Small Extracellular Vesicles Secreted by Nigrostriatal Astrocytes Rescue Cell Death and Preserve Mitochondrial Function in Parkinson's Disease.

Extracellular vesicles (EVs) are emerging as powerful players in cell-to-cell communication both in healthy and diseased brain. In Parkinson's disease (PD)-characterized by selective dopaminergic neuron death in ventral midbrain (VMB) and degeneration of their terminals in striatum (STR)-astrocytes exert dual harmful/protective functions, with mechanisms not fully elucidated. Here, this study shows that astrocytes from the VMB-, STR-, and VMB/STR-depleted brains release a population of small EVs  in a region-specific manner. Interestingly, VMB-astrocytes secreted the highest rate of EVs, which is further exclusively increased in response to CCL3, a chemokine that promotes robust dopaminergic neuroprotection in different PD models. The neuroprotective potential of nigrostriatal astrocyte-EVs is investigated in differentiated versus undifferentiated SH-SY5Y cells exposed to oxidative stress and mitochondrial toxicity. EVs from both VMB- and STR-astrocytes counteract H2 O2 -induced caspase-3 activation specifically in differentiated cells, with EVs from CCL3-treated astrocytes showing a higher protective effect. High resolution respirometry further reveals that nigrostriatal astrocyte-EVs rescue neuronal mitochondrial complex I function impaired by the neurotoxin MPP+ . Notably, only EVs from VMB-astrocyte fully restore ATP production, again specifically in differentiated SH-SY5Y. These results highlight a regional diversity in the nigrostriatal system for the secretion and activities of astrocyte-EVs, with neuroprotective implications for PD.

High-Resolution Respirometry Reveals MPP+ Mitochondrial Toxicity Mechanism in a Cellular Model of Parkinson's Disease.

MPP+ is the active metabolite of MPTP, a molecule structurally similar to the herbicide Paraquat, known to injure the dopaminergic neurons of the nigrostriatal system in Parkinson's disease models. Within the cells, MPP+ accumulates in mitochondria where it inhibits complex I of the electron transport chain, resulting in ATP depletion and neuronal impairment/death. So far, MPP+ is recognized as a valuable tool to mimic dopaminergic degeneration in various cell lines. However, despite a large number of studies, a detailed characterization of mitochondrial respiration in neuronal cells upon MPP+ treatment is still missing. By using high-resolution respirometry, we deeply investigated oxygen consumption related to each respiratory state in differentiated neuroblastoma cells exposed to the neurotoxin. Our results indicated the presence of extended mitochondrial damage at the inner membrane level, supported by increased LEAK respiration, and a drastic drop in oxygen flow devoted to ADP phosphorylation in respirometry measurements. Furthermore, prior to complex I inhibition, an enhancement of complex II activity was observed, suggesting the occurrence of some compensatory effect. Overall our findings provide a mechanistic insight on the mitochondrial toxicity mediated by MPP+, relevant for the standardization of studies that employ this neurotoxin as a disease model.

Parkinson's disease, aging and adult neurogenesis: Wnt/ß-catenin signalling as the key to unlock the mystery of endogenous brain repair.

A common hallmark of age-dependent neurodegenerative diseases is an impairment of adult neurogenesis. Wingless-type mouse mammary tumor virus integration site (Wnt)/ß-catenin (WßC) signalling is a vital pathway for dopaminergic (DAergic) neurogenesis and an essential signalling system during embryonic development and aging, the most critical risk factor for Parkinson's disease (PD). To date, there is no known cause or cure for PD. Here we focus on the potential to reawaken the impaired neurogenic niches to rejuvenate and repair the aged PD brain. Specifically, we highlight WßC-signalling in the plasticity of the subventricular zone (SVZ), the largest germinal region in the mature brain innervated by nigrostriatal DAergic terminals, and the mesencephalic aqueduct-periventricular region (Aq-PVR) Wnt-sensitive niche, which is in proximity to the SNpc and harbors neural stem progenitor cells (NSCs) with DAergic potential. The hallmark of the WßC pathway is the cytosolic accumulation of ß-catenin, which enters the nucleus and associates with T cell factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors, leading to the transcription of Wnt target genes. Here, we underscore the dynamic interplay between DAergic innervation and astroglial-derived factors regulating WßC-dependent transcription of key genes orchestrating NSC proliferation, survival, migration and differentiation. Aging, inflammation and oxidative stress synergize with neurotoxin exposure in "turning off" the WßC neurogenic switch via down-regulation of the nuclear factor erythroid-2-related factor 2/Wnt-regulated signalosome, a key player in the maintenance of antioxidant self-defense mechanisms and NSC homeostasis. Harnessing WßC-signalling in the aged PD brain can thus restore neurogenesis, rejuvenate the microenvironment, and promote neurorescue and regeneration.

Wnt/ß-Catenin Signaling Pathway Governs a Full Program for Dopaminergic Neuron Survival, Neurorescue and Regeneration in the MPTP Mouse Model of Parkinson's Disease.

Wingless-type mouse mammary tumor virus (MMTV) integration site (Wnt) signaling is one of the most critical pathways in developing and adult tissues. In the brain, Wnt signaling contributes to different neurodevelopmental aspects ranging from differentiation to axonal extension, synapse formation, neurogenesis, and neuroprotection. Canonical Wnt signaling is mediated mainly by the multifunctional ß-catenin protein which is a potent co-activator of transcription factors such as lymphoid enhancer factor (LEF) and T-cell factor (TCF). Accumulating evidence points to dysregulation of Wnt/ß-catenin signaling in major neurodegenerative disorders. This review highlights a Wnt/ß-catenin/glial connection in Parkinson's disease (PD), the most common movement disorder characterized by the selective death of midbrain dopaminergic (mDAergic) neuronal cell bodies in the subtantia nigra pars compacta (SNpc) and gliosis. Major findings of the last decade document that Wnt/ß-catenin signaling in partnership with glial cells is critically involved in each step and at every level in the regulation of nigrostriatal DAergic neuronal health, protection, and regeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, focusing on Wnt/ß-catenin signaling to boost a full neurorestorative program in PD.

Neural Stem Cell Grafts Promote Astroglia-Driven Neurorestoration in the Aged Parkinsonian Brain via Wnt/ß-Catenin Signaling.

During aging-one the most potent risk factors for Parkinson's disease (PD)-both astrocytes and microglia undergo functional changes that ultimately hamper homoeostasis, defense, and repair of substantia nigra pars compacta (SNpc) midbrain dopaminergic (mDA) neurons. We tested the possibility of rejuvenating the host microenvironment and boosting SNpc DA neuronal plasticity via the unilateral transplantation of syngeneic neural stem/progenitor cells (NSCs) in the SNpc of aged mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced experimental PD. Transplanted NSCs within the aged SNpc engrafted and migrated in large proportions to the tegmental aqueduct mDA niche, with 30% acquiring an astroglial phenotype. Both graft-derived exogenous (ex-Astro) and endogenous astrocytes (en-Astro) expressed Wnt1. Both ex-Astro and en-Astro were key triggers of Wnt/ß-catenin signaling in SNpc-mDA neurons and microglia, which was associated with mDA neurorescue and immunomodulation. At the aqueduct-ventral tegmental area level, NSC grafts recapitulated a genetic Wnt1-dependent mDA developmental program, inciting the acquisition of a mature Nurr1+ TH+ neuronal phenotype. Wnt/ß-catenin signaling antagonism abolished mDA neurorestoration and immune modulatory effects of NSC grafts. Our work implicates an unprecedented therapeutic potential for somatic NSC grafts in the restoration of mDA neuronal function in the aged Parkinsonian brain. Stem Cells 2018;36:1179-1197.

Wnt3a promotes pro-angiogenic features in macrophages in vitro: Implications for stroke pathology.

Wnt3a is implicated in several key cellular processes and its expression has been reported in different cell types. Here, we report a novel function for Wnt3a in macrophages, whose exposure to this ligand shifts them towards a pro-angiogenic phenotype capable, under oxygen and glucose deprivation, of inducing in vitro tubular pattern structures in endothelial cells resembling capillary-like vasculature. These newly acquired angiogenetic features also include increased proliferation and migration and surprisingly, an increase in cell death. This work provides a new link between Wnt3a and macrophage-mediated angiogenesis under glucose and oxygen deprivation in vitro, which are worth further investigation in pathological conditions including stroke, where the stimulation of the angiogenic process might help to recovery after tissue injury Impact statement This work provides a new link between Wnt3a and macrophage-mediated angiogenesis under glucose and oxygen deprivation in vitro. Our results reveal how Wnt3a shifts macrophages towards a pro-angiogenic phenotype, which is able-in absence of both glucose and oxygen-of inducing angiogenesis in vitro, thus pointing to a synergy between the activation of the pathway and the hypoxia scenario. This work also demonstrates that modulation of cell death is key in order to explain the observed angiogenic effects. We consider all these findings of significant importance, since no connection between Wnt3a, macrophages, and angiogenesis has been established so far. Furthermore, we do believe that this work provides new and interesting results, with Wnt signaling pathway emerging as an interesting target mediating beneficial outcomes during the inflammatory response undoubtedly linked to stroke pathology, where angiogenesis has been already proposed as a potential mechanism to promote recovery after the injury.

Uncovering novel actors in astrocyte-neuron crosstalk in Parkinson's disease: the Wnt/ß-catenin signaling cascade as the common final pathway for neuroprotection and self-repair.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by progressive loss of dopaminergic (DAergic) neuronal cell bodies in the substantia nigra pars compacta and gliosis. The cause and mechanisms underlying the demise of nigrostriatal DAergic neurons are ill-defined, but interactions between genes and environmental factors are recognized to play a critical role in modulating the vulnerability to PD. Current evidence points to reactive glia as a pivotal factor in PD pathophysiology, playing both protective and destructive roles. Here, the contribution of reactive astrocytes and their ability to modulate DAergic neurodegeneration, neuroprotection and neurorepair in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) rodent model of PD will be discussed in the light of novel emerging evidence implicating wingless-type mouse mammary tumor virus integration site (Wnt)/ß-catenin signaling as a strong candidate in MPTP-induced nigrostriatal DAergic plasticity. In this work, we highlight an intrinsic Wnt1/frizzled-1/ß-catenin tone that critically contributes to the survival and protection of adult midbrain DAergic neurons, with potential implications for drug design or drug action in PD. The dynamic interplay between astrocyte-derived factors and neurogenic signals in MPTP-induced nigrostriatal DAergic neurotoxicity and repair will be summarized, together with recent findings showing a critical role of glia-neural stem/progenitor cell (NPC) interactions aimed at overcoming neurodegeneration and inducing neurorestoration. Understanding the intrinsic plasticity of nigrostriatal DAergic neurons and deciphering the signals facilitating the crosstalk between astrocytes, microglia, DAergic neurons and NPCs may have major implications for the role of stem cell technology in PD, and for identifying potential therapeutic targets to induce endogenous neurorepair.

Wnt your brain be inflamed? Yes, it Wnt!

The roles of Wnts in neural development, synaptogenesis, and cancer are generally well characterized. Nonetheless, evidence exists that interactions between the immune and nervous systems control major brain regenerative processes ranging from physiological or pathological (reparative) regeneration to neurogenesis and synaptic plasticity. Recent studies describe deregulated Wnt-Fzd signaling in degenerative and inflammatory central nervous system (CNS) disorders, and the expression of Wnt signaling components in the immune system, and in immune-like cells of the mammalian CNS. This would suggest a likely involvement of Wnts in inflammation-driven brain damage and inflammation-directed brain repair. Here, we review how Wnts modulate neuroimmune interactions and offer a perspective on the most challenging therapeutic opportunities for those CNS diseases where injury-reactive Wnt-flavored inflammation precedes secondary neurodegeneration.

The MPTP mouse model: cues on DA release and neural stem cell restorative role.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to cause parkinsonism in humans and this fact is a major incentive for using this toxin as an animal model to study the pathogenesis of Parkinson's disease (PD). Although the monkey MPTP model remains the best, most studies have been performed in mice. The so-called acute and sub-acute regimens are commonly used. Both induce tissue striatal dopamine (DA) depletion and nigral neuron death. Tissue striatal DA depletion does not necessarily correlate with impairment of striatal dopaminergic functioning. In freely moving mice, systemic acute or sub-acute MPTP directly induces prolonged release of striatal DA. Such DA release may be considered the first step in MPTP-induced striatal DA depletion. Reportedly, neural stem cells improve symptoms in the MPTP model of PD by interacting with the MPTP-induced pathological nigrostriatal milieu.

Loss of aromatase cytochrome P450 function as a risk factor for Parkinson's disease?

The final step in the physiological synthesis of 17beta estradiol (E(2)) is aromatization of precursor testosterone by a CYP19 gene product, cytochrome P450 estrogen aromatase in the C19 steroid metabolic pathway. Within the central nervous system (CNS) the presence, distribution, and activity of aromatase have been well characterized. Developmental stage and injury are known modulators of brain enzyme activity, where both neurons and glial cells reportedly have the capability to synthesize this key estrogenic enzyme. The gonadal steroid E(2) is a critical survival, neurotrophic and neuroprotective factor for dopaminergic neurons of the substantia nigra pars compacta (SNpc), the cells that degenerate in Parkinson's disease (PD). In previous studies we underlined a crucial role for the estrogenic status at the time of injury in dictating vulnerability to the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our ongoing studies address the contribution of brain aromatase and extragonadal E(2) as vulnerability factors for PD pathology in female brain, by exposing aromatase knockout (ArKO, -/-) female mice which are unable to synthesize estrogens to MPTP. Our initial results indicate that aromatase deficiency from early embryonic life significantly impairs the functional integrity of SNpc tyrosine hydroxylase-positive neurons and dopamine transporter innervation of the caudate-putamen in adulthood. In addition, ArKO females exhibited a far greater vulnerability to MPTP-induced nigrostriatal damage as compared to their Wt type gonadally intact and gonadectomized counterparts. Characterization of this novel implication of P450 aromatase as determining factor for PD vulnerability may unravel new avenues for the understanding and development of novel therapeutic approaches for Parkinson's disease.

Multiple sclerosis and anti-Plasmodium falciparum innate immune response.

Several epidemiological investigations conducted in Sardinia, insular Italy, indicate that the strong selective pressure of malaria along the centuries may have concurred to the elevated genetic MS-risk in this region. To test such hypothesis in an experimental setting, we have compared the immune response to P. falciparum (the causative agent of malaria) in Sardinian MS patients relative to their ethnic healthy controls and control MS patients of different ethnicity. To this purpose, the P. falciparum-driven peripheral mononuclear cell proliferation, the production of pro-inflammatory cytokines of the innate immunity such as TNF-alpha, IL-6 and IL-12 and the ability to inhibit the parasite growth have been tested in relation to HLA-DR alleles and TNF promoter polymorphisms known of being associated to MS. We found that P. falciparum-induced proliferation, cytokine production and parasite killing are significantly augmented in Sardinian MS patients as compared to controls (p<0.01). Additionally, a correlation is found with genes associated to Sardinian MS, namely the TNF(-376A) promoter polymorphism and the class II HLA-DRB1*0405 allele. In conclusion, we have found evidences that some genetic traits formerly selected to confer a protective responses to P. falciparum now partially contribute to the elevated MS susceptibility amongst Sardinians.

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.

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 microm 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 microm) or melatonin (50 microm) 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 Parkinson's disease.

Chitotriosidase in patients with acute ischemic stroke.

BACKGROUND: Following an acute brain ischemia, local endothelia allow monocyte chemoattraction into the lesion site which contributes to brain damage through a group of neurotoxic factors. A relationship exists between the extent of brain damage and the plasma level of monocyte products, including chitotriosidase, though usually strictly related to preexisting infectious-inflammatory diseases. PURPOSE: Since chitotriosidase activity is also elevated in pathogen-free conditions, we tested whether chitotriosidase upregulation might be specifically related to stroke and unrelated to clinically relevant infectious diseases. METHODS: We studied the plasma level of chitotriosidase activity, TNF-alpha and IL-6 in 44 consecutive patients with acute brain ischemia without concomitant symptoms or signs of inflammatory-infectious diseases. Results were compared with stroke severity and outcome as detected by brain CT and NIH scale. Blood samples were collected, on average, 11 h after stroke onset. RESULTS: Chitotriosidase activity positively correlates with stroke severity, as measured by NIH scale (r = 0.69, p < 0.01), to the extent of brain damage as documented by CT (r = 0.75, p < or = 0.001) and the TNF-alpha level (r = 0.76, p < 0.001); it also inversely correlates with the IL-6 level (r = -0.43, p < or = 0.05). CONCLUSION: Our results indicate that chitotriosidase is a specific marker of macrophage activation occurring in stroke which directly correlates with stroke severity independently of preexisting inflammatory or infectious conditions.

Role of endogenous melatonin in the oxidative homeostasis of the extracellular striatal compartment: a microdialysis study in PC12 cells in vitro and in the striatum of freely moving rats.

A capillary apparatus for in vitro microdialysis was used to investigate melatonin and ascorbic acid effects on dopamine (DA) autoxidation or nitric oxide (NO)-mediated oxidation in suspended PC12 cells. Following high K+ (KCl 75 mm) infusion, secreted DA underwent a partial autoxidation or peroxynitrite-mediated oxidation when the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mm) was co-infused with KCl. Ascorbic acid was supplied to the medium by means of intracellular reduction of infused dehydroascorbic acid (DHAA) (5.0 mm). Melatonin (50 microm) and DHAA showed a synergistic effect in inhibiting DA autoxidation and peroxynitrite-mediated DA oxidation. Moreover, melatonin increased dialysate recovery of ascorbic acid released from PC12 cells. Endogenous melatonin was depleted in rats maintained on a 24-hr light cycle for 1 wk. In melatonin-depleted rats, baseline levels of dialysate ascorbic acid were lower than controls, while those of DA were unaffected. In these rats, intrastriatal infusion of 5.0 mm SIN-1 induced DA increases significantly lower than in controls; in addition, dialysate ascorbic acid concentrations exhibited significant decreases. Melatonin co-infusion restored SIN-1 effects on dialysate DA and antagonized SIN-1-induced ascorbic acid decreases. Melatonin-depleted rats were allowed to recover. In these rats, striatal baseline ascorbic acid, as well as SIN-1-induced increases in dialysate DA did not differ from controls. Taken together, these findings suggest that endogenous melatonin is an active component of the striatal extracellular antioxidant pool, as it maintains endogenous ascorbic acid in its reduced status and co-operates with ascorbic acid in protecting extracellular DA from exogenous NO-mediated oxidation.

Signaling pathways in the nitric oxide and iron-induced dopamine release in the striatum of freely moving rats: role of extracellular Ca2+ and L-type Ca2+ channels.

We showed previously that exogenous iron potentiated nitric oxide (NO) donor-induced release of striatal dopamine (DA) in freely moving rats, using microdialysis. In this study, the increase in dialysate DA induced by intrastriatal infusion of the NO-donor 3-morpholinosydnonimine (SIN-1, 1.0 mM for 180 min) was scarcely affected by Ca2+ omission. N-methyl-d-glucamine dithiocarbamate (MGD) is a thiol compound whose NO trapping activity is potentiated by iron(II). Intrastriatal co-infusion of MGD either alone or associated with iron(II), however, potentiated SIN-1-induced increases in dialysate DA. In contrast, co-infusion of the NO trapper 4-(carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) significantly attenuated the increase in dialysate DA induced by SIN-1 (5.0 mM for 180 min). SIN-1+MGD+iron(II)-induced increases in dialysate DA were inhibited by Ca2+ omission or co-infusion of either deferoxamine or the L-type (Ca(v) 1.1-1.3) Ca2+ channel inhibitor nifedipine; in contrast, the increase was scarcely affected by co-infusion of the N-type (Ca(v) 2.2) Ca2+ channel inhibitor omega-conotoxin GVIA. These results demonstrate that exogenous NO-induced release of striatal DA is independent on extracellular Ca2+; however, in presence of the NO trapper MGD, NO may preferentially react with either endogenous or exogenous iron to form a complex which releases striatal DA with an extracellular Ca2+-dependent and nifedipine-sensitive mechanism.

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 Parkinson's 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 (E(2)) 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.

High frequency of TNF alleles -238A and -376A in individuals from northern Sardinia.

The G to A single nucleotide polymorphisms (SNPs), at position -376, -308 and -238 in the promoter of the tumor necrosis factor alpha (TNF) gene, have been independently correlated with numerous diseases. Alleles TNF(-376A) and TNF(-238A) are normally found throughout the world with very low frequencies. We investigated the frequency of these SNPs in Sicilian subjects hospitalized after traumatic brain injury and in three groups of subjects from northern Sardinia: healthy subjects and individuals with multiple sclerosis or ischemic stroke. While no significant difference was found between healthy and disease subjects, the frequency of TNF(-376A) and TNF(-238A) was elevated up to 10 times in Sardinia compared to Sicily and other populations throughout the world. These elevated frequencies may be the result of genetic drift or of selective pressure on TNF itself or on neighboring genes, including the HLA. Malaria, endemic to Sardinia until the end of the 1940s, and the bubonic plague, are among the possible causes of selection. These findings indicate that Sardinia is an ideal location to further elucidate the correlation between TNF or HLA polymorphisms and diseases, including multiple sclerosis and type-I diabetes, present with an unusually high frequency and co-morbidity in Sardinia.

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 Parkinson's 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.

Apoptotic cell death and amyloid precursor protein signaling in neuroblastoma SH-SY5Y cells.

We have recently shown that the amyloid precursor protein (APP) and a subset of its C-terminal fragments (CTFs) are tyrosine phosphorylated in human brain and in cultured cells. Tyrosine phosphorylation generates a substrate that is sequentially bound by the adaptor proteins ShcA and Grb2, and this interaction is significantly enhanced in Alzheimer's disease brains. Here we have studied the APP/CTFs phosphorylation and ShcA activation in a human neuroblastoma cell line, SH-SY5Y, under basal and apoptotic conditions. To commit these cells to apoptosis, we used staurosporin, a well-known apoptotic inducer and protein kinase C blocker. Our data suggest the following: (1) in normally proliferating SH-SY5Y cells, full-length APP is complexed with Grb2[Q3], likely through its SH2 domain; (2) upon induction of apoptosis, APP is degraded and ShcA-Grb2 coimmunoprecipitates with CTFs recognized by anti-APP antibodies; and (3) caspase inhibitors partially block the degradation of APP and the coprecipitation of CTFs with ShcA-Grb2 adaptors. In summary, our data suggest that in SH-SY5Y cells, tyrosine-phosphorylated APP is involved in a complex with ShcA-Grb2 adaptors that is disrupted during apoptosis. The abnormal degradation of APP and consequent increased levels of CTFs (as has been observed in Alzheimer's disease and Down's syndrome) generate a complex between tyrosine-phosphorylated CTFs and intracellular adaptors. The signaling through APP and its CTFs may have significant relevance for apoptotic cell death in Alzheimer's disease.

Exposure to a dysfunctional glucocorticoid receptor from early embryonic life programs the resistance to experimental autoimmune encephalomyelitis via nitric oxide-induced immunosuppression.

Glucocorticoid (GC) hormones play a central role in the bidirectional communication between the neuroendocrine and the immune systems and exert, via GC receptors (GR), potent immunosuppressive and anti-inflammatory effects. In this study, we report that GR deficiency of transgenic mice expressing GR antisense RNA from early embryonic life has a dramatic impact in programming the susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. GR deficiency renders mice resistant to myelin oligodendrocyte glycoprotein-induced EAE, and such mice do not develop clinical or histological signs of disease compared with EAE-susceptible wild-type mice. Resistance to EAE in GR-deficient mice is associated not with endogenous GC levels, but with a significant reduction in spleen and lymph node cell proliferation. The use of NO inhibitors in vitro indicates that NO is the candidate immunosuppressor molecule. GR-deficient mice develop 3- to 6-fold higher nitrite levels in the periphery and are resistant to NO inhibition by GCs. Specific inhibition of NO production in vivo by treatment with the inducible NO synthase inhibitor, L-N(6)-(1-iminoethyl)-lysine, suppressed circulating nitrites, increased myelin oligodendrocyte glycoprotein-specific cell proliferation, and rendered GR-deficient mice susceptible to EAE. Thus, life-long GR deficiency triggers inducible NO synthase induction and NO generation with consequent down-regulation of effector cell proliferation. These findings identify a novel link among GR, NO, and EAE susceptibility and highlight NO as critical signaling molecule in bidirectional communication between the hypothalamic-pituitary-adrenocortical axis and the immune system.