Shannon Dallas

Multidrug Resistance-Associated Proteins: Expression and Function in the Central Nervous System

Drug delivery to the brain is highly restricted, since compounds must cross a series of structural and metabolic barriers to reach their final destination, often a cellular compartment such as neurons, microglia, or astrocytes. The primary barriers to the central nervous system are the blood-brain and blood-cerebrospinal fluid barriers. Through structural modifications, including the presence of tight junctions that greatly limit paracellular transport, the cells that make up these barriers restrict diffusion of many pharmaceutically active compounds. In addition, the cells that comprise the blood-brain and blood-cerebrospinal fluid barriers express multiple ATP-dependent, membrane-bound, efflux transporters, such as members of the multidrug resistance-associated protein (MRP) family, which contribute to lowered drug accumulation. A relatively new concept in brain drug distribution just beginning to be explored is the possibility that cellular components of the brain parenchyma could act as a “second” barrier to brain permeation of pharmacological agents via expression of many of the same transporters. Indeed, efflux transporters expressed in brain parenchyma may facilitate the overall export of xenobiotics from the central nervous system, essentially handing themoff to the barrier tissues. We propose that these primary and secondary barriers work in tandem to limit overall accumulation and distribution of xenobiotics in the central nervous system. The present review summarizes recent knowledge in this area and emphasizes the clinical significance of MRP transporter expression in a variety of neurological disorders.

Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons

Parkinsons disease (PD) is characterized by the selective and progressive loss of dopaminergic (DA) neurons in the midbrain substantia nigra. Currently, available treatment is unable to alter PD progression. Previously, we demonstrated that valproic acid (VPA), a mood stabilizer, anticonvulsant and histone deacetylase (HDAC) inhibitor, increases the expression of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in astrocytes to protect DA neurons in midbrain neuron-glia cultures. The present study investigated whether these effects are due to HDAC inhibition and histone acetylation. Here, we show that two additional HDAC inhibitors, sodium butyrate (SB) and trichostatin A (TSA), mimic the survival-promoting and protective effects of VPA on DA neurons in neuron-glia cultures. Similar to VPA, both SB and TSA increased GDNF and BDNF transcripts in astrocytes in a time-dependent manner. Furthermore, marked increases in GDNF promoter activity and promoter-associated histone H3 acetylation were noted in astrocytes treated with all three compounds, where the time-course for acetylation was similar to that for gene transcription. Taken together, our results indicate that HDAC inhibitors up-regulate GDNF and BDNF expression in astrocytes and protect DA neurons, at least in part, through HDAC inhibition. This study indicates that astrocytes may be a critical neuroprotective mechanism of HDAC inhibitors, revealing a novel target for the treatment of psychiatric and neurodegenerative diseases.

Microglial PHOX and Mac-1 are essential to the enhanced dopaminergic neurodegeneration elicited by A30P and A53T mutant alpha-synuclein.

α‐Synuclein, a gene whose mutations, duplication, and triplication has been linked to autosomal dominant familial Parkinsons disease (fPD), appears to play a central role in the pathogenesis of sporadic PD (sPD) as well. Enhancement of neurodegeneration induced by mutant α‐synuclein has been attributed to date largely to faster formation of α‐synuclein aggregates in neurons. Recently, we reported that microglial activation enhances wild type (WT) α‐synuclein‐elicited dopaminergic neurodegeneration. In the present study, using a primary mesencephalic culture system, we tested whether mutated α‐synuclein could activate microglia more powerfully than WT α‐synuclein, thereby contributing to the accelerated neurodegeneration observed in fPD. The results showed that α‐synuclein with the A30P or A53T mutations caused greater microglial activation than WT α‐synuclein. Furthermore, the extent of microglial activation paralleled the degree of dopaminergic neurotoxicity induced by WT and mutant α‐synuclein. Mutant α‐synuclein also induced greater production of reactive oxygen species than WT α‐synuclein by NADPH oxidase (PHOX), and PHOX activation was linked to direct activation of macrophage antigen‐1 (Mac‐1) receptor, rather than α‐synuclein internalization via scavenger receptors. These results have, for the first time, demonstrated that microglia are also critical in enhanced neurotoxicity induced by mutant α‐synuclein.

Reactive microgliosis: extracellular μ-calpain and microglia-mediated dopaminergic neurotoxicity

Microglia, the innate immune cells in the brain, can become chronically activated in response to dopaminergic neuron death, fuelling a self-renewing cycle of microglial activation followed by further neuron damage (reactive microgliosis), which is implicated in the progressive nature of Parkinsons disease. Here, we use an in vitro approach to separate neuron injury factors from the cellular actors of reactive microgliosis and discover molecular signals responsible for chronic and toxic microglial activation. Upon injury with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium, N27 cells (dopaminergic neuron cell line) released soluble neuron injury factors that activated microglia and were selectively toxic to dopaminergic neurons in mixed mesencephalic neuron-glia cultures through nicotinamide adenine dinucleotide phosphate oxidase. mu-Calpain was identified as a key signal released from damaged neurons, causing selective dopaminergic neuron death through activation of microglial nicotinamide adenine dinucleotide phosphate oxidase and superoxide production. These findings suggest that dopaminergic neurons may be inherently susceptible to the pro-inflammatory effects of neuron damage, i.e. reactive microgliosis, providing much needed insight into the chronic nature of Parkinsons disease.

MAC1 mediates LPS-induced production of superoxide by microglia: The role of pattern recognition receptors in dopaminergic neurotoxicity

Microglia‐derived superoxide is critical for the inflammation‐induced selective loss of dopaminergic (DA) neurons, but the underlying mechanisms of microglial activation remain poorly defined. Using neuron‐glia and microglia‐enriched cultures from mice deficient in the MAC1 receptor (MAC1−/−), we demonstrate that lipopolysaccharide (LPS) treatment results in lower TNFα response, attenuated loss of DA neurons, and absence of extracellular superoxide production in MAC1−/− cultures. Microglia accumulated fluorescently labeled LPS in punctate compartments associated with the plasma membrane, intracellular vesicles, and the Golgi apparatus. Cytochalasin D (CD), an inhibitor of phagocytosis, blocked LPS internalization. However, microglia derived from Toll‐like receptor 4 deficient mice and MAC1−/− mice failed to show a significant decrease in intracellular accumulation of labeled LPS, when compared with controls. Pretreatment with the scavenger receptor inhibitor, fucoidan, inhibited 79% of LPS accumulation in microglia without affecting superoxide, indicating that LPS internalization and superoxide production are mediated by separate phagocytosis receptors. Together, these data demonstrate that MAC1 is essential for LPS‐induced superoxide from microglia, implicating MAC1 as a critical trigger of microglial‐derived oxidative stress during inflammation‐mediated neurodegeneration.

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes.

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.

Critical Review of Preclinical Approaches to Investigate Cytochrome P450–Mediated Therapeutic Protein Drug-Drug Interactions and Recommendations for Best Practices: A White Paper

Drug-drug interactions (DDIs) between therapeutic proteins (TPs) and small-molecule drugs have recently drawn the attention of regulatory agencies, the pharmaceutical industry, and academia. TP-DDIs are mainly caused by proinflammatory cytokine or cytokine modulator–mediated effects on the expression of cytochrome P450 enzymes. To build consensus among industry and regulatory agencies on expectations and challenges in this area, a working group was initiated to review the preclinical state of the art. This white paper represents the observations and recommendations of the working group on the value of in vitro human hepatocyte studies for the prediction of clinical TP-DDI. The white paper was developed following a “Workshop on Recent Advances in the Investigation of Therapeutic Protein Drug-Drug Interactions: Preclinical and Clinical Approaches” held at the Food and Drug Administration White Oak Conference Center on June 4 and 5, 2012. Results of a workshop poll, cross-laboratory data comparisons, and the overall recommendations of the in vitro working group are presented herein. The working group observed that evaluation of TP-DDI for anticytokine monoclonal antibodies is currently best accomplished with a clinical study in patients with inflammatory disease. Treatment-induced changes in appropriate biomarkers in phase 2 and 3 studies may indicate the potential for a clinically measurable treatment effect on cytochrome P450 enzymes. Cytokine-mediated DDIs observed with anti-inflammatory TPs cannot currently be predicted using in vitro data. Future success in predicting clinical TP-DDIs will require an understanding of disease biology, physiologically relevant in vitro systems, and more examples of well conducted clinical TP-DDI trials.

Tethered spheroids as an in vitro hepatocyte model for drug safety screening

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.

Galactosylated cellulosic sponge for multi-well drug safety testing

Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 μm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell-cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to β-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H(2)O(2) outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H(2)O(2) further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H(2)O(2) to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.