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

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

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

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

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

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

The Effects of Amphetamine and Methamphetamine on the Release of Norepinephrine, Dopamine and Acetylcholine From the Brainstem Reticular Formation

Amphetamine (AMPH) and methamphetamine (METH) are widely abused psychostimulants, which produce a variety of psychomotor, autonomic and neurotoxic effects. The behavioral and neurotoxic effects of both compounds (from now on defined as AMPHs) stem from a fair molecular and anatomical specificity for catecholamine-containing neurons, which are placed in the brainstem reticular formation (RF). In fact, the structural cross-affinity joined with the presence of shared molecular targets between AMPHs and catecholamine provides the basis for a quite selective recruitment of brainstem catecholamine neurons following AMPHs administration. A great amount of investigations, commentary manuscripts and books reported a pivotal role of mesencephalic dopamine (DA)-containing neurons in producing behavioral and neurotoxic effects of AMPHs. Instead, the present review article focuses on catecholamine reticular neurons of the low brainstem. In fact, these nuclei add on DA mesencephalic cells to mediate the effects of AMPHs. Among these, we also include two pontine cholinergic nuclei. Finally, we discuss the conundrum of a mixed neuronal population, which extends from the pons to the periaqueductal gray (PAG). In this way, a number of reticular nuclei beyond classic DA mesencephalic cells are considered to extend the scenario underlying the neurobiology of AMPHs abuse. The mechanistic approach followed here to describe the action of AMPHs within the RF is rooted on the fine anatomy of this region of the brainstem. This is exemplified by a few medullary catecholamine neurons, which play a pivotal role compared with the bulk of peripheral sympathetic neurons in sustaining most of the cardiovascular effects induced by AMPHs

Protective effects of the combination Bifidobacterium longum plus lactoferrin against NSAID-induced enteropathy

Objectives Non-steroidal anti-inflammatory drugs can exert detrimental effects in the lower digestive tract. This study examined the protective effects of a combination of the probiotic Bifidobacterium longum BB536 (Bifidobacterium) with the prebiotic lactoferrin in a rat model of diclofenac-induced enteropathy. Methods Enteropathy was induced in 40-week-old male rats by intragastric diclofenac (4 mg/kg BID, 14 days). Lactoferrin (100 mg/kg BID), Bifidobacterium (2.5\u2022106 CFU/rat BID) or their combination were administered 1 hour before diclofenac. At the end of treatments, the ileum was processed for the evaluation of histological damage, myeloperoxidase (MPO) and malondialdehyde (MDA) levels, as well as the expression of toll-like receptors 2 and 4 (TLR-2/-4) and the activation of downstream signaling molecules (MyD88 and NF-kB p65). Blood hemoglobin and fecal calprotectin were also assessed. Results Diclofenac induced intestinal damage, along with increments of MPO and MDA, overexpression of TLR-2, TLR-4, MyD88 and NF-kB p65, increase in fecal calprotectin and decrease in blood hemoglobin levels. Lactoferrin or Bifidobacterium alone prevented diclofenac-induced enteric damage, and the changes in blood hemoglobin, MPO, MDA, fecal calprotectin and NF-kB p65. Bifidobacterium, but not lactoferrin, decreased TLR-4 expression, while none of them affected MyD88 overexpression. TLR-2 expression was slightly enhanced by all treatments. The combined administration of lactoferrin and Bifidobacterium reduced further the intestinal damage, and restored MPO and blood hemoglobin levels. Conclusions Diclofenac induced ileal mucosal lesions by activation of inflammatory and pro-oxidant mechanisms. These detrimental actions were prevented by the combination of lactoferrin with Bifidobacterium likely through the modulation of TLR-2/-4/NF-kB pro-inflammatory pathways

The Autophagy-Related Organelle Autophagoproteasome Is Suppressed within Ischemic Penumbra

The peri-infarct region, which surrounds the irreversible ischemic stroke area is named ischemic penumbra. This term emphasizes the borderline conditions for neurons placed within such a critical region. Area penumbra separates the ischemic core, where frank cell loss occurs, from the surrounding healthy brain tissue. Within such a brain region, nervous matter, and mostly neurons are impaired concerning metabolic conditions. The classic biochemical marker, which reliably marks area penumbra is the over-expression of the heat shock protein 70 (HSP70). However, other proteins related to cell clearing pathways are modified within area penumbra. Among these, autophagy proteins like LC3 increase in a way, which recapitulates Hsp70. In contrast, components, such as P20S, markedly decrease. Despite apparent discrepancies, the present study indicates remarkable overlapping between LC3 and P20S redistribution within area penumbra. In fact, the amount of both proteins is markedly reduced within vacuoles. Specifically, a massive loss of LC3 + P20S immuno-positive vacuoles (autophagoproteasomes) is reported here. This represents the most relevant sub-cellular alteration here described in cell clearing pathways within area penumbra. The functional significance of these findings remains to be determined and it will take a novel experimental stream to decipher the fine-tuning of such a phenomenon