Deletion of the BDNF Truncated Receptor TrkB.T1 Delays Disease Onset in a Mouse Model of Amyotrophic Lateral Sclerosis

Brain Derived Neurotrophic Factor (BDNF) exerts strong pro-survival effects on developing and injured motoneurons. However, in clinical trials, BDNF has failed to benefit patients with amyotrophic lateral sclerosis (ALS). To date, the cause of this failure remains unclear. Motoneurons express the TrkB kinase receptor but also high levels of the truncated TrkB.T1 receptor isoform. Thus, we investigated whether the presence of this receptor may affect the response of diseased motoneurons to endogenous BDNF. We deleted TrkB.T1 in the hSOD1G93A ALS mouse model and evaluated the impact of this mutation on motoneuron death, muscle weakness and disease progression. We found that TrkB.T1 deletion significantly slowed the onset of motor neuron degeneration. Moreover, it delayed the development of muscle weakness by 33 days. Although the life span of the animals was not affected we observed an overall improvement in the neurological score at the late stage of the disease. To investigate the effectiveness of strategies aimed at bypassing the TrkB.T1 limit to BDNF signaling we treated SOD1 mutant mice with the adenosine A2A receptor agonist CGS21680, which can activate motoneuron TrkB receptor signaling independent of neurotrophins. We found that CGS21680 treatment slowed the onset of motor neuron degeneration and muscle weakness similarly to TrkB.T1 removal. Together, our data provide evidence that endogenous TrkB.T1 limits motoneuron responsiveness to BDNF in vivo and suggest that new strategies such as Trk receptor transactivation may be used for therapeutic intervention in ALS or other neurodegenerative disorders

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

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 Scaffold Protein Cybr Is Required for Cytokine-Modulated Trafficking of Leukocytes In Vivo

Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions

Chemotherapy-induced bone marrow nerve injury impairs hematopoietic regeneration

Anti-cancer chemotherapy drugs challenge hematopoietic tissues to regenerate, but commonly produce long-term sequelae. Deficits in hematopoietic stem or stromal cell function have been described, but the mechanisms mediating chemotherapy-induced hematopoietic dysfunction remain unclear. Administration of multiple cycles of cisplatin chemotherapy causes significant sensory neuropathy. Here, we demonstrate that chemotherapy-induced nerve injury in the bone marrow is a critical lesion impairing hematopoietic regeneration. We show using various pharmacological and genetic models that the selective loss of adrenergic innervation in the BM alters its regeneration following genotoxic insult. Sympathetic nerves in the marrow promote the survival of stem cell niche constituents that initiate recovery. Neuroprotection by deletion of Trp53 in sympathetic neurons or neuro-regeneration using 4-methylcatechol or glial-derived neurotrophic factor (GDNF) administration can restore hematopoietic recovery. Thus, these results shed light on the potential benefit of adrenergic nerve protection to shield hematopoietic niches from injury