Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5.

Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3beta. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases.

Voltage-gated K+ channel beta subunits: expression and distribution of Kv beta 1 and Kv beta 2 in adult rat brain.

Recent cloning of K+ channel beta subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming alpha subunits. Here, we examined the expression, distribution, and association of two of these beta subunits, Kv beta 1 and Kv beta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta-subunit genes are heterogeneously expressed, with high densities of Kv beta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv beta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv beta 1 and Kv beta 2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kv beta 2 is the major beta subunit present in rat brain membranes, and that most K+ channel complexes containing Kv beta 1 also contain Kv beta 2. Taken together, these data suggest that Kv beta 2 is a component of almost all K+ channel complexes containing Kv 1 alpha subunits, and that individual channels may contain two or more biochemically and functionally distinct beta-subunit polypeptides.

Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain.

Recent cloning of auxiliary subunits associated with voltage-gated ion channels and their subsequent coexpression with the channel forming alpha-subunits has revealed that the expression level, gating and conductance properties of the expressed channels can be profoundly affected by the presence of an auxiliary subunit polypeptide. In the present study, we raised antibodies against the beta-subunit associated with the bovine dendrotoxin sensitive K(+)-channel complex and used these antibodies to characterize the related beta-subunit polypeptides in rat brain. The anti-beta-subunit antibodies displayed a specific reaction on immunoblots of rat brain membranes with a major 38 kDa polypeptide, and a minor 41 kDa polypeptide, which correspond closely to the predicted sizes of the Kv beta 2 and Kv beta 1 beta-subunit polypeptides, respectively, recently cloned from rat brain. Reciprocal coimmunoprecipitation experiments revealed that the beta-subunit polypeptides are associated with Kv1.2 and Kv1.4, but not Kv2.1, alpha-subunits. Immunohistochemical staining revealed that the beta-subunit polypeptides were widely distributed in adult rat brain. Moreover, the cellular distribution of beta-subunit immunoreactivity corresponded closely with immunoreactivity for Kv1.2, and to a lesser extent Kv1.4, but not with Kv2.1. These results suggest that neuronal mechanisms may exist to direct the selective interaction of K+ channel alpha- and beta-subunit polypeptides, and that the properties of K+ channels in specific subcellular domains may be regulated by the formation of heteromultimeric K+ channel complexes containing specific combinations of alpha- and beta-subunits.

Ehk-1 and Ehk-2: two novel members of the Eph receptor-like tyrosine kinase family with distinctive structures and neuronal expression.

We have identified two novel members of the Eph RTK family, termed Ehk (eph homology kinase) -1 and -2. Compared to the amino acid sequences of various Eph family members, Ehk-1 and Ehk-2 are closest to the Sek and Cek-4/Mek-4/Hek kinases, and both are more similar to the Elk kinase than they are to the Eck or Eph kinases. Analysis of Ehk-1 cDNAs from various brain libraries reveals alternatively spliced transcripts that can encode five different forms of Ehk-1 transmembrane proteins. By contrast, Ehk-2 cDNAs revealed only a single form of protein coding region. However, the structure of Ehk-2 differs from all known members of the Eph family based on a 42 amino acid insert positioned between homology regions IV and V in the kinase domain. Ehk-1 and Ehk-2 are almost exclusively expressed in the nervous system. RNA in situ hybridization analyses on adult brain show that the Ehks are predominantly expressed in neurons and display overlapping, but distinct patterns of expression in various neuronal populations.

The alpha component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development.

We recently proposed that ciliary neurotrophic factor (CNTF) shares two receptor components with a generally acting cytokine, leukemia inhibitory factor (LIF), but that CNTF also requires a third receptor component (CNTFR alpha) that is mostly restricted to the nervous system in its expression. Here we demonstrate that a transfected CNTFR alpha gene is sufficient to confer CNTF responsiveness upon hemopoietic cells normally responsive only to LIF, providing evidence that CNTFR alpha is a required receptor component that uniquely characterizes CNTF-responding cells. Consistent with this notion, CNTFR alpha expression could be localized to neurons within all known peripheral targets of CNTF. CNTFR alpha was also widely expressed within neurons of the CNS, suggesting that CNTF has broader CNS actions than previously appreciated. However, in vivo localization of CNTFR alpha, as well as of CNTF itself, is consistent with a particularly important role for CNTF in motor function as well as during neuropoiesis.

Gonadotropin-releasing hormone and chorionic gonadotropin gene expression in human placental development.

Control of human chorionic gonadotropin (hCG) synthesis during pregnancy is poorly understood, although in vitro data suggest a role for placental gonadotropin releasing hormone (GnRH) in its regulation. To study GnRH regulation during placental development, placental tissue of different gestational ages was analyzed for GnRH and beta hCG mRNA content. cRNA probes to exonic/intronic sequences of GnRH and beta hCG transcripts were constructed and used to perform solution hybridization/nuclease protection and in situ hybridization assays. The levels of GnRH mRNA were approximately 0.1-1% of that of beta hCG mRNA, in agreement with its suggested paracrine, rather than endocrine, role. While beta hCG mRNA content decreased significantly from first trimester to term (643 to 21.6 pg/microgram RNA), there was no significant change in GnRH mRNA (0.179 to 0.155 pg/microgram RNA). While beta hCG mRNA was localized almost exclusively in syncytiotrophoblasts, GnRH mRNA was present in all cell types of the placenta, including the stroma. In the course of performing sense-strand controls in the in situ hybridization, we noted that the placenta appeared to express more antisense GnRH than sense GnRH mRNA, again, in all cell types. Solution hybridization/nuclease protection analysis with exon 1 and exon 3 probes confirmed this observation, showing that there is two to three times more antisense GnRH RNA than sense GnRH mRNA. These studies suggest that GnRH gene expression and its role in regulating hCG production in human placenta is complex and does not fit a simple model for paracrine regulation of hCG.

The pathologic effects of photodynamic therapy on the larynx. Experimental study.

Using an argon pump dye laser at a wavelength of 630 nm, varying amounts of energy (10 to 170 J/cm2) were delivered to the presensitized (hematoporphyrin derivative) canine larynx. There appear to be four energy zones that result in progressive degrees of tissue response. Between 10 and 40 J/cm2 the larynx appears normal, with only mild erythema noted at 60 J/cm2. Above 100 J/cm2 moderate to severe erythema and edema were seen, with two upper-airway deaths observed. Transmission pattern analysis revealed that by either increasing the total energy applied and/or positioning the cylindrical diffuser tip at different levels of the larynx, the entire structure including the undersurface of the vocal cord and subglottic region could be exposed to red light.

Thermal effects of photodynamic therapy on the larynx. Experimental study.

Whether cell death in carcinoma of the larynx during photodynamic therapy is due to singlet oxygen formation or mild hyperthermia is not resolved. When various experimental tumors were previously heated within the range of 41 degrees C to 43 degrees C, selective destruction was found. A canine model was used by placing a thermocouple probe into the vocal cord and then varying the total energy (10 to 150 J/cm2), using a 630-nm dye laser; a temperature rise of only 0.5 degrees C to 2.1 degrees C was found. Thus, the thermal effects generated within the larynx (final temperature, 34.9 degrees C to 37.1 degrees C) should be insufficient to act alone in achieving a therapeutic response in either tumor tissue or destruction of normal epithelium.