Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP^C) into disease-specific forms, termed PrP^Sc, that have the ability to interact with PrP^C promoting its conversion to PrP^Sc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP^C region involved in the interaction with PrP^Sc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP^Sc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.     

Administration of PLP139-151 primes T cells distinct from those spontaneously responsive in vitro to this antigen

We examined the TCR repertoire used by naive SJL mice in their in vitro spontaneous response to proteolipid protein (PLP) 139-151 by Vbeta-Jbeta spectratyping and compared it to that used after immunization with the peptide. T cells from immunized mice use the public rearrangement Vbeta10-Jbeta1.1, but naive mice do not; in contrast, TCR CDR3-beta rearrangements of Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 consistently are associated with the spontaneous response. T cells involved in spontaneous and induced responses can each recognize PLP(139-151) presented in vivo, but its s.c. administration has different consequences for the two repertoires. Four days after immunization, T cells associated with spontaneous responsiveness appear in the draining lymph nodes but disappear by day 10 and never appear elsewhere. Simultaneously, Vbeta10-Jbeta1.1 T cells are likewise activated in the lymph nodes by day 4 and spread to the spleen by day 10. Eight- to 10-wk-old naive mice use a narrower repertoire of TCRs than do immunized age-matched mice. Induced Vbeta10-Jbeta1.1 T cells home to the CNS during experimental autoimmune encephalomyelitis, whereas we failed to detect Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 TCR rearrangements in the CNS. Thus, we observe that administration of PLP(139-151) primes a T cell repertoire distinct from the one responsible for spontaneous responsiveness. This "immunized" repertoire substitutes for the naive one and becomes dominant at the time of disease onset.     

Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.     

Neuromelanin can protect against iron-mediated oxidative damage in system modeling iron overload of brain aging and Parkinson's disease

In Parkinson's disease (PD), dopamine neurons containing neuromelanin selectively degenerate. Neuromelanin binds iron and accumulates in aging. Iron accumulates in reactive form during aging, PD, and is involved in neurodegeneration. It is not clear how the interaction of neuromelanin and iron can be protective or toxic by modulating redox processes. Here, we investigated the interaction of neuromelanin from human substantia nigra with iron in the presence of ascorbic acid, dopamine, and hydrogen peroxide. We observed that neuromelanin blocks hydroxyl radical production by Fenton's reaction, in a dose-dependent manner. Neuromelanin also inhibited the iron-mediated oxidation of ascorbic acid, thus sparing this major antioxidant molecule in brain. The protective effect of neuromelanin on ascorbate oxidation occurs even in conditions of iron overload into neuromelanin. The blockade of iron into a stable iron–neuromelanin complex prevents dopamine oxidation, inhibiting the formation of neurotoxic dopamine quinones. The above processes occur intraneuronally in aging and PD, thus showing that neuromelanin is neuroprotective. The iron–neuromelanin complex is completely decomposed by hydrogen peroxide and its degradation rate increases with the amount of iron bound to neuromelanin. This occurs in PD when extraneuronal iron–neuromelanin is phagocytosed by microglia and iron–neuromelanin degradation releases reactive/toxic iron.     

Involvement of nuclear factor-kappa B in bcl-xL-induced interleukin 8 expression in glioblastoma

We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkBalpha and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkBalpha protein, paralleled by an increase in the phosphorylation of the same IkBalpha and IKKalpha/beta. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkBalpha in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells.     

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X₇ receptors, it is still unclear whether neuronal functions can be attributed to P2X₇ receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X₇ receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non-neuronal cells and allows exposure of 'nude' release-regulating pre-synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X₇ receptors. Together, our findings suggest that (i) P2X₇ receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X₇ receptors are localized on glutamatergic nerve terminals; (iii) P2X₇ receptors play a significant role in ATP-evoked glutamate efflux, which involves Ca²⁺-dependent vesicular release; and (iv) the P2X₇ receptor itself constitutes a significant Ca²⁺-independent mode of exit for glutamate.     

Serotonin-1A receptor function in the dorsal raphe nucleus following chronic administration of the selective serotonin reuptake inhibitor sertraline

Serotonin-1A (5-HT_1A) receptors in the dorsal raphe nucleus (DRN) function as somatodendritic autoreceptors, and therefore play a critical role in controlling serotonergic cell firing and serotonergic neurotransmission. We hypothesized that a decrease in the capacity of 5-HT_1A receptors to activate G proteins was a general mechanism by which 5-HT_1A receptors in the DRN are desensitized following chronic administration of selective serotonin reuptake inhibitors (SSRIs). Using in vivo microdialysis, we found that the ability of the 5-HT_1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in striatum was attenuated following chronic treatment of rats with the SSRIs sertraline or fluoxetine. This apparent desensitization of somatodendritic 5-HT_1A autoreceptor function was not accompanied by a decrease in 5-HT_1A receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT. In marked contrast to what was observed following chronic administration of fluoxetine, 5-HT_1A receptor-stimulated [³⁵S]GTPγS binding in the DRN was not altered following chronic sertraline treatment. Thus, desensitization of 5-HT_1A somatodendritic autoreceptor function following chronic sertraline administration appears not to be due to a decrease in the capacity 5-HT_1A receptors to activate G proteins in the DRN. Our findings suggest that the SSRIs may not be a homogeneous class of antidepressant drug with regard to the mechanism by which the function of somatodendritic 5-HT_1A autoreceptors is regulated.