A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions

The aim of this study was to develop a three-dimensional (3D) model of the human blood-brain barrier in vitro, which mimics the cellular architecture of the CNS and could be used to analyse the delivery of nanoparticles to cells of the CNS. The model includes human astrocytes set in a collagen gel, which is overlaid by a monolayer of human brain endothelium (hCMEC/D3 cell line). The model was characterised by transmission electron microscopy (TEM), immunofluorescence microscopy and flow cytometry. A collagenase digestion method could recover the two cell types separately at 92-96% purity. Astrocytes grown in the gel matrix do not divide and they have reduced expression of aquaporin-4 and the endothelin receptor, type B compared to two-dimensional cultures, but maintain their expression of glial fibrillary acidic protein. The effects of conditioned media from these astrocytes on the barrier phenotype of the endothelium was compared with media from astrocytes grown conventionally on a two-dimensional (2D) substratum. Both induce the expression of tight junction proteins zonula occludens-1 and claudin-5 in hCMEC/D3 cells, but there was no difference between the induced expression levels by the two media. The model has been used to assess the transport of glucose-coated 4nm gold nanoparticles and for leukocyte migration. TEM was used to trace and quantitate the movement of the nanoparticles across the endothelium and into the astrocytes. This blood-brain barrier model is very suitable for assessing delivery of nanoparticles and larger biomolecules to cells of the CNS, following transport across the endothelium

Systemic α-synuclein injection triggers selective neuronal pathology as seen in patients with Parkinson’s disease

Abstract: Parkinson’s disease (PD) is an α-synucleinopathy characterized by the progressive loss of specific neuronal populations. Here, we develop a novel approach to transvascularly deliver proteins of complex quaternary structures, including α-synuclein preformed fibrils (pff). We show that a single systemic administration of α-synuclein pff triggers pathological transformation of endogenous α-synuclein in non-transgenic rats, which leads to neurodegeneration in discrete brain regions. Specifically, pff-exposed animals displayed a progressive deterioration in gastrointestinal and olfactory functions, which corresponded with the presence of cellular pathology in the central and enteric nervous systems. The α-synuclein pathology generated was both time dependent and region specific. Interestingly, the most significant neuropathological changes were observed in those brain regions affected in the early stages of PD. Our data therefore demonstrate for the first time that a single, transvascular administration of α-synuclein pff can lead to selective regional neuropathology resembling the premotor stage of idiopathic PD. Furthermore, this novel delivery approach could also be used to deliver a range of other pathogenic, as well as therapeutic, protein cargos transvascularly to the brain

RNA knockdown as a potential therapeutic strategy in Parkinson\u27s disease

Parkinson\u27s disease is a prevalent progressive degenerative disorder of the elderly. There is a current need for novel therapeutic strategies because the standard levodopa pharmacotherapy is only temporarily efficacious. Recently, there have been some high-profile successful preclinical results obtained in animal models of neurological disorders using small interfering RNAs delivered by viral vectors. RNA interference can theoretically be applied to Parkinson\u27s disease since over-expression of various proteins is known to kill the dopamine neurons of the substantia nigra in animal models and in familial forms of Parkinson\u27s disease. Potential RNA interfering strategies and caveats are discussed in this review. © 2006 Nature Publishing Group All rights reserved

Introducing a Novel Method of Intravascular Adeno-associated Virus-mediated Gene Delivery.

Introduction: Adeno-associated virus (AAV) has shown therapeutic potential as a viral vector in various studies of gene therapy. However, research on its use in targeting intravascular cells in a localized manner is lacking. We introduce a novel method to deliver various AAV serotypes intravascularly and examine their efficiency in transducing cells of the murine carotid artery. Objective: The study aimed to examine the transduction efficiency of AAV-mediated gene delivery in cells of the murine carotid artery both with and without a fully-formed aneurysm. Results of infection were visualized with green fluorescence protein (GFP) reporter gene. Methods: Naïve murine carotid artery or experimentally-induced murine carotid aneurysm was ligated distally and proximally. A small incision was made and 5 uL AAV2, AAV5, AAV8, or AAV9 was microsurgically injected and allowed to incubate for 30 min. Incision was closed and tissue was excised three weeks following AAV injection. Carotid artery or aneurysm tissue was excised and fixed in 4% paraformaldehyde solution. On both naïve carotid artery tissue and aneurysm tissue, GFP was visualized by immunofluorescence using antibody against GFP. Results: Three out of four serotypes of AAV successfully transduced cells within both the murine aneurysm tissue and the naïve carotid artery tissue. AAV5- and AAV9-transduced aneurysm tissue showed the greatest presence of GFP, with AAV8 showing less overall fluorescence. AAV2 showed no fluorescence. Conclusion: AAV-mediated gene delivery is an effective way to transduce cells intravascularly with a transgene of interest. Our method can be generalized across a wide variety of studies to further research or treat other vascular disease

rAAV-mediated nigral human parkin over-expression partially ameliorates motor deficits via enhanced dopamine neurotransmission in a rat model of Parkinson\u27s disease

We hypothesized that over-expressing the E3 ligase, parkin, whose functional loss leads to Parkinson\u27s disease, in the nigrostriatal tract might be protective in the unilateral 6-hydroxydopamine (6-OHDA) rat lesion model. Recombinant adeno-associated virus (rAAV) encoding human parkin or green fluorescent protein (GFP) was injected into the rat substantia nigra 6 weeks prior to a four-site striatal 6-OHDA lesion. Vector-mediated parkin over-expression significantly ameliorated motor deficits as measured by amphetamine-induced rotational behavior and spontaneous behavior in the cylinder test but forelimb akinesia as assessed by the stepping test was unaffected. rAAV-mediated human parkin was expressed in the nigrostriatal tract, the substantia pars reticulata, and the subthalamic nucleus. However, in lesioned animals, there was no difference between nigral parkin and GFP-transduction on lesion-induced striatal tyrosine hydroxylase (TH) innervation or nigral TH positive surviving neurons. A second lesion experiment was performed to determine if striatal dopamine (DA) neurotransmission was enhanced as measured biochemically. In this second group of parkin and GFP treated rats, behavioral improvement was again observed. In addition, striatal TH and DA levels were slightly increased in the parkin-transduced group. In a third experiment, we evaluated parkin and GFP transduced rats 6 weeks after vector injection without DA depletion. When challenged with amphetamine, parkin treated rats tended to display asymmetries biased away from the treated hemisphere. Nigral parkin over-expression induced increases in both striatal TH and DA levels. Therefore, while parkin over-expression exerted no protective effect on the nigrostriatal DA system, parkin appeared to enhance the efficiency of nigrostriatal DA transmission in intact nigral DA neurons likely due to the observed increases in TH

Neonatal Nicotine Exposure Primes Midbrain Neurons to a Dopaminergic Phenotype and Increases Adult Drug Consumption

Background: Nicotine intake induces addiction through neuroplasticity of the reward circuitry, altering the activity of dopaminergic neurons of the ventral tegmental area. Prior work demonstrated that altered circuit activity can change neurotransmitter expression in the developing and adult brain. Here we investigated the effects of neonatal nicotine exposure on the dopaminergic system and nicotine consumption in adulthood. Methods: Male and female mice were used for two-bottle-choice test, progressive ratio breakpoint test, immunohistochemistry, RNAscope, quantitative polymerase chain reaction, calcium imaging, and DREADD (designer receptor exclusively activated by designer drugs)-mediated chemogenic activation/inhibition experiments. Results: Neonatal nicotine exposure potentiates drug preference in adult mice, induces alterations in calcium spike activity of midbrain neurons, and increases the number of dopamine-expressing neurons in the ventral tegmental area. Specifically, glutamatergic neurons are first primed to express transcription factor Nurr1, then acquire the dopaminergic phenotype following nicotine re-exposure in adulthood. Enhanced neuronal activity combined with Nurr1 expression is both necessary and sufficient for the nicotine-mediated neurotransmitter plasticity to occur. Conclusions: Our findings illuminate a new mechanism of neuroplasticity by which early nicotine exposure primes the reward system to display increased susceptibility to drug consumption in adulthood

Neonatal Nicotine Exposure Primes Midbrain Neurons to a Dopaminergic Phenotype and Increases Adult Drug Consumption.

BackgroundNicotine intake induces addiction through neuroplasticity of the reward circuitry, altering the activity of dopaminergic neurons of the ventral tegmental area. Prior work demonstrated that altered circuit activity can change neurotransmitter expression in the developing and adult brain. Here we investigated the effects of neonatal nicotine exposure on the dopaminergic system and nicotine consumption in adulthood.MethodsMale and female mice were used for two-bottle-choice test, progressive ratio breakpoint test, immunohistochemistry, RNAscope, quantitative polymerase chain reaction, calcium imaging, and DREADD (designer receptor exclusively activated by designer drugs)-mediated chemogenic activation/inhibition experiments.ResultsNeonatal nicotine exposure potentiates drug preference in adult mice, induces alterations in calcium spike activity of midbrain neurons, and increases the number of dopamine-expressing neurons in the ventral tegmental area. Specifically, glutamatergic neurons are first primed to express transcription factor Nurr1, then acquire the dopaminergic phenotype following nicotine re-exposure in adulthood. Enhanced neuronal activity combined with Nurr1 expression is both necessary and sufficient for the nicotine-mediated neurotransmitter plasticity to occur.ConclusionsOur findings illuminate a new mechanism of neuroplasticity by which early nicotine exposure primes the reward system to display increased susceptibility to drug consumption in adulthood

The phosphorylation state of Ser-129 in human α-synuclein determines neurodegeneration in a rat model of Parkinson disease

Studies have shown that α-synuclein (α-syn) deposited in Lewy bodies in brain tissue from patients with Parkinson disease (PD) is extensively phosphorylated at Ser-129. We used recombinant Adeno-associated virus (rAAV) to overexpress human wild-type (wt) α-syn and two human α-syn mutants with site-directed replacement of Ser-129 to alanine (S129A) or to aspartate (S129D) in the nigrostriatal tract of the rat to investigate the effect of Ser-129 phosphorylation state on dopaminergic neuron pathology. Rats were injected with rAAV2/5 vectors in the substantia nigra pars compacta (SNc) on one side of the brain; the other side remained as a nontransduced control. The level of human wt or mutant α-syn expressed on the injected side was about four times the endogenous rat α-syn. There was a significant reduction of dopaminergic neurons in the SNc and dopamine (DA) and tyrosine hydroxylase (TH) levels in the striatum of all S129A-treated rats as early as 4 wk postinjection. Nigral DA pathology occurred more slowly in the wt-injected animals, but by 26 wk the wt α-syn group lost nigral TH neurons equivalent to the mutated S129A group at 8 wk. In stark contrast, we did not observe any pathological changes in S129D-treated animals. Therefore, the nonphosphorylated form of S129 exacerbates α-syn-induced nigral pathology, whereas Ser-129 phosphorylation eliminates α-syn-induced nigrostriatal degeneration. This suggests possible new therapeutic targets for Parkinson Disease. © 2008 by The National Academy of Sciences of the USA