Effect of TrkB.T1 deletion on motoneuron degeneration.

<p>(A) Representative immunofluorescent images of lumbar spinal cord showing ChAT-positive motoneurons in 8-week-old animals. In comparison to age-matched WT controls, SOD1 transgenic mice show a small but significant loss of motoneurons. This loss was prevented by deletion of TrkB.T1 in these mice (SOD1T1−/−). (B) Histogram showing number of motor neurons at 8, 12, 16 and 20 weeks. Cell counts show a progressive decrease in number of motoneurons in SOD1 animals as compared to WT controls. The deletion of TrkB.T1 in SOD1 transgenic mice completely rescues this loss at 8 and 12 weeks. At 16 weeks this neuroprotection is partial and significant whereas at 20 weeks it is completely lost such that both the SOD1 and SOD1T1−/− groups show severe reduction in motoneuron numbers as compared to WT animals. The data are the Mean ± SEM. * indicates P<0.05. N = 6 in each group. Statistical analysis by ANOVA followed by post-hoc Tukey test.</p

The adenosine A2A receptor agonist CGS21680 delays disease onset in SOD1 mutant mice.

<p>CGS treatment at 12 weeks of age significantly rescues motor neuron degeneration in SOD1 mice (A; p<0.05). Analysis of rotarod data showing that treatment of SOD1 mice with CGS delays the impairment in rotarod performance by 7 days compared to untreated SOD1 mice (B; Kaplan Meier Analysis, p<0.01). CGS treatment also prolongs the duration of the early phase of the disease by 12 days (C; p<0.05) although it does not affect the mean life span of the SOD1 mutant animals (D).</p

Deletion of TrkB.T1 improves the motor performance of SOD1 transgenic mice on rotarod in the early phase of the disease.

<p>(A) Histogram showing the rotarod performance of WT, SOD1 and SOD1T1−/− animals at 8, 12, 16 and 20 weeks. At 8 weeks, both SOD1 and SOD1 T1−/− animals perform similarly to controls although SOD1 mice show some impairments. With disease progression, the SOD1 transgenic mice show a significant reduction in the amount of time spent on the rotarod at 12 and 16 weeks as compared to controls. A slight loss of motor performance is evident only at 16 weeks in the SOD1T1 −/− animals, although by 20 weeks the SOD1 and SOD1;T1−/− groups are indistinguishable. The data are the Mean ± SEM. * P<0.05. N≥7. Statistical analysis by ANOVA followed by post-hoc Tukey test. (B) Kaplan-Meier analysis of the SOD1 and SOD1T1−/− mice rotarod performance in relation to their age. Note that deletion of TrkB.T1 in the SOD1 transgenic mice delays the impairment in rotarod performance by 34 days (81.6±5.87 days, n = 10 in SOD1 versus 115.8±6.80, n = 7 in SOD1 T1−/−, p<0.05).</p

Exon 1 Disruption Alters Tissue-Specific Expression of Mouse p53 and Results in Selective Development of B Cell Lymphomas

<div><p>p53 is a tumor suppressor gene mutated in >50% of human cancers, while p53 deficiency in mice results in cancers and accelerated mortality. Thymic T cell lymphoma is the most common malignancy in p53-deficient mice, making it difficult to study the role of p53 in other malignancies. To overcome this limitation, we attempted to generate mice with a reversible p53 knockout (p53^rev/rev) by inserting a floxed transcriptional stop into the first exon of p53, anticipating that this would allow tissue-specific Cre-mediated expression of p53. Contrary to expectations, functional p53 protein was expressed in the thymus and multiple other tissues of p53^rev/rev mice in the absence of Cre, whereas B cells expressed p53 protein only in the presence of B cell-specific CD19-Cre. In the absence of Cre, 76% of p53^rev/rev mice developed splenic marginal zone B cell lymphomas, indicating sensitivity of this B cell subset to transformation caused by p53 deficiency. 5′-RACE identified p53 mRNA transcribed from a novel start site utilized in thymocytes but not normal B cells or B cell lymphomas from p53^rev/rev mice. The p53^rev/rev mouse thus demonstrates an effect of p53 deficiency in development of splenic marginal zone lymphomas and provides a model for study of p53-deficient human B cell lymphomas.</p> </div

Generation of p53^rev/rev mice.

<p>A) Gene targeting strategy and restriction map of the p53 gene. Filled boxes indicate exons; labeled boxes indicate neomycin resistance or herpesvirus thymidine kinase (tk) genes; and arrows indicate loxP sites. B) Southern blot analysis of ES cell DNA. The 5 kb band represents the germ line allele and 7 kb band represents the targeted allele after Kpn I digestion and hybridization with probe A. C) PCR analysis for the p53^rev/rev mouse genotype. The 300 and 500-bp PCR products represent the wt and gene-targeted alleles, respectively.</p

B cell lymphomas developed in p53^rev/rev mice.

<p>A) Survival of p53^rev/rev and control (p53^+/rev and p53^+/+) mice. B) Pie chart shows percentages of cause of death for p53^rev/rev mice. C, E, G, I) Structure of splenic marginal zone for normal B6, normal p53^rev/rev, and two individual tumor-bearing p53^rev/rev mice, respectively (H&E, 10×). D, F, H, J) High magnification of red pulp of and marginal zone for the spleen of a normal B6 mouse, normal p53^rev/rev, and lymphoma-affected spleens from two different p53^rev/rev mice, respectively (H&E, 63×).</p

B cell lymphomas do not develop in p53^rev/revCD19-Cre⁺ mice.

<p>A) The B cells of p53^rev/revCD19-Cre⁺ and p53^rev/revCD19-Cre⁻ mice were isolated from splenocytes with MACS systems. The purified B cells were irradiated and cultured for three hours at 37°C. The lysates of B cells were immunoblotted with anti-p53 antibodies. B) B cell lymphoma incidence was determined in p53^rev/revCD19-Cre⁺ and p53^rev/revCD19-Cre⁻ mice followed for 9 months.</p

Expression of p53 in lymphocytes of p53^rev/rev mice.

<p>A) Western blot analysis was used to determine p53 protein expression in thymocytes of wt and p53^rev/rev mice as indicated. Actin protein expression was used as a loading control. The results shown are representative of four independent experiments. B) Western blot analysis was used to determine p53 protein expression in spleen cells of wt and p53^rev/rev mice as indicated. Actin protein expression was used as a loading control. The results shown are representative of four independent experiments. C) Western blot analysis was used to determine p53 protein expression in total spleen cells (SP), purified splenic B cells (B) and purified splenic T cells (T) of p53^+/+ and p53^rev/rev mice with (IR) or without (C) irradiation as indicated. Actin protein expression was used as a loading control. The results shown are representative of two independent experiments.</p

Apoptosis of thymocytes and splenocytes in response to irradiation.

<p>A) Apoptosis of littermate p53^−/− and p53^+/+ thymocytes after irradiation and overnight culture. The results shown are representative of three independent experiments. B) Apoptosis of littermate p53^rev/rev and wt thymocytes after irradiation and overnight culture. The results shown are representative of three independent experiments. C) Apoptosis of littermate p53^−/− and p53^+/+ spleen cells after irradiation and overnight culture. The results shown are representative of two independent experiments. D) Apoptosis of littermate p53^rev/rev and wt spleen cells after irradiation and overnight culture. The results shown are representative of two independent experiments.</p

SYK analysis of p53^rev/rev tumors.

<p>SYK analysis of p53^rev/rev tumors.</p