Physiological and Pathological Factors Affecting Drug Delivery to the Brain by Nanoparticles.

The prevalence of neurological/neurodegenerative diseases, such as Alzheimer's disease is known to be increasing due to an aging population and is anticipated to further grow in the decades ahead. The treatment of brain diseases is challenging partly due to the inaccessibility of therapeutic agents to the brain. An increasingly important observation is that the physiology of the brain alters during many brain diseases, and aging adds even more to the complexity of the disease. There is a notion that the permeability of the blood-brain barrier (BBB) increases with aging or disease, however, the body has a defense mechanism that still retains the separation of the brain from harmful chemicals in the blood. This makes drug delivery to the diseased brain, even more challenging and complex task. Here, the physiological changes to the diseased brain and aged brain are covered in the context of drug delivery to the brain using nanoparticles. Also, recent and novel approaches are discussed for the delivery of therapeutic agents to the diseased brain using nanoparticle based or magnetic resonance imaging guided systems. Furthermore, the complement activation, toxicity, and immunogenicity of brain targeting nanoparticles as well as novel in vitro BBB models are discussed

Neocortex and allocortex respond differentially to cellular stress in vitro and aging in vivo.

In Parkinson's and Alzheimer's diseases, the allocortex accumulates aggregated proteins such as synuclein and tau well before neocortex. We present a new high-throughput model of this topographic difference by microdissecting neocortex and allocortex from the postnatal rat and treating them in parallel fashion with toxins. Allocortical cultures were more vulnerable to low concentrations of the proteasome inhibitors MG132 and PSI but not the oxidative poison H2O2. The proteasome appeared to be more impaired in allocortex because MG132 raised ubiquitin-conjugated proteins and lowered proteasome activity in allocortex more than neocortex. Allocortex cultures were more vulnerable to MG132 despite greater MG132-induced rises in heat shock protein 70, heme oxygenase 1, and catalase. Proteasome subunits PA700 and PA28 were also higher in allocortex cultures, suggesting compensatory adaptations to greater proteasome impairment. Glutathione and ceruloplasmin were not robustly MG132-responsive and were basally higher in neocortical cultures. Notably, neocortex cultures became as vulnerable to MG132 as allocortex when glutathione synthesis or autophagic defenses were inhibited. Conversely, the glutathione precursor N-acetyl cysteine rendered allocortex resilient to MG132. Glutathione and ceruloplasmin levels were then examined in vivo as a function of age because aging is a natural model of proteasome inhibition and oxidative stress. Allocortical glutathione levels rose linearly with age but were similar to neocortex in whole tissue lysates. In contrast, ceruloplasmin levels were strikingly higher in neocortex at all ages and rose linearly until middle age. PA28 levels rose with age and were higher in allocortex in vivo, also paralleling in vitro data. These neo- and allocortical differences have implications for the many studies that treat the telencephalic mantle as a single unit. Our observations suggest that the topographic progression of protein aggregations through the cerebrum may reflect differential responses to low level protein-misfolding stress but also reveal impressive compensatory adaptations in allocortex

Correction: Neocortex and Allocortex Respond Differentially to Cellular Stress In Vitro and Aging In Vivo

In Parkinson’s and Alzheimer’s diseases, the allocortex accumulates aggregated proteins such as synuclein and tau well before neocortex. We present a new high-throughput model of this topographic difference by microdissecting neocortex and allocortex from the postnatal rat and treating them in parallel fashion with toxins. Allocortical cultures were more vulnerable to low concentrations of the proteasome inhibitors MG132 and PSI but not the oxidative poison H(2)O(2). The proteasome appeared to be more impaired in allocortex because MG132 raised ubiquitin-conjugated proteins and lowered proteasome activity in allocortex more than neocortex. Allocortex cultures were more vulnerable to MG132 despite greater MG132-induced rises in heat shock protein 70, heme oxygenase 1, and catalase. Proteasome subunits PA700 and PA28 were also higher in allocortex cultures, suggesting compensatory adaptations to greater proteasome impairment. Glutathione and ceruloplasmin were not robustly MG132-responsive and were basally higher in neocortical cultures. Notably, neocortex cultures became as vulnerable to MG132 as allocortex when glutathione synthesis or autophagic defenses were inhibited. Conversely, the glutathione precursor N-acetyl cysteine rendered allocortex resilient to MG132. Glutathione and ceruloplasmin levels were then examined in vivo as a function of age because aging is a natural model of proteasome inhibition and oxidative stress. Allocortical glutathione levels rose linearly with age but were similar to neocortex in whole tissue lysates. In contrast, ceruloplasmin levels were strikingly higher in neocortex at all ages and rose linearly until middle age. PA28 levels rose with age and were higher in allocortex in vivo, also paralleling in vitro data. These neo- and allocortical differences have implications for the many studies that treat the telencephalic mantle as a single unit. Our observations suggest that the topographic progression of protein aggregations through the cerebrum may reflect differential responses to low level protein-misfolding stress but also reveal impressive compensatory adaptations in allocortex

Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes

Role of glutathione in protection of neo- and allocortex against proteasome inhibition.

<p><b>A:</b> Glutathione (GSH) levels as a function of total MAP2 levels in neocortex and allocortex in the presence or absence of MG132. Allocortical cells exhibited less glutathione than neocortical cells in both conditions <i>in vitro</i>. <b>B–C:</b> MAP2 levels (<b>B</b>) and glutathione levels (<b>C</b>) as a function of indicated concentrations of buthionine sulfoximine (BSO). Neocortical cells exhibited much greater glutathione loss with buthionine sulfoximine than allocortical cells. Buthionine sulfoximine was not lethal to neurons at the indicated concentrations. <b>D–E:</b> Neocortical and allocortical neurons were treated with vehicle or MG132 (0.25 µM) and vehicle or buthionine sulfoximine (12.5 µM). Both allocortical neurons and neocortical neurons were more vulnerable to combined treatment with buthionine sulfoximine and MG132 than either toxin alone (<b>D</b>). The glutathione assay revealed again that neocortical cells lost more glutathione with buthionine sulfoximine than allocortical cells, but that allocortical cells had overall less glutathione than neocortical cells in all four groups (<b>E</b>). <b>F–G</b>: N-acetyl cysteine (3 mM) completely prevented the toxicity of 0.25 µM MG132 in allocortical cultures (<b>F</b>) and considerably raised glutathione levels both with and without MG132 on board (<b>G</b>). Shown are the mean and standard error of the mean of at least 3 independent experiments. For all panels, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001, or ****<i>p</i> ≤ 0.0001 allocortex versus neocortex; ^∧<i>p</i> ≤ 0.05, ^∧∧<i>p</i> ≤ 0.01, ^∧∧∧<i>p</i> ≤ 0.001, or ^∧∧∧∧<i>p</i> ≤ 0.0001 MG132 versus vehicle;+<i>p</i> ≤ 0.05,++<i>p</i> ≤ 0.01,+++<i>p</i> ≤ 0.001,++++<i>p</i> ≤ 0.0001 buthionine sulfoximine versus water or N-acetyl cysteine versus water; Bonferroni <i>post hoc</i> correction following two-way ANOVA.</p

Involvement of autophagic defenses and ubiquitin-proteasome system.

<p><b>A–B</b>: Infrared Western immunoblotting for macroautophagy-related molecule Beclin 1 following treatment of neo- and allocortical cultures with 0.25 and 1 µM MG132 is shown. β-actin was used as a loading control. <b>C–D</b>: Ammonium chloride (20 mM NH₄Cl) was used to inhibit all forms of autophagy and wortmannin (50 nM) was used to inhibit macroautophagy in neo- and allocortical cultures subjected to vehicle or 0.25 µM MG132. Neocortical neurons became as vulnerable to MG132 as allocortical neurons in response to NH₄Cl. Wortmannin failed to elicit an effect. Shown are the mean and standard error of the mean of at least 3 independent experiments. *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001 allocortex vs neocortex; ^∧<i>p</i> ≤ 0.05 or ^∧∧∧<i>p</i> ≤ 0.001 MG132 vs vehicle; +<i>p</i> ≤ 0.05 NH₄Cl versus vehicle; Bonferroni <i>post hoc</i> correction following two-way ANOVA. <b>E–F:</b> Infrared Western immunoblotting for ubiquitin-conjugated proteins revealed that allocortex exhibited higher levels of this measure of proteotoxic stress in response to MG132. <b>G:</b> Proteasome activity was measured in the presence or absence of MG132 in a fluorogenic assay. Allocortical proteasomes were more inhibited by MG132 than those from neocortex. Shown are the mean and standard error of the mean of at least 3 independent experiments. For F and G, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01 allocortex versus neocortex;+<i>p</i> ≤ 0.05,+++<i>p</i> ≤ 0.001,++++<i>p</i> ≤ 0.0001 MG132 versus vehicle (0 µM MG132); Bonferroni <i>post hoc</i> correction following two-way ANOVA.</p

Characterization of <i>in vitro</i> model.

<p><b>A–F:</b> Neocortical and allocortical tissue was microdissected from postnatal rat brains, dissociated, and plated in 96-well plates at 100,000 cells per well. Cells were treated with 2.5 µM cytosine arabinofuranoside to suppress, but not eliminate, glial proliferation on day in vitro 2 (DIV2). Plates were immunostained on DIV4 for the neuronal marker microtubule associated protein 2 (MAP2, shown in red). Nuclei were stained blue with Hoechst. Merged images reveal that the majority of the Hoechst-stained nuclei were neuronal. However, arrowheads point to large nuclei that were possibly glial. Long stemmed arrows point to condensed, potentially apoptotic nuclei that may have lost their neuronal phenotype. <b>G:</b> Blind cell counts revealed that nearly 75% of neocortical and allocortical Hoechst-positive cells expressed the MAP2 neuronal phenotype. <b>H and I:</b> Basal survival on DIV4 was measured by an infrared In-Cell Western assay for MAP2 and by the Cell Titer Glo assay for ATP. Both assays revealed that allocortex survived <i>in vitro</i> conditions at approximately twice the rate of neocortex. A grayscale inset of a representative infrared MAP2 stain of neo- versus allocortical neurons is included in <b>H.</b> Shown are the mean and standard error of the mean of at least 3 independent experiments. **<i>p</i> ≤ 0.01 or ***<i>p</i> ≤ 0.001 by the two tailed <i>t</i>-test. <b>J:</b> Brains were fixed following tissue dissections, cut in the sagittal plane, and stained for the infrared nuclear marker DRAQ5. Neocortical dissections (arrow) were centered in primary motor and sensory cortex (Ctx), dorsal to hippocampus (HP) and caudoputamen (CP). Allocortical dissections (arrow) were centered much more laterally in the brain ventral to hippocampus in the entorhinal and piriform cortices.</p

Impact of natural aging on neocortex and allocortex <i>in vivo</i>.

<p><b>A:</b> Glutathione levels in whole tissue lysates of rat neo- and allocortex as a function of age. Allocortical glutathione levels were similar to those in neocortex but rose in an age-dependent manner and were significantly higher at 19–22 months of age relative to the youngest group (2–4 months). Neocortical glutathione did not change significantly as a function of age. <b>B–C:</b> Western immunoblotting for ceruloplasmin as a function of age and brain region. Neocortex had much more ceruloplasmin than allocortex at every age examined. Furthermore, neocortical ceruloplasmin rose in an age-dependent manner until rats were 16–19 months old. <b>D–E:</b> PA28 levels as a function of age in neo- and allocortex. PA28 levels rose in neocortex at 19–22 months and in allocortex at 16–19 months relative to the youngest age group. Allocortical PA28 levels were significantly higher than in neocortex at 16–19 months of age. Data are expressed as mean and standard error of the mean from 4–5 rats per group. For all panels, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001, allocortex versus neocortex;+<i>p</i> ≤ 0.05,+++<i>p</i> ≤ 0.001 versus levels in the 2–4 month old group; Bonferroni <i>post hoc</i> correction following two-way ANOVA.</p

Differential vulnerability of neo- and allocortex to toxins.

<p><b>A–C:</b> Neo- and allocortical cells were plated at cell densities that varied only by 20,000 cells per well (40K, 60K, 80K, 100K, and 120K cells per well). Plates were immunostained for MAP2 (<b>A and B</b>) or assayed for ATP (<b>C</b>). A linear correlation between signal and plating density was apparent with both assays at all densities except for ATP assessments of 120,000 allocortical cells. See Results section for <i>p</i> values. <b>D–F:</b> Neo- and allocortical cultures were treated with indicated concentrations of hydrogen peroxide. Allocortex was more resistant to oxidative stress by both MAP2 (<b>D and E</b>) and ATP assays (<b>F</b>). <b>G–I:</b> Neo- and allocortical cultures were treated with indicated concentrations of MG132 and assayed for MAP2 (<b>G and H</b>) and ATP (<b>I</b>). Both assays revealed that neocortex was less sensitive to low concentrations of this proteasome inhibitor. <b>J–M:</b> Neo- and allocortical cultures were treated with indicated concentrations of PSI and assayed for MAP2 (<b>J, K, and M</b>) and ATP (<b>L</b>). Allocortex was more vulnerable to low, but not high concentrations of PSI. Shown are the mean and standard error of the mean of at least 3 independent experiments. *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001, or ****<i>p</i> ≤ 0.0001 versus neocortex at the same MG132 concentration, Bonferroni <i>post hoc</i> correction following two-way ANOVA. <b>N:</b> Representative higher resolution photomontage of MAP2 immunostained neocortical and allocortical neurons treated with 0.25 µM MG132 or vehicle (dimethyl sulfoxide, DMSO). Results were quantified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058596#pone-0058596-g002" target="_blank">Figure 2O</a>. <b>O:</b> Neurons were counted by a blind observer at 200x magnification in a 0.213 mm² field of view (three fields per well). Raw cell counts are shown to illustrate that there were more allocortical neurons under basal conditions. However, allocortical neurons were more vulnerable to 0.25 µM MG132. Shown are the mean and standard error of the mean of four independent experiments. **<i>p</i> ≤ 0.01, ****<i>p</i> ≤ 0.0001 versus neocortex; ^∧∧∧∧<i>p</i> ≤ 0.0001 MG132 versus vehicle (0 µM MG132); Bonferroni <i>post hoc</i> correction following two-way ANOVA.</p