effects of different drugs on brain neurochemical systems.

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity.

The apparent Michaelis constant, K(M), for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOx-based amperometric biosensors in the linear response region (100+/-11 nA cm(-2)microM(-1) at pH 7.4; SD, n=23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design.

Endogenous melatonin protects L-DOPA from autoxidation in the striatal extracellular compartment of the freely moving rat: potential implication for long-term L-DOPA therapy in Parkinson's disease

Abstract:  We previously showed, using microdialysis, that autoxidation of exogenous L‐dihydroxyphenylalanine (l‐DOPA) occurs in vivo in the extracellular compartment of the freely moving rat, with a consequent formation of l‐DOPA semiquinone (l‐DOPA‐SQ). In the present study, intrastriatal infusion of l‐DOPA (1.0 μm for 200 min) increased dialysate l‐DOPA concentrations (maximum increases up to 116‐fold baseline values); moreover, l‐DOPA‐SQ was detected in dialysates. Individual dialysate concentrations of l‐DOPA were negatively correlated with those of l‐DOPA‐SQ. Co‐infusion of N‐acetylcysteine (100 μm) or melatonin (50 μm) increased l‐DOPA (up to 151‐ and 246‐fold, respectively) and decreased l‐DOPA‐SQ (by about 53% and 87%, respectively) dialysate concentrations. Systemic l‐DOPA [25 mg/kg intraperitoneally (i.p.) twice in a 12‐h interval] significantly increased striatal baseline dialysate concentrations of l‐DOPA and decreased dopamine (DA) and ascorbic acid (AsAc) concentrations, when compared with controls. Following systemic l‐DOPA, l‐DOPA‐SQ was detected in dialysates. Endogenous melatonin was depleted in rats maintained on a 24‐h light cycle for 1 wk. In melatonin‐depleted rats, systemic l‐DOPA induced a smaller increase in dialysate l‐DOPA, a greater increase in l‐DOPA‐SQ formation, and a greater reduction in DA and AsAc dialysate concentrations. Co‐administration of melatonin (5.0 mg/kg, i.p., twice in a 12‐h interval) with l‐DOPA, in control as well as in light‐exposed rats, significantly increased dialysate l‐DOPA concentrations, greatly inhibited l‐DOPA‐SQ formation, and restored up to the control values dialysate DA and AsAc concentrations. These findings demonstrate that endogenous melatonin protects exogenous l‐DOPA from autoxidation in the extracellular compartment of the striatum of freely moving rats; moreover, systemic co‐administration of melatonin with l‐DOPA markedly increases striatal l‐DOPA bioavailability in control as well as in melatonin‐depleted rats. These results may be of relevance to the long‐term l‐DOPA therapy of Parkinsons disease.

Enzyme Biosensors for Biomedical Applications: Strategies for Safeguarding Analytical Performances in Biological Fluids.

Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.

LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.