Different Effects of p52SHC1 and p52SHC3 on the Cell Cycle of Neurons and Neural Stem Cells.

SHC3 is exclusively expressed in postmitotic neurons, while SHC1 is found in neural stem cells and neural precursor cells but absent in mature neurons. In this study, we discovered that suppression of p52SHC1 expression by RNA interference resulted in proliferation defects in neural stem cells, along with significantly reduced protein levels of cyclin E and cyclin A. At the same time, p52SHC3 RNAi caused cell cycle re-entry (9.54% in S phase and 5.70% in G2-M phase) in primary neurons with significantly up-regulated expression of cyclin D1, cyclin E, cyclin A, CDK2, and phosphorylated CDK2. When p52SHC3 was overexpressed, the cell cycle of neural stem cells was arrested with reduced protein levels of cyclin D1, cyclin E, and cyclin A, while overexpression of p52SHC1 did not result in significant changes in postmitotic neurons. Our results indicate that p52SHC3 plays an important role in maintaining the mitotic quiescence of neurons, while p52SHC1 regulates the proliferation of neural stem cells.

Transcription factor SCIRR69 involved in the activation of brain-derived neurotrophic factor gene promoter II in mechanically injured neurons.

The spinal cord injury and regeneration-related gene #69 (SCIRR69), which was identified in our screen for genes upregulated after spinal cord injury, encode a protein belonging to the cAMP response element-binding protein (CREB)/ATF family of transcription factors. Our previous study showed that SCIRR69 functions as a transcriptional activator. However, the target gene regulated by SCIRR69 and its roles in injured neurons remain unknown. In this study, we showed that SCIRR69 is widely distributed in the central nervous system. Full-length SCIRR69 is an endoplasmic reticulum-bound protein. Following mechanical injury to neurons, SCIRR69 was induced and proteolytically cleaved by site-1 and site-2 proteases, and the proteolytically cleaved SCIRR69 (p60-SCIRR69) was translocated to the nucleus where it bound to brain-derived neurotrophic factor (BDNF) gene promoter II. In addition, loss- and gain-of-function studies confirmed that SCIRR69 is involved in the regulation of BDNF expression in injured neurons. As expected, the culture supernatants of PC12 cells stably expressing p60-SCIRR69 contained higher levels of BDNF, and more remarkably promoted neurite outgrowth in a spinal cord slice culture model in vitro than the supernatants of control cells. These results suggest that SCIRR69 is a novel regulator of the BDNF gene and may play an important role in the repair and/or regeneration of damaged neural tissues by specifically activating BDNF promoter II.

Immunogenicity assessment of therapeutic proteins and peptides.

Assessment of immunogenicity is a major aspect in evaluating the safety of biological therapeutic proteins. It is important to evaluate the immunogenic potential of the biologics in an appropriate fashion using clearly defined strategy and clinical trials. The studies must include the appropriate risk assessment procedures using validated methods. The immune responses against the therapeutic biologics can be studied using various methodologies. These include enzyme linked immunoassays (ELISA), surface plasmon resonance (SPR), chemiluminescence, and flowcytometry assays for binding antibodies and cell based assays for neutralizing antibodies. The immune responses to the biologics can widely vary in various cross section of the population, thus a combination of techniques are necessary to fully evaluate the immunogenic potential of the biologics. This review outlines various commonly used technology platforms, its merits and shortcomings for the evaluation of the immune responses.

A non-radioactive method for detecting neutralizing antibodies against therapeutic proteins in serum.

The presence of neutralizing antibodies against protein therapeutics continues to cause concern in the biomedical field. These antibodies not only reduce the efficacy of the protein therapeutics, but may also block the normal function of their endogenous counterparts, which can result in serious health risks to the patient. To date, a limited number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. However, many of the existing assays involve the use of radioactive materials. We have established a novel and non-radioactive bioassay system for detecting neutralizing antibodies in patient serum samples. Our assay measures the cell metabolic activities that are closely associated with cell proliferation and apoptosis. The biologic effect of the therapeutic protein and the capability of the antibodies to neutralize the therapeutics are reflected by changes of the cellular metabolic activities triggered by the administration of the therapeutics or presence of the anti-therapeutic protein antibodies. Compared with existing assays, this new assay is equally or more sensitive, and completely eliminates the use of radioactive materials.

Identification and characterization of scirr1, a novel gene up-regulated after spinal cord injury.

Spinal cord injury and regeneration involves transcriptional activity of many genes, of which many remain unknown. Using the rat spinal cord full- transection model, bioinformatics, cloning, expression assays, fusion proteins, and transfection techniques, we identified and characterized one such differentially expressed gene, termed scirr1 (spinal cord injury and/or regeneration related gene 1). Fourteen orthologs were found in 13 species from echinoderm to insect and human by Blast search of NCBI protein reference sequence database. However, no further information is available for these homologues. Using whole-mount in situ hybridization, mouse scirr1 mRNA was expressed temporally and spatially in accordance with the early development sequence of the central nervous system. In adult rat spinal cord, expression of scirr1 mRNA was localized to neurons of gray matter by in situ hybridization. Using immunohistochemistry, SCIRR1 protein was found to be up-regulated and expressed more highly in spinal cord neurons farther from the epicenter of injury. Although the precise function of SCIRR1 is unknown, its unique pattern of expression during CNS early development and up-regulation after spinal cord injury suggest that SCIRR1 should be involved in the succeeding injury and/or repair processes of the injured spinal cord. Also, the typical F-box and leucine-rich repeat (LRR) architecture of rat SCIRR1 indicated that it may play an important substrate recruiting role in the pleiotropic ubiquitin/proteasome pathway. All these make scirr1 a new interesting start to study the spinal cord injury and regeneration mechanism.

A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression.

The presence of neutralizing antibodies against protein therapeutics is a concern in the biomedical field. Such antibodies not only reduce the efficacy of protein therapeutics, but also impose potential dangers to the patients receiving them. To date, a small number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. Most of the existing assays, however, either involve the use of radioactive materials or have limited sensitivities and/or poor specificities. With advances in mRNA profiling and detection techniques, we have established a novel and non-radioactive bioassay system using branched DNA (bDNA) technology for detecting protein-therapeutic neutralizing antibodies in patient serum. Our assay measures the variations of target gene expression that reflect the biologic effect of the therapeutic agent and the capability of the antibodies, if present, to neutralize the therapeutics. Compared with most existing assays, the new assay is more sensitive and specific, and completely eliminates the use of radioactive materials. Application of the new assay system can be widely expanded if new target genes and responding cell lines for other therapeutics are identified or engineered.

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury.

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.

Expression of GFRalpha1 receptor splicing variants with different biochemical properties is modulated during kidney development.

The glial cell line-derived neurotrophic factor (GDNF) family coreceptor alpha1 (GFRalpha1) is a critical component of the RET receptor kinase signal-transducing complex. The activity of this multicomponent receptor is stimulated by the glial cell line-derived neurotrophic factor (GDNF) and is involved in neuronal cells survival and kidney development. GFRalpha1 pre-mRNA is alternatively spliced and produces two isoforms: GFRalpha1a, which includes the exon 5; and GFRalpha1b, which excludes it. Here we show that the Gfralpha1a isoform is predominantly expressed in neuronal tissues and in PC12 cells differentiated toward a neuronal phenotype. GFRalpha1 splicing is also regulated during kidney development, GFRalpha1a is the minor isoform before birth and then rapidly becomes the major form after birth. We established cell lines expressing either GFRalpha1 isoforms and demonstrated that the GFRalpha1b isoform binds GDNF more efficiently than GFRalpha1a. Consistently, GFRalpha1b promotes a stronger RET phosphorylation than GFRalpha1a. These results indicate that specific inclusion of the GFRalpha1 exon 5 in neuronal tissues or during kidney development may alter the binding properties of GDNF to GFRalpha1, and thus could constitute an additional regulatory mechanism of the RET signaling pathway.

The candidate neuroprotective agent artemin induces autonomic neural dysplasia without preventing peripheral nerve dysfunction.

Artemin (ART) signals through the GFR alpha-3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfr alpha-3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.

Transgenic overexpression of human IL-17E results in eosinophilia, B-lymphocyte hyperplasia, and altered antibody production.

We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3(+) eosinophils increased in the blood and lymph nodes of the transgenic mice by 50- and 300-fold, respectively. Eosinophils also increased 8- to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19(+) B cells increased 2- to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4(+) T lymphocytes increased 2-fold in both blood and spleen. High serum levels of the cytokines IL-2, IL-4, IL-5, granulocyte colony-stimulating factor, eotaxin, and interferon gamma were observed. Consistent with B-lymphocyte increases, serum immunoglobulin (Ig) M, IgG, and IgE were significantly elevated. Antigenic challenge of the transgenic mice with keyhole limpet hemocyanin (KLH) resulted in a decrease in anti-KLH IgG accompanied by increases of anti-KLH IgA and IgE. In situ hybridization of transgenic tissues revealed that IL-17Rh1 (IL-17BR/Evi27), a receptor that binds IL-17E, is up-regulated. Taken together, these data indicate that IL-17E regulates hematopoietic and immune functions, stimulating the development of eosinophils and B lymphocytes. The fact that hIL-17E overexpression results in high levels of circulating eosinophils, IL-4, IL-5, eotaxin, and IgE suggests that IL-17E may be a proinflammatory cytokine favoring Th2-type immune responses.