Palmitic and Stearic Acids Inhibit Chaperone-Mediated Autophagy (CMA) in POMC-like Neurons In Vitro.

The intake of food with high levels of saturated fatty acids (SatFAs) is associated with the development of obesity and insulin resistance. SatFAs, such as palmitic (PA) and stearic (SA) acids, have been shown to accumulate in the hypothalamus, causing several pathological consequences. Autophagy is a lysosomal-degrading pathway that can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Previous studies showed that PA impairs macroautophagy function and insulin response in hypothalamic proopiomelanocortin (POMC) neurons. Here, we show in vitro that the exposure of POMC neurons to PA or SA also inhibits CMA, possibly by decreasing the total and lysosomal LAMP2A protein levels. Proteomics of lysosomes from PA- and SA-treated cells showed that the inhibition of CMA could impact vesicle formation and trafficking, mitochondrial components, and insulin response, among others. Finally, we show that CMA activity is important for regulating the insulin response in POMC hypothalamic neurons. These in vitro results demonstrate that CMA is inhibited by PA and SA in POMC-like neurons, giving an overview of the CMA-dependent cellular pathways that could be affected by such inhibition and opening a door for in vivo studies of CMA in the context of the hypothalamus and obesity.

Chaperone mediated autophagy contributes to the newly synthesized histones H3 and H4 quality control.

Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis.

Connexin-46 Contained in Extracellular Vesicles Enhance Malignancy Features in Breast Cancer Cells.

Under normal conditions, almost all cell types communicate with their neighboring cells through gap junction channels (GJC), facilitating cellular and tissue homeostasis. A GJC is formed by the interaction of two hemichannels; each one of these hemichannels in turn is formed by six subunits of transmembrane proteins called connexins (Cx). For many years, it was believed that the loss of GJC-mediated intercellular communication was a hallmark in cancer development. However, nowadays this paradigm is changing. The connexin 46 (Cx46), which is almost exclusively expressed in the eye lens, is upregulated in human breast cancer, and is correlated with tumor growth in a Xenograft mouse model. On the other hand, extracellular vesicles (EVs) have an important role in long-distance communication under physiological conditions. In the last decade, EVs also have been recognized as key players in cancer aggressiveness. The aim of this work was to explore the involvement of Cx46 in EV-mediated intercellular communication. Here, we demonstrated for the first time, that Cx46 is contained in EVs released from breast cancer cells overexpressing Cx46 (EVs-Cx46). This EV-Cx46 facilitates the interaction between EVs and the recipient cell resulting in an increase in their migration and invasion properties. Our results suggest that EV-Cx46 could be a marker of cancer malignancy and open the possibility to consider Cx46 as a new therapeutic target in cancer treatment.

Chaperone Mediated Autophagy Degrades TDP-43 Protein and Is Affected by TDP-43 Aggregation.

TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver's CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain's CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.

Temporal pattern of neuronal insulin release during Caenorhabditis elegans aging: Role of redox homeostasis.

The insulin-IGF-1/DAF-2 pathway has a central role in the determination of aging and longevity in Caenorhabditis elegans and other organisms. In this paper, we measured neuronal insulin secretion (using INS-22::Venus) during C. elegans lifespan and monitored how this secretion is modified by redox homeostasis. We showed that INS-22::Venus secretion fluctuates during the organism lifetime reaching maximum levels in the active reproductive stage. We also demonstrate that long-lived daf-2 insulin receptor mutants show remarkable low levels of INS-22::Venus secretion. In contrast, we found that short-lived mutant worms that lack the oxidation repair enzyme MSRA-1 show increased levels of INS-22::Venus secretion, specifically during the reproductive stage. MSRA-1 is a target of the insulin-IGF-1/DAF-2 pathway, and the expression of this antioxidant enzyme exclusively in the nervous system rescues the mutant insulin release phenotype and longevity. The msra-1 mutant phenotype can also be reverted by antioxidant treatment during the active reproductive stage. We showed for the first time that there is a pattern of neuronal insulin release with a noticeable increment during the peak of reproduction. Our results suggest that redox homeostasis can modulate longevity through the regulation of insulin secretion, and that the insulin-IGF-1/DAF-2 pathway could be regulated, at least in part, by a feedback loop. These findings highlight the importance of timing for therapeutic interventions aimed at improving health span.

Chaperone Mediated Autophagy in the Crosstalk of Neurodegenerative Diseases and Metabolic Disorders.

Chaperone Mediated Autophagy (CMA) is a lysosomal-dependent protein degradation pathway. At least 30% of cytosolic proteins can be degraded by this process. The two major protein players of CMA are LAMP-2A and HSC70. While LAMP-2A works as a receptor for protein substrates at the lysosomal membrane, HSC70 specifically binds protein targets and takes them for CMA degradation. Because of the broad spectrum of proteins able to be degraded by CMA, this pathway has been involved in physiological and pathological processes such as lipid and carbohydrate metabolism, and neurodegenerative diseases, respectively. Both, CMA, and the mentioned processes, are affected by aging and by inadequate nutritional habits such as a high fat diet or a high carbohydrate diet. Little is known regarding about CMA, which is considered a common regulation factor that links metabolism with neurodegenerative disorders. This review summarizes what is known about CMA, focusing on its molecular mechanism, its role in protein, lipid and carbohydrate metabolism. In addition, the review will discuss how CMA could be linked to protein, lipids and carbohydrate metabolism within neurodegenerative diseases. Furthermore, it will be discussed how aging and inadequate nutritional habits can have an impact on both CMA activity and neurodegenerative disorders.

PP32 and SET/TAF-Iß proteins regulate the acetylation of newly synthesized histone H4.

Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iß. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iß protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iß depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iß and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.

Design, synthesis and optimization of bis-amide derivatives as CSF1R inhibitors.

Signaling via the receptor tyrosine kinase CSF1R is thought to play an important role in recruitment and differentiation of tumor-associated macrophages (TAMs). TAMs play pro-tumorigenic roles, including the suppression of anti-tumor immune response, promotion of angiogenesis and tumor cell metastasis. Because of the role of this signaling pathway in the tumor microenvironment, several small molecule CSF1R kinase inhibitors are undergoing clinical evaluation for cancer therapy, either as a single agent or in combination with other cancer therapies, including immune checkpoint inhibitors. Herein we describe our lead optimization effort that resulted in the identification of a potent, cellular active and orally bioavailable bis-amide CSF1R inhibitor. Docking and biochemical analysis allowed the removal of a metabolically labile and poorly permeable methyl piperazine group from an early lead compound. Optimization led to improved metabolic stability and Caco2 permeability, which in turn resulted in good oral bioavailability in mice.

Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production.

Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons.

The ROR2 tyrosine kinase receptor regulates dendritic spine morphogenesis in hippocampal neurons.

Wnt signaling regulates synaptic development and function and contributes to the fine-tuning of the molecular and morphological differentiation of synapses. We have shown previously that Wnt5a activates non-canonical Wnt signaling to stimulate postsynaptic differentiation in excitatory hippocampal neurons promoting the clustering of the postsynaptic scaffold protein PSD-95 and the development of dendritic spines. At least three different kinds of Wnt receptors have been associated with Wnt5a signaling: seven trans-membrane Frizzled receptors and the tyrosine kinase receptors Ryk and ROR2. We report here that ROR2 is distributed in the dendrites of hippocampal neurons in close proximity to synaptic contacts and it is contained in dendritic spine protrusions. We demonstrate that ROR2 is necessary to maintain dendritic spine number and morphological distribution in cultured hippocampal neurons. ROR2 overexpression increased dendritic spine growth without affecting the density of dendritic spine protrusions in a form dependent on its extracellular Wnt binding cysteine rich domain (CRD) and kinase domain. Overexpression of dominant negative ROR2 lacking the extracellular CRD decreased spine density and the proportion of mushroom like spines, while ROR2 lacking the C-terminal and active kinase domains only affected spine morphology. Our results indicate a crucial role of the ROR2 in the formation and maturation of the postsynaptic dendritic spines in hippocampal neurons.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer.

BACKGROUND: Enzalutamide (formerly MDV3100 and available commercially as Xtandi), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: binding of androgens to AR, AR nuclear translocation, and association of AR with DNA. Here, we investigate the effects of enzalutamide on AR signaling, AR-dependent gene expression and cell apoptosis. METHODS: The expression of AR target gene prostate-specific antigen (PSA) was measured in LnCaP and C4-2 cells. AR nuclear translocation was assessed in HEK-293 cells stably transfected with AR-yellow fluorescent protein. The in vivo effects of enzalutamide were determined in a mouse xenograft model of CRPC. Differential gene expression in LNCaP cells was measured using Affymetrix human genome microarray technology. RESULTS: We found that unlike bicalutamide, enzalutamide lacked AR agonistic activity at effective doses and did not induce PSA expression or AR nuclear translocation. Additionally, it is more effective than bicalutamide at inhibiting agonist-induced AR nuclear translocation. Enzalutamide induced the regression of tumor volume in a CRPC xenograft model and apoptosis in AR-over-expressing prostate cancer cells. Finally, gene expression profiling in LNCaP cells indicated that enzalutamide opposes agonist-induced changes in genes involved in processes such as cell adhesion, angiogenesis, and apoptosis. CONCLUSIONS: These results indicate that enzalutamide efficiently inhibits AR signaling, and we suggest that its lack of AR agonist activity may be important for these effects.

Wingless-type family member 5A (Wnt-5a) stimulates synaptic differentiation and function of glutamatergic synapses.

Growing evidence indicates that Wingless-type (Wnt) signaling plays an important role in the maturation of the central nervous system. We report here that Wingless-type family member 5A (Wnt-5a) is expressed early in development and stimulates dendrite spine morphogenesis, inducing de novo formation of spines and increasing the size of the preexisting ones in hippocampal neurons. Wnt-5a increased intracellular calcium concentration in dendritic processes and the amplitude of NMDA spontaneous miniature currents. Acute application of Wnt-5a increased the amplitude of field excitatory postsynaptic potentials (fEPSP) in hippocampal slices, an effect that was prevented by calcium-channel blockers. The physiological relevance of our findings is supported by studies showing that Wnt scavengers decreased spine density, miniature excitatory postsynaptic currents, and fEPSP amplitude. We conclude that Wnt-5a stimulates different aspects of synaptic differentiation and plasticity in the mammalian central nervous system.

Cognition-enhancing properties of Dimebon in a rat novel object recognition task are unlikely to be associated with acetylcholinesterase inhibition or N-methyl-D-aspartate receptor antagonism.

Dimebon (dimebolin) treatment enhances cognition in patients with Alzheimer's disease (AD) or Huntington's disease. Although Dimebon was originally thought to improve cognition and memory through inhibition of acetylcholinesterase (AChE) and the N-methyl-d-aspartate (NMDA) receptor, the low in vitro affinity for these targets suggests that these mechanisms may not contribute to its clinical effects. To test this hypothesis, we assessed whether Dimebon enhances cognition in rats and if such an action is related to either mechanism or additional candidate mechanisms. Acute oral administration of Dimebon to rats (0.05, 0.5, and 5 mg/kg) enhanced cognition in a novel object recognition task and produced Dimebon brain concentrations of 1.7 +/- 0.43, 14 +/- 5.1, and 172 +/- 94 nM, respectively. At these concentrations, Dimebon did not alter the activity of recombinant human or rat brain AChE. Unlike the AChE inhibitors donepezil and galantamine, Dimebon did not change acetylcholine levels in the hippocampus or prefrontal cortex of freely moving rats. Dimebon displays affinity for the NMDA receptor (K(i) = 105 +/- 18 microM) that is considerably higher than brain concentrations associated with cognition enhancement in the novel object recognition task and 200-fold weaker than that of memantine (K(i) = 0.54 +/- 0.05 microM). Dimebon did not block NMDA-induced calcium influx in primary neuronal cells (IC(50) > 50 microM), consistent with a lack of significant effect on this pathway. The cognition-enhancing effects of Dimebon are unlikely to be mediated by AChE inhibition or NMDA receptor antagonism, and its mechanism of action appears to be distinct from currently approved medications for AD.

Role of the Wnt receptor Frizzled-1 in presynaptic differentiation and function.

BACKGROUND: The Wnt signaling pathway regulates several fundamental developmental processes and recently has been shown to be involved in different aspects of synaptic differentiation and plasticity. Some Wnt signaling components are localized at central synapses, and it is thus possible that this pathway could be activated at the synapse. RESULTS: We examined the distribution of the Wnt receptor Frizzled-1 in cultured hippocampal neurons and determined that this receptor is located at synaptic contacts co-localizing with presynaptic proteins. Frizzled-1 was found in functional synapses detected with FM1-43 staining and in synaptic terminals from adult rat brain. Interestingly, overexpression of Frizzled-1 increased the number of clusters of Bassoon, a component of the active zone, while treatment with the extracellular cysteine-rich domain (CRD) of Frizzled-1 decreased Bassoon clustering, suggesting a role for this receptor in presynaptic differentiation. Consistent with this, treatment with the Frizzled-1 ligand Wnt-3a induced presynaptic protein clustering and increased functional presynaptic recycling sites, and these effects were prevented by co-treatment with the CRD of Frizzled-1. Moreover, in synaptically mature neurons Wnt-3a was able to modulate the kinetics of neurotransmitter release. CONCLUSION: Our results indicate that the activation of the Wnt pathway through Frizzled-1 occurs at the presynaptic level, and suggest that the synaptic effects of the Wnt signaling pathway could be modulated by local activation through synaptic Frizzled receptors.

Wnt-5a/JNK signaling promotes the clustering of PSD-95 in hippocampal neurons.

During the formation of synapses, specific regions of pre- and postsynaptic cells associate to form a single functional transmission unit. In this process, synaptogenic factors are necessary to modulate pre- and postsynaptic differentiation. In mammals, different Wnt ligands operate through canonical and non-canonical Wnt pathways, and their precise functions to coordinate synapse structure and function in the mature central nervous system are still largely unknown. Here, we studied the effect of different Wnt ligands on postsynaptic organization. We found that Wnt-5a induces short term changes in the clustering of PSD-95, without affecting its total levels. Wnt-5a promotes the recruitment of PSD-95 from a diffuse dendritic cytoplasmic pool to form new PSD-95 clusters in dendritic spines. Moreover, Wnt-5a acting as a non-canonical ligand regulates PSD-95 distribution through a JNK-dependent signaling pathway, as demonstrated by using the TAT-TI-JIP peptide in mature hippocampal neurons. Finally, using adult rat hippocampal slices, we found that Wnt-5a modulates glutamatergic synaptic transmission through a postsynaptic mechanism. Our studies indicate that the Wnt-5a/JNK pathway modulates the postsynaptic region of mammalian synapse directing the clustering and distribution of the physiologically relevant scaffold protein, PSD-95.

Kinetics, molecular basis, and differentiation of L-lactate transport in spermatogenic cells.

Round spermatid energy metabolism is closely dependent on the presence of L-lactate in the external medium. This L-lactate has been proposed to be supplied by Sertoli cells in the seminiferous tubules. L-Lactate, in conjunction with glucose, modulates intracellular Ca(2+) concentration in round spermatids and pachytene spermatocytes. In spite of this central role of L-lactate in spermatogenic cell physiology, the mechanism of L-lactate transport, as well as possible differentiation during spermatogenesis, has not been studied in these cells. By measuring radioactive L-lactate transport and intracellular pH (pH(i)) changes with pH(i) fluorescent probes, we show that these cells transport L-lactate using monocarboxylate-H(+) transport (MCT) systems. RT-PCR, in situ mRNA hybridization, and immunocyto- and immunohistochemistry data show that pachytene spermatocytes express mainly the MCT1 and MCT4 isoforms of the transporter (intermediate- and low-affinity transporters, respectively), while round spermatids, besides MCT1 and MCT4, also show expression of the MCT2 isoform (high-affinity transporter). These molecular data are consistent with the kinetic data of L-lactate transport in these cells demonstrating at least two transport components for L-lactate. These separate transport components reflect the ability of these cells to switch between the generation of glycolytic L-lactate in the presence of external glucose and the use of L-lactate when this substrate is available in the external environment. The supply of these substrates is regulated by the hormonal control of Sertoli cell glycolytic activity.