Advances in Nanomedicine Design: Multidisciplinary Strategies for Unmet Medical Needs.

Globally, a rising burden of complex diseases takes a heavy toll on human lives and poses substantial clinical and economic challenges. This review covers nanomedicine and nanotechnology-enabled advanced drug delivery systems (DDS) designed to address various unmet medical needs. Key nanomedicine and DDSs, currently employed in the clinic to tackle some of these diseases, are discussed focusing on their versatility in diagnostics, anticancer therapy, and diabetes management. First-hand experiences from our own laboratory and the work of others are presented to provide insights into strategies to design and optimize nanomedicine- and nanotechnology-enabled DDS for enhancing therapeutic outcomes. Computational analysis is also briefly reviewed as a technology for rational design of controlled release DDS. Further explorations of DDS have illuminated the interplay of physiological barriers and their impact on DDS. It is demonstrated how such delivery systems can overcome these barriers for enhanced therapeutic efficacy and how new perspectives of next-generation DDS can be applied clinically.

Necroptotic-Apoptotic Regulation in an Endothelin-1 Model of Cerebral Ischemia.

The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia-reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death.

Cell-based high-throughput screen for small molecule inhibitors of Bax translocation.

Aberrant regulation of programmed cell death (PCD) has been tied to an array of human pathologies ranging from cancers to autoimmune disorders to diverse forms of neurodegeneration. Pharmacologic modulation of PCD signalling is therefore of central interest to a number of clinical and biomedical applications. A key component of PCD signalling involves the modulation of pro- and anti-apoptotic Bcl-2 family members. Among these, Bax translocation represents a critical regulatory phase in PCD. In the present study, we have employed a high-content high-throughput screen to identify small molecules which inhibit the cellular process of Bax re-distribution to the mitochondria following commitment of the cell to die. Screening of 6246 Generally Recognized As Safe compounds from four chemical libraries post-induction of cisplatin-mediated PCD resulted in the identification of 18 compounds which significantly reduced levels of Bax translocation. Further examination revealed protective effects via reduction of executioner caspase activity and enhanced mitochondrial function. Consistent with their effects on Bax translocation, these compounds exhibited significant rescue against in vitro and in vivo cisplatin-induced apoptosis. Altogether, our findings identify a new set of clinically useful small molecules PCD inhibitors and highlight the role which cAMP plays in regulating Bax-mediated PCD.

Endothelin-1-mediated cerebral ischemia in mice: early cellular events and the role of caspase-3.

Over the past 30 years a number of animal models of cerebral ischemic injury have been developed. Middle cerebral artery occlusion (MCAO) in particular reproduces both ischemic and reperfusion elements and is widely utilized as a model of ischemic stroke in rodents. However substantial variability exists in this model even in clonal inbred mice due to stochastic elements of the cerebral vasculature. Models such as MCAO thus exhibit significant irreducible variabilities with respect to their zone of injury as well as inducing a sizable volume of injury to the cerebrum with damage to sub-cortical structures, conditions not typically seen for the majority of human clinical strokes. An alternative model utilizes endothelin-1 application focally to cerebral vasculature, resulting in an ischemic reperfusion injury which more closely mimics that seen in human clinical stroke. In order to further define this model we demonstrate that intra-cortical administration of ET-1 results in a highly reproducible pattern of tissue injury which is limited to the cerebral cortex, characterizing the early cellular and molecular events which occur during the first 24 h post-injury. In addition we demonstrate that caspase-3 is both necessary and sufficient to regulate a majority of cortical cell death observed during this period. The enhanced survival effects seen upon genetic deletion of caspase-3 appear to arise as a result of direct modification of cell autonomous PCD signaling as opposed to secondary effectors such as granulocyte infiltration or microglia activation. Taken together these findings detail the early mechanistic features regulating endothelin-1-mediated ischemic injury.

1α,25-Dihydroxyvitamin D3 reduces cerebral amyloid-ß accumulation and improves cognition in mouse models of Alzheimer's disease.

We demonstrate a role of the vitamin D receptor (VDR) in reducing cerebral soluble and insoluble amyloid-ß (Aß) peptides. Short-term treatment of two human amyloid precursor protein-expressing models, Tg2576 and TgCRND8 mice, with 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], the endogenous active ligand of VDR, resulted in higher brain P-glycoprotein (P-gp) and lower soluble Aß levels, effects negated with coadministration of elacridar, a P-gp inhibitor. Long-term treatment of TgCRND8 mice with 1,25(OH)2D3 during the period of plaque formation reduced soluble and insoluble plaque-associated Aß, particularly in the hippocampus in which the VDR is abundant and P-gp induction is greatest after 1,25(OH)2D3 treatment, and this led to improved conditioned fear memory. In mice fed a vitamin D-deficient diet, lower cerebral P-gp expression was observed, but levels were restored on replenishment with VDR ligands. The composite data suggest that the VDR is an important therapeutic target in the prevention and treatment of Alzheimer's disease.

The use of PD-1 functional knockout rats to study idiosyncratic adverse reactions to nevirapine.

Idiosyncratic drug reactions (IDRs) are associated with significant patient morbidity/mortality and lead to considerable drug candidate attrition in drug development. Their idiosyncratic nature makes the study of IDRs difficult. In particular, nevirapine is associated with a relatively high risk of serious skin rash and liver injury. We previously found that nevirapine causes a similar skin rash in female Brown Norway rats, but these animals do not develop significant liver injury. Programmed cell death protein-1 (PD-1) is an immune checkpoint involved in immune tolerance, and anti-PD-1 antibodies have been used to treat cancer. However, they increase the risk of liver injury caused by co-administered drugs. We found that PD-1-/- mice are more susceptible to drug-induced liver injury, but PD-1-/- mice are not a good model for all drugs. In particular, they do not develop a skin rash when treated with nevirapine, at least in part because they lack the sulfotransferase in their skin that forms the reactive metabolite responsible for the rash. Therefore, we developed a PD-1 mutant (PD-1m/m) rat, with an excision in the ligand-binding domain of PD-1, to test whether nevirapine would cause a more serious skin rash in these animals. The PD-1m/m rat was based on a Sprague Dawley background, which has a lower incidence of skin rash than Brown Norway rats. The treated PD-1m/m rats developed more severe liver injury than PD-1-/- mice, but in contrast to expectations, they did not develop a skin rash. Functional knockouts provide a unique tool to study the mechanisms of IDRs.

An efficient low cost means of biophysical gene transfection in primary cells.

Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1-10 µl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections.

Cellular transfection using rapid decrease in hydrostatic pressure.

Of all methods exercised in modern molecular biology, modification of cellular properties through the introduction or removal of nucleic acids is one of the most fundamental. As such, several methods have arisen to promote this process; these include the condensation of nucleic acids with calcium, polyethylenimine or modified lipids, electroporation, viral production, biolistics, and microinjection. An ideal transfection method would be (1) low cost, (2) exhibit high levels of biological safety, (3) offer improved efficacy over existing methods, (4) lack requirements for ongoing consumables, (5) work efficiently at any scale, (6) work efficiently on cells that are difficult to transfect by other methods, and (7) be capable of utilizing the widest array of existing genetic resources to facilitate its utility in research, biotechnical and clinical settings. To address such issues, we describe here Pressure-jump-poration (PJP), a method using rapid depressurization to transfect even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner. Finally, PJP-induced transfection in primary versus transformed cells reveals a surprising dichotomy between these classes which may provide further insight into the process of cellular transformation.

The development and characterization of a CRISPR/Cas9-mediated PD-1 functional knockout rat as a tool to study idiosyncratic drug reactions.

Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine.

Evaluation of the biodistribution and preliminary safety profile of a novel brain-targeted manganese dioxide-based nanotheranostic system for Alzheimer's disease.

A novel brain-targeted and reactive oxygen species-activatable manganese dioxide containing nanoparticle system functionalized with anti-amyloid-ß antibody (named aAß-BTRA-NC) developed by our group has shown great promise as a highly selective magnetic resonance imaging (MRI) contrast agent for early detection and multitargeted disease-modifying treatment of Alzheimer's disease (AD). To further evaluate the suitability of the formulation for future clinical application, we investigated the safety, biodistribution, and pharmacokinetic profile of aAß-BTRA-NC in a transgenic TgCRND8 mouse AD model, wild type (WT) littermate, and CD-1 mice. Dose-ascending studies demonstrated that aAß-BTRA-NC was well-tolerated by the animals up to 300 µmol Mn/kg body weight [b.w.], 3 times the efficacious dose for early AD detection without apparent adverse effects; Histopathological, hematological, and biochemical analyses indicated that a single dose of aAß-BTRA-NC did not cause any toxicity in major organs. Immunotoxicity data showed that aAß-BTRA-NC was safer than commercially available gadolinium-based MRI contrast agents at an equivalent dose of 100 µmol/kg b.w. of metal ions. Intravenously administered aAß-BTRA-NC was taken up by main organs with the order of liver, kidneys, intestines, spleen, followed by other organs, and cleared after one day to one week post injection. Pharmacokinetic analysis indicated that the plasma concentration profile of aAß-BTRA-NC followed a 2-compartmental model with faster clearance in the AD mice than in the WT mice. The results suggest that aAß-BTRA-NC exhibits a strong safety profile as a nanotheranostic agent which warrants more robust preclinical development for future clinical applications.

Matrigel alters the pathophysiology of orthotopic human breast adenocarcinoma xenografts with implications for nanomedicine evaluation.

Matrigel, a mouse sarcoma-derived basement membrane protein mixture, is frequently used to facilitate human tumor xenograft growth in rodents. Despite its known effects on tumor growth and metastasis, its impact on tumor pathophysiology and preclinical evaluation of nanomedicines in tumor xenografts has not been reported previously. Herein bilateral MDA435 tumors were established orthotopically with (Mat+) or without (Mat-) co-injection of Matrigel. Tumor perfusion, morphology and nanoparticle retention were evaluated. As compared to Mat- tumors, Mat+tumors exhibited enhanced vascular perfusion and lymphatic flow, greater blood vessel and lymphatic growth within the tumor core, and more deformation and collapse of lymphatics in tumor-associated lymph nodes. These changes were accompanied by reduced nanoparticle retention in Mat+tumors. The results suggest that Matrigel is not a passive medium for tumor growth, but rather significantly alters long-term tumor architecture. These findings have significant implications for the evaluation of therapeutic nanomedicine in xenograft mouse models. FROM THE CLINICAL EDITOR: Matrigel is utilized in facilitating human tumor xenograft growth in rodents. The authors demonstrate that Matrigel is not a passive medium for tumor growth; instead it significantly alters long-term tumor architecture, with major implications in the evaluation of therapeutic nanomedicine in xenograft mouse models.

Dysregulation of Amino Acid Transporters in a Rat Model of TLR7-Mediated Maternal Immune Activation.

Maternal immune activation (MIA) during pregnancy is linked to neurodevelopmental disorders in humans. Similarly, the TLR7 agonist imiquimod alters neurodevelopment in rodents. While the mechanisms underlying MIA-mediated neurodevelopmental changes are unknown, they could involve dysregulation of amino acid transporters essential for neurodevelopment. Therefore, we sought to determine the nature of such transporter changes in both imiquimod-treated rats and human placentas during infection. Pregnant rats received imiquimod on gestational day (GD)14. Transporter expression was measured in placentas and fetal brains via qPCR (GD14.5) and immunoblotting (GD16). To monitor function, fetal brain amino acid levels were measured by HPLC on GD16. Gene expression in the cortex of female fetal brains was further examined by RNAseq on GD19. In human placentas, suspected active infection was associated with decreased ASCT1 and SNAT2 protein expression. Similarly, in imiquimod-treated rats, ASCT1 and SNAT2 protein was also decreased in male placentas, while EAAT2 was decreased in female placentas. CAT3 was increased in female fetal brains. Consistent with this, imiquimod altered amino acid levels in fetal brains, while RNAseq demonstrated changes in expression of several genes implicated in autism. Thus, imiquimod alters amino acid transporter levels in pregnant rats, and similar changes occur in human placentas during active infection. This suggests that changes in expression of amino acid transporters may contribute to effects mediated by MIA toward altered neurodevelopment.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Brain-Penetrating and Disease Site-Targeting Manganese Dioxide-Polymer-Lipid Hybrid Nanoparticles Remodel Microenvironment of Alzheimer's Disease by Regulating Multiple Pathological Pathways.

Finding effective disease-modifying treatment for Alzheimer's disease remains challenging due to an array of factors contributing to the loss of neural function. The current study demonstrates a new strategy, using multitargeted bioactive nanoparticles to modify the brain microenvironment to achieve therapeutic benefits in a well-characterized mouse model of Alzheimer's disease. The application of brain-penetrating manganese dioxide nanoparticles significantly reduces hypoxia, neuroinflammation, and oxidative stress; ultimately reducing levels of amyloid ß plaques within the neocortex. Analyses of molecular biomarkers and magnetic resonance imaging-based functional studies indicate that these effects improve microvessel integrity, cerebral blood flow, and cerebral lymphatic clearance of amyloid ß. These changes collectively shift the brain microenvironment toward conditions more favorable to continued neural function as demonstrated by improved cognitive function following treatment. Such multimodal disease-modifying treatment may bridge critical gaps in the therapeutic treatment of neurodegenerative disease.

Mutation I810N in the alpha3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS.

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na(+),K(+)-ATPase alpha3 isoform inactive. Total Na(+),K(+)-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na(+),K(+)-ATPase alpha3 by transgenesis, which also rescued Na(+),K(+)-ATPase activity. Our findings reveal the functional significance of the Na(+),K(+)-ATPase alpha3 isoform in the control of epileptiform activity and seizure behavior.

Novel localization of folate transport systems in the murine central nervous system.

BACKGROUND: Folates are a family of B9 vitamins that serve as one-carbon donors critical to biosynthetic processes required for the development and function of the central nervous system (CNS) in mammals. Folate transport is mediated by three highly specific systems: (1) folate receptor alpha (FRα; FOLR1/Folr1), (2) the reduced folate-carrier (RFC; SLC19A1/Slc19a1) and (3) the proton-coupled folate transporter (PCFT; SLC46A1/Slc46a1). Folate transport into and out of the CNS occurs at the blood-cerebrospinal fluid barrier (BCSFB), mediated by FRα and PCFT. Impairment of folate transport at the BCSFB results in cerebral folate deficiency in infants characterized by severe neurological deficiencies and seizures. In contrast to the BCSFB, CNS folate transport at other brain barriers and brain parenchymal cells has not been extensively investigated. The aim of this study is to characterize folate transport systems in the murine CNS at several known barriers encompassing the BCSFB, arachnoid barrier (AB), blood-brain barrier (BBB) and parenchymal cells (astrocytes, microglia, neurons). METHODS: Applying immunohistochemistry, localization of folate transport systems (RFC, PCFT, FRα) was examined at CNS barriers and parenchymal sites in wildtype (C57BL6/N) mice. Subcellular localization of the folate transport systems was further assessed in an in vitro model of the mouse AB. Gene and protein expression was analyzed in several in vitro models of brain barriers and parenchyma by qPCR and western blot analysis. RESULTS: RFC, PCFT, and FRα expression was localized within the BCSFB and BBB consistent with previous reports. Only RFC and PCFT expression was detected at the AB. Varied levels of RFC and PCFT expression were detected in neuronal and glial cells. CONCLUSIONS: Localization of RFC and PCFT within the AB, described here for the first time, suggest that AB may contribute to folate transport between the peripheral circulation and the CSF. RFC and PCFT expression observed in astrocytes and microglia is consistent with the role that one or both of these transporters may play in delivering folates into cells within brain parenchyma. These studies provide insights into mechanisms of folate transport in the CNS and may enhance our understanding of the critical role folates play in neurodevelopment and in the development of novel treatment strategies for disorders of brain folate deficiency due to impaired transporter function.

Cisplatin toxicity in the developing brain displays an absolute requirement for caspase-3.

Cisplatin is a member of a widely utilized class of chemotherapeutic agent that initiates DNA damage response, cell cycle arrest, and p53-dependent apoptotic cell death in concert with DNA­platinum adduct formation. While normal programmed cell death (PCD) can occur in the developing neuroepithelium in the absence of caspase-3 within certain genetic backgrounds, we observed an absolute dependency upon this executioner caspase with respect to cisplatin-induced PCD in the developing central nervous system (CNS). We therefore examined the nature of this genotoxic injury in the CNS in vivo, in which cisplatin treatment causes widespread cellular injury consistent with hallmarks of apoptosis which are averted upon caspase-3 inhibition. Examination of cisplatin-mediated injury as a function of time revealed the presence of an alternative, delayed form of necroptosis-like cell death which manifests in Casp3-/- neuroepithelia for several days following the normal pattern of apoptosis. Together, these findings suggest a coordinated regulation of these disparate PCD pathways in response to genotoxic stress in vivo and highlight the unique and critical role which caspase-3 plays among executioner caspases in coordinating apoptotic versus necroptotic responsiveness of the developing CNS to genotoxic injury.

Exploring the transformability of polymer-lipid hybrid nanoparticles and nanomaterial-biology interplay to facilitate tumor penetration, cellular uptake and intracellular targeting of anticancer drugs.

BACKGROUND: Successful delivery of anticancer drugs to intracellular targets requires different properties of the nanocarrier to overcome multiple transport barriers. However, few nanocarrier systems, to date, possess such properties, despite knowledge about the biological fate of inorganic and polymeric nanocarriers in relation to their fixed size, shape and surface properties. Herein, a polymer-lipid hybrid nanoparticle (PLN) system is described with size and shape transformability and its mechanisms of cellular uptake and intracellular trafficking are studied. METHODS: Pharmaceutical lipids were screened for use in transformable PLN. Mechanisms of cellular uptake and the role of fatty acid-binding proteins in intracellular trafficking of PLN were investigated in breast cancer cells. Intra-tumoral penetration and retention of doxorubicin (DOX) were evaluated by confocal microscopy. RESULTS: The lead PLNs showed time-dependent size reduction and shape change from spherical to spiky shape. This transformability of PLNs and lipid trafficking pathways facilitated intracellular transport of DOX-loaded PLN (DOX-PLN) into mitochondria and nuclei. DOX-PLN significantly increased DOX penetration and retention over free DOX or non-transformable liposomal DOX particles at 4 h post-intravenous administration. CONCLUSION: Transformability of PLN and lipid-biology interplay can be exploited to design new nanocarriers for effective drug delivery to tumor cells and intracellular targets.

Oxidative DNA damage and repair in teratogenesis and neurodevelopmental deficits.

Several teratogenic agents, including ionizing radiation and xenobiotics such as phenytoin, benzo[a]pyrene, thalidomide, and methamphetamine, can initiate the formation of reactive oxygen species (ROS) that oxidatively damage cellular macromolecules including DNA. Oxidative DNA damage, and particularly the most prevalent 8-oxoguanine lesion, may adversely affect development, likely via alterations in gene transcription rather than via a mutational mechanism. Contributions from oxidative DNA damage do not exclude roles for alternative mechanisms of initiation like receptor-mediated processes or the formation of covalent xenobiotic-macromolecular adducts, damage to other macromolecular targets like proteins and lipids, and other effects of ROS like altered signal transduction. Even in the absence of teratogen exposure, endogenous developmental oxidative stress can have embryopathic consequences in the absence of key pathways for detoxifying ROS or repairing DNA damage. Critical proteins in pathways for DNA damage detection/repair signaling, like p53 and ataxia telangiectasia mutated, and DNA repair itself, like oxoguanine glycosylase 1 and Cockayne syndrome B, can often, but not always, protect the embryo from ROS-initiating teratogens. Protection may be variably dependent upon such factors as the nature of the teratogen and its concentration within the embryo, the stage of development, the species, strain, gender, target tissue and cell type, among other factors.

Calcineurin inhibition enhances motor neuron survival following injury.

The immunosuppressive agents cyclosporin A (CsA) and FK-506 have previously been shown to exhibit neurotrophic and neuroprotective properties in vivo. Given that significant clinical expertise exists for both drugs, they represent an attractive starting point for treatment of acute neural injuries. One putative mechanism for neuroprotection by these drugs relates to inhibition of calcineurin activity. However each drug-immunophilin complex can potentially influence additional signal transduction pathways. Furthermore, several non-immunosuppressive immunophilin ligands have been described as possessing neuroprotective properties, suggesting that neuroprotection may be separable from calcineurin inhibition. In the present study, we examined the mechanism of this neuroprotection in facial motor neurons following axotomy-induced injury. Similar to previous studies in rats, CsA and FK-506 enhanced motor neuron survival in mice following acute injury. To examine the mechanism responsible for neuroprotection by these agents, pharmacologic inhibitors of several potential alternate signalling pathways (17-(allylamino)-17-demethoxygeldanamycin, rapamycin, cypermethrin) were evaluated with respect to neuroprotection. Of these, only cypermethrin, a direct calcineurin inhibitor not previously associated with neuronal survival properties, was observed to significantly enhance motor neuron survival following injury. The results demonstrate for the first time that direct inhibition of calcineurin is neuroprotective in vivo. These data support a model in which calcineurin inhibition promotes neuronal survival, distinct from effects upon neurite outgrowth.