Toxicology Evaluation of Drugs Administered via Uncommon Routes: Intranasal, Intraocular, Intrathecal/Intraspinal, and Intra-Articular.

As the need for nasal, ocular, spinal, and articular therapeutic compounds increases, toxicology assessments of drugs administered via these routes play an important role in human safety. This symposium outlined the local and systemic evaluation to support safety during the development of these drugs in nonclinical models with some case studies. Discussions included selection of appropriate species for the intended route; conducting nonclinical studies that closely mimic the intended use with adequate duration; functional assessment, if deemed necessary; evaluation of local tissues with special histological staining procedure; and evaluations of safety margins based on local and systemic toxicity.

Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain.

Adult neurogenesis occurs throughout life in discrete regions of the adult mammalian brain. Little is known about the mechanism governing the sequential developmental process that leads to integration of new neurons from adult neural stem cells into the existing circuitry. Here, we investigated roles of Disrupted-In-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, in adult hippocampal neurogenesis. Unexpectedly, downregulation of DISC1 leads to accelerated neuronal integration, resulting in aberrant morphological development and mispositioning of new dentate granule cells in a cell-autonomous fashion. Functionally, newborn neurons with DISC1 knockdown exhibit enhanced excitability and accelerated dendritic development and synapse formation. Furthermore, DISC1 cooperates with its binding partner NDEL1 in regulating adult neurogenesis. Taken together, our study identifies DISC1 as a key regulator that orchestrates the tempo of functional neuronal integration in the adult brain and demonstrates essential roles of a susceptibility gene for major mental illness in neuronal development, including adult neurogenesis.

Sumoylation in axons triggers retrograde transport of the RNA-binding protein La.

A surprisingly large population of mRNAs has been shown to localize to sensory axons, but few RNA-binding proteins have been detected in these axons. These axonal mRNAs include several potential binding targets for the La RNA chaperone protein. La is transported into axonal processes in both culture and peripheral nerve. Interestingly, La is posttranslationally modified in sensory neurons by sumoylation. In axons, small ubiquitin-like modifying polypeptides (SUMO)-La interacts with dynein, whereas native La interacts with kinesin. Lysine 41 is required for sumoylation, and sumoylation-incompetent La(K41R) shows only anterograde transport, whereas WT La shows both anterograde and retrograde transport in axons. Thus, sumoylation of La determines the directionality of its transport within the axonal compartment, with SUMO-La likely recycling to the cell body.

Pathophysiological mechanisms for actions of the neurotrophins.

Neurotrophins provide trophic and tropic support for different neuronal subpopulations in the developing and adult nervous systems. Expression of the neurotrophins and their receptors can be altered in several different disease or injury states that impact upon the functions in the central and peripheral nervous systems. The intracellular signals used by the neurotrophins are triggered by ligand binding to the cell surface Trk and p75NTR receptors. In general, signals emanating from Trk receptors support survival, growth and synaptic strengthening, while those emanating from p75NTR induce apoptosis, attenuate growth and weaken synaptic signaling. Mature neurotrophins are the preferred ligand for Trk proteins while p75NTR binds preferentially to the proneurotrophins and serves as a signaling component of the receptor complex for growth inhibitory molecules of central nervous system myelin [ie, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgP) and Nogo]. The functional antagonism between Trk and p75NTR signaling may significantly impact the pathogenesis of human neurodevelopmental and neurodegenerative diseases and further complicate therapeutic uses of exogenous neurotrophins. The potential for each is discussed in this review.

PC12 cells regulate inducible cyclic AMP (cAMP) element repressor expression to differentially control cAMP response element-dependent transcription in response to nerve growth factor and cAMP.

Both cyclic AMP (cAMP) and nerve growth factor (NGF) have been shown to cause rapid activation of cAMP response element-binding protein (CREB) by phosphorylation of serine 133, but additional regulatory events contribute to CREB-targeted gene expression. Here, we have used stable transfection with a simple cAMP response element (CRE)-driven reporter to address the kinetics of CRE-dependent transcription during neuronal differentiation of PC12 cells. In naive cells, dibutyryl cAMP (dbcAMP) generated a rapid increase in CRE-driven luciferase activity by 5 h that returned to naive levels by 24 h. Luciferase induction after NGF treatment was delayed until 48 h when CRE-driven luciferase expression became TrkA dependent. Blocking histone deacetylase (HDAC) activity accelerated NGF-dependent CRE-driven luciferase expression by at least 24 h and resulted in a sustained cAMP-dependent expression of CRE-driven luciferase beyond 24 h. Inhibition of protein synthesis before stimulation with NGF or dbcAMP indicated that both stimuli induce expression of a transcriptional repressor that delays NGF-dependent and attenuates cAMP-dependent CRE-driven transcription. NGF caused a rapid but transient HDAC-dependent increase in inducible cAMP element repressor (ICER) expression, but ICER expression was sustained with increased cAMP. Depletion of ICER from PC12 cells indicated that HDAC-dependent ICER induction is responsible for the delay in CRE-dependent transcription after NGF treatment.

Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons.

Recent studies have begun to focus on the signals that regulate axonal protein synthesis and the functional significance of localized protein synthesis. However, identification of proteins that are synthesized in mammalian axons has been mainly based on predictions. Here, we used axons purified from cultures of injury-conditioned adult dorsal root ganglion (DRG) neurons and proteomics methodology to identify axonally synthesized proteins. Reverse transcription (RT)-PCR from axonal preparations was used to confirm that the mRNA for each identified protein extended into the DRG axons. Proteins and the encoding mRNAs for the cytoskeletal proteins beta-actin, peripherin, vimentin, gamma-tropomyosin 3, and cofilin 1 were present in the axonal preparations. In addition to the cytoskeletal elements, several heat shock proteins (HSP27, HSP60, HSP70, grp75, alphaB crystallin), resident endoplasmic reticulum (ER) proteins (calreticulin, grp78/BiP, ERp29), proteins associated with neurodegenerative diseases (ubiquitin C-terminal hydrolase L1, rat ortholog of human DJ-1/Park7, gamma-synuclein, superoxide dismutase 1), anti-oxidant proteins (peroxiredoxins 1 and 6), and metabolic proteins (e.g., phosphoglycerate kinase 1 (PGK 1), alpha enolase, aldolase C/Zebrin II) were included among the axonally synthesized proteins. Detection of the mRNAs encoding each of the axonally synthesized proteins identified by mass spectrometry in the axonal compartment indicates that the DRG axons have the potential to synthesize a complex population of proteins. Local treatment of the DRG axons with NGF or BDNF increased levels of cytoskeletal mRNAs into the axonal compartment by twofold to fivefold but had no effect on levels of the other axonal mRNAs studied. Neurotrophins selectively increased transport of beta-actin, peripherin, and vimentin mRNAs from the cell body into the axons rather than changing transcription or mRNA survival in the axonal compartment.

Repression of Na,K-ATPase beta1-subunit by the transcription factor snail in carcinoma.

The Na,K-ATPase consists of two essential alpha- and beta-subunits and regulates the intracellular Na+ and K+ homeostasis. Although the alpha-subunit contains the catalytic activity, it is not active without functional beta-subunit. Here, we report that poorly differentiated carcinoma cell lines derived from colon, breast, kidney, and pancreas show reduced expression of the Na,K-ATPase beta1-subunit. Decreased expression of beta1-subunit in poorly differentiated carcinoma cell lines correlated with increased expression of the transcription factor Snail known to down-regulate E-cadherin. Ectopic expression of Snail in well-differentiated epithelial cell lines reduced the protein levels of E-cadherin and beta1-subunit and induced a mesenchymal phenotype. Reduction of Snail expression in a poorly differentiated carcinoma cell line by RNA interference increased the levels of Na,K-ATPase beta1-subunit. Furthermore, Snail binds to a noncanonical E-box in the Na,K-ATPase beta1-subunit promoter and suppresses its promoter activity. These results suggest that down-regulation of Na,K-ATPase beta1-subunit and E-cadherin by Snail are associated with events leading to epithelial to mesenchymal transition.

Regulation of neurogenesis by neurotrophins: implications in hippocampus-dependent memory.

Neurogenesis, the generation of new neurons from neural precursor cells (NPCs), is a multi-step process that includes the proliferation of NPCs, fate determination, migration, and neuronal maturation. Neurogenesis is regulated by several extrinsic factors,such as enriched environment, physical exercise, hormones and stress, many of which also induce the expression of neurotrophins.In this review, we summarize studies on the role of neurotrophins in neurogenesis during development and in adults.We discuss the functional significance of neurogenesis in learning and memory, and how neurotrophins regulate this process.In this context, we describe recent experiments linking adult neurogenesis to long-term synaptic plasticity in the hippocampal dentate gyrus. Further study of the relationship between neurotrophins, adult neurogenesis and dentate synaptic plasticity might provide new insights into the mechanisms by which gene-environment interactions control cognition and brain plasticity.

Persistent TrkA activity is necessary to maintain transcription in neuronally differentiated PC12 cells.

Neurotrophins are required for the differentiation and survival of several different neuronal subpopulations in the developing nervous system. The PC12 cell line responds to nerve growth factor (NGF) by withdrawing from the cell cycle and acquiring a sympathetic neuron-like phenotype. Previous studies have shown that the activation kinetics of the NGF receptor, TrkA, and downstream protein kinases appear rapid and seemingly transient after NGF treatment of naive PC12 cells. However, maintenance of the neuronal phenotype and survival of differentiated PC12 cells under serum-free conditions require constant NGF exposure. In this study we have addressed the mechanisms that NGF uses to maintain neuronal PC12 cells. We show that TrkA remains phosphorylated at a basal level throughout differentiation of the PC12 cells. The phospho-TrkA levels in the differentiated PC12 cells were diminished by both complete NGF withdrawal and pharmacological inhibition of Trk kinase activity. Intracellular sequestration of the majority of TrkA molecules (both phosphorylated and non-phosphorylated TrkA) and persistent dephosphorylation of the small pool of cell surface TrkA renders the persistent phospho-TrkA signal in the differentiated PC12 cells resistant to partial NGF withdrawal as well as exposure to additional NGF. NGF regulated both extracellular-regulated kinases 1/2 and Akt activity in the differentiated PC12 cells via sustained TrkA activity. Moreover, analysis of transcription using activating protein 1-, serum response element-, and cyclic AMP response element-Luc reporter constructs showed that NGF regulated these promoters through TrkA activity in differentiated PC12 cells. Interestingly, the initial response of the cyclic AMP response element promoter to NGF was delayed, becoming Trk-dependent well beyond the peaks in TrkA and downstream protein kinase signal transduction.