fMRI and Sleep Correlates of the Age-Related Impairment in Motor Memory Consolidation

Behavioral studies indicate that older adults exhibit normal motor sequence learning (MSL), but paradoxically, show impaired consolidation of the new memory trace. However, the neural and physiological mechanisms underlying this impairment are entirely unknown. Here, we sought to identify, through functional magnetic resonance imaging during MSL and electroencephalographic (EEG) recordings during daytime sleep, the functional correlates and physiological characteristics of this age-related motor memory deficit. As predicted, older subjects did not exhibit sleep-dependent gains in performance (i.e., behavioral changes that reflect consolidation) and had reduced sleep spindles compared with young subjects. Brain imaging analyses also revealed that changes in activity across the retention interval in the putamen and related brain regions were associated with sleep spindles. This change in striatal activity was increased in young subjects, but reduced by comparison in older subjects. These findings suggest that the deficit in sleep-dependent motor memory consolidation in elderly individuals is related to a reduction in sleep spindle oscillations and to an associated decrease of activity in the cortico-striatal network.status: publishe

Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway

<div><p>Constitutive activation of the Rearranged during Transfection (RET) proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC). The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.</p></div

Components of an active RET signaling pathway are observed in MTC and correlate to DNA-PKcs.

<p>Immunohistochemistry of MTC array samples for phosphorylated ERK 1/2 and phosphorylated AKT indicated the presence of both of these components of an active RET signaling pathway in MTC similar to staining observed for ps2056 DNA-PKcs. 10x images of whole tissue array sample are shown.</p

List of proteins found to be significantly altered in RET 9 and RET 51 lines compared to control lines.

<p>Fold change ≤1.5 is considered increased expression and between 0.2 and 0.6 is decreased expression.</p><p>List of proteins found to be significantly altered in RET 9 and RET 51 lines compared to control lines.</p

Inhibition of RET reduces phosphorylation of DNA-PKcs s2056 and blocking DNA-PKcs activity increases chemosensitivity.

<p>A) TT cells were treated with RPI-1 (5 μM) to inhibit RET for 24 hrs prior to nuclei isolation. Western blot analysis indicated that RET inhibition reduces DNA-PKcs s2056 phosphorylation. Total DNA-PKcs and Lamin B1 were used as loading controls. B) TT cells treated with Nu7441 (NU) (1 μM) to inhibit DNA-PKcs had a greater decrease in cell viability than DOX alone. *p ≤ 0.05, error bars = s.d.</p

Phosphorylation of DNA-PKcs at s2056 is elevated in RET 9 and RET 51 cells.

<p>A) Western blot analysis shows phospho-s2056 (ps2056) (460 Kda) to be elevated in RET9 and RET 51 cell lysates. Total DNA-PKcs was used as loading control. B) Immunocytochemistry of cells plated in chamber slides and stained for ps2056 (red) and Dapi as counterstain (blue). C) ICC revealed a significant increase in ps2056 located in the nuclei of RET 9 and RET 51 cells compared to EV. * p ≤ 0.05, error bars = s.d.</p

DNA-PKcs ps2056 is present in human medullary thyroid cancer and correlates with RET signaling.

<p>Immunohistochemistry analysis of tissue microarrays containing normal and MTC samples for phospho-s2056 (ps2056) and phospho-RET (pRET). Each tissue sample was given a score of 0 (no signal), 1 (weak), 2 (moderate), 3 (high) for ps2056 levels. Images represent a score of 3. *chi test p ≤ 0.01 for normal vs tumor ps2056, ** p ≤ 0.01 for normal vs. tumor +ps2056 +RET.</p

Inhibition of DNA-PKcs reduces chemoresistance in RET 9 and RET 51 cells.

<p>EV, RET 9 and RET 51 cells were treated with the DNA-PKcs inhibitor Nu7441 (NU) alone or with doxorubicin (DOX). EV proliferation was set at 100 and the % of EV for 9 and 51 were plotted. NU increased sensitivity to DOX in both the RET 9 and 51 cells. *p ≤ 0.05, **p ≤ 0.01, error bars = s.d. B) Western blot analysis of the DNA-PKcs target gene AKT showed decreased AKT phosphorylation with NU treatment indicating inhibition of DNA-PKcs activity.</p

RET inhibition reduces phosphorylation of DNA-PKcs.

<p>EV, RET 9 and RET 51 cells were treated with the RET inhibitor RPI-1 for 24 hours before cells were harvested and analyzed by western blot. RPI-1 treatment significantly reduced phosphorylation of DNA-PKcs at site s2056 according to Image J analysis. GAPDH was used as loading control. **p ≤ 0.005, error bars = s.d.</p