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•We stimulated primary motor cortex (M1) in awake rhesus macaques using sp-TMS.•Recruitment curves (RC) were measured with a motor evoked potential (MEP) readout.•Traditional motor threshold (tradMT, at 100 µV) was near the RC inflection point.•A physiologically relevant motor response threshold was found at 90 % of the tradMT.•Plateau of the RC appears at smaller amplitudes in macaques compared to humans. Cortical excitability (CE) is commonly assessed via motor evoked potentials (MEPs) elicited by single-pulse transcranial magnetic stimulation (sp-TMS). While the motor threshold (MT) remains the most widely used measure of CE, it provides a limited, one-dimensional measure based on a fixed MEP amplitude criterion. In contrast, the recruitment curve (RC) offers a more comprehensive characterization of corticospinal recruitment dynamics. To date, the few available preclinical TMS studies measuring RC in non-human primates have been conducted under anaesthesia with limited translational relevance. Hence, we characterised CE in 20 sessions of 4 awake rhesus macaques by recording RCs at nine stimulation intensity levels and parametrising them using exponentiated sigmoid functions. The traditional 100 µV MEP MT criterion level (SI100µV) aligned most closely with the inflection point of the RC sigmoid fit and was consistent with relative frequency-based traditional MT (tradMT) measured in separate sessions. The onset of the logarithmic recruitment phase of the sigmoid (lower ankle point) was found at 0.9 × SI100µV/tradMT. Well-formed MEPs were measured below the SI100µV/tradMT, but not below the lower ankle point, which is a physiologically relevant response threshold. Thus, in rhesus macaques the 100-µV criterion may be suitable to approximate the RC inflection point, but not the physiological motor threshold. The overall RC shape was consistent with previous human data, however, plateau MEP amplitudes were substantially smaller than those reported in humans. These results lay the groundwork for the adaptation of TMS protocols and CE metrics to non-human primates that is necessary for translationally valid research.

Cortical excitability (CE) is commonly assessed by recording motor evoked potentials (MEPs) in response to single-pulse transcranial magnetic stimulation (sp-TMS). While the motor threshold (MT) remains the most widely used measure of CE, it provides a one-dimensional, criterion-based assessment. In contrast, the recruitment curve (RC) offers a more comprehensive characterization of the full dynamics of cortical recruitment. Yet, only a few preclinical studies involving translationally relevant non-human primates were conducted, and most were under anaesthesia. Hence, we aimed to characterise CE in awake rhesus macaques by recording traditionally defined MT and RCs. Traditional MT with a 100 µV MEP criterion (‘tradMT’) was measured in 8 awake adult male rhesus macaques using C-B65 coil and MagVenture stimulator. RCs were recorded at nine relative intensity levels (0.5 – 1.5 × tradMT) in 4 macaques. A sigmoid function was fitted to obtain key CE parameters: the inflection point, lower ankle point, and plateau. TradMT values were stable and replicable, and aligned most closely with the inflection point of the RC. The lower ankle points were found around at 0.9 × tradMT, marking the transition from a constant to a logarithmic phase, representing a physiologically relevant threshold. Plateau MEP amplitudes were substantially smaller compared to those reported in humans. Fitted RC parameters revealed a distinction between tradMT and the physiologically relevant threshold. The overall RC shape was consistent with human data, suggesting similar recruitment processes, leading to high translational validity. However, the marked difference in maximal MEP magnitude emphasises the importance of species-specific adaptations.

Background Remodeling of Ca 2+ signaling is an important step in cancer progression, and altered expression of members of the Ca 2+ signaling toolkit including the plasma membrane Ca 2+ ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors. Methods In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps. Results Three distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca 2+ pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca 2+ extrusion capacity in response to 17β-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein. Conclusions Our results suggest that the expression of Ca 2+ pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression.

Loss of epithelial cell polarity and tissue disorganization are hallmarks of carcinogenesis, in which Ca 2+ signaling plays a significant role. Here we demonstrate that the plasma membrane Ca 2+ pump PMCA4 (ATP2B4) is downregulated in luminal breast cancer, and this is associated with shorter relapse-free survival in patients with luminal A and B1 subtype tumors. Using the MCF-7 breast cancer cell model we show that PMCA4 silencing results in the loss of cell polarity while a forced increase in PMCA4b expression induces cell polarization and promotes lumen formation. We identify Arf6 as a regulator of PMCA4b endocytic recycling essential for PMCA4-mediated lumen formation. Silencing of the single pmca gene in Drosophila melanogaster larval salivary gland destroys lumen morphology suggesting a conserved role of PMCAs in lumen morphogenesis. Our findings point to a role of PMCA4 in controlling epithelial cell polarity, and in the maintenance of normal glandular tissue architecture. PMCA4 promotes epithelial polarization and lumen formation, while its loss induces partial EMT in luminal breast cancer cells. Its downregulation in LUMA/B1 breast cancer correlates with poor survival and may therefore serve as a prognostic marker.

Loss of epithelial cell polarity is a hallmark of carcinogenesis, in which Ca2+ signaling plays a role. Here we demonstrate that the plasma membrane Ca2+ pump PMCA4 is downregulated in luminal breast cancer, and this is associated with shorter relapse-free survival of patients with luminal A and B1 sub-type tumors. We find that in vitro silencing of PMCA4 in MCF-7 breast cancer cells induces internalization of E-cadherin and Dlg1 leading to a severe loss of cell polarity while re-expression of the b variant of PMCA4 restores cell polarity and promotes lumen formation mediated by the Arf6-trafficking pathway. We also demonstrate via an in vivo model using Drosophila melanogaster that silencing of the single pmca gene destroys lumen morphology in the larval salivary gland suggesting a conserved role of PMCAs in lumen morphogenesis. Additionally, enhanced secretory vesicle accumulation in PMCA(4)-deficient cells indicates a profound secretion defect both in vitro and in vivo. Our findings point to a novel role of PMCA4 in restoring epithelial cell polarity, and maintaining normal glandular tissue architecture.