ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

Modeling Parkinson's disease-related symptoms in alpha-synuclein overexpressing mice.

BACKGROUND: Intracellular deposition of alpha-synuclein (α-syn) as Lewy bodies and Lewy neurites is a central event in the pathogenesis of Parkinson's disease (PD) and other α-synucleinopathies. Transgenic mouse models overexpressing human α-syn, are useful research tools in preclinical studies of pathogenetic mechanisms. Such mice develop α-syn inclusions as well as neurodegeneration with a topographical distribution that varies depending on the choice of promoter and which form of α-syn that is overexpressed. Moreover, they display motor symptoms and cognitive disturbances that to some extent resemble the human conditions. PURPOSE: One of the main motives for assessing behavior in these mouse models is to evaluate the potential of new treatment strategies, including their impact on motor and cognitive symptoms. However, due to a high within-group variability with respect to such features, the behavioral studies need to be applied with caution. In this review, we discuss how to make appropriate choices in the experimental design and which tests that are most suitable for the evaluation of PD-related symptoms in such studies. METHODS: We have evaluated published results on two selected transgenic mouse models overexpressing wild type (L61) and mutated (A30P) α-syn in the context of their validity and utility for different types of behavioral studies. CONCLUSIONS: By applying appropriate behavioral tests, α-syn transgenic mouse models provide an appropriate experimental platform for studies of symptoms related to PD and other α-synucleinopathies.

Age-related increase of alpha-synuclein oligomers is associated with motor disturbances in L61 transgenic mice.

The pathogenesis of Parkinson's disease involves fibrillization and deposition of alpha-synuclein (α-syn) into Lewy bodies. Accumulating evidence suggests that α-syn oligomers are particularly neurotoxic. Transgenic (tg) mice overexpressing wild-type human α-syn under the Thy-1 promoter (L61) reproduce many Parkinson's disease features, but the pathogenetic relevance of α-syn oligomers in this mouse model has not been studied in detail. Here, we report an age progressive increase of α-syn oligomers in the brain of L61 tg mice. Interestingly, more profound motor symptoms were observed in animals with higher levels of membrane-bound oligomers. As this tg model is X-linked, we also performed subset analyses, indicating that both sexes display a similar age-related increase in α-syn oligomers. However, compared with females, males featured increased brain levels of oligomers from an earlier age, in addition to a more severe behavioral phenotype with hyperactivity and thigmotaxis in the open field test. Taken together, our data indicate that α-syn oligomers are central to the development of brain pathology and behavioral deficits in the L61 tg α-syn mouse model.

Aged Opossums Show Alterations in Spatial Learning Behavior and Reduced Neurogenesis in the Dentate Gyrus.

In many mammalian species including opossums, adult neurogenesis, the function of which is not completely understood, declines with aging. Aging also causes impairment of cognition. To understand whether new neurons contribute to learning and memory, we performed experiments on young and aged laboratory opossums, Monodelphis domestica, and examined the association between spatial memory using the Morris water maze test and the rate of adult neurogenesis in the dentate gyrus (DG). Modification of this test allowed us to assess how both young and aged opossums learn and remember the location of the platform in the water maze. We found that both young and aged opossums were motivated to perform this task. However, aged opossums needed more time to achieve the test than young opossums. Classical parameters measuring spatial learning in a water maze during a probe test showed that young opossums spent more time in the platform zone crossing it more often than aged opossums. Additionally, hippocampal neurogenesis was lower in the aged opossums than in the young animals but new neurons were still generated in the DG of aged opossums. Our data revealed individual differences in the levels of doublecortin in relation to memory performance across aged opossums. These differences were correlated with distinct behaviors, particularly, aged opossums with high levels of DCX achieved high performance levels in the water maze task. We, therefore suggest that new neurons in the DG of Monodelphis opossums contribute to learning and memory.

Stress-Dependent Changes in the CacyBP/SIP Interacting Protein S100A6 in the Mouse Brain.

The CacyBP/SIP target S100A6 is widely present in the nervous system, and its up-regulation is associated with certain neurodegenerative diseases. Here, we examined the involvement of S100A6 protein in stress responses in mice. Using Western blotting, we observed a marked change in brainstem structures, whereby stressed mice showed approximately one-third the protein level produced in the control group. A decreased level of S100A6 protein in stressed animals was also detected in the olfactory bulb and the cerebellum and stress-related structures such as the hippocampus and the hypothalamus. Additionally, using immunohistochemistry, high levels of S100A6 expression were observed in astrocytes localized in the border zones of all brain ventricles, tanycytes of the ventro-lateral walls of the hypothalamus, including the arcuate nucleus (ARH) and low levels of this protein were in neurons of the olfactory bulb, the hippocampus, the thalamus, the cerebral cortex, the brainstem and the cerebellum. Although S100A6-expressing cells in all these brain structures did not change their phenotype in response to stress, the intensity of immunofluorescent labeling in all studied structures was lower in stressed mice than in control animals. For example, in the ARH, where extremely strong immunostaining was observed, the number of immunolabeled fibers was decreased by approximately half in the stressed group compared with the controls. Although these results are descriptive and do not give clue about functional role of S100A6 in stress, they indicate that the level of S100A6 decreases in several brain structures in response to chronic mild stress, suggesting that this protein may modify stress responses.

Distribution and function of TrkB receptors in the developing brain of the opossum Monodelphis domestica.

The expression, development pattern, spatiotemporal distribution, and function of TrkB receptors were investigated during the postnatal brain development of the opossum. Full-length TrkB receptor expression was detectable in the newborn opossum, whereas three different short forms that are expressed in the adult brain were almost undetectable in the newborn opossum brain. The highest level of full-length TrkB receptor expression was observed at P35, which corresponds to the time of eye opening. We found that in different brain structures, TrkB receptors were localized in various compartments of cells. The hypothalamus was distinguished by the presence of TrkB receptors not only in cell bodies but also in the neuropil. Double immunofluroscent staining for TrkB and a marker for the identification of the cell phenotype in several brain regions such as the olfactory bulb, hippocampus, thalamus, and cerebellum showed that unlike in eutherians, in the opossum, TrkB receptors were predominantly expressed in neurons. A lack of TrkB receptors in glial cells, particularly astrocytes and oligodendrocytes, provides evidence that TrkB receptors can play a functionally different role in marsupials than in eutherians. The effects of TrkB signaling on the development of cortical progenitor cells were examined in vitro using shRNAs. Blockade of the endogenous TrkB receptor expression induced a decrease in the number of progenitor cells proliferation, whereas the number of apoptotic progenitor cells increased. These changes were statistically significant but relatively small. In contrast, TrkB signaling was strongly involved in regulation of the cortical progenitor cell differentiation process.

Interleukin 6 deficiency affects spontaneous activity of mice in age- and sex-dependent manner.

We analyzed the role of interleukin 6 (IL-6) in modulation of the pattern of mice spontaneous activity. Wild type (WT) and IL-6 deficient mice of both sexes, young and aging, were housed individually and various types of their activity were recorded and analyzed with the Phenorack system in their home cages during 72 hours-long sessions. All investigated groups of mice were active mainly during the dark phase of the 24-hours cycle. Generally, the IL-6 deficient animals were more active than their WT controls and females of both genotypes more active than males. Aging mice were less active than the sex and genotype-matched young animals. The independent variables (age, sex and genotype) strongly interacted, which suggests that the modulatory influence of IL-6 on mice behavior may be different in males and females and that it changes during aging. We conclude that under normal physiological conditions signaling of IL-6 via its receptor participates in modulation of the basic pattern of activity. This modulation differs in males and females and changes with aging.