Deficient signaling by insulin, as occurs in diabetes, is associated with impaired brain function, and diabetes is associated with an increased prevalence of Alzheimer's disease. One of the hallmark pathological characteristics of Alzheimer's disease is the presence of neurofibrillary tangles containing hyperphosphorylated tau, a microtubuleassociated protein. Therefore, we tested the hypothesis that insulin depletion caused by administration of streptozotocin may cause tau hyperphosphorylation in mouse brain by using site-specific phosphorylation-dependent tau antibodies to obtain precise identification of the phosphorylation of tau on individual residues. A massive (fivefold average increase) and widespread at multiple residues (detected with eight different phosphorylation-dependent tau antibodies) increase in the phosphorylation of tau was found in mouse cerebral cortex and hippocampus within 3 days of insulin depletion by streptozotocin treatment. This hyperphosphorylation of tau at some sites was rapidly reversible by peripheral insulin administration. Examination of several kinases that phosphorylate tau indicated that they were unlikely to account for the widespread hyperphosphorylation of tau caused by streptozotocin treatment, but there was a large decrease in mouse brain protein phosphatase 2A activity, which is known to mediate tau phosphorylation. These results show that insulin deficiency causes rapid and large increases in tau phosphorylation, a condition that could prime tau for the neuropathology of Alzheimer's disease, thereby contributing to the increased susceptibility to Alzheimer's disease caused by diabetes. Diabetes 55:3320 -3325, 2006

Insulin regulates the phosphorylation and activities of Akt and glycogen synthase kinase-3 (GSK3) in peripheral tissues, but in the brain it is less clear how this signaling pathway is regulated in vivo and whether it is affected by diabetes. We found that Akt and GSK3 are sensitive to glucose, because fasting decreased and glucose administration increased by severalfold the phosphorylation of Akt and GSK3 in the cerebral cortex and hippocampus of non-diabetic mice. Brain Akt and GSK3 phosphorylation also increased after streptozotocin administration (3 days), which increased blood glucose and depleted blood insulin, indicating regulation by glucose availability even with deficient insulin. Changes in Akt and GSK3 phosphorylation and activities in epididymal fat were opposite to those of brain after streptozotocin treatment. Streptozotocin-induced hyperglycemia and increased brain Akt and GSK3 phosphorylation were reversed by lowering blood glucose with insulin administration. Long term hyperglycemia also increased brain Akt and GSK3 phosphorylation, both 4 weeks after streptozotocin and in db/db insulin-resistant mice. Thus, the Akt-GSK3 signaling pathway is regulated in mouse brain in vivo in response to physiological and pathological changes in insulin and glucose.

Pelvic nerve (PN) bladder primary afferent neurons were retrogradely labeled by intraparenchymal (IPar) microinjection of fluorescent tracer or intravesical (IVes) infusion of tracer into the bladder lumen. IPar and IVes techniques labeled two distinct populations of PN bladder neurons differentiated on the basis of dorsal root ganglion (DRG) soma labeling, dye distribution within the bladder, and intrinsic electrophysiological properties. IPar (Fast blue)- and IVes (DiI)-labeled neurons accounted for 91.5% (378.3 ± 32.3) and 8% (33.0 ± 26.0) of all labeled neurons, respectively (p<0.01), with only 2.0 ± 1.2 neurons labeled by both techniques. When dyes were switched, IPar (DiI)- and IVes (Fast blue) labeled neurons accounted for 77.6% (103.0 ± 25.8) and 22.4% (29.8 ± 10.5), respectively (P<0.05), with 6.0 ± 1.5 double-labeled neurons. Following IPar labeling, DiI was distributed throughout non-urothelial layers of the bladder. In contrast, dye was contained within the urothelium and occasionally the submucosa after IVes labeling. Electrophysiological properties of DiI-labeled IPar and IVes DRG neurons were characterized by whole-mount, in situ patch-clamp recordings. IPar- and IVes-labeled neurons differed significantly with respect to rheobase, input resistance, membrane capacitance, amplitude of inactivating and sustained K+ currents, and rebound action potential firing, suggesting that the IVes population is more excitable. This study is the first to demonstrate that IVes labeling is a minimally invasive approach for retrograde labeling of PN bladder afferent neurons, to selectively identify urothelial versus non-urothelial bladder DRG neurons, and to elucidate electrophysiological properties of urothelial and non-urothelial afferents in an intact DRG soma preparation.

Malignant peripheral nerve sheath tumors (MPNSTs) are the most common malignancy associated with neurofibromatosis type 1 (NF1). These Schwann cell lineage-derived sarcomas aggressively invade adjacent nerve and soft tissue, frequently precluding surgical resection. Little is known regarding the mechanisms underlying this invasive behavior. We have shown that MPNSTs express neuregulin-1 (NRG-1) β isoforms, which promote Schwann cell migration during development, and NRG-1α isoforms, whose effects on Schwann cells are poorly understood. Hypothesizing that NRG-1β and/or NRG-1α promote MPNST invasion, we found that NRG-1β promoted MPNST migration in a substrate-specific manner, markedly enhancing migration on laminin but not on collagen type I or fibronectin. The NRG-1 receptors erbB3 and erbB4 were present in MPNST invadopodia (processes mediating invasion), partially colocalized with focal adhesion kinase and the laminin receptor β1-integrin and coimmunoprecipitated with β1-integrin. NRG-1β stimulated human and murine MPNST cell migration and invasion in a concentration-dependent manner in three-dimensional migration assays, acting as a chemotactic factor. Both baseline and NRG-1β induced migration were erbB-dependent and required the action of MEK 1/2, SAPK/JNK, PI-3 kinase, Src family kinases and ROCK-I/II. In contrast, NRG-1α had no effect on the migration and invasion of some MPNST lines and inhibited the migration of others. While NRG-1β potently and persistently activated Erk 1/2, SAPK/JNK, Akt and Src family kinases, NRG-1α did not activate Akt and activated these other kinases with kinetics distinct from those evident in NRG-1β stimulated cells. These findings suggest that NRG-1β enhances MPNST migration and that NRG-1β and NRG-1α differentially modulate this process.

Few therapeutic options are available for malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1 (NF1). Guided by clinical observations suggesting that some NF1-associated nerve sheath tumors are hormonally responsive, we hypothesized that the selective estrogen receptor (ER) modulator tamoxifen would inhibit MPNST tumorigenesis in vitro and in vivo. To test this hypothesis, we examined tamoxifen effects on MPNST cell proliferation and survival, MPNST xenograft growth, and the mechanism by which tamoxifen impeded these processes. We found that 1-5 μM 4-hydroxy-tamoxifen induced MPNST cell death, whereas 0.01-0.1 μM 4-hydroxy-tamoxifen inhibited mitogenesis. Dermal and plexiform neurofibromas, MPNSTs, and MPNST cell lines expressed ERβ and G-protein-coupled ER-1 (GPER); MPNSTs also expressed estrogen biosynthetic enzymes. However, MPNST cells did not secrete 17β-estradiol, exogenous 17β-estradiol did not stimulate mitogenesis or rescue 4-hydroxy-tamoxifen effects on MPNST cells, and the steroidal antiestrogen ICI-182,780 did not mimic tamoxifen effects on MPNST cells. Further, ablation of ERβ and GPER had no effect on MPNST proliferation, survival, or tamoxifen sensitivity, indicating that tamoxifen acts via an ER-independent mechanism. Consistent with this hypothesis, inhibitors of calmodulin (trifluoperazine, W-7), another known tamoxifen target, recapitulated 4-hydroxy-tamoxifen effects on MPNST cells. Tamoxifen was also effective in vivo, demonstrating potent antitumor activity in mice orthotopically xenografted with human MPNST cells. We conclude that 4-hydroxy-tamoxifen inhibits MPNST cell proliferation and survival via an ER-independent mechanism. The in vivo effectiveness of tamoxifen provides a rationale for clinical trials in cases of MPNSTs.

Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC-MS/MS analysis using a 10 μm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ~1000 cells, was needed per LC-MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow.

GSK3β is prominent for its role in apoptosis signaling and has been shown to be involved in Parkinson's disease (PD) pathogenesis. The overall effects of GSK3β activity on cell fate are wellestablished, but the effects of mitochondrial GSK3β activity on mitochondrial function and cell fate are unknown. Here we selectively expressed constitutively active GSK3β within the mitochondria and found that this enhanced the apoptosis signaling activated by the PD-mimetic NADH:ubiquinone oxidoreductase (complex I) inhibitors 1-methyl-4-phenylpyridinium ion (MPP + ) and rotenone. Additionally, expression of GSK3β in the mitochondria itself caused a significant decrease in complex I activity and ATP production. Increased mitochondrial GSK3β activity also increased reactive oxygen species production and perturbed the mitochondrial morphology. Conversely, chemical inhibitors of GSK3β inhibited MPP + -and rotenone-induced apoptosis, and attenuated the mitochondrial GSK3β-mediated impairment in complex I. These results indicate that unregulated mitochondrial GSK3β activity can mimic some of the mitochondrial insufficiencies found in PD pathology.

Genetic changes occurring in different stages of pre-cancer lesions reflect causal events initiating and promoting the progression to cancer. Co-existing pre-cancerous lesions including low- and high-grade squamous intraepithelial lesion (LGSIL and HGSIL), and adjacent “normal” cervical epithelium from six formalin-fixed paraffin-embedded samples were selected. Tissues from these 18 samples were isolated using laser-capture microdissection, RNA was extracted and sequenced. RNA-sequencing generated 2.4 billion raw reads in 18 samples, of which ~50.1% mapped to known and annotated genes in the human genome. There were 40 genes up-regulated and 3 down-regulated (normal to LGSIL) in at least one-third of the sample pairs (same direction and FDR p < 0.05) including S100A7 and KLK6. Previous studies have shown that S110A7 and KLK7 are up-regulated in several other cancers, whereas CCL18, CFTR, and SLC6A14, also differentially expressed in two samples, are up-regulated specifically in cervical cancer. These differentially expressed genes in normal to LGSIL progression were enriched in pathways related to epithelial cell differentiation, keratinocyte differentiation, peptidase, and extracellular activities. In progression from LGSIL to HGSIL, two genes were up-regulated and five down-regulated in at least two samples. Further investigations using co-existing samples, which account for all internal confounders, will provide insights to better understand progression of cervical pre-cancer.