Fabiana Geraci

Hsp70 and Its Molecular Role in Nervous System Diseases

Heat shock proteins (HSPs) are induced in response to many injuries including stroke, neurodegenerative disease, epilepsy, and trauma. The overexpression of one HSP in particular, Hsp70, serves a protective role in several different models of nervous system injury, but has also been linked to a deleterious role in some diseases. Hsp70 functions as a chaperone and protects neurons from protein aggregation and toxicity (Parkinson disease, Alzheimer disease, polyglutamine diseases, and amyotrophic lateral sclerosis), protects cells from apoptosis (Parkinson disease), is a stress marker (temporal lobe epilepsy), protects cells from inflammation (cerebral ischemic injury), has an adjuvant role in antigen presentation and is involved in the immune response in autoimmune disease (multiple sclerosis). The worldwide incidence of neurodegenerative diseases is high. As neurodegenerative diseases disproportionately affect older individuals, disease-related morbidity has increased along with the general increase in longevity. An understanding of the underlying mechanisms that lead to neurodegeneration is key to identifying methods of prevention and treatment. Investigators have observed protective effects of HSPs induced by preconditioning, overexpression, or drugs in a variety of models of brain disease. Experimental data suggest that manipulation of the cellular stress response may offer strategies to protect the brain during progression of neurodegenerative disease.

Extracellular membrane vesicles as a mechanism of cell-to-cell communication: advantages and disadvantages

Microvesicles represent a newly identified mechanism of intercellular communication. Two different types of microvesicles have been identified: membrane-derived vesicles (EVs) and exosomes. EVs originate by direct budding from the plasma membrane, while exosomes arise from ectocytosis of multivesicular bodies. Recent attention has focused on the capacity of EVs to alter the phenotype of neighboring cells to make them resemble EV-producing cells. Stem cells are an abundant source of EVs, and the interaction between stem cells and the microenvironment (i.e., stem cell niche) plays a critical role in determining stem cell phenotype. The stem cell niche hypothesis predicts that stem cell number is limited by the availability of niches releasing the necessary signals for self-renewal and survival, and the niche thus provides a mechanism for controlling and limiting stem cell numbers. EVs may play a fundamental role in this context by transferring genetic information between cells. EVs can transfer mRNA and microRNA to target cells, both of which may be involved in the change in target-cell phenotype towards that of EV-producing cells. The exchange of genetic information may be bidirectional, and EV-mediated transfer of genetic information after tissue damage may reprogram stem cells to acquire the phenotypic features of the injured tissue cells. In addition, stem cell-derived EVs may induce the de-differentiation of cells that survive injury by promoting their reentry into the cell cycle and subsequently increasing the possibility of tissue regeneration.

Bergamot Reduces Plasma Lipids, Atherogenic Small Dense LDL, and Subclinical Atherosclerosis in Subjects with Moderate Hypercholesterolemia: A 6 Months Prospective Study.

Background: Some patients experience statin-induced side effects or prefer nutraceutical approaches for the treatment of dyslipidemia. This has led to a search for alternative therapeutic approaches for dyslipidemia management. In recent studies Citrus bergamia (known as Bergamot) juice was able to reduce serum levels of lipids. Such benefit may be attributed to high amounts of flavonoids contained in Bergamot fruit juice (neoeriocitrin, neohesperidin, naringin). The aim of the present study was to fully investigate the effects of a Bergamot extract on cardio-metabolic parameters, including plasma lipids, atherogenic lipoproteins and subclinical atherosclerosis. Methods: Eighty subjects (42 men and 38 women, mean age: 55 ± 13 years) with moderate hypercholesterolemia [e.g., with plasma LDL-cholesterol concentrations between 160 and 190 mg/dl (between 4.1 and 4.9 mmol/l)] were included. A Bergamot-derived extract (Bergavit R®) was given at a fixed dose daily (150 mg of flavonoids, with 16% of neoeriocitrin, 47% of neohesperidin and 37% of naringin) for 6 months. Lipoprotein subfractions were assessed by gel electrophoresis. With this methodology low density lipoprotein (LDL) subclasses are distributed as seven bands (LDL-1 and -2 as large LDL, and LDL-3 to -7 as atherogenic small, dense LDL). Subclinical atherosclerosis was assessed by carotid intima-media thickness (cIMT) using B-mode ultrasound. Results: After 6 months, Bergavit R® reduced total cholesterol (from 6.6 ± 0.4 to 5.8 ± 1.1 mmol/l, p < 0.0001), triglycerides (from 1.8 ± 0.6 to 1.5 ± 0.9 mmol/l, p = 0.0020), and LDL-cholesterol (from 4.6 ± 0.2 to 3.7 ± 1.0 mmol/l, p < 0.0001), while HDL- cholesterol increased (from 1.3 ± 0.2 to 1.4 ± 0.4 mmol/l, p < 0.0007). In addition, a significant increase in LDL-1 (from 41.2 ± 0.2 to 49.6 ± 0.2%, p < 0.0001) was accompanied by decreased small, dense LDL-3, -4, and 5 particles (from 14.5 ± 0.1 to 9.0 ± 0.1% p < 0.0001; 3.2 ± 0.1 to 1.5 ± 0.1% p = 0.0053; 0.3 ± 0.0% to 0.1 ± 0.0% p = 0.0133, respectively). cIMT also decreased from 1.2 ± 0.4 to 0.9 ± 0.1 mm (p < 0.0001). Conclusion: This is the first study investigating the effects of Bergamot flavonoids supplementation on cardio-metabolic risk in dyslipidemic subjects. Bergavit R® (Bergamot juice extract) supplementation significantly reduced plasma lipids and improved the lipoprotein profile. cIMT was also reduced significantly over a relatively short time frame of 6 months.

A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.

Protective role of heat shock proteins in Parkinson's disease.

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. Despite a large amount of research, the pathogenetic mechanism of these diseases has not yet been clarified. Abnormal protein folding, oxidative stress, mitochondrial dysfunction, and apoptotic mechanisms have all been reported as causes of neurodegenerative diseases in association with neuroinflammatory mechanisms which, by generating deleterious molecules, could promote the cascade of events leading to neurodegeneration. Heat shock proteins (HSPs) play a central role in preventing protein misfolding and inhibiting apoptotic activity, and represent a class of proteins potentially involved in PD pathogenesis. The present review will focus on two HSPs, HSP70 and HSP90, with the aim of specifying their role in PD pathogenesis.

Stress response in mesoangioblast stem cells

Stem cells are presumed to survive various stresses, since they are recruited to areas of tissue damage and regeneration, where inflammatory cytokines and cytotoxic cells may result in severe cell injury. We explored the ability of mesoangioblasts to respond to different cell stresses such as heat, heavy metals and osmotic stress, by analyzing heat shock protein (HSP)70 synthesis as a stress indicator. We found that the A6 mesoangioblast stem cells constitutively synthesize HSP70 in a heat shock transcription factor (HSF)-independent way. However, A6 respond to heat shock and cadmium treatment by synthesizing HSP70 over the constitutive expression and this synthesis is HSF1 dependent. The exposure of A6 to copper or to a hypertonic medium does neither induce HSP70 synthesis nor activation of HSF1, while a constitutive binding of constitutive heat shock element binding factor was found. Together, these data suggest that mesoangioblasts constitutively express HSP70 as an ‘a priori’ activation mechanism, while they maintain the ability to respond to stress stimuli.

Membrane vesicles containing matrix metalloproteinase‐9 and fibroblast growth factor‐2 are released into the extracellular space from mouse mesoangioblast stem cells

Certain proteins, including fibroblast growth factor‐2 (FGF‐2) and matrix metalloproteinase‐9 (MMP‐9), have proved very effective in increasing the efficacy of mesoangioblast stem cell therapy in repairing damaged tissue. We provide the first evidence that mouse mesoangioblast stem cells release FGF‐2 and MMP‐9 in their active form through the production of membrane vesicles. These vesicles are produced and turned over continuously, but are stable for some time in the extracellular milieu. Mesoangioblasts shed membrane vesicles even under oxygen tensions that are lower than those typically used for cell culture and more like those of mouse tissues. These findings suggest that mesoangioblasts may themselves secrete paracrine signals and factors that make damaged tissues more amenable to cell therapy through the release of membrane vesicles. J. Cell. Physiol. 224:144–151, 2010

Hsp70 localizes differently from chaperone Hsc70 in mouse mesoangioblasts under physiological growth conditions

Mouse A6 mesoangioblasts express Hsp70 even in the absence of cellular stress. Its expression and its intracellular localization were investigated under normal growth conditions and under hyperthermic stress. Immunofluorescence assays indicated that without any stress a fraction of Hsp70 co-localized with actin microfilaments, in the cell cortex and in the contractile ring of dividing cells, while the Hsc70 chaperone did not. Hsp70 immunoprecipitation assays confirmed that a portion of Hsp70 binds actin. Immunoblot assays showed that both proteins were present in the nucleus. After heat treatment Hsp70 and actin continued to co-localize in the leading edge of A6 cells but not on microfilaments. Although Hsp70 and Hsc70 are both basally synthesized they showed different cellular distribution, suggesting an Hsp70 different activity respect to the Hsc70 chaperone. Moreover, we found Hsp70 in the culture medium as it has been described in other cell types.

EFFECT OF THE IMPase INHIBITOR L690,330 ON SEA URCHIN DEVELOPMENT

A variety of concentrations of the IMPase inhibitor L690,330 were added to sea urchin embryos. Immediate arrest of development was obtained for concentrations from 7.5mmon. Concentrations lower than 3.5mmpermitted gastrulation but inhibited skeletogenesis and disturbed elongation along the animal—vegetal axis. The latter results are similar to those obtained by counteracting lithium effect with myoinositol, which are suggested to be due to partial relief of IMPase inhibition.

Extracellular Hsp70 Enhances Mesoangioblast Migration via an Autocrine Signaling Pathway

Mouse mesoangioblasts are vessel‐associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. Therefore, they represent a promising tool in the regeneration of injured tissues. Several studies have demonstrated that homing of mesoangioblasts into blood and injured tissues are mainly controlled by cytokines/chemokines and other inflammatory factors. However, little is known about the molecular mechanisms regulating their ability to traverse the extracellular matrix (ECM). Here, we demonstrate that membrane vesicles released by mesoangioblasts contain Hsp70, and that the released Hsp70 is able to interact by an autocrine mechanism with Toll‐like receptor 4 (TLR4) and CD91 to stimulate migration. We further demonstrate that Hsp70 has a positive role in regulating matrix metalloproteinase 2 (MMP2) and MMP9 expression and that MMP2 has a more pronounced effect on cell migration, as compared to MMP9. In addition, the analysis of the intracellular pathways implicated in Hsp70 regulated signal transduction showed the involvement of both PI3K/AKT and NF‐κB. Taken together, our findings present a paradigm shift in our understanding of the molecular mechanisms that regulate mesoangioblast stem cells ability to traverse the extracellular matrix (ECM). J. Cell. Physiol. 232: 1845–1861, 2017.