Chaperone-mediated autophagy and disease: Implications for cancer and neurodegeneration.

Chaperone-mediated autophagy (CMA) is a proteolytic process whereby selected intracellular proteins are degraded inside lysosomes. Owing to its selectivity, CMA participates in the modulation of specific regulatory proteins, thereby playing an important role in multiple cellular processes. Studies conducted over the last two decades have enabled the molecular characterization of this autophagic pathway and the design of specific experimental models, and have underscored the importance of CMA in a range of physiological processes beyond mere protein quality control. Those findings also indicate that decreases in CMA function with increasing age may contribute to the pathogenesis of age-associated diseases, including neurodegeneration and cancer. In the context of neurological diseases, CMA impairment is thought to contribute to the accumulation of misfolded/aggregated proteins, a process central to the pathogenesis of neurodegenerative diseases. CMA therefore constitutes a potential therapeutic target, as its induction accelerates the clearance of pathogenic proteins, promoting cell survival. More recent evidence has highlighted the important and complex role of CMA in cancer biology. While CMA induction may limit tumor development, experimental evidence also indicates that inhibition of this pathway can attenuate the growth of established tumors and improve the response to cancer therapeutics. Here, we describe and discuss the evidence supporting a role of impaired CMA function in neurodegeneration and cancer, as well as future research directions to evaluate the potential of this pathway as a target for the prevention and treatment of these diseases.

PKCλ/ι inhibition activates an ULK2-mediated interferon response to repress tumorigenesis.

The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.

PKCλ/ι Loss Induces Autophagy, Oxidative Phosphorylation, and NRF2 to Promote Liver Cancer Progression.

Oxidative stress plays a critical role in liver tissue damage and in hepatocellular carcinoma (HCC) initiation and progression. However, the mechanisms that regulate autophagy and metabolic reprogramming during reactive oxygen species (ROS) generation, and how ROS promote tumorigenesis, still need to be fully understood. We show that protein kinase C (PKC) λ/ι loss in hepatocytes promotes autophagy and oxidative phosphorylation. This results in ROS generation, which through NRF2 drives HCC through cell-autonomous and non-autonomous mechanisms. Although PKCλ/ι promotes tumorigenesis in oncogene-driven cancer models, emerging evidence demonstrate that it is a tumor suppressor in more complex carcinogenic processes. Consistently, PKCλ/ι levels negatively correlate with HCC histological tumor grade, establishing this kinase as a tumor suppressor in liver cancer.

Pros and Cons of Chaperone-Mediated Autophagy in Cancer Biology.

Autophagy contributes to cellular quality control and energetics through lysosomal breakdown and recycling of essential cellular components. Chaperone-mediated autophagy (CMA) adds to these autophagic functions the ability to timely and selectively degrade single tagged proteins to terminate their cellular function and, in this way, participate in the regulation of multiple cellular processes. Many cancer cells upregulate CMA for protumorigenic and prosurvival purposes. However, growing evidence supports a physiological role for CMA in limiting malignant transformation. Understanding the mechanisms behind this functional switch of CMA from antioncogenic to pro-oncogenic is fundamental for targeting CMA in cancer treatment. We summarize current understanding of CMA functions in cancer biology and discuss the basis for its context-dependent dual role in oncogenesis.

Factors influencing family care by immigrant women in Spain: a qualitative study / Fatores que influem no cuidado familiar prestado por mulheres imigrantes na Espanha: um estudo qualitativo / Factores que influyen en el cuidado familiar prestado por mujeres inmigrantes en España: un estudio cualitativo

Abstract Objective. To identify and understand factors that influence the relationships in the environment of family care provided by live-in immigrant caregivers. Methods. Interpretive qualitative study from a phenomenological perspective, using in-depth interviews, discussion groups and participant observation. The observation unit was the Sevillian families (Spain) with elderly dependents and a live-in female immigrant caregiver. Analysis units considered were health, care, dependence, gender, ethnicity and social class. Categories were analysed using QSR-NUD*ISTVivo9. After saturation, we triangulated between researchers, disciplines, sources and techniques to validate the results. Results. Factors of cultural discovery or clash were: language, religion, food, concept of space and time, caregiver's name and the attitudes held by both the hiring family, related to its social class, and by caregivers. Conclusion. Interpersonal relationships are the most important factor: an egalitarian relationship based on good treatment is beneficial to all involved. Knowing these codes will improve the quality of professional care in the family.

Resumo Objetivo. Identificar e compreender os fatores que influem nas relações no contexto do cuidado familiar prestado por mulheres imigrantes internas contratadas. Métodos. Estudo qualitativo interpretativo desde uma perspectiva fenomenológica, se utilizaram entrevistas em profundidade, grupos de discussão e observação participante. A unidade de observação a constituem famílias sevilhanas (Espanha) com adultos maiores dependentes a seu cargo e cuidadora imigrante interna contratada. Como unidades de análise se contemplaram: saúde, cuidados, dependência, gênero, etnia, classe social. As categorias se analisaram com o programa informático QSR NUD*ISTVivo9. Após chegar à saturação, se triangularam investigadoras, disciplinas, fontes e técnicas, para validar os resultados. Resultados. Como elementos de encontro/choque cultural se encontraram: a linguagem, a religião, a alimentação, a noção espaço-tempo, o nome da cuidadora e as atitudes, tanto da família que contratadora, relacionada com sua classe social, como das cuidadoras. Conclusão. As relações interpessoais é o fator mais importante: uma relação baseada no bom trato beneficia a todas as pessoas implicadas. Conhecer estas chaves pode favorecer a qualidade do cuidado profissional enfermeiro prestado à família.

Resumen Objetivo. Identificar y comprender los factores que influyen en las relaciones en el contexto del cuidado familiar prestado por mujeres inmigrantes internas contratadas. Métodos . Estudio cualitativo interpretativo desde una perspectiva fenomenológica. Para el efecto, se utilizaron entrevistas en profundidad, grupos de discusión y observación participante. La unidad de observación la constituyen familias sevillanas (España) con adultos mayores dependientes a su cargo y con una cuidadora inmigrante interna contratada. Como unidades de análisis se contemplaron: salud, cuidados, dependencia, género, etnicidad, clase social. Las categorías se analizaron con el programa informático QSR NUD*ISTVivo9. Tras llegar a la saturación, se triangularon investigadoras, disciplinas, fuentes y técnicas para validar los resultados. Resultados . Como elementos de encuentro/choque cultural se han encontrado: el lenguaje, la religión, la alimentación, la noción espacio-tiempo, el nombre de la cuidadora y las actitudes, tanto de la familia que contrata -relacionada con su clase social-, así como de las cuidadoras. Conclusión. Las relaciones interpersonales son el factor más importante: una relación basada en el buen trato beneficia a todas las personas implicadas. Conocer estas claves puede favorecer la calidad del cuidado profesional enfermero prestado a la familia.

Lysosomal mTORC2/PHLPP1/Akt Regulate Chaperone-Mediated Autophagy.

Chaperone-mediated autophagy (CMA), a selective form of degradation of cytosolic proteins in lysosomes, contributes to maintenance of proteostasis and to the cellular adaptation to stress. CMA substrates are delivered by a cytosolic chaperone to the lysosomal surface, where, upon unfolding, they are internalized through a membrane translocation complex. The molecular components that participate in CMA substrate targeting and translocation are well characterized, but those involved in CMA regulation remain mostly unknown. In this study, we have identified that CMA is under the positive control of the phosphatase PHLPP1 that associates with the lysosomal membrane and counteracts the inhibitory effect of mTORC2 on CMA. Lysosomal Akt, a target of the mTORC2/PHLPP1 kinase-phosphatase pair, modulates CMA activity by controlling the dynamics of assembly and disassembly of the CMA translocation complex at the lysosomal membrane. The lysosomal mTORC2/PHLPP1/Akt axis could become a target to restore CMA dysfunction in aging and disease.

Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease.

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington's disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Can cholinesterase inhibitors provide additional effects to cholinergic neurotransmission enhancement?

The most frequent of the primary degenerative dementias is Alzheimer's disease (AD). The gradual loss of memory and attention in patients suffering from this illness are accompanied by aphasia, apraxia, agnosia, and alterations in visual-spatial perception. This group of symptoms is completed by emotional alterations, psychic instability, and changes in personality that appear in advanced phases of the illness. Different histopathological alterations have been described, like marked atrophy of the cerebral cortex with loss of cortical and subcortical neurons. Other histopathological hallmarks are the formation of senile plaques composed of beta-amyloid (Abeta) and neuro fibrillary tangles composed of hyperphosphorylation of tau protein.

Depolarization preconditioning produces cytoprotection against veratridine-induced chromaffin cell death.

The hypothesis that K(+) channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K(+) solutions or with tetraethylammonium (TEA), a K(+) channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K(+) (20-80 mM) or TEA (10-30 mM) for 24 h caused 20-60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca(2+) ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K(+) or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca(2+) signal. High K(+) shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K(+) channels that depolarize the cell and cause a cytosolic Ca(2+) signal, leading to enhanced expression of BDNF and Bcl-2.

Effect of amyloid peptides on the increase in TrkA receptor expression induced by nicotine in vitro and in vivo.

The ability of nicotine to induce a cytoprotective or neuroprotective action occurs through several downstream mechanisms. One possibility is that the drug increases the expression of tyrosine kinase A (TrkA) nerve growth factor (NGF) receptors. Certain beta-amyloid peptides (e.g., Abeta1-42) have been shown to bind with high affinity to alpha7 nicotinic receptors and thus interfere with a potentially neurotrophic influence. Treatment of differentiated PC-12 cells with nicotine produced a concentration-dependent increase in cell-surface TrkA receptors that occurred concomitantly with cytoprotection. The effect of nicotine was blocked by either of the alpha7 receptor antagonists alpha-bungarotoxin (alpha-BTX) or methyllycaconatine. The cytoprotective action of nicotine also was inhibited by pretreatment with 10-100 nM Abeta1-42. Nicotine also was administered (four injections of 30 microg, spaced evenly over 24 h) to rats by direct injection into a lateral cerebral ventricle. Brain TrkA expression was increased significantly in hippocampus and entorhinal cortex (up to 32% above control), with no changes found in cerebral cortex or hypothalamus. The nicotine-induced increases in TrKA expression in hippocampus and entorhinal cortex were significantly inhibited by 10 microg alpha-BTXor by 10 nmol Abeta1-42. Therefore, physiologically relevant concentrations of Abeta1-42 can prevent nicotine-induced TrkA receptor expression in brain regions containing cholinergic neurons susceptible to the neurotoxicity associated with Alzheimer's disease.

Unequal neuroprotection afforded by the acetylcholinesterase inhibitors galantamine, donepezil, and rivastigmine in SH-SY5Y neuroblastoma cells: role of nicotinic receptors.

Donepezil, rivastigmine, and galantamine are three drugs with acetylcholinesterase (AChE)-inhibiting activity that are currently being used to treat patients suffering from Alzheimer's disease. We have studied the neuroprotective effects of these drugs, in comparison with nicotine, on cell death caused by beta-amyloid (Abeta) and okadaic acid, two models that are relevant to Alzheimer's pathology, in the human neuroblastoma cell line SH-SY5Y. Galantamine and donepezil showed a U-shaped neuroprotective curve against okadaic acid toxicity; maximum protection was achieved at 0.3 microM galantamine and at 1 microM donepezil; at higher concentrations, protection was diminished. Rivastigmine showed a concentration-dependent effect; maximum protection was achieved at 3 microM. When apoptosis was induced by Abeta25-35, galantamine, donepezil, and rivastigmine showed maximum protection at the same concentrations: 0.3, 1, and 3 microM, respectively. Nicotine also afforded protection against Abeta- and okadaic acid-induced toxicity. The neuroprotective effects of galantamine, donepezil, and nicotine were reversed by the alpha7 nicotinic antagonist methyllycaconitine but not by the alpha4beta2 nicotinic antagonist dihydro-beta-erythroidine. The phosphoinositide 3-kinase (PI3K)-Akt blocker 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) reversed the protective effects of galantamine, donepezil, and nicotine but not that of rivastigmine. In contrast, the bcl-2 antagonist ethyl[2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA 14-1) reversed the protective effects of the three AChE inhibitors and that of nicotine. Our results show that galantamine, donepezil, and rivastigmine afford neuroprotection through a mechanism that is likely unrelated to AChE inhibition. Such neuroprotection seemed to be linked to alpha7 nicotinic receptors and the PI3K-Akt pathway in the case of galantamine and donepezil but not for rivastigmine.

Albumin prevents mitochondrial depolarization and apoptosis elicited by endoplasmic reticulum calcium depletion of neuroblastoma cells.

Serum albumin protects against cell death elicited by various cytotoxic agents; however, conflicting views on the protective mechanism still remain. Hence, we have studied the ability of serum albumin to prevent apoptosis of human neuroblastoma SH-SY 5 Y cells elicited by four compounds known to release Ca(2+) from the endoplasmic reticulum, i.e. dotarizine, flunarizine, thapsigargin and cyclopiazonic acid. Spontaneous basal apoptosis, after 24 h incubation in Dulbecco's Modified Eagle Medium (DMEM) containing 10% serum, was 5%. Dotarizine (30--50 microM) enhanced basal apoptosis to 18--43%, flunarizine (30--50 microM) to 15%, thapsigargin (1--10 microM) to 21--35%, and cyclopiazonic acid (100 microM) to 10%. Serum deprivation augmented basal apoptosis to 20%. Under serum-free medium, 30 microM dotarizine or flunarizine drastically enhanced apoptosis to 63% and 68%, respectively; the increase was milder with 1 microM thapsigargin (37%) and 30 microM cyclopiazonic acid (27%). In serum-free medium, albumin (29 or 49 mg/ml) fully prevented the apoptotic effects of dotarizine, flunarizine and cyclopiazonic acid. The four compounds increased the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in fluo-4 loaded cells; such increase developed slowly to reach a plateau after several minutes, followed by a slow decline. Albumin did not modify the kinetic parameters of such increase. In the absence of serum, dotarizine, flunarizine, thapsigargin, and cyclopiazonic acid caused mitochondrial depolarization in tetramethylrhodamine ethyl ester (TMRE)-loaded cells; depolarization was inhibited by cytoprotective concentrations of albumin. These results suggest that albumin protects cells from entering into apoptosis by preventing mitochondrial depolarization. They also suggest that inhibition of mitochondrial depolarization might become a target to develop new anti-apoptotic compounds with therapeutic neuroprotective potential in stroke, Alzheimer's disease, and other neurodegenerative diseases.

ITH4012 (ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-phenylbenzol[1,8]naphthyridine-3-carboxylate), a novel acetylcholinesterase inhibitor with "calcium promotor" and neuroprotective properties.

Ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-phenylbenzol[1,8] naphthyridine-3-carboxylate (ITH4012) is a novel tacrine derivative that can reduce cell death induced by various compounds with different mechanisms of action, such as thapsigargin (reticular stress), H2O2 (free radicals), and veratridine (calcium overload), in bovine chromaffin cell. Cell viability, quantified as lactic dehydrogenase release, was significantly reduced by ITH4012 at concentrations ranging from 0.01 to 3 microM. In the human neuroblastoma cell line SH-SY5Y, ITH4012 also reduced amyloid beta25-35-induced apoptosis, determined by flow cytometry. ITH4012 caused a slight elevation in the cytosolic concentration of Ca2+ in fura 2-loaded bovine chromaffin cells, which could be related to the induction of protein synthesis relevant for cell survival. Blockade of protein synthesis by cycloheximide or blockade of Bcl-2's active site with HA14-1 (ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate) reversed the cytoprotective action of ITH4012. Furthermore, exposure of bovine chromaffin cells for 24 or 48 h to neuroprotective concentrations of this compound enhanced, nearly 3-fold, the expression of the antiapoptotic protein Bcl-2. In conclusion, ITH4012 is a tacrine derivative that maintains acetylcholinesterase-inhibiting activity (IC50=0.8 microM) but has the additional property of acting as a calcium promotor, a property leading to neuroprotection through the induction of antiapoptotic proteins.

Galantamine prevents apoptosis induced by beta-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors.

Galantamine is currently used to treat Alzheimer's disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer's disease.