SH-SY5Y Cell Line In Vitro Models for Parkinson Disease Research-Old Practice for New Trends.

The SH-SY5Y cell line is a simple and inexpensive in vitro experimental model for studying Parkinson disease (PD). This experimental model is a useful tool for elucidating pathophysiological mechanisms of PD and in the development of new pharmacological therapies. In this review, we aim to summarize current protocols for SH-SY5Y cell culturing and differentiation and PD experimental designs derived from the SH-SY5Y cell line. The most efficient protocol for differentiation of the SH-SY5Y cell line into dopaminergic neurons seems to be the addition of retinoic acid to the growth medium, followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) addition in a low concentration of fetal bovine serum. PD pathological changes, such as neuronal apoptosis and the intraneuronal alpha-synuclein aggregation, can be reproduced in the SH-SY5Y cell line either by the use of neurotoxic agents [such as rotenone, 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine] or by genetic modification (transfection of the alpha-synuclein wild-type or mutant gene, genetic manipulation of other genes involved in PD). In addition, compounds with a potential neuroprotective role may be tested on neurotoxicity-induced SH-SY5Y models. The cell line can also be used for testing PD pathophysiological mechanisms such as the prion-like neuronal transmission of alpha-synuclein or the microbiota influence in PD. In conclusion, the use of the SH-SY5Y cell line represents a basic but consistent first step in experiments related to PD, but which must be followed by the confirmation of the results through more complex in vitro and in vivo experimental models.

Application of Droplet Digital PCR Technology in Muscular Dystrophies Research.

Although they are considered rare disorders, muscular dystrophies have a strong impact on people's health. Increased disease severity with age, frequently accompanied by the loss of ability to walk in some people, and the lack of treatment, have directed the researchers towards the development of more effective therapeutic strategies aimed to improve the quality of life and life expectancy, slow down the progression, and delay the onset or convert a severe phenotype into a milder one. Improved understanding of the complex pathology of these diseases together with the tremendous advances in molecular biology technologies has led to personalized therapeutic procedures. Different approaches that are currently under extensive investigation require more efficient, sensitive, and less invasive methods. Due to its remarkable analytical sensitivity, droplet digital PCR has become a promising tool for accurate measurement of biomarkers that monitor disease progression and quantification of various therapeutic efficiency and can be considered a tool for non-invasive prenatal diagnosis and newborn screening. Here, we summarize the recent applications of droplet digital PCR in muscular dystrophy research and discuss the factors that should be considered to get the best performance with this technology.

CD36 - A novel molecular target in the neurovascular unit.

CD36 is an integral membrane protein primarily known for its function as a fatty acid transporter, yet also playing other biological roles from lipid metabolism to inflammation modulation. These pleiotropic effects are explained by the existence of multiple different ligands and the extensive distribution in numerous cell types. Moreover, the receptor is related to various pathologies and it may prove to be a good target for prospective therapeutic strategies. In the neurovascular unit (NVU), CD36 is expressed in cells like microglia, microvascular endothelial cells, astrocytes and neurons. In the normal brain, CD36 was proven to be involved in phagocytosis of apoptotic cells, oro-sensory detection of dietary lipids, and fatty acid transport across the blood brain barrier (BBB). CD36 was also acknowledged as a potentially important player in central nervous system (CNS) disorders, such as Alzheimer Disease-associated vascular dysfunction and oxidative stress and the neuroinflammatory response in stroke. Despite continuous efforts, the therapeutic arsenal for such diseases is still scarce and there is an increasing interest in discovering new molecular targets for more specific therapeutic approaches. In this review, we summarize the role of CD36 in the normal function of the NVU and in several CNS disorders, focusing on the dysregulation of the NVU and the potential therapeutic modulation.

Transcriptomic Crosstalk between Gliomas and Telencephalic Neural Stem and Progenitor Cells for Defining Heterogeneity and Targeted Signaling Pathways.

Recent studies have begun to reveal surprising levels of cell diversity in the human brain, both in adults and during development. Distinctive cellular phenotypes point to complex molecular profiles, cellular hierarchies and signaling pathways in neural stem cells, progenitor cells, neuronal and glial cells. Several recent reports have suggested that neural stem and progenitor cell types found in the developing and adult brain share several properties and phenotypes with cells from brain primary tumors, such as gliomas. This transcriptomic crosstalk may help us to better understand the cell hierarchies and signaling pathways in both gliomas and the normal brain, and, by clarifying the phenotypes of cells at the origin of the tumor, to therapeutically address their most relevant signaling pathways.

Role of 5-HT1A and 5-HT3 receptors in serotonergic activation of sensory neurons in relation to itch and pain behavior in the rat.

Serotonin (5-hydroxytryptamine, 5-HT) released by platelets, mast cells, and immunocytes is a potent inflammatory mediator which modulates pain and itch sensing in the peripheral nervous system. The serotonergic receptors expressed by primary afferent neurons involved in these sensory functions are not fully identified and appear to be to a large extent species dependent. Moreover, the mechanisms through which 5-HT receptor activation is coupled to changes in neuronal excitability have not been completely revealed. Using a combination of in vitro (calcium and voltage imaging and patch-clamp) and in vivo behavioral methods, we used both male and female Wistar rats to provide evidence for the involvement of two 5-HT receptor subtypes, 5-HT1A and 5-HT3, in mediating the sustained and transient effects, respectively, of 5-HT on rat primary afferent neurons involved in pain and itch processing. In addition, our results are consistent with a model in which sustained serotonergic responses triggered via the 5-HT1A receptor are due to closure of background potassium channels, followed by membrane depolarization and action potentials, during which the activation of voltage-gated calcium channels leads to calcium entry. Our results may provide a better understanding of mammalian serotonergic itch signaling.

Age-related ultrastructural changes of the basement membrane in the mouse blood-brain barrier.

The blood-brain barrier (BBB) is essential for a functional neurovascular unit. Most studies focused on the cells forming the BBB, but very few studied the basement membrane (BM) of brain capillaries in ageing. We used transmission electron microscopy and electron tomography to investigate the BM of the BBB in ageing C57BL/6J mice. The thickness of the BM of the BBB from 24-month-old mice was double as compared with that of 6-month-old mice (107 nm vs 56 nm). The aged BBB showed lipid droplets gathering within the BM which further increased its thickness (up to 572 nm) and altered its structure. The lipids appeared to accumulate toward the glial side of the BM. Electron tomography showed that the lipid-rich BM regions are located in small pockets formed by the end-feet of astrocytes. These findings suggest an imbalance of the lipid metabolism and that may precede the structural alteration of the BM. These alterations may favour the accretion of abnormal proteins that lead to neurodegeneration in ageing. These findings warrant further investigation of the BM of brain capillaries and of adjoining cells as potential targets for future therapies.

Studies on the Synthesis, Photophysical and Biological Evaluation of Some Unsymmetrical Meso-Tetrasubstituted Phenyl Porphyrins.

Abstract: We designed three unsymmetrical meso-tetrasubstituted phenyl porphyrins for further development as theranostic agents for cancer photodynamic therapy (PDT): 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (P2.2), Zn(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Zn(II)2.2) and Cu(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Cu(II)2.2). The porphyrinic compounds were synthesized and their structures were confirmed by elemental analysis, FT-IR, UV-Vis, EPR and NMR. The compounds had a good solubility in polar/nonpolar media. P2.2 and, to a lesser extent, Zn(II)2.2 were fluorescent, albeit with low fluoresence quantum yields. P2.2 and Zn(II)2.2 exhibited PDT-acceptable values of singlet oxygen generation. A "dark" cytotoxicity study was performed using cells that are relevant for the tumor niche (HT-29 colon carcinoma cells and L929 fibroblasts) and for blood (peripheral mononuclear cells). Cellular uptake of fluorescent compounds, cell viability/proliferation and death were evaluated. P2.2 was highlighted as a promising theranostic agent for PDT in solid tumors considering that P2.2 generated PDT-acceptable singlet oxygen yields, accumulated into tumor cells and less in blood cells, exhibited good fluorescence within cells for imagistic detection, and had no significant cytotoxicity in vitro against tumor and normal cells. Complexing of P2.2 with Zn(II) or Cu(II) altered several of its PDT-relevant properties. These are consistent arguments for further developing P2.2 in animal models of solid tumors for in vivo PDT.

Juxtacerebral Tissue Regeneration Potential: Telocytes Contribution.

It is well proved already that neurogenesis does take place in mammals' brain, including human brain. However, neurogenesis by itself is not able to compensate for brain tissue loss in serious neurological diseases, such as stroke, brain trauma or neurodegenerative disorders. Recent evidences show that neural stem cell niches are present not only in classical locations, such as subventricularor subgranular zones, but in other areas as well, including tissues contiguous to the brain (meninges and choroid plexus).In this chapter we revise the relationship of neural stem cells with interstitial cells (mainly telocytes), which we think is significant, and we describe what is known about the juxtacerebral tissue neurogenesis potential.

Isolated human uterine telocytes: immunocytochemistry and electrophysiology of T-type calcium channels.

Recently, telocytes (TCs) were described as a new cell type in the interstitial space of many organs, including myometrium. TCs are cells with very long, distinctive extensions named telopodes (Tps). It is suggested that TCs play a major role in intercellular signaling, as well as in morphogenesis, especially in morphogenetic bioelectrical signaling. However, TC plasma membrane is yet unexplored regarding the presence and activity of ion channels and pumps. Here, we used a combination of in vitro immunofluorescence and patch-clamp technique to characterize T-type calcium channels in TCs. Myometrial TCs were identified in cell culture (non-pregnant and pregnant myometrium) as cells having very long Tps and which were positive for CD34 and platelet-derived growth factor receptor-α. Immunofluorescence analysis of the subfamily of T-type (transient) calcium channels CaV3.1 and CaV3.2 presence revealed the expression of these ion channels on the cell body and Tps of non-pregnant and pregnant myometrium TCs. The expression in TCs from the non-pregnant myometrium is less intense, being confined to the cell body for CaV3.2, while CaV3.1 was expressed both on the cell body and in Tps. Moreover, the presence of T-type calcium channels in TCs from non-pregnant myometrium is also confirmed by applying brief ramp depolarization protocols. In conclusion, our results show that T-type calcium channels are present in TCs from human myometrium and could participate in the generation of endogenous bioelectric signals responsible for the regulation of the surrounding cell behavior, during pregnancy and labor.

Platelet-Rich Plasma in Dermatology: New Insights on the Cellular Mechanism of Skin Repair and Regeneration.

The skin's recognised functions may undergo physiological alterations due to ageing, manifesting as varying degrees of facial wrinkles, diminished tautness, density, and volume. Additionally, these functions can be disrupted (patho)physiologically through various physical and chemical injuries, including surgical trauma, accidents, or chronic conditions like ulcers associated with diabetes mellitus, venous insufficiency, or obesity. Advancements in therapeutic interventions that boost the skin's innate regenerative abilities could significantly enhance patient care protocols. The application of Platelet-Rich Plasma (PRP) is widely recognized for its aesthetic and functional benefits to the skin. Yet, the endorsement of PRP's advantages often borders on the dogmatic, with its efficacy commonly ascribed solely to the activation of fibroblasts by the factors contained within platelet granules. PRP therapy is a cornerstone of regenerative medicine which involves the autologous delivery of conditioned plasma enriched by platelets. This is achieved by centrifugation, removing erythrocytes while retaining platelets and their granules. Despite its widespread use, the precise sequences of cellular activation, the specific cellular players, and the molecular machinery that drive PRP-facilitated healing are still enigmatic. There is still a paucity of definitive and robust studies elucidating these mechanisms. In recent years, telocytes (TCs)-a unique dermal cell population-have shown promising potential for tissue regeneration in various organs, including the dermis. TCs' participation in neo-angiogenesis, akin to that attributed to PRP, and their role in tissue remodelling and repair processes within the interstitia of several organs (including the dermis), offer intriguing insights. Their potential to contribute to, or possibly orchestrate, the skin regeneration process following PRP treatment has elicited considerable interest. Therefore, pursuing a comprehensive understanding of the cellular and molecular mechanisms at work, particularly those involving TCs, their temporal involvement in structural recovery following injury, and the interconnected biological events in skin wound healing and regeneration represents a compelling field of study.

Dermal Telocytes: A Different Viewpoint of Skin Repairing and Regeneration.

Fifteen years after their discovery, telocytes (TCs) are yet perceived as a new stromal cell type. Their presence was initially documented peri-digestively, and gradually throughout the interstitia of many (non-)cavitary mammalian, human, and avian organs, including skin. Each time, TCs proved to be involved in diverse spatial relations with elements of interstitial (ultra)structure (blood vessels, nerves, immune cells, etc.). To date, transmission electron microscopy (TEM) remained the single main microscopic technique able to correctly and certainly attest TCs by their well-acknowledged (ultra)structure. In skin, dermal TCs reiterate almost all (ultra)structural features ascribed to TCs in other locations, with apparent direct implications in skin physiology and/or pathology. TCs' uneven distribution within skin, mainly located in stem cell niches, suggests involvement in either skin homeostasis or dermatological pathologies. On the other hand, different skin diseases involve different patterns of disruption of TCs' structure and ultrastructure. TCs' cellular cooperation with other interstitial elements, their immunological profile, and their changes during remission of diseases suggest their role(s) in tissue regeneration/repair processes. Thus, expanding the knowledge on dermal TCs could offer new insights into the natural skin capacity of self-repairing. Moreover, it would become attractive to consider that augmenting dermal TCs' presence/density could become an attractive therapeutic alternative for treating various skin defects.

Lipid Handling Protein Gene Expression in Colorectal Cancer: CD36 and Targeting miRNAs.

The reprogramming of lipid metabolism has been highlighted in colorectal cancer (CRC) studies, suggesting a critical role for the scavenger receptor CD36 and fatty acid synthase (FASN) in this malignancy. In this study, we analyzed the gene expression levels of CD36, FASN, the cell surface glypican 4 (GPC4), and the two transporters SLC27A3 and SLC27A4 in 39 paired tumoral and peritumoral tissues from patients with CRC compared with 18 normal colonic mucosae. Moreover, the levels of seven miRNAs targeting CD36 and most of the analyzed genes were evaluated. We found a significant impairment of the expression of all the analyzed genes except GPC4 as well as the differential expression of miR-16-5p, miR-26b-5p, miR-107, miR-195-5p, and miR-27a-3p in the colonic mucosa of CRC patients. Interestingly, CD36 and miR-27a-3p were downregulated and upregulated, respectively, in tumoral tissues compared to peritumoral and control tissues, with a significant negative correlation in the group of patients developing lymph node metastasis. Our results sustain the relationship between CRC and fatty acid metabolism and emphasize the importance of related miRNAs in developing new therapeutic strategies.

Serum and Fecal Markers of Intestinal Inflammation and Intestinal Barrier Permeability Are Elevated in Parkinson's Disease.

Parkinson's disease (PD) is characterized by alpha-synuclein misfolding with subsequent intraneuronal amyloid formation and accumulation, low grade neuroinflammatory changes, and selective neurodegeneration. Available evidence suggests that the pathology usually begins in the gut and olfactory mucosa, spreading to the brain via the vagus and olfactory nerves, by a prion-like mechanism. A causal relationship has not been established, but gut dysbiosis is prevalent in PD and may lead to intestinal inflammation and barrier dysfunction. Additionally, epidemiological data indicate a link between inflammatory bowel diseases and PD. Calprotectin and zonulin are markers of intestinal inflammation and barrier permeability, respectively. We evaluated their serum and fecal levels in 22 patients with sporadic PD and 16 unmatched healthy controls. Mean calprotectin was higher in PD, both in serum (14.26 mcg/ml ± 4.50 vs. 5.94 mcg/ml ± 3.80, p = 0.0125) and stool (164.54 mcg/g ± 54.19 vs. 56.19 mcg/g ± 35.88, p = 0.0048). Mean zonulin was also higher in PD serum (26.69 ng/ml ± 3.55 vs. 19.43 ng/ml ± 2.56, p = 0.0046) and stool (100.19 ng/ml ± 28.25 vs. 37.3 ng/ml ± 13.26, p = 0.0012). Calprotectin was above the upper reference limit in 19 PD serums and 6 controls (OR = 10.56, 95% CI = 2.17-51.42, p = 0.0025) and in 20 PD stool samples and 4 controls (OR = 30, 95% CI = 4.75-189.30, p = 0.000045). Increased zonulin was found only in the stool samples of 8 PD patients. Despite the small sample size, our findings are robust, complementing and supporting other recently published results. The relation between serum and fecal calprotectin and zonulin levels and sporadic PD warrants further investigation in larger cohorts.

Macrophages and Stem Cells-Two to Tango for Tissue Repair?

Macrophages (MCs) are present in all tissues, not only supporting homeostasis, but also playing an important role in organogenesis, post-injury regeneration, and diseases. They are a heterogeneous cell population due to their origin, tissue specificity, and polarization in response to aggression factors, depending on environmental cues. Thus, as pro-inflammatory M1 phagocytic MCs, they contribute to tissue damage and even fibrosis, but the anti-inflammatory M2 phenotype participates in repairing processes and wound healing through a molecular interplay with most cells in adult stem cell niches. In this review, we emphasize MC phenotypic heterogeneity in health and disease, highlighting their systemic and systematic contribution to tissue homeostasis and repair. Unraveling the intervention of both resident and migrated MCs on the behavior of stem cells and the regulation of the stem cell niche is crucial for opening new perspectives for novel therapeutic strategies in different diseases.

Sea-Buckthorn Seed Oil Induces Proliferation of both Normal and Dysplastic Keratinocytes in Basal Conditions and under UVA Irradiation.

Past decades demonstrate an increasing interest in herbal remedies in the public eye, with as many as 80% of people worldwide using these remedies as healthcare products, including those for skin health. Sea buckthorn and its derived products (oil; alcoholic extracts), rich in flavonoids and essential fatty acids, are among these healthcare products. Specifically, sea buckthorn and its derivatives are reported to have antioxidant and antitumor activity in dysplastic skin cells. On the other hand, evidence suggests that the alteration of lipid metabolism is related to increased malignant behavior. Given the paradoxical involvement of lipids in health and disease, we investigated how sea-buckthorn seed oil, rich in long-chain fatty acids, modifies the proliferation of normal and dysplastic skin cells in basal conditions, as well as under ultraviolet A (UVA) radiation. Using real-time analysis of normal and dysplastic human keratinocytes, we showed that sea-buckthorn seed oil stimulated the proliferation of dysplastic cells, while it also impaired the ability of both normal and dysplastic cells to migrate over a denuded area. Furthermore, UVA exposure increased the expression of CD36/SR-B2, a long-chain fatty acid translocator that is related to the metastatic behavior of tumor cells.

Skeletal muscle regeneration involves macrophage-myoblast bonding.

Regeneration in adult skeletal muscle relies on the activation, proliferation, and fusion of myogenic precursor cells (MPC), mostly resident satellite cells (SC). However, the regulatory mechanism during this process is still under evaluation, with the final aim to manipulate regeneration when the intrinsic mechanism is corrupted. Furthermore, intercellular connections during skeletal muscle regeneration have not been previously thoroughly documented. Our hypothesis was that a direct and close cellular interaction between SC/MPC and invading myeloid cells is a key step to control regeneration. We tested this hypothesis during different steps of skeletal muscle regeneration: (a) the recruitment of activated SC; (b) the differentiation of MPC; (c) myotubes growth, in a mouse model of crush injury. Samples harvested (3 and 5 days) post-injury were screened by light and confocal microscopy. Ultrastructural analysis was performed by conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) followed by 3D modeling of electron tomography (ET) data. This revealed a new type of interaction between macrophages and myogenic cells by direct heterocellular surface apposition over large areas and long linear distances. In the analyzed volume, regions spaced below 20 nm, within molecular range, represented 31% of the macrophage membrane surface and more than 27% of the myotube membrane. The constant interaction throughout all stages of myogenesis suggests a potential new type of regulatory mechanism for the myogenic process. Thus, deciphering structural and molecular mechanisms of SC-macrophage interaction following injury might open promising perspectives for improving muscle healing.

Targeting CD36 as Biomarker for Metastasis Prognostic: How Far from Translation into Clinical Practice?

Metastasis requires cellular changes related to cell-to-cell and cell-to-matrix adhesion, immune surveillance, activation of growth and survival signalling pathways, and epigenetic modifications. In addition to tumour cells, tumour stroma is also modified in relationship to the primary tumour as well as to distant metastatic sites (forming a metastatic niche). A common denominator of most stromal partners in tumour progression is CD36, a scavenger receptor for fatty acid uptake that modulates cell-to-extracellular matrix attachment, stromal cell fate (for adipocytes, endothelial cells), TGFß activation, and immune signalling. CD36 has been repeatedly proposed as a prognostic marker in various cancers, mostly of epithelial origin (breast, prostate, ovary, and colon) and also for hepatic carcinoma and gliomas. Data gathered in preclinical models of various cancers have shown that blocking CD36 might prove beneficial in stopping metastasis spread. However, targeting the receptor in clinical trials with thrombospondin mimetic peptides has proven ineffective, and monoclonal antibodies are not yet available for patient use. This review presents data to support CD36 as a potential prognostic biomarker in cancer, its current stage towards achieving bona fide biomarker status, and knowledge gaps that must be filled before further advancement towards clinical practice.

Heterocellular molecular contacts in the mammalian stem cell niche.

Adult tissue homeostasis and repair relies on prompt and appropriate intervention by tissue-specific adult stem cells (SCs). SCs have the ability to self-renew; upon appropriate stimulation, they proliferate and give rise to specialized cells. An array of environmental signals is important for maintenance of the SC pool and SC survival, behavior, and fate. Within this special microenvironment, commonly known as the stem cell niche (SCN), SC behavior and fate are regulated by soluble molecules and direct molecular contacts via adhesion molecules providing connections to local supporting cells and the extracellular matrix. Besides the extensively discussed array of soluble molecules, the expression of adhesion molecules and molecular contacts is another fundamental mechanism regulating niche occupancy and SC mobilization upon activation. Some adhesion molecules are differentially expressed and have tissue-specific consequences, likely reflecting the structural differences in niche composition and design, especially the presence or absence of a stromal counterpart. However, the distribution and identity of intercellular molecular contacts for adhesion and adhesion-mediated signaling within stromal and non-stromal SCN have not been thoroughly studied. This review highlights common details or significant differences in cell-to-cell contacts within representative stromal and non-stromal niches that could unveil new standpoints for stem cell biology and therapy.

Oxidative Stress and the Microbiota-Gut-Brain Axis.

The gut-brain axis is increasingly recognized as an important pathway of communication and of physiological regulation, and gut microbiota seems to play a significant role in this mutual relationship. Oxidative stress is one of the most important pathogenic mechanisms for both neurodegenerative diseases, such as Alzheimer's or Parkinson's, and acute conditions, such as stroke or traumatic brain injury. A peculiar microbiota type might increase brain inflammation and reactive oxygen species levels and might favor abnormal aggregation of proteins. Reversely, brain lesions of various etiologies result in alteration of gut properties and microbiota. These recent hypotheses could open a door for new therapeutic approaches in various neurological diseases.

Claudin-1 and occludin expression in demyelinating peripheral neuropathies.

In the last years, only few studies focused on the role of tight junctions in human peripheral nerve. This type of junction is found between apposed membranes of myelinating Schwann cells, between perineurial cells and between endothelial cells (of the epi-, peri- and endo-neurium vessels). We investigated the presence of claudin-1 and occludin in peripheral nerve biopsy of patients with demyelinating peripheral neuropathies by immunolabeling (immunohistochemistry and immunofluorescence) and Western blot. Immunolabeling highlighted claudin-1 expression mostly in the tight junctions of the perineurial cells and in the autotypic junctions of the Schwann cells of the patients with demyelinating peripheral neuropathies. Occludin was mostly expressed in the tight junctions of perineurial cells and endothelial cells and only faintly in autotypic junctions of Schwann cells. The Western blot data showed significant differences in claudin-1 and occludin expression levels in patients with demyelinating peripheral neuropathies versus normal unaffected control. Our results show that autotypic tight junctions molecular composition, like claudin-1 and occludin expression could influence the demyelinating process by altering the permeability of the blood-nerve barrier.