JNK Activation Correlates with Cognitive Impairment and Alteration of the Post-Synaptic Element in the 5xFAD AD Mouse Model

The c-Jun N-terminal kinases (JNKs) are a family of proteins that, once activated by stress stimuli, can alter neuronal functions and survival. The JNK cascade plays a crucial role in the post-synaptic neuronal compartment by altering its structural organization and leading, at worst, to an overall impairment of neuronal communication. Increasing evidence suggests that synaptic impairment is the first neurodegenerative event in Alzheimer’s disease (AD). To better elucidate this mechanism, we longitudinally studied 5xFAD mice at three selected time points representative of human AD symptom progression. We tested the mice cognitive performance by using the radial arm water maze (RAWM) in parallel with biochemical evaluations of post-synaptic enriched protein fraction and total cortical parenchyma. We found that 5xFAD mice presented a strong JNK activation at 3.5 months of age in the post-synaptic enriched protein fraction. This JNK activation correlates with a structural alteration of the post-synaptic density area and with memory impairment at this early stage of the disease that progressively declines to cause cell death. These findings pave the way for future studies on JNK as a key player in early neurodegeneration and as an important therapeutic target for the development of new compounds able to tackle synaptic impairment in the early phase of AD pathology

Novel Medicinal Mushroom Blend as a Promising Supplement in Integrative Oncology: A Multi-Tiered Study using 4T1 Triple-Negative Mouse Breast Cancer Model

Abstract: Although medicinal mushroomextracts have been proposed as promising anti-cancer agents, their precise impacts on metastatic breast cancer are still to be clarified. For this purpose, the present study exploited the eect of a novel medicinal mushroom blend, namely Micotherapy U-care, in a 4T1 triple-negative mouse breast cancer model. Mice were orally administered with Micotherapy U-care, consisting of a mixture of Agaricus blazei, Ophiocordyceps sinensis, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes. The syngeneic tumor-bearing mice were generated by injecting 4T1 cells in both supplemented and non-supplemented mice. After sacrifice 35 days later, specific endpoints and pathological outcomes of the murine pulmonary tissue were evaluated. (i) Histopathological and ultrastructural analysis and (ii) immunohistochemical assessment of TGF-ß1, IL-6 and NOS2, COX2, SOD1 as markers of inflammation and oxidative stress were performed. The QoL was comparatively evaluated. Micotherapy U-care supplementation, starting before 4T1 injection and lasting until the end of the experiment, dramatically reduced the pulmonary metastases density, also triggering a decrease of fibrotic response, and reducing IL-6, NOS, and COX2 expression. SOD1 and TGF-ß1 results were also discussed. These findings support the valuable potential of Micotherapy U-care as adjuvant therapy in the critical management of triple-negative breast cancer

Oxidative stress and neuroinflammation effects in hippocampal development, aging, and disease

This PhD thesis aims to evaluate oxidative stress and inflammation pathways considering three pathological models such as Prolidase Deficiency, Physiological Aging and Anorexia Nervosa. Furthermore, preliminary experiments were performed to better understand the involvement of different cell death pathways i.e. apoptosis, autophagy and mitophagy. Prolidase deficiency (PD) is a rare genetic disorder caused by a mutation in the PEPD gene that codes for the prolidase enzyme. This enzyme can selectively cut proline dimers present at the carboxy-terminal end of a polypeptide and participates in the turnover of collagen. Recently published data have demonstrated the presence of cerebellar morphological alterations in PD mice, extracellular matrix disorganization, and consequently tissue damage and anomalies in cell migration [1]. Ageing is a condition closely related to oxidative stress and inflammation that physiologically affects everyone. Recently, it has been shown that the medicinal mushroom Hericium erinaceus (He1) has antioxidant activity and partially reactivates cell proliferation in some brain areas of physiologically aged mice and animals subjected to a diet supplemented with the mushroom [2] Eating Disorders are common, complex, and difficult-to-cure diseases that mostly arise during adolescence. Among the various disorders, Anorexia Nervosa (AN) was considered a pathology characterized by an insufficient energy supply for survival, with consequences that affect the whole organism and affect endocrine, cardiovascular, gastrointestinal, haematological, and skeletal muscle functions. Oxidative stress and consequently inflammation seem to be a common thread in these three pathological models and could trigger several types of cell death. Morphological and immunohistochemical reactions were conducted on mouse and human hippocampal sections considering the following antibodies: superoxide dismutase 1 and 2 (SOD1, SOD2), cyclooxygenase 2 (COX2), nitric oxide synthase 2 (NOS2), interleukin 6 (IL-6), and transforming growth factor β (TGF-β). In addition, for the cell death pathways Bax, Bcl2, p62, PINK1 and PARKIN were evaluated. Haematoxylin and eosin staining, and Nissl staining were also performed for cytoarchitectural analyses. The purpose of this thesis was to determine differences in the expression of antioxidant proteins, inflammatory cytokines and molecules and cell death markers in the hippocampal formation of mice affected by PD (dal) at 10 days after birth (P10), P21 and P60, aged mice supplemented for 2 months with 1 mg/day of standardized extracts of He1 and human brain biopsies of a control and AN patient. The results were obtained by using microtome cutting techniques, immunohistochemistry and immunofluorescence experiments and optical microscope analysis followed by statistical evaluations. First, the morphological differences in the dentate gyrus of the hippocampus were evaluated by Haematoxylin/eosin and Nissl staining and further the expression of specific markers for oxidative stress and inflammation, i.e. SOD1, SOD2, COX4, NOS2, COX2, IL- 6, TGF-β and cell death phenomena due to apoptosis, i.e. Bcl-2 and Bax, autophagy, i.e. p62 and mitophagy, i.e. PINK1 and PARKIN were evaluated. From these analyses, it was possible to observe how a strong oxidative stress and inflammation environment is present in pathological conditions and contributes to activate mechanisms of regulated cell death. Most of the observed cells expressed markers of autophagy and mitophagy rather than apoptosis, a sign, however, of an attempt by the cells to eliminate the harmful components without causing further damage to the tissue. In conclusion, we can therefore think that these data can provide insights to improve an antioxidative and antiinflammation treatment through activation of programmed cell death for cell survival.This PhD thesis aims to evaluate oxidative stress and inflammation pathways considering three pathological models such as Prolidase Deficiency, Physiological Aging and Anorexia Nervosa. Furthermore, preliminary experiments were performed to better understand the involvement of different cell death pathways i.e. apoptosis, autophagy and mitophagy. Prolidase deficiency (PD) is a rare genetic disorder caused by a mutation in the PEPD gene that codes for the prolidase enzyme. This enzyme can selectively cut proline dimers present at the carboxy-terminal end of a polypeptide and participates in the turnover of collagen. Recently published data have demonstrated the presence of cerebellar morphological alterations in PD mice, extracellular matrix disorganization, and consequently tissue damage and anomalies in cell migration [1]. Ageing is a condition closely related to oxidative stress and inflammation that physiologically affects everyone. Recently, it has been shown that the medicinal mushroom Hericium erinaceus (He1) has antioxidant activity and partially reactivates cell proliferation in some brain areas of physiologically aged mice and animals subjected to a diet supplemented with the mushroom [2] Eating Disorders are common, complex, and difficult-to-cure diseases that mostly arise during adolescence. Among the various disorders, Anorexia Nervosa (AN) was considered a pathology characterized by an insufficient energy supply for survival, with consequences that affect the whole organism and affect endocrine, cardiovascular, gastrointestinal, haematological, and skeletal muscle functions. Oxidative stress and consequently inflammation seem to be a common thread in these three pathological models and could trigger several types of cell death. Morphological and immunohistochemical reactions were conducted on mouse and human hippocampal sections considering the following antibodies: superoxide dismutase 1 and 2 (SOD1, SOD2), cyclooxygenase 2 (COX2), nitric oxide synthase 2 (NOS2), interleukin 6 (IL-6), and transforming growth factor β (TGF-β). In addition, for the cell death pathways Bax, Bcl2, p62, PINK1 and PARKIN were evaluated. Haematoxylin and eosin staining, and Nissl staining were also performed for cytoarchitectural analyses. The purpose of this thesis was to determine differences in the expression of antioxidant proteins, inflammatory cytokines and molecules and cell death markers in the hippocampal formation of mice affected by PD (dal) at 10 days after birth (P10), P21 and P60, aged mice supplemented for 2 months with 1 mg/day of standardized extracts of He1 and human brain biopsies of a control and AN patient. The results were obtained by using microtome cutting techniques, immunohistochemistry and immunofluorescence experiments and optical microscope analysis followed by statistical evaluations. First, the morphological differences in the dentate gyrus of the hippocampus were evaluated by Haematoxylin/eosin and Nissl staining and further the expression of specific markers for oxidative stress and inflammation, i.e. SOD1, SOD2, COX4, NOS2, COX2, IL- 6, TGF-β and cell death phenomena due to apoptosis, i.e. Bcl-2 and Bax, autophagy, i.e. p62 and mitophagy, i.e. PINK1 and PARKIN were evaluated. From these analyses, it was possible to observe how a strong oxidative stress and inflammation environment is present in pathological conditions and contributes to activate mechanisms of regulated cell death. Most of the observed cells expressed markers of autophagy and mitophagy rather than apoptosis, a sign, however, of an attempt by the cells to eliminate the harmful components without causing further damage to the tissue. In conclusion, we can therefore think that these data can provide insights to improve an antioxidative and antiinflammation treatment through activation of programmed cell death for cell survival

Cognitive Healthy Aging in Mice: Boosting Memory by an Ergothioneine-Rich <i>Hericium erinaceus</i> Primordium Extract

Brain aging is a crucial risk factor for several neurodegenerative disorders and dementia. The most affected cognitive function is memory, worsening early during aging. Inflammation and oxidative stress are known to have a role in pathogenesis of cognitive impairments, and a link exists between aging/frailty and immunosenescence/inflammaging. Based on anti-aging properties, medicinal mushrooms represent a source to develop medicines and functional foods. In particular, Hericium erinaceus (He) displays several actions ranging from boosting the immune system to fighting senescence, due to its active ingredients/metabolites. Among these, Ergothioneine (ERGO) is known as the longevity vitamin. Currently, we demonstrated the efficacy of an ERGO-rich He primordium extract (He2) in preventing cognitive decline in a murine model of aging. We focused on recognition memory deterioration during aging, monitored through spontaneous behavioral tests assessing both memory components and frailty index. A parallel significant decrease in key markers of inflammation and oxidative stress, i.e., IL6, TGFβ1, GFAP, Nrf2, SOD1, COX2, NOS2, was revealed in the hippocampus by immunohistochemistry, accompanied by an enhancement of NMDAR1and mGluR2, crucially involved in glutamatergic neurotransmission. In summary, we disclosed a selective, preventive and neuroprotective effect of He2 on aged hippocampus, both on recognition memory as well on inflammation/oxidative stress/glutamate receptors expression

Role of Na⁺/Ca²⁺ Exchanger (NCX) in Glioblastoma Cell Migration (In Vitro)

Glioblastoma (GBM) is the most malignant form of primary brain tumor. It is characterized by the presence of highly invasive cancer cells infiltrating the brain by hijacking neuronal mechanisms and interacting with non-neuronal cell types, such as astrocytes and endothelial cells. To enter the interstitial space of the brain parenchyma, GBM cells significantly shrink their volume and extend the invadopodia and lamellipodia by modulating their membrane conductance repertoire. However, the changes in the compartment-specific ionic dynamics involved in this process are still not fully understood. Here, using noninvasive perforated patch-clamp and live imaging approaches on various GBM cell lines during a wound-healing assay, we demonstrate that the sodium-calcium exchanger (NCX) is highly expressed in the lamellipodia compartment, is functionally active during GBM cell migration, and correlates with the overexpression of large conductance K+ channel (BK) potassium channels. Furthermore, a NCX blockade impairs lamellipodia formation and maintenance, as well as GBM cell migration. In conclusion, the functional expression of the NCX in the lamellipodia of GBM cells at the migrating front is a conditio sine qua non for the invasion strategy of these malignant cells and thus represents a potential target for brain tumor treatment

Neuroprotective Metabolites of Hericium erinaceus Promote Neuro-Healthy Aging

Abstract: Frailty is a geriatric syndrome associated with both locomotor and cognitive decline, typically linked to chronic systemic inflammation, i.e., inflammaging. In the current study, we investigated the effect of a two-month oral supplementation with standardized extracts of H. erinaceus, containing a known amount of Erinacine A, Hericenone C, Hericenone D, and L-ergothioneine, on locomotor frailty and cerebellum of aged mice. Locomotor performances were monitored comparing healthy aging and frail mice. Cerebellar volume and cytoarchitecture, together with inflammatory and oxidative stress pathways, were assessed focusing on senescent frail animals. H. erinaceus partially recovered the aged-related decline of locomotor performances. Histopathological analyses paralleled by immunocytochemical evaluation of specific molecules strengthened the neuroprotective role of H. erinaceus able to ameliorate cerebellar alterations, i.e., milder volume reduction, slighter molecular layer thickness decrease and minor percentage of shrunken Purkinje neurons, also diminishing inflammation and oxidative stress in frail mice while increasing a key longevity regulator and a neuroprotective molecule. Thus, our present findings demonstrated the efficacy of a nonpharmacological approach, based on the dietary supplementation using H. erinaceus extract, which represent a promising adjuvant therapy to be associated with conventional geriatric treatments

The Designer Drug αPHP Affected Cell Proliferation and Triggered Deathly Mechanisms in Murine Neural Stem/Progenitor Cells

In the present study, the potential functional properties of the extracts from the edible part of Capsicum annuum L. var. Peperone di Voghera (VP) were studied. The phytochemical analysis revealed a high amount of ascorbic acid, paralleled by a low carotenoid content. Normal human diploid fibroblasts (NHDF) were chosen as the in vitro model models to investigate the effects of the VP extract on oxidative stress and aging pathways. The extract of Carmagnola pepper (CP), another important Italian variety, was used as the reference vegetable. The cytotoxicity evaluation was performed firstly, using a 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, while the VP potential antioxidant and antiaging activity was investigated by immunofluorescence staining focusing on specifically selected proteins. The MTT data revealed the highest cell viability at a concentration of up to 1 mg/mL. The immunocytochemical analyses highlighted an increased expression of transcription factors and enzymes involved in redox homeostasis (Nrf2, SOD2, catalase), improved mitochondrial functionality, and the up-regulation of the longevity gene SIRT1. The present results supported the functional role of the VP pepper ecotype, suggesting a feasible use of its derived products as valuable food supplements

<i>Hericium erinaceus</i> Improves Recognition Memory and Induces Hippocampal and Cerebellar Neurogenesis in Frail Mice during Aging

Frailty is a geriatric syndrome associated with both locomotor and cognitive decline, implicated in both poor quality of life and negative health outcomes. One central question surrounding frailty is whether phenotypic frailty is associated with the cognitive impairment during aging. Using spontaneous behavioral tests and by studying the dynamic change during aging, we demonstrated that the two form of vulnerability, locomotor and recognition memory decline, develop in parallel and therefore, integration of the motoric and cognitive evaluations are imperative. We developed an integrated frailty index based on both phenotypic and recognition memory performances. Hericium erinaceus (H. erinaceus) is a medicinal mushroom that improves recognition memory in mice. By using HPLC-UV-ESI/MS analyses we obtained standardized amounts of erinacine A and hericenones C and D in H. erinaceus extracts, that were tested in our animal model of physiological aging. Two-month oral supplementation with H. erinaceus reversed the age-decline of recognition memory. Proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) immunohistochemistry in the hippocampus and cerebellum in treated mice supported a positive effect of an H. erinaceus on neurogenesis in frail mice