Effetti della rapamicina in un modello in vitro di glioblastoma multiforme

I gliomi sono neoplasie che originano dalle cellule gliali (astrociti e oligodendrociti) e, tra i tumori primitivi dell’encefalo, sono le forme più frequenti. Tra questi il glioblastoma (GB, astrocitoma di grado IV) è la forma più aggressiva, caratterizzata da molteplici alterazioni molecolari, particolare tendenza all’invasione ed elevata resistenza a chemioterapia e radioterapia. Dal punto di vista istologico il GB è costituito da una popolazione eterogenea di cellule tumorali scarsamente differenziate e presenta regioni dense di focolai necrotici “a pseudo-palizzata”. Inoltre mostra intensa proliferazione microvascolare e infiltrazione nei tessuti circostanti, entrambe sostenute dalla presenza di nicchie contenenti cellule staminali/progenitrici del GB (GSPCs). A livello molecolare, recidiva e infiltrazione sono correlati con l’incremento del complesso molecolare mTOR (mammalian Target Of Rapamycin), dotato di attività chinasica che controlla la vitalità cellulare. In particolare, mTOR agisce da modulatore negativo dell’autofagia, la principale via di rimozione di macromolecole ed organelli danneggiati, la cui alterazione è alla base di molte malattie. Sebbene sia noto da tempo che la rapamicina e i suoi analoghi strutturali agiscono come potenti inibitori di mTOR, non vi sono studi definitivi riguardanti il loro effetto sul GB. A tale scopo nel presente studio si è valutato l’effetto indotto da varie dosi di rapamicina in un modello sperimentale in vitro di GB, utilizzando una linea cellulare di glioma umano (U87MG). A queste cellule è stata somministrata rapamicina a varie concentrazioni, da 1 fino a 1000 nM, per un tempo di esposizione di 24 ore. I risultati ottenuti hanno evidenziato che, a basse dosi, la rapamicina non ha nessun effetto sulla vitalità cellulare, mentre alte dosi sono citotossiche. In particolare, la rapamicina induce morte cellulare in maniera dose-dipendente a partire da 10 nM, fino a raggiungere un plateau alla dose di 100 nM (50% di morte cellulare). Dosi di rapamicina comprese tra 1 nM e 10 nM promuovono invece differenziamento cellulare. Questo effetto è stato valutato sia dal punto di vista istologico che immunocitochimico. In particolare, all’aumentare della concentrazione di rapamicina si osservano modificazioni della morfologia cellulare, che consistono nell’aumento del numero e della lunghezza dei prolungamenti cellulari. Allo stesso tempo, dosi crescenti di rapamicina provocano una riduzione dell’immunopositività per gli antigeni di staminalità (nestina) e un aumento dell’immunopositività per antigeni di differenziamento neuronale (β-tubulina). Infine è stata misurata l’espressione di α-sinucleina, un substrato dell’autofagia (mTOR-dipendente), che rappresenta un marker in numerose malattie neurodegenerative. L’espressione dell’α-sinucleina è stata indagata attraverso western-blot e immunocitochimica. Il trattamento con rapamicina promuove una riduzione dose-dipendente della proteina. Questi risultati sono stati confermati da studi preliminari, in corso di completamento in un modello in vivo di GB. In questo modello basse dosi di rapamicina, prolungano la sopravvivenza e inibiscono la crescita tumorale

Egas Moniz: 90 years (1927-2017) from cerebral angiography

In June 2017 we celebrate the 90th anniversary of the pioneer discovery of cerebral angiography, the seminal imaging technique used for visualizing cerebral blood vessels and vascular alterations as well as other intracranial disorders. Egas Moniz (1874-1955) was the first to describe the use of this revolutionary technique which, until 1975 (when computed tomography, CT, scan was introduced in the clinical practice), was the sole diagnostic tool to provide an imaging of cerebral vessels and therefore alterations due to intracranial pathology. Moniz introduced in the clinical practice this fundamental and important diagnostic tool. The present contribution wishes to pay a tribute to the Portuguese neurosurgeon, who was also a distinguished neurologist and statesman. Despite his tremendous contribution in modern brain imaging, Egas Moniz was awarded the Nobel Prize in Physiology or Medicine in 1949 for prefrontal leucotomy, the neurosurgical intervention nowadays unacceptable, but should rather be remembered for his key contribution to modern brain imaging. KEYWORDS

MTOR modulates intercellular signals for enlargement and infiltration in glioblastoma multiforme

Recently, exosomal release has been related to the acquisition of a malignant phenotype in glioblastoma cancer stem cells (GSCs). Remarkably, intriguing reports demonstrate that GSC-derived extracellular vesicles (EVs) contribute to glioblastoma multiforme (GBM) tumorigenesis via multiple pathways by regulating tumor growth, infiltration, and immune invasion. In fact, GSCs release tumor-promoting macrovesicles that can disseminate as paracrine factors to induce phenotypic alterations in glioma-associated parenchymal cells. In this way, GBM can actively recruit different stromal cells, which, in turn, may participate in tumor microenvironment (TME) remodeling and, thus, alter tumor progression. Vice versa, parenchymal cells can transfer their protein and genetic contents to GSCs by EVs; thus, promoting GSCs tumorigenicity. Moreover, GBM was shown to hijack EV-mediated cell-to-cell communication for self-maintenance. The present review examines the role of the mammalian Target of Rapamycin (mTOR) pathway in altering EVs/exosome-based cell-to-cell communication, thus modulating GBM infiltration and volume growth. In fact, exosomes have been implicated in GSC niche maintenance trough the modulation of GSCs stem cell-like properties, thus, affecting GBM infiltration and relapse. The present manuscript will focus on how EVs, and mostly exosomes, may act on GSCs and neighbor non tumorigenic stromal cells to modify their expression and translational profile, while making the TME surrounding the GSC niche more favorable for GBM growth and infiltration. Novel insights into the mTOR-dependent mechanisms regulating EV-mediated intercellular communication within GBM TME hold promising directions for future therapeutic applications

The effects of proteasome on baseline and methamphetamine-dependent dopamine transmission.

Abstract The Ubiquitin Proteasome System (UPS) is a major multi-catalytic machinery, which guarantees cellular proteolysis and turnover. Beyond cytosolic and nuclear cell compartments, the UPS operates at the synapse to modulate neurotransmission and plasticity. In fact, dysregulations of the UPS are linked with early synaptic alterations occurring in a variety of dopamine (DA)-related brain disorders. This is the case of psychiatric conditions such as methamphetamine (METH) addiction. While being an extremely powerful DA releaser, METH impairs UPS activity, which is largely due to DA itself. In turn, pre- and post- synaptic neurons of the DA circuitry show a high vulnerability to UPS inhibition. Thus, alterations of DA transmission and UPS activity are intermingled within a chain of events underlying behavioral alterations produced by METH. These findings, which allow escaping the view of a mere implication of the UPS in protein toxicity-related mechanisms, indicate a more physiological role for the UPS in modulating DA-related behavior. This is seminal for those plasticity mechanisms which underlie overlapping psychiatric disorders such as METH addiction and schizophrenia

The multi‐faceted effect of curcumin in glioblastoma from rescuing cell clearance to autophagy‐independent effects

The present review focuses on the multi‐faceted effects of curcumin on the neurobiology glioblastoma multiforme (GBM), with a special emphasis on autophagy (ATG)‐dependent molecular pathways activated by such a natural polyphenol. This is consistent with the effects of curcumin in a variety of experimental models of neurodegeneration, where the molecular events partially overlap with GBM. In fact, curcumin broadly affects various signaling pathways, which are similarly affected in cell degeneration and cell differentiation. The antitumoral effects of curcumin include growth inhibition, cell cycle arrest, anti‐migration and anti‐invasion, as well as chemo‐ and radio‐sensitizing activity. Remarkably, most of these effects rely on mammalian target of rapamycin (mTOR)‐dependent ATG induction. In addition, curcumin targets undifferentiated and highly tumorigenic GBM cancer stem cells (GSCs). When rescuing ATG with curcumin, the tumorigenic feature of GSCs is suppressed, thus counteracting GBM establishment and growth. It is noteworthy that targeting GSCs may also help overcome therapeutic resistance and reduce tumor relapse, which may lead to a significant improvement of GBM prognosis. The present review focuses on the multi‐faceted effects of curcumin on GBM neurobiology, which represents an extension to its neuroprotective efficacy

mTOR-Dependent Cell Proliferation in the Brain

The mammalian Target of Rapamycin (mTOR) is a molecular complex equipped with kinase activity which controls cell viability being key in the PI3K/PTEN/Akt pathway. mTOR acts by integrating a number of environmental stimuli to regulate cell growth, proliferation, autophagy, and protein synthesis. These effects are based on the modulation of different metabolic pathways. Upregulation of mTOR associates with various pathological conditions, such as obesity, neurodegeneration, and brain tumors. This is the case of high-grade gliomas with a high propensity to proliferation and tissue invasion. Glioblastoma Multiforme (GBM) is a WHO grade IV malignant, aggressive, and lethal glioma. To date, a few treatments are available although the outcome of GBM patients remains poor. Experimental and pathological findings suggest that mTOR upregulation plays a major role in determining an aggressive phenotype, thus determining relapse and chemoresistance. Among several activities, mTOR-induced autophagy suppression is key in GBM malignancy. In this article, we discuss recent evidence about mTOR signaling and its role in normal brain development and pathological conditions, with a special emphasis on its role in GBM

Ambiguous Effects of Autophagy Activation Following Hypoperfusion/Ischemia

Autophagy primarily works to counteract nutrient deprivation that is strongly engaged during starvation and hypoxia, which happens in hypoperfusion. Nonetheless, autophagy is slightly active even in baseline conditions, when it is useful to remove aged proteins and organelles. This is critical when the mitochondria and/or proteins are damaged by toxic stimuli. In the present review, we discuss to that extent the recruitment of autophagy is beneficial in counteracting brain hypoperfusion or, vice-versa, its overactivity may per se be detrimental for cell survival. While analyzing these opposite effects, it turns out that the autophagy activity is likely not to be simply good or bad for cell survival, but its role varies depending on the timing and amount of autophagy activation. This calls for the need for an appropriate autophagy tuning to guarantee a beneficial effect on cell survival. Therefore, the present article draws a theoretical pattern of autophagy activation, which is hypothesized to define the appropriate timing and intensity, which should mirrors the duration and severity of brain hypoperfusion. The need for a fine tuning of the autophagy activation may explain why confounding outcomes occur when autophagy is studied using a rather simplistic approach

The Role of Cellular Prion Protein in Promoting Stemness and Differentiation in Cancer

Cellular prion protein (PrPC) is seminal to modulate a variety of baseline cell functions to grant homeostasis. The classic role of such a protein was defined as a chaperone-like molecule being able to rescue cell survival. Nonetheless, PrPC also represents the precursor of the deleterious misfolded variant known as scrapie prion protein (PrPSc). This variant is detrimental in a variety of prion disorders. This multi-faceted role of PrP is greatly increased by recent findings showing how PrPC in its folded conformation may foster tumor progression by acting at multiple levels. The present review focuses on such a cancer-promoting effect. The manuscript analyzes recent findings on the occurrence of PrPC in various cancers and discusses the multiple effects, which sustain cancer progression. Within this frame, the effects of PrPC on stemness and differentiation are discussed. A special emphasis is provided on the spreading of PrPC and the epigenetic effects, which are induced in neighboring cells to activate cancer-related genes. These detrimental effects are further discussed in relation to the aberrancy of its physiological and beneficial role on cell homeostasis. A specific paragraph is dedicated to the role of PrPC beyond its effects in the biology of cancer to represent a potential biomarker in the follow up of patients following surgical resection

Parallelism between central and enteric nervous system damage in experimental parkinsonism

Parkinson’s disease (PD) is a neurodegenerative condition which affects dopaminergic neurons of the substantia nigra (SN), leading to a movement disorder. Non motor alterations occur in several viscera, in particular the gastrointestinal tract. In 9-week old C57BL mice we examined the effects of the parkinsonism-inducing neurotoxin 1-methyl, 4-phenyl, 1,2,3,6,-tetrahydropyridine (MPTP, administered either acutely or chronically) in SN and striatum, as well as in duodenum. Motor tests (open field and PaGE) were performed. One week after treatment with MPTP (acute: 20 mg/KgX3, 2h apart or chonic: 5 mg/kg x2/die, for 3 weeks), histological investigations, immunohistochemistry and immunoblotting for tyrosine hydroxylase (TH), and α-synuclein (α-syn) were carried out. Immunocytochemical investigations were analyzed under electron microscopy. Motor tests showed a failure of the PaGE test in all MPTP-treated animals, whereas no difference was found in open field test in comparison with controls. Analysis of histological sections showed some alterations consisting of slight atrophy of duodenal mucosa and glandular disarrangement only after chronic treatment. Under electron microscopy the brush border appeared discontinuous. In all MPTP-administered mice, TH immunopositivity was reduced in SN and striatum, confirming its central dopaminergic neurotoxicity. At duodenal level, TH immunostaining was lost following all MPTP treatments with a slight variation in chronic compared with acute administrations. This was confirmed by semiquantitative immunoblotting. Moreover, α-syn immunostaining was enhanced by MPTP treatment but this was way more evident following chronic administration both at central and peripheral level. Following chronic treatment α-Syn immunopositive structures were investigated under electron microscopy. Our study shows that chronic more than acute administration of MPTP induces alterations at duodenal level reminiscent of dopaminergic damage in SN and striatum. Moreover, this experimental model of parkinsonism features gastrointestinal dysfunction observed in PD patients. These findings lend substance to the concept of the enteric nervous system as a double brain which recapitulates and is an ancestry of the central nervous system

The Neuroanatomy of the Reticular Nucleus Locus Coeruleus in Alzheimer's Disease.

Alzheimer's Disease (AD) features the accumulation of β-amyloid and Tau aggregates, which deposit as extracellular plaques and intracellular neurofibrillary tangles (NFTs), respectively. Neuronal Tau aggregates may appear early in life, in the absence of clinical symptoms. This occurs in the brainstem reticular formation and mostly within Locus Coeruleus (LC), which is consistently affected during AD. LC is the main source of forebrain norepinephrine (NE) and it modulates a variety of functions including sleep-waking cycle, alertness, synaptic plasticity, and memory. The iso-dendritic nature of LC neurons allows their axons to spread NE throughout the whole forebrain. Likewise, a prion-like hypothesis suggests that Tau aggregates may travel along LC axons to reach out cortical neurons. Despite this timing is compatible with cross-sectional studies, there is no actual evidence for a causal relationship between these events. In the present mini-review, we dedicate special emphasis to those various mechanisms that may link degeneration of LC neurons to the onset of AD pathology. This includes the hypothesis that a damage to LC neurons contributes to the onset of dementia due to a loss of neuroprotective effects or, even the chance that, LC degenerates independently from cortical pathology. At the same time, since LC neurons are lost in a variety of neuropsychiatric disorders we considered which molecular mechanism may render these brainstem neurons so vulnerable