Axoplasmic importins enable retrograde injury signaling in lesioned nerve.

Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the importin/karyopherin alpha and beta families underlie this process. We show that importins are found in axons at significant distances from the cell body and that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.

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.

PKC isozymes in the enhanced regrowth of retinal neurites after optic nerve injury.

PURPOSE: To establish an in vitro model of axonal regeneration from mammalian retinal ganglion cells and to evaluate the role of PKC isozymes in promoting such retinal axon regeneration. METHODS: Postnatal day-3 mice were subjected to optic nerve crush, and then retinal ganglion cells (RGCs) were used for culture 5 days later. RGCs were selected using anti-Thy 1.2-coated magnetic beads and plated onto a merosin substrate. Changes in axonal localization of PKC and axonal regeneration were examined in cultured RGCs by immunofluorescence. Changes in PKC isozyme mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The role of PKC in RGC neurite outgrowth was examined by treatment with activators or pharmacological inhibitors of PKC activity. RESULTS: RGCs subjected to optic nerve crush injury demonstrated more rapid neurite outgrowth in vitro when compared with RGCs isolated from naïve retina. The neurites of these injury-conditioned RGCs showed both an increased rate of extension and enhanced PKC localization in culture. Injury-conditioned RGCs had elevated PKC isozyme mRNA levels, which probably contributed to the increased level of PKC protein in injury-conditioned RGC axons. Pharmacological activation of PKC enhanced neurite growth, whereas inhibition of PKC suppressed neurite growth in both the conditioned and naïve RGCs. CONCLUSIONS: RGCs actively respond to axonal injury by regulating expression of genes that promote neurite outgrowth. PKC-alpha and -beta isozymes are among the growth-associated proteins that are upregulated after injury. Results of pharmacological manipulation of PKC activity support the argument that increased PKC levels enhance neurite regrowth after axonal injury.

Nimotuzumab combined with radiotherapy for esophageal cancer: preliminary study of a Phase II clinical trial.

OBJECTIVE: To determine the safety and therapeutic effects of nimotuzumab (h-R3) combined with radiotherapy in esophageal cancer. METHODS: This Phase II clinical trial involved 42 patients with stage II (inoperable or refused surgery) to stage IV (supraclavicular lymph node metastasis only) esophageal cancers treated between November 2008 and July 2010. All patients had squamous cell carcinomas, and all received three-dimensional conformal radiotherapy and 200 mg nimotuzumab per week during radiotherapy. RESULTS: There were 9, 25, and 8 patients with stage II, III and IV disease, respectively. All except two patients received 50-70 Gy radiation; 37 patients (88.1%) received more than five nimotuzumab doses. Grade III toxicities (21.4% of all adverse events) included esophagitis and gastrointestinal, dermatological and hematological toxicities. Complete response, partial response, stable disease, and progressive disease were observed in 0, 22 (52.4%), 17 (40.5%) and 3 (7.1%) patients at 1 month after the treatment. The epidermal growth factor receptor (EGFR) overexpression rate was 95.2%. After a median follow-up of 37 months, the median survival time (MST) was 14 months. The 2 year and 3 year overall survival (OS) rates were 33.3% and 26.2%, respectively. The median progression-free survival (PFS) time was 10 months. The 2 year and 3 year PFS rates were 24.5% and 22.1%, respectively. The MST in the 13 patients with (+++) EGFR expression (group A) and 7 patients with (++) EGFR expression (group B) was 15 and 11 months, respectively. The 2 year and 3 year OS rates were 46.2% and 38.5% in group A and 28.6% and 28.6% in group B, respectively (P = 0.405). CONCLUSION: Although concurrent chemoradiotherapy was the standard care for locally advanced esophageal cancer, radiotherapy was the choice for those who were refused or could not tolerate chemoradiotherapy. Our study shows that nimotuzumab combined with radiotherapy was well tolerated in patients with esophageal cancer. EGFR overexpression was more common than previously reported. OS was higher after combined therapy than after historical control radiotherapy alone. Further studies are required to confirm the therapeutic efficacy of nimotuzumab in esophageal cancer.

Voluntary exercise increases axonal regeneration from sensory neurons.

Recent advances in understanding the role of neurotrophins on activity-dependent plasticity have provided insight into how behavior can affect specific aspects of neuronal biology. We present evidence that voluntary exercise can prime adult dorsal root ganglion neurons for increased axonal regeneration through a neurotrophin-dependent mechanism. Dorsal root ganglion neurons showed an increase in neurite outgrowth when cultured from animals that had undergone 3 or 7 days of exercise compared with sedentary animals. Neurite length over 18-22 h in culture correlated directly with the distance that animals ran. The exercise-conditioned animals also showed enhanced regrowth of axons after an in vivo nerve crush injury. Sensory ganglia from the 3- and 7-day-exercised animals contained higher brain-derived neurotrophic factor, neurotrophin 3, synapsin I, and GAP43 mRNA levels than those from sedentary animals. Consistent with the rise in brain-derived neurotrophic factor and neurotrophin 3 during exercise, the increased growth potential of the exercise-conditioned animals required activation of the neurotrophin signaling in vivo during the exercise period but did not require new mRNA synthesis in culture.

Effects of altered expression and localization of cyclophilin A on differentiation of p19 embryonic carcinoma cells.

1. The retinoblastoma susceptibility gene product, p105Rb (RB), is an important regulator in the control of cell proliferation, differentiation, and apoptosis. Several cellular factors that complex with RB and exert their cellular regulatory functions have been identified, such as the RB:cyclophilin A (CypA) complex. 2. CypA is a cytoplasmic immunophilin and known for its involvement in T-cell differentiation and proliferation. Although CypA has a pivotal role in the immune response, its function in other signaling pathways is largely unknown. 3. In this study, we used a model of neuronal differentiation to demonstrate that the nuclear translocation of CypA, the appearance of hypophosphorylated RB and the enhancement of RB: CypA complex formation correlates with retinoic acid induced neuronal differentiation. 4. Inhibition of CypA expression results in repression of both the hypophosphorylated RB and the neuron-specific differentiation marker, class III beta tubulin. 5. The evidence of enriched CypA and colocalization of RB with CypA in the nucleus of primary adult sensory neurons substantiated the important event of RB-mediated neuronal differentiation of p19 EC cells.