Anti-Amyloid Effects of Small Molecule Aß-Binding Agents in PS1/APP Mice.

AIMS: One promising approach for treatment of Alzheimer's disease (AD) is use of anti-amyloid therapies, based on the hypothesis that increases in amyloid-beta (Aß) deposits in brain are a major cause of AD. Several groups have focused on Aß immunotherapy with some success. Small molecules derivatives of Congo red have been shown to inhibit Aß aggregation and protect against Aß neurotoxicity in vitro. The agents described here are all small molecule Aß-binding agents (SMAßBA's) derivatives of Congo red. MAIN METHODS: Here, we have explored the anti-amyloid properties of these SMAßBA's in mice doubly transgenic for human prensenilin-1 (PS1) and APP gene mutations that cause early-onset AD. Mice were treated with either methoxy-X04, X:EE:B34 and X:034-3-OMe1. After treatment, brains were examined for Aß-deposition, using histochemistry, and soluble and insoluble Aß levels were determined using ELISA. KEY FINDINGS: A range of anti-amyloid activity was observed with these three compounds. PS1/APP mice treated with methoxy-X04 and X:EE:B34 showed decrease in total Aß load, a decrease in Aß fibril load, and a decrease in average plaque size. Treatment with methoxy-X04 also resulted in a decrease in insoluble Aß levels. The structurally similar compound, X:034:3-OMe1, showed no significant effect on any of these measures. The effectiveness of the SMAßBA's may be related to a combination of binding affinity for Aß and entry into brain, but other factors appear to apply as well. SIGNIFICANCE: These data suggest that SMAßBA's may significantly decrease amyloid burden in brain during the pathogenesis of AD and could be useful therapeutics alone, or in combination with immunotherapy.

Chronic dizocilpine (MK-801) reversibly delays GABA(A) receptor maturation in cerebellar granule neurons in vitro.

We investigated the effect of chronically blocking NMDA receptor stimulation to examine changes in GABA(A) receptor expression and pharmacology in cerebellar granule cells at different stages of maturation. We have previously shown that NMDA-selective glutamate receptor stimulation alters GABA(A) receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunits. When NMDA receptor stimulation is blocked with MK-801 during the first week in vitro, a decrease in the alpha1, gamma2S, and gamma2L receptor subunit mRNAs occurred. When present only during the second week, changes were limited to the alpha1 and gamma2L mRNAs. Finally, if MK-801 was present during the first week and removed during the second week, these changes reversed. Whole-cell voltage-clamp recordings showed that treatment with MK-801 during either the first or second week increased the EC50 of the receptors for GABA and attenuated the potentiation mediated by flunitrazepam. Last, these properties were reversed if MK-801 was removed after the first week in vitro. Our results suggest that MK-801 reversibly inhibits GABA(A) receptor maturation by modulating receptor subunit expression and that the altered pharmacological responses appear to be dominated by changes in the levels of allosteric modulation mediated by the gamma2 receptor subunit.

Hypothermia-enhanced human tumor cell radiosensitivity.

Ablation of neoplastic disease by freezing has found increasing utility as a potential therapeutic modality. To assess the effect of cooling temperatures on cellular radiation response, an established human cervical carcinoma cell line (HTB35) was subjected to holding temperatures of 0, 5, or 15 degrees C for up to 24 h before irradiation. Survival was measured by in vitro clonogenic assay of colonies containing at least 50 cells. Cooling for up to 12 h did not significantly decrease survival, but after 24 h survival fell to 75% of control cultures grown at 37 degrees C. X-irradiation immediately after cooling for 24 h resulted in 1.6-fold enhanced radiosensitivity. However, the radiosensitizing effect decayed rapidly if the cooled cells were returned to normal growth temperature for 6 h or longer before irradiation and subculture. Both temperature and cooling duration influenced the radiation response. With 0, 5, or 15 degrees C, radiosensitivity increased after 3, 6, or 12 h, respectively, and progressively rose with up to 24 h of cooling. By flow cytometric analysis, no statistically significant difference was observed in the S-phase fraction between control cells and those cooled to 0 degree C for 24 h. These data demonstrate cooling-enhanced in vitro radiation sensitivity which is dependent upon cooling temperature, duration, and rewarming interval before irradiation. While cell cycle redistribution does not appear to be a factor in the increased radiosensitivity, differences in the radiation survival curves between cooled versus normothermic cells suggest that diminished capacity for sublethal damage repair may be a significant influence on the changes which were observed.

Radiation survival of two human cervical carcinoma cell lines after multifraction irradiation.

PURPOSE: Multifraction irradiation may contribute to radiation therapy treatment failure if selection of radiation resistant subpopulations occurs. We sought to determine whether surviving cells following daily fraction irradiation of two human cervical squamous cell carcinoma lines would express different radiation survival characteristics compared to the unirradiated parent. METHODS AND MATERIALS: A late-passage line (HTB35) and an early-passage line (RECA) received daily 2 Gy x-irradiation. Two new stable HTB35 cell lines were established after 40 and 60 Gy (HTB35-40 and HTB35-60). A single line was established from RECA after 30 Gy (RECA-30). High dose rate (74 cGy/min) acute radiation survival curves were prepared from the three new lines and the unirradiated parents. Potentially lethal damage repair (PLDR) and sublethal damage repair (SLDR) responses were detailed for HTB35, HTB35-40 and HTB35-60. Low dose rate (1.27 cGy/min) survival was measured for HTB35 and HTB35-60. Clones were derived from HTB35 and from HTB35-60 and the surviving fraction at 2 Gy (SF2) values were determined. RESULTS: The two parent lines (HTB35 and RECA) differed in acute radiation survival. The surviving lines following multifraction irradiation (HTB35-40, HTB35-60, and RECA-30) showed no change in acute radiation response compared to the appropriate parent. HTB35-40 and HTB35-60 were repair proficient, demonstrating similar PLDR and SLDR recovery ratios as the parent. Likewise, acute, low dose rate survival of HTB35 and HTB35-60 was similar. Nine clones derived from HTB35 lacked a consistent difference in SF2 compared to the original culture. A single clone of seven derived from HTB35-60 was consistently radiation resistant (SF2 = 0.81 +/- 0.06) compared to the original culture (SF2 = 0.50 +/- 0.09). CONCLUSION: No evidence was obtained that cell lines generated following multiple daily fractions of x-irradiation in vitro possessed acute radiation survival or repair characteristics that were different from the unirradiated parent.

Thyroid hormone receptor messenger ribonucleic acid in human granulosa and ovarian stromal cells.

OBJECTIVE: To examine whether mRNA for thyroid hormone receptors alpha and beta is present in human granulosa cells in nonstimulated ovaries. DESIGN: Paraffin-embedded sections of ovaries from normally cycling women were analyzed by in situ hybridization with oligonucleotide probes for thyroid hormone receptors alpha and beta. The sense strand oligonucleotide was used as a control for each of the probes. RESULTS: Granulosa cells from the preovulatory antral follicles examined showed positive staining for both the thyroid hormone receptor alpha and beta probes. Positive staining of ovarian stromal cells also was observed for both probes. CONCLUSION: Thyroid hormone receptor mRNAs are expressed in both granulosa cells and ovarian stromal cells found in nonstimulated ovaries. It is, therefore, conceivable that thyroid hormone may play a direct role in human ovarian physiology.