Peculiar Localization Research Articles

Effects of Anionic Liposome Delivery of All-Trans-Retinoic Acid on Neuroblastoma Cell Differentiation.

All-trans-retinoic acid (ATRA) has long been known to affect cell growth and differentiation. To improve ATRA's therapeutic efficacy and pharmacodynamics, several delivery systems have been used. In this study, free ATRA and anionic-liposome-encapsulated ATRA were compared for their effects on SK-N-SH human neuroblastoma cell growth and differentiation. Anionic liposomes made of L-α-phosphatidylcholine (PC) and L-α-phosphatidic acid (PA), empty (PC-PA) and loaded with ATRA (PC-PA-ATRA), were characterized by dynamic light scattering (DLS) and electrophoretic mobility measurements, and drug entrapment efficiency (EE%) was measured to evaluate the applicability of the new colloidal formulation. The results of brightfield microscopy and cell growth curves indicated that ATRA, whether free or encapsulated, reduced growth and induced differentiation, resulting in SK-N-SH cells changing from epithelioid to neuronal-like morphologies, and producing a significant increase in neurite growth. To further characterize the neuro-differentiation of SK-N-SH cells, the expression of βIII-Tubulin and synaptophysin and mitochondria localization were analyzed via immunofluorescence. Increased expression of neuronal markers and a peculiar localization of mitochondria in the neuritic extensions were apparent both in ATRA- and PC-PA-ATRA-differentiated cells. As a whole, our results strongly indicate that ATRA treatment, by any means, can induce the differentiation of parent SK-N-SH, and they highlight that its encapsulation in anionic liposomes increases its differentiation ability in terms of the percentage of neurite-bearing cells. Interestingly, our data also suggest an unexpected differentiation capability of anionic liposomes per se. This work highlights the importance of developing and carefully testing novel delivery nanocarriers, which are a necessary first "step" in the development of new therapeutic settings.

G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes.

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

Impact of 5-HT6 Receptor Subcellular Localization on Its Signaling and Its Pathophysiological Roles.

The serotonin (5-HT)6 receptor still raises particular interest given its unique spatio-temporal pattern of expression among the serotonin receptor subtypes. It is the only serotonin receptor specifically expressed in the central nervous system, where it is detected very early in embryonic life and modulates key neurodevelopmental processes, from neuronal migration to brain circuit refinement. Its predominant localization in the primary cilium of neurons and astrocytes is also unique among the serotonin receptor subtypes. Consistent with the high expression levels of the 5-HT6 receptor in brain regions involved in the control of cognitive processes, it is now well-established that the pharmacological inhibition of the receptor induces pro-cognitive effects in several paradigms of cognitive impairment in rodents, including models of neurodevelopmental psychiatric disorders and neurodegenerative diseases. The 5-HT6 receptor can engage several signaling pathways in addition to the canonical Gs signaling, but there is still uncertainty surrounding the signaling pathways that underly its modulation of cognition, as well as how the receptor's coupling is dependent on its cellular compartmentation. Here, we describe recent findings showing how the proper subcellular localization of the receptor is achieved, how this peculiar localization determines signaling pathways engaged by the receptor, and their pathophysiological influence.

Commentary on “Type II Toxin-antitoxin Systems Have a Peculiar Localization in Escherichia Coli Cells

Bacterial Toxin-Antitoxin (TA) modules were extensively studied. They are located on extra chromosomal genetic elements as well as on bacterial chromosomes. They carry two genes. One encoding for the toxin, and the other for the antitoxin. The toxin is a stable protein, while the antitoxin is either RNA or a labile protein.

Abstract 903: Metformin antitumoral activity is exclusively mediated by the membrane functional expression of the Chloride Intracellular Channel 1 in glioblastoma stem cells

Abstract Glioblastoma (GB) is the deadliest brain tumor, representing a major clinical challenge in the field. Despite standard of care together with cutting-edge strategies, life expectancy stands invariantly poor. Tumor heterogeneity and its high-grade of invasiveness make it difficult to be surgically resected, and recurrence is inevitable. There is scientific consensus that tumor relapse originates from progenitor/stem-like cells known as glioblastoma stem cells (GSCs) displaying properties of self-renewal and multi-lineage differentiation that contribute to tumor mass heterogeneity. Thus, GSCs represent the target of newly designed therapies. In recent years, it has been highlighted the anti-cancer properties of metformin, the first-line drug in Type-2 diabetes (T2D) treatment. Metformin assumption in T2D patients has been positively linked with a decrease in the risk of several cancer types and cancer-related mortality, including glioblastoma. Additionally, numerous studies demonstrated metformin's anticancer properties against GSCs. Despite metformin's mechanism of action on cancer cells has not been clarified yet, its effect has been proposed to target the oxidative phosphorylation (OXPHOS) pathway. Considering that GSCs have been reported to shuttle between glycolysis and OXPHOS energetic pathways, their metabolic plasticity should prevent the antiproliferative effect of metformin. However, metformin-treated GSCs show a phenotype superimposable to that caused by the blockage of the transmembrane form of the chloride intracellular channel 1 (tmCLIC1). tmCLIC1 involvement in the progression of GB has been assessed deeply in vitro as well as in vivo. In addition, its peculiar localization and enrichment on GSCs' membrane renders it a valuable pharmacological target for GB. Here, we show that in glioblastoma, the transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) works as a privileged and unique metformin membrane receptor. Nuclear magnetic resonance experiments on live cells confirm the binding to tmCLIC1. Metformin impairs tmCLIC1 activity by a specific binding on a single amino acid, the arginine29. Its mutation demonstrates that metformin-depending tmCLIC1 block is prevented, failing to reduce glioblastoma cells proliferation in 2D and 3D models as well. Moreover, in vivo experiments on zebrafish embryos closely mirror the behavior observed in vitro. Our results demonstrate that tmCLIC1 is essential for metformin antineoplastic effect in glioblastoma and prove a direct binding between the drug and its target. Considering tmCLIC1 contribution to glioblastoma progression, the present work paves the way for future investigations intended to develop a strategy aimed to enhance metformin-tmCLIC1 interaction. Ivan Verduci was supported by a AIRC fellowship for Italy. Citation Format: Ivan Verduci, Francesca Cianci, Riccardo Cazzoli, Gaetano Cannavale, Stefania Castiglione, Matteo Ranucci, Luca Palloni, Beatrice Balboni, Stefania Faletti, Stefania Girotto, Giuliana Pelicci, Saverio Minucci, Alessandro Fantin, Tullio Florio, Michele Mazzanti. Metformin antitumoral activity is exclusively mediated by the membrane functional expression of the Chloride Intracellular Channel 1 in glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 903.

Amphiphilicity-Controlled Localization of Red Emitting Bicationic Fluorophores in Tumor Cells Acting as Bio-Probes and Anticancer Drugs.

Small organic molecules arouse lively interest for their plethora of possible biological applications, such as anticancer therapy, for their ability to interact with nucleic acids, or bioimaging, thanks to their fluorescence emission. Here, a panchromatic series of styryl-azinium bicationic dyes, which have already proved to exhibit high water-solubility and significant red fluorescence in water, were investigated through spectrofluorimetric titrations to assess the extent of their association constants with DNA and RNA. Femtosecond-resolved transient absorption spectroscopy was also employed to characterize the changes in the photophysical properties of these fluorophores upon interaction with their biological targets. Finally, in vitro experiments conducted on tumor cell lines revealed that some of the bicationic fluorophores had a peculiar localization within cell nuclei exerting important antiproliferative effects, others were instead found to localize in the cytoplasm without leading to cell death, being useful to mark specific organelles in light of live cell bioimaging. Interestingly, this molecule-dependent behavior matched the different amphiphilicity featured by these bioactive compounds, which are thus expected to be caught in a tug-of-war between lipophilicity, ensured by the presence of aromatic rings and needed to pass cell membranes, and hydrophilicity, granted by charged groups and necessary for stability in aqueous media.

All that reddens is not psoriasis: a peculiar localization of pigmented purpuric dermatosis in two psoriatic patients.

article: All that reddens is not psoriasis: a peculiar localization of pigmented purpuric dermatosis in two psoriatic patients - Italian Journal of Dermatology and Venereology 2022 August;157(4):376-7 - Minerva Medica - Journals

Spatial distribution of single guest molecules along thickness of thin films of poly(2-hydroxyethyl acrylate).

We have investigated three-dimensional distribution and diffusion behaviors of single guest dyes in 1-µm thick films of poly(2-hydroxyethyl acrylate) (PHEA) by using astigmatism imaging method. Perylene diimide derivative (BP-PDI) in the PHEA films localized along the Z-axis at ca. Z = 600-700nm distant from the interface (Z = 0) between PHEA and glass substrate. This Z-localization was not observed in different polymer films of poly(methyl methacrylate) (PMMA), poly(methyl acrylate) (PMA), and polystyrene (PSt). Because the glass transition temperature of the PHEA is lower than the room temperature, BP-PDI in the PHEA films exhibited Brownian motion, normal diffusion on the XY plane and confined motion along the Z-direction. For elucidating the mechanism of the peculiar localization of the guest dyes along film thickness in the PHEA films, we measured diffusion behaviors of different dyes, R6G and Atto 488, in 1-µm thick PHEA films, obtaining result that the Z-distributions of the dyes were overall similar to that of BP-PDI. The result indicates that the Z-localization of the guest dyes should be ascribed not to the interaction between glass surface and guest dye but mainly to the Z-dependent property of the PHEA film. Indeed, the lateral diffusion coefficients of the guest dyes depended on their Z-positions.

Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles.

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Combining Electrospray Mass Spectrometry (ESI-MS) and Computational Techniques in the Assessment of G-Quadruplex Ligands: A Hybrid Approach to Optimize Hit Discovery.

Guanine-rich sequences forming G-quadruplexes (GQs) are present in several genomes, ranging from viral to human. Given their peculiar localization, the induction of GQ formation or GQ stabilization with small molecules represents a strategy for interfering with crucial biological functions. Investigating the recognition event at the molecular level, with the aim of fully understanding the triggered pharmacological effects, is challenging. Native electrospray ionization mass spectrometry (ESI-MS) is being optimized to study these noncovalent assemblies. Quantitative parameters retrieved from ESI-MS studies, such as binding affinity, the equilibrium binding constant, and sequence selectivity, will be overviewed. Computational experiments supporting the ESI-MS investigation and boosting its efficiency in the search for GQ ligands will also be discussed with practical examples. The combination of ESI-MS and in silico techniques in a hybrid high-throughput-screening workflow represents a valuable tool for the medicinal chemist, providing data on the quantitative and structural aspects of ligand–GQ interactions.

Abstract 3079: The antitumoral activity of metformin in glioblastoma stem cells is mediated by tmCLIC1 protein

Abstract Glioblastoma (GB) is the deadliest brain tumor, representing a major clinical challenge in the field. Despite standard of care together with cutting-edge strategies, life expectancy stands invariantly poor. Tumor heterogeneity and its high-grade of invasiveness make it difficult to be surgically resected and recurrence is inevitable. There is scientific consensus that tumor relapse originates from progenitor/stem-like cells known as glioblastoma stem cells (GSCs) displaying properties of self-renewal and multi-lineage differentiation that contribute to tumor mass heterogeneity. Thus, GSCs represent the target of newly designed therapies. In recent years, it has been highlighted the anti-cancer properties of metformin, the first-line drug in Type-2 diabetes (T2D) treatment. Metformin assumption in T2D patients has been positively linked with a decrease in the risk of several cancer types and cancer-related mortality, including glioblastoma. Additionally, numerous studies demonstrated metformin's anticancer properties against GSCs. Despite metformin's mechanism of action on cancer cells has not been clarified yet, its effect has been proposed to target the oxidative phosphorylation (OXPHOS) pathway. Considering that GSCs have been reported to shuttle between glycolysis and OXPHOS energetic pathways, their metabolic plasticity should prevent the antiproliferative effect of metformin. However, metformin-treated GSCs show a phenotype superimposable to that caused by the blockage of the transmembrane form of the chloride intracellular channel 1 (tmCLIC1). tmCLIC1 involvement in the progression of GB has been assessed deeply in vitro as well as in vivo. In addition, its peculiar localization and enrichment on GSCs' membrane renders it a valuable pharmacological target for GB. The goal of the present work is to attest tmCLIC1 as the solely interactor with metformin in glioblastoma stem cells. Ivan Verduci was supported by a AIRC fellowship for Italy. Citation Format: Ivan Verduci, Francesca Cianci, Gaetano Cannavale, Stefania Castiglione, Matteo Ranucci, Michele Mazzanti. The antitumoral activity of metformin in glioblastoma stem cells is mediated by tmCLIC1 protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3079.

Cytokinesis: An Anillin-RhoGEF Module Sets the Stage for Septin Double Ring Assembly.

In many eukaryotes septin filaments form an hourglass-like structure at the division site that rearranges into a double ring at cytokinesis. A new study elucidates how an anillin-RhoGEF complex guides the assembly of the septin double ring in budding yeast.

When bacteria meet mitochondria: The strange case of the tick symbiont Midichloria mitochondrii†.

Mitochondria are key eukaryotic organelles that perform several essential functions. Not surprisingly, many intracellular bacteria directly or indirectly target mitochondria, interfering with innate immunity, energy production or apoptosis, to make the host cell a more hospitable niche for bacterial replication. The alphaproteobacterium Midichloria mitochondrii has taken mitochondrial targeting to another level by physically colonising mitochondria, as shown by transmission electron micrographs of bacteria residing in the mitochondrial intermembrane space. This unique localization provokes a number of questions around the mechanisms allowing, and reasons driving intramitochondrial tropism. We suggest possible scenarios that could lead to this peculiar localization and hypothesize potential costs and benefits of mitochondrial colonisation for the bacterium and its host.

The Microtubule-Depolymerizing Kinesin-13 Klp10A Is Enriched in the Transition Zone of the Ciliary Structures of Drosophila melanogaster.

The precursor of the flagellar axoneme is already present in the primary spermatocytes of Drosophila melanogaster. During spermatogenesis each primary spermatocyte shows a centriole pair that moves to the cell membrane and organizes an axoneme-based structure, the cilium-like region (CLR). The CLRs persist through the meiotic divisions and are inherited by young spermatids. During spermatid differentiation the ciliary caps elongate giving rise to the sperm axoneme. Mutations in Klp10A, a kinesin-13 of Drosophila, results in defects of centriole/CLR organization in spermatocytes and of ciliary cap assembly in elongating spermatids. Reduced Klp10A expression also results in strong structural defects of sensory type I neurons. We show, here, that this protein displays a peculiar localization during male gametogenesis. The Klp10A signal is first detected at the distal ends of the centrioles when they dock to the plasma membrane of young primary spermatocytes. At the onset of the first meiotic prometaphase, when the CLRs reach their full size, Klp10A is enriched in a distinct narrow area at the distal end of the centrioles and persists in elongating spermatids at the base of the ciliary cap. We conclude that Klp10A could be a core component of the ciliary transition zone in Drosophila.

Quantitative MRI analysis of infrapatellar and suprapatellar fat pads in normal controls, moderate and end-stage osteoarthritis

The aim of this study was to analyze the magnetic resonance imaging (MRI) volumetric and morphometric characteristics of the infrapatellar fat pad (IFP) and the suprapatellar fat pad (SFP) in normal controls, moderate and end-stage osteoarthritis (OA) patients. Forty-four MRI images of the three groups were collected: a) 17 patients undergoing meniscectomy with Outerbridge score 0 (control group); b) 15 patients undergoing meniscectomy with Outerbridge score 3/4 (moderate OA group); and c) 12 patients undergoing total knee replacement (end-stage OA group). Volume, depth, femoral and tibial arch lengths of IFP were quantified. The hypointense IFP signals were also scored. The SFP volume, oblique, antero-posterior and cranio-caudal lengths were determined. IFP and SFP characteristics were compared between groups. A decrease of IFP volume, depth, femoral, and tibial arch lengths in moderate and end-stage OA compared to controls were observed. A difference in IFP hypointense signal was found between groups. No differences were found in SFP characteristics between the groups. In controls and moderate OA patients, correlations were found among the different MRI characteristics of both IFP and SFP, while in the end-stage OA group correlations were found only in SFP. We evidenced differences of the IFP MRI morphometric characteristics between the groups analyzed, supporting an important role of IFP in OA pathology and progression. On the contrary, no differences were highlighted in SFP analysis suggesting that this fat pad is not clearly involved in OA, probably due to its peculiar localization and different function.

GM1 Ganglioside Promotes Osteogenic Differentiation of Human Tendon Stem Cells.

Gangliosides, the sialic acid-conjugated glycosphingolipids present in the lipid rafts, have been recognized as important regulators of cell proliferation, migration, and apoptosis. Due to their peculiar localization in the cell membrane, they modulate the activity of several key cell receptors, and increasing evidence supports their involvement also in stem cell differentiation. In this context, herein we report the role played by the ganglioside GM1 in the osteogenic differentiation of human tendon stem cells (hTSCs). In particular, we found an increase of GM1 levels during osteogenesis that is instrumental for driving the process. In fact, supplementation of the ganglioside in the medium significantly increased the osteogenic differentiation capability of hTSCs. Mechanistically, we found that GM1 supplementation caused a reduction in the phosphorylation of the platelet-derived growth factor receptor-β (PDGFR-β), which is a known inhibitor of osteogenic commitment. These results were further corroborated by the observation that GM1 supplementation was able to revert the inhibitory effects on osteogenesis when the process was inhibited with exogenous PDGF.

The Effect of Hypothermia on Phospholipid and Fatty Acid Composition of Synaptic Membranes in the Rat Brain

The effect of moderate and deeper hypothermia on the phospholipid (PL) and fatty acid (FA) composition of synaptic membranes (synaptosomes) in the rat brain was investigated. As hypothermia deepened, phosphatidylcholine (PC) and phosphatidylserine (PS) levels decreased while those of phosphatidylethanolamine (PEA) remained intact. We attribute the differences both to a peculiar localization of these PL in the synaptic membrane and to a specificity of their function. Under hypothermal exposure, the saturated FA (SFA) level in the FA repertoire of total synaptosomal PL slightly decreased (by 9%) while that of polyunsaturated FA (PUFA) considerably increased, leading to a rise in the lipid unsaturation index (LUI) (by 47%) and promoting the maintenance of synaptic membrane fluidity. For three basic PL (PC, PS and PEA), the tendency was opposite: the SFA level increased while that of PUFA decreased, leading to a fall in the LUI and promoting a higher packing order of PL within the synaptic membrane. In the FA repertoire of the plasmalogen form of PEA (p-PEA), enforced hypothermia led to elevated levels both of SFA and PUFA as well as to a particularly high LUI, typical for this PL. These changes are supposed to be aimed at maintaining optimal membrane fluidity. We consider all the observed changes in lipid characteristics as adaptive, allowing the synaptic function in homeotherms to be supported as body temperature falls.

A rice class-XIV kinesin enters the nucleus in response to cold

Higher plants possess a large number of kinesins, but lack the minus-end directed dynein motors. However, the kinesin class XIV has strongly expanded, and minus-end directed motors from this class may have taken over functions of cytoplasmic dyneins. In this study, we address the functional aspects of a novel rice homologue of the Arabidopsis class-XIV kinesins ATK1 and ATK5. Since a loss-of-function rice mutant of this kinesin is not viable, the function was studied in tobacco BY-2 as heterologous system. OsDLK-GFP stably expressed in BY-2 cells decorates cortical microtubules, but also can shift into the nucleus of interphase cells. Because of this peculiar localisation, we coined the name Dual Localisation Kinesin (DLK). The nuclear import of this protein is strongly and reversibly promoted in response to cold. During mitosis, OsDLK is repartitioned between spindle and phragmoplast. Motility assays in vitro using show that OsDLK can convey mutual sliding of microtubules and moves at a velocity comparable to other class-XIV kinesins. When tobacco cells overexpressing OsDLK are synchronised, they exhibit a delayed entry into metaphase, while the later phases of mitosis are accelerated. The data are discussed in relation to additional functions of this kinesin type, beyond their transport along microtubules.

Localization of soft modes at the depinning transition.

We characterize the soft modes of the dynamical matrix at the depinning transition, and compare the matrix with the properties of the Anderson model (and long-range generalizations). The density of states at the edge of the spectrum displays a universal linear tail, different from the Lifshitz tails. The eigenvectors are instead very similar in the two matrix ensembles. We focus on the ground state (soft mode), which represents the epicenter of avalanche instabilities. We expect it to be localized in all finite dimensions, and make a clear connection between its localization length and the Larkin length of the depinning model. In the fully connected model, we show that the weak-strong pinning transition coincides with a peculiar localization transition of the ground state.

Cerebellar lesions associated to Tuberous Sclerosis: New insights from an Italian paediatric series

Objectives: To evaluate: a) the cerebellar involvement in a pediatric population with tuberous sclerosis; b) the possible clinical and neuroradiological differences comparing patients with cerebellar lesions and those without; c) whether the cerebellar involvement may be considered a prognostic factor for neurological and neuropsychiatric outcomes in patients with tuberous sclerosis. Methods: Retrospective analysis of a pediatric population of 51 patients (21 males, 30 females) affected by tuberous sclerosis. For each patient we evaluated the clinical characteristics and in particular the neurological and neuropsychiatric phenotypes and all the available brain MRI. Results: 25.5% (13/51) of patients had cerebellar lesions (unilateral in 9/13 cases and bilateral in 4/13 cases). 91% of patients with cerebellar lesions had a TSC2 gene mutation (p=0.11) and in all cases mutations were de novo (p=0.02). Patients with cerebellar involvement compared to those without had a more severe phenotype (drug-resistant epilepsy: 46.2% vs 28.9%; intellectual disability: 61.5% vs 39.5%; neuropsychiatric disorders: 61.5% vs 50%; pervasive developmental disorder: 23.1% vs 10.5%; a higher brain tuber load tuberous, a higher frequency of subependymal nodules and subependymal giant cell astrocytomas). All cerebellar lesions involved the posterior lobe. Conclusions: A quarter of our patients with tuberous sclerosis have cerebellar lesions. Cases with cerebellar lesions, compared to those without, have more severe clinical phenotypes and neuroradiologic findings. The cerebellar lesions have a constant and peculiar localization in the posterior lobe of cerebellum, that is considered the seat of the cognitive functions of the cerebellum. Our data support the hypothesis that cerebellar lesions could represent a negative prognostic biomarker in patients with tuberous sclerosis. Their early detection could suggest to screen more carefully for neuropsychiatric disorders and neuropsychological problems and they could facilitate the selection of patients amenable to early treatment with mTOR inhibitors.