Combined light and electron microscopy (CLEM) to quantify methamphetamine-induced alpha-synuclein-related pathology.

Methamphetamine (METH) produces a cytopathology, which is rather specific within catecholamine neurons both in vitro and ex vivo, in animal models and chronic METH abusers. This led some authors to postulate a sort of parallelism between METH cytopathology and cell damage in Parkinson's disease (PD). In fact, METH increases and aggregates alpha-syn proto-fibrils along with producing spreading of alpha-syn. Although alpha-syn is considered to be the major component of aggregates and inclusions developing within diseased catecholamine neurons including classic Lewy body (LB), at present, no study provided a quantitative assessment of this protein in situ, neither following METH nor in LB occurring in PD. Similarly, no study addressed the quantitative comparison between occurrence of alpha-syn and other key proteins and no investigation measured the protein compared with non-protein structure within catecholamine cytopathology. Therefore, the present study addresses these issues using an oversimplified model consisting of a catecholamine cell line where the novel approach of combined light and electron microscopy (CLEM) was used measuring the amount of alpha-syn, which is lower compared with p62 or poly-ubiquitin within pathological cell domains. The scenario provided by electron microscopy reveals unexpected findings, which are similar to those recently described in the pathology of PD featuring packing of autophagosome-like vesicles and key proteins shuttling autophagy substrates. Remarkably, small seed-like areas, densely packed with p62 molecules attached to poly-ubiquitin within wide vesicular domains occurred. The present data shed new light about quantitative morphometry of catecholamine cell damage in PD and within the addicted brain.

Application of the NSE score (Neurology-Stability-Epidural compression assessment) to establish the need for surgery in spinal metastases of elderly patients: a multicenter investigation.

PURPOSE: This retropective multicentric study aims to investigate the clinical applicability of the NSE score in the elderly, to verify the role of this tool as an easy help for decision making also for this class of patients. METHODS: All elderly patients (> 65 years) suffering from spinal metastases undergoing surgical or non-surgical treatment at the authors' Institutions between 2015 and 2022 were recruited. An agreement group (AG) and non-agreement group (NAG) were identified accordingly to the agreement between the NSE score indication and the performed treatment. Neurological status and axial pain were evaluated for both groups at follow-up (3 and 6 months). The same analysis was conducted specifically grouping patients older than 75 years. RESULTS: A strong association with improvement or preservation of clinical status (p < 0.001) at follow-up was obtained in AG. The association was not statistically significant in NAG at the 3-month follow-up (p 1.00 and 0.07 respectively) and at 6 months (p 0.293 and 0.09 respectively). The group of patients over 75 years old showed similar results in terms of statistical association between the agreement group and better outcomes. CONCLUSION: Far from the need or the aim to build dogmatic algorithms, the goal of preserving a proper performance status plays a key role in a modern oncological management: functional outcomes of the multicentric study group showed that the NSE score represents a reliable tool to establish the need for surgery also for elderly patients.

Is There a Place for Lewy Bodies before and beyond Alpha-Synuclein Accumulation? Provocative Issues in Need of Solid Explanations.

In the last two decades, alpha-synuclein (alpha-syn) assumed a prominent role as a major component and seeding structure of Lewy bodies (LBs). This concept is driving ongoing research on the pathophysiology of Parkinson's disease (PD). In line with this, alpha-syn is considered to be the guilty protein in the disease process, and it may be targeted through precision medicine to modify disease progression. Therefore, designing specific tools to block the aggregation and spreading of alpha-syn represents a major effort in the development of disease-modifying therapies in PD. The present article analyzes concrete evidence about the significance of alpha-syn within LBs. In this effort, some dogmas are challenged. This concerns the question of whether alpha-syn is more abundant compared with other proteins within LBs. Again, the occurrence of alpha-syn compared with non-protein constituents is scrutinized. Finally, the prominent role of alpha-syn in seeding LBs as the guilty structure causing PD is questioned. These revisited concepts may be helpful in the process of validating which proteins, organelles, and pathways are likely to be involved in the damage to meso-striatal dopamine neurons and other brain regions involved in PD.

Damage to the Locus Coeruleus Alters the Expression of Key Proteins in Limbic Neurodegeneration.

The present investigation was designed based on the evidence that, in neurodegenerative disorders, such as Alzheimer's dementia (AD) and Parkinson's disease (PD), damage to the locus coeruleus (LC) arising norepinephrine (NE) axons (LC-NE) is documented and hypothesized to foster the onset and progression of neurodegeneration within target regions. Specifically, the present experiments were designed to assess whether selective damage to LC-NE axons may alter key proteins involved in neurodegeneration within specific limbic regions, such as the hippocampus and piriform cortex, compared with the dorsal striatum. To achieve this, a loss of LC-NE axons was induced by the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) in C57 Black mice, as assessed by a loss of NE and dopamine-beta-hydroxylase within target regions. In these experimental conditions, the amount of alpha-synuclein (alpha-syn) protein levels were increased along with alpha-syn expressing neurons within the hippocampus and piriform cortex. Similar findings were obtained concerning phospho-Tau immunoblotting. In contrast, a decrease in inducible HSP70-expressing neurons and a loss of sequestosome (p62)-expressing cells, along with a loss of these proteins at immunoblotting, were reported. The present data provide further evidence to understand why a loss of LC-NE axons may foster limbic neurodegeneration in AD and limbic engagement during PD.

Percutaneous pedicle screw fixation without arthrodesis of 368 thoracolumbar fractures: long-term clinical and radiological outcomes in a single institution.

PURPOSE: Traumatic thoracolumbar (TL) fractures are the most common vertebral fractures. Although a consensus on the preferred treatment is missing, percutaneous pedicle screw fixation (PPSF) has been progressively accepted as treatment option, since it is related to lower soft tissues surgical-injury and perioperative complications rate. This study aims to evaluate the long-term clinical-radiological outcomes after PPSF for TL fractures at a single tertiary academic hospital. METHODS: This is a retrospective cohort study. Back pain was obtained at preoperative, postoperative and final follow-up using Visual Analog Scale. Patient-reported outcomes, the Oswestry Disability Index and the 36-Item Short Form, were obtained to asses disability during follow-up. Radiological measures included Cobb angle, mid-sagittal index, sagittal index (SI) and vertebral body height loss. A multivariate regression analysis on preoperative radiological features was performed to investigate independent risk factors for implant failure. RESULTS: A total of 296 patients with 368 TL fractures met inclusion criteria. Mean follow-up was 124.3 months. The clinical and radiological parameters significantly improved from preoperative to last follow-up measurements. The multivariate analysis showed that Cobb angle (OR = 1.3, p < 0.001), SI (OR = 1.5, p < 0.001) and number of fractures (OR = 1.1, p = 0.05), were independent risk factors for implant failure. The overall complication rate was 5.1%, while the reoperation rate for implant failure was 3.4%. CONCLUSIONS: In our case series, PPSF for TL injuries demonstrated good long-term clinical-radiological outcomes, along with low complication and reoperation rates. Accordingly, PPSF could be considered as a valuable treatment option for neurologically intact patients with TL fractures. Additionally, in this cohort, number of fractures ≥ 2, Cobb angle ≥ 15° and sagittal index ≥ 21° were independent risk factors for implant failure.

Extreme Lateral Interbody Fusion (XLIF) with Lateral Modular Plate Fixation: Preliminary Report on Clinical and Radiological Outcomes.

The lateral transpsoas approach (extreme lateral interbody fusion, or XLIF) allows surgeons to use various lordotic cage sizes to help restore intervertebral disk height, correct sagittal alignment, and improve fusion rates. The use of standalone devices has consistently raised doubts due to the high risk of complications and inadequate functional recovery that a circumferential arthrodesis can support. The recent introduction of a novel XLIF cage with adapted lateral plate fixation (XLPF) may further enhance the structural rigidity, consolidating the cage and plate into a singular modular entity. Nine patients from our surgical centers underwent a procedure of 1-level XLIF with XLPF in selected cases. We observed that XLPF does not extend the intraoperative footprint and provides immediate rigidity to the anterior column without any additional risk of complications and with minimal increased time compared to the traditional cage implant procedure. Although it has been shown that the use of interbody fusion cages with supplemental posterior fixation improves stabilization in all directions, the technique of standalone lateral cages may also have a place in spine surgery in that the stability may be sufficient in selected cases, such as junctional syndrome and in some forms of degenerative scoliosis.

The Role of Cellular Prion Protein in Glioma Tumorigenesis Could Be through the Autophagic Mechanisms: A Narrative Review.

The carcinogenesis of glial tumors appears complex because of the many genetic and epigenetic phenomena involved. Among these, cellular prion protein (PrPC) is considered a key factor in cell-death resistance and important aspect implicated in tumorigenesis. Autophagy also plays an important role in cell death in various pathological conditions. These two cellular phenomena are related and share the same activation by specific alterations in the cellular microenvironment. Furthermore, there is an interdependence between autophagy and prion activity in glioma tumorigenesis. Glioma is one of the most aggressive known cancers, and the fact that such poorly studied processes as autophagy and PrPC activity are so strongly involved in its carcinogenesis suggests that by better understanding their interaction, more can be understood about its origin and treatment. Few studies in the literature relate these two cellular phenomena, much less try to explain their combined activity and role in glioma carcinogenesis. In this study, we explored the recent findings on the molecular mechanism and regulation pathways of autophagy, examining the role of PrPC in autophagy processes and how they may play a central role in glioma tumorigenesis. Among the many molecular interactions that PrP physiologically performs, it appears that processes shared with autophagy activity are those most implicated in glial tumor carcinogeneses such as activity on MAP kinases, PI3K, and mTOR. This work can be supportive and valuable as a basis for further future studies on this topic.

Post-traumatic intracranial pseudo-aneurysms of posterior circulation: a comprehensive review of an under-diagnosed and rare entity.

Traumatic aneurysms are rare and the total number of cases involving the posterior circulation (TIPC) is even smaller. Traumatic brain injury (TBI) may be responsible not only of rupture in brain aneurysm (BrA) pre-existing to trauma, but it has been identified also as a possible pathogenetic cause of TIPC formation in patients not affected by intracranial vascular lesions. A complete literature review was performed of all reported cases regarding rupture of BrA with SAH resulting from TIPC not previously identified at the first radiological screening. A representative case of a left posterior inferior cerebellar artery (PICA) pseudo-aneurysm caused by left vertebral artery's dissection is reported. We show a unique complete collection of all 34 cases. Despite their rarity, TIPCs are associated with a significant morbidity and mortality rate, as high as 40-60%. Of the 22 patients with good neurological status (64.7%), we did not notice a significant correlation with regard to the location of the aneurysm, type of treatment, or clinical onset. Early recognition of a pseudo-aneurysm and adequate treatment seem to be the most important prognostic factor for these patients. Despite their rarity, TIPCs are associated with a significant morbidity and mortality rate. A TIPC should be suspected in case of delayed deterioration in head-injured patient and should be investigated with angiography. Conservative management is worsened by poor prognosis and the goal of treatment is to exclude the aneurysm from circulation with surgical or endovascular methods as soon as possible.

Alterations of Mitochondrial Structure in Methamphetamine Toxicity.

Recent evidence shows that methamphetamine (METH) produces mitochondrial alterations that contribute to neurotoxicity. Nonetheless, most of these studies focus on mitochondrial activity, whereas mitochondrial morphology remains poorly investigated. In fact, morphological evidence about the fine structure of mitochondria during METH toxicity is not available. Thus, in the present study we analyzed dose-dependent mitochondrial structural alterations during METH exposure. Light and transmission electron microscopy were used, along with ultrastructural stoichiometry of catecholamine cells following various doses of METH. In the first part of the study cell death and cell degeneration were assessed and they were correlated with mitochondrial alterations observed using light microscopy. In the second part of the study, ultrastructural evidence of specific mitochondrial alterations of crests, inner and outer membranes and matrix were quantified, along with in situ alterations of mitochondrial proteins. Neurodegeneration induced by METH correlates significantly with specific mitochondrial damage, which allows definition of a scoring system for mitochondrial integrity. In turn, mitochondrial alterations are concomitant with a decrease in fission/mitophagy protein Fis1 and DRP1 and an increase in Pink1 and Parkin in situ, at the mitochondrial level. These findings provide structural evidence that mitochondria represent both direct and indirect targets of METH-induced toxicity.

Within the Ischemic Penumbra, Sub-Cellular Compartmentalization of Heat Shock Protein 70 Overlaps with Autophagy Proteins and Fails to Merge with Lysosomes.

The brain area which surrounds the frankly ischemic region is named the area penumbra. In this area, most cells are spared although their oxidative metabolism is impaired. area penumbra is routinely detected by immunostaining of a molecule named Heat Shock Protein 70 (HSP70). Within the area penumbra, autophagy-related proteins also increase. Therefore, in the present study, the autophagy-related microtubule-associated protein I/II-Light Chain 3 (LC3) was investigated within the area penumbra along with HSP70. In C57 black mice, ischemia was induced by permanent occlusion of the distal part of the middle cerebral artery. Immunofluorescence and electron microscopy show that LC3 and HSP70 are overexpressed and co-localize within the area penumbra in the same cells and within similar subcellular compartments. In the area penumbra, marked loss of co-localization of HSP70 and LC3-positive autophagy vacuoles, with lysosomal-associated membrane protein 1 (LAMP1) or cathepsin-D-positive lysosome vacuoles occurs. This study indicates that, within the area penumbra, a failure of autophagolysosomes depends on defective compartmentalization of LC3, LAMP1 and cathepsin-D and a defect in merging between autophagosomes and lysosomes. Such a deleterious effect is likely to induce a depletion of autophagolysosomes and cell clearing systems, which needs to be rescued in the process of improving neuronal survival.

Bacopa Protects against Neurotoxicity Induced by MPP+ and Methamphetamine.

The neurotoxins methamphetamine (METH) and 1-methyl-4-phenylpyridinium (MPP+) damage catecholamine neurons. Although sharing the same mechanism to enter within these neurons, METH neurotoxicity mostly depends on oxidative species, while MPP+ toxicity depends on the inhibition of mitochondrial activity. This explains why only a few compounds protect against both neurotoxins. Identifying a final common pathway that is shared by these neurotoxins is key to prompting novel remedies for spontaneous neurodegeneration. In the present study we assessed whether natural extracts from Bacopa monnieri (BM) may provide a dual protection against METH- and MPP+-induced cell damage as measured by light and electron microscopy. The protection induced by BM against catecholamine cell death and degeneration was dose-dependently related to the suppression of reactive oxygen species (ROS) formation and mitochondrial alterations. These were measured by light and electron microscopy with MitoTracker Red and Green as well as by the ultrastructural morphometry of specific mitochondrial structures. In fact, BM suppresses the damage of mitochondrial crests and matrix dilution and increases the amount of healthy and total mitochondria. The present data provide evidence for a natural compound, which protects catecholamine cells independently by the type of experimental toxicity. This may be useful to counteract spontaneous degenerations of catecholamine cells.

Diffuse Axonal Injury: Clinical Prognostic Factors, Molecular Experimental Models and the Impact of the Trauma Related Oxidative Stress. An Extensive Review Concerning Milestones and Advances.

Traumatic brain injury (TBI) is a condition burdened by an extremely high rate of morbidity and mortality and can result in an overall disability rate as high as 50% in affected individuals. Therefore, the importance of identifying clinical prognostic factors for diffuse axonal injury (DAI) in (TBI) is commonly recognized as critical. The aim of the present review paper is to evaluate the most recent contributions from the relevant literature in order to understand how each single prognostic factor determinates the severity of the clinical syndrome associated with DAI. The main clinical factors with an important impact on prognosis in case of DAI are glycemia, early GCS, the peripheral oxygen saturation, blood pressure, and time to recover consciousness. In addition, the severity of the lesion, classified on the ground of the cerebral anatomical structures involved after the trauma, has a strong correlation with survival after DAI. In conclusion, modern findings concerning the role of reactive oxygen species (ROS) and oxidative stress in DAI suggest that biomarkers such as GFAP, pNF-H, NF-L, microtubule associated protein tau, Aß42, S-100ß, NSE, AQP4, Drp-1, and NCX represent a possible critical target for future pharmaceutical treatments to prevent the damages caused by DAI.

Rapamycin Ameliorates Defects in Mitochondrial Fission and Mitophagy in Glioblastoma Cells.

Glioblastoma (GBM) cells feature mitochondrial alterations, which are documented and quantified in the present study, by using ultrastructural morphometry. Mitochondrial impairment, which roughly occurs in half of the organelles, is shown to be related to mTOR overexpression and autophagy suppression. The novelty of the present study consists of detailing an mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission. These phenomena contribute to explain the increase in altered mitochondria reported here. Administration of the mTOR inhibitor rapamycin rescues mitochondrial alterations. In detail, rapamycin induces the expression of genes promoting mitophagy (PINK1, PARKIN, ULK1, AMBRA1) and mitochondrial fission (FIS1, DRP1). This occurs along with over-expression of VPS34, an early gene placed upstream in the autophagy pathway. The topographic stoichiometry of proteins coded by these genes within mitochondria indicates that, a remarkable polarization of proteins involved in fission and mitophagy within mitochondria including LC3 takes place. Co-localization of these proteins within mitochondria, persists for weeks following rapamycin, which produces long-lasting mitochondrial plasticity. Thus, rapamycin restores mitochondrial status in GBM cells. These findings add novel evidence about mitochondria and GBM, while fostering a novel therapeutic approach to restore healthy mitochondria through mTOR inhibition.

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.

Stoichiometric Analysis of Shifting in Subcellular Compartmentalization of HSP70 within Ischemic Penumbra.

The heat shock protein (HSP) 70 is considered the main hallmark in preclinical studies to stain the peri-infarct region defined area penumbra in preclinical models of brain ischemia. This protein is also considered as a potential disease modifier, which may improve the outcome of ischemic damage. In fact, the molecule HSP70 acts as a chaperonine being able to impact at several level the homeostasis of neurons. Despite being used routinely to stain area penumbra in light microscopy, the subcellular placement of this protein within area penumbra neurons, to our knowledge, remains undefined. This is key mostly when considering studies aimed at deciphering the functional role of this protein as a determinant of neuronal survival. The general subcellular placement of HSP70 was grossly reported in studies using confocal microscopy, although no direct visualization of this molecule at electron microscopy was carried out. The present study aims to provide a direct evidence of HSP70 within various subcellular compartments. In detail, by using ultrastructural morphometry to quantify HSP70 stoichiometrically detected by immuno-gold within specific organelles we could compare the compartmentalization of the molecule within area penumbra compared with control brain areas. The study indicates that two cell compartments in control conditions own a high density of HSP70, cytosolic vacuoles and mitochondria. In these organelles, HSP70 is present in amount exceeding several-fold the presence in the cytosol. Remarkably, within area penumbra a loss of such a specific polarization is documented. This leads to the depletion of HSP70 from mitochondria and mostly cell vacuoles. Such an effect is expected to lead to significant variations in the ability of HSP70 to exert its physiological roles. The present findings, beyond defining the neuronal compartmentalization of HSP70 within area penumbra may lead to a better comprehension of its beneficial/detrimental role in promoting neuronal survival.

EGFR amplification is a real independent prognostic impact factor between young adults and adults over 45yo with wild-type glioblastoma?

BACKGROUND: In 2019 a group of University of Pennsylvania (Hoffman et al., J Neurooncol 145: 321-328, 2019) aimed to explore the prognostic impact of expression of epidermal growth factor receptor (EGFR), one of the most common genetic alterations in WT-GBM, in young adults with IDH-WT GBM, suggesting an inferior outcomes in young adults (< 45yo) with newly diagnosed, IDH-WT GBM. At the same time, our group were considering the dimension of this subpopulation treated in our centre, and we performed the same analysis, comparing datas with affected elderly adults. METHODS: We explore the prognostic impact of EGFR expression status in young adults with IDH-WT GBM, and compare this impact with the affected elderly adults. We therefore analyzed clinical characteristics, tumor genetics, and clinical outcomes in a cohort of adults aged 18-45 years with newly diagnosed WT GBM. We selected a total of 146 patients affected by newly diagnosed IDH-WT GBM who underwent surgery, radiation, and chemotherapy in our Institution in the period ranging between January 2014 and December 2016. We focused primarily on the prognostic impact of EGFR expression. RESULTS: We confirmed through a Bivariate Analysis that the Age of the Patients, the Volume of the lesions, were statistically strongly associated with the survival parameters; The general OS of the cohort presented a breakthrough point between the patients who were respectively younger and older than 45 years, EGFR mutation was per se not associated to a survival reduction in all the cohort patients. When analyzing exclusively the Survival parameters of the patients whose age was under 40, it was possible to outline a non statistically significant trend towards a lesser OS in younger patients harboring an EGFR expression. CONCLUSIONS: Once again the main difference in terms of OS in GBM is shown in a EOR and in Age. To our knowledge, ours is the second study (Hoffman et al., J Neurooncol 145: 321-328, 2019) to evaluate the prognostic impact of EGFR CN gain specifically in young adults with IDH-WT GBM and in the era of modern radiation and Temozolomide, but is the first one to compares this impact with a population of adults over 45, and correlates this date with clinical onset, dimension and localization of disease between this groups. We suggest other centers to evaluate this important finding with a larger number of patients and we are inclined to accept collaborations to increase the power of this study.

Promiscuous Roles of Autophagy and Proteasome in Neurodegenerative Proteinopathies.

Alterations in autophagy and the ubiquitin proteasome system (UPS) are commonly implicated in protein aggregation and toxicity which manifest in a number of neurological disorders. In fact, both UPS and autophagy alterations are bound to the aggregation, spreading and toxicity of the so-called prionoid proteins, including alpha synuclein (α-syn), amyloid-beta (Aß), tau, huntingtin, superoxide dismutase-1 (SOD-1), TAR-DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS). Recent biochemical and morphological studies add to this scenario, focusing on the coordinated, either synergistic or compensatory, interplay that occurs between autophagy and the UPS. In fact, a number of biochemical pathways such as mammalian target of rapamycin (mTOR), transcription factor EB (TFEB), Bcl2-associated athanogene 1/3 (BAG3/1) and glycogen synthase kinase beta (GSk3ß), which are widely explored as potential targets in neurodegenerative proteinopathies, operate at the crossroad between autophagy and UPS. These biochemical steps are key in orchestrating the specificity and magnitude of the two degradation systems for effective protein homeostasis, while intermingling with intracellular secretory/trafficking and inflammatory pathways. The findings discussed in the present manuscript are supposed to add novel viewpoints which may further enrich our insight on the complex interactions occurring between cell-clearing systems, protein misfolding and propagation. Discovering novel mechanisms enabling a cross-talk between the UPS and autophagy is expected to provide novel potential molecular targets in proteinopathies.

mTOR-Related Cell-Clearing Systems in Epileptic Seizures, an Update.

Recent evidence suggests that autophagy impairment is implicated in the epileptogenic mechanisms downstream of mTOR hyperactivation. This holds true for a variety of genetic and acquired epileptic syndromes besides malformations of cortical development which are classically known as mTORopathies. Autophagy suppression is sufficient to induce epilepsy in experimental models, while rescuing autophagy prevents epileptogenesis, improves behavioral alterations, and provides neuroprotection in seizure-induced neuronal damage. The implication of autophagy in epileptogenesis and maturation phenomena related to seizure activity is supported by evidence indicating that autophagy is involved in the molecular mechanisms which are implicated in epilepsy. In general, mTOR-dependent autophagy regulates the proliferation and migration of inter-/neuronal cortical progenitors, synapse development, vesicular release, synaptic plasticity, and importantly, synaptic clustering of GABAA receptors and subsequent excitatory/inhibitory balance in the brain. Similar to autophagy, the ubiquitin-proteasome system is regulated downstream of mTOR, and it is implicated in epileptogenesis. Thus, mTOR-dependent cell-clearing systems are now taking center stage in the field of epilepsy. In the present review, we discuss such evidence in a variety of seizure-related disorders and models. This is expected to provide a deeper insight into the molecular mechanisms underlying seizure activity.

Quantitative Ultrastructural Morphometry and Gene Expression of mTOR-Related Mitochondriogenesis within Glioblastoma Cells.

In glioblastoma (GBM) cells, an impairment of mitochondrial activity along with autophagy suppression occurs. Autophagy suppression in GBM promotes stemness, invasion, and poor prognosis. The autophagy deficit seems to be due, at least in part, to an abnormal up-regulation of the mammalian target of rapamycin (mTOR), which may be counteracted by pharmacological mTORC1 inhibition. Since autophagy activation is tightly bound to increased mitochondriogenesis, a defect in the synthesis of novel mitochondria is expected to occur in GBM cells. In an effort to measure a baseline deficit in mitochondria and promote mitochondriogenesis, the present study used two different GBM cell lines, both featuring mTOR hyperactivity. mTORC1 inhibition increases the expression of genes and proteins related to autophagy, mitophagy, and mitochondriogenesis. Autophagy activation was counted by RT-PCR of autophagy genes, LC3- immune-fluorescent puncta and immune-gold, as well as specific mitophagy-dependent BNIP3 stoichiometric increase in situ, within mitochondria. The activation of autophagy-related molecules and organelles after rapamycin exposure occurs concomitantly with progression of autophagosomes towards lysosomes. Remarkably, mitochondrial biogenesis and plasticity (increased mitochondrial number, integrity, and density as well as decreased mitochondrial area) was long- lasting for weeks following rapamycin withdrawal.

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.