Migraine with aura white matter lesions: preliminary data on clinical aspects.

A few clinic-based magnetic resonance imaging studies report an increased risk of signal abnormalities in migraineurs brain's white matter, especially in migraine with aura subjects. A vascular genesis has been hypnotized and migraine with aura was considered an independent risk factor for stroke. Available data of magnetic resonance imaging alterations are often nonspecific and sometimes controversial. The aim of our study is to investigate migraine with aura patients with standardized brain magnetic resonance imaging to detect and to quantify the presence of white matter lesions and to analyze their relation with clinical data. We report preliminary data about first 90 subjects. We did not recognize any clinical aspect in close relationship with these alterations. The only clinical feature that seems to play a role in the presence of alterations is the age, and only in migraineurs women.

Pathogenesis of chronic cluster headache and bouts: role of tryptamine, arginine metabolism and α1-agonists.

The aim of this study was to explore the possible role of tryptamine in the pathogenesis of chronic cluster headache along with that of adrenaline and noradrenaline (α-agonists) together with arginine metabolism in the origin of cluster bouts. Plasma levels of tyramine, tryptamine, serotonin, 5-hydroxyindolacetic acid, noradrenalin, adrenalin and the markers of arginine metabolism such as arginine, homoarginine, citrulline, ADMA and NMMA, were measured in 23 chronic cluster headache patients (10 chronic cluster ab initio and 13 transformed from episodic cluster) and 28 control subjects. The plasma levels of tyramine, tryptamine, noradrenalin and adrenalin were found several times higher in chronic cluster headache patients compared to controls, whereas the plasma levels of arginine, homoarginine and citrulline were significantly lower. No differences were found in the plasma levels of serotonin, 5-hydroxyindolacetic, ADMA and NMMA between chronic cluster headache patients and control subjects. These results provide support for a role of tryptamine in the pathogenesis of chronic cluster headache and, in particular, in the duration of the cluster bouts. In addition, the low levels of the nitric oxide substrates together with the high levels of noradrenalin and adrenalin suggest an activation of endothelial TAAR1 receptors followed by the release of nitric oxide in the circulation that may constitute the final step of the physiopathology of cluster crisis.

Difficulties in work activities and the pervasive effect over disability in patients with episodic and chronic migraine.

Migraine is associated with reduced productivity in work-related activities. The degree to which problems with work are, in turn, associated to the level of migraine-related disability as well as to headache frequency has been poorly explored. The aim of the study was to assess if migraine patients with different degrees of work difficulties showed a different level of migraine-related disability. A consecutive sample of patients with episodic migraine (EM) or with chronic migraine (CM) with medication overuse (MO) attending the Headache Centre of the Neurological Institute C. Besta of Milan was studied. All patients completed the MIDAS and the WHODAS 2.0 questionnaires. The total scores of both questionnaires, frequency of headaches, average pain intensity, and the scores of each subscale of the WHODAS 2.0 were calculated separately for EM and CM patients. The score of WHODAS 2.0 "Work difficulties" subscale was used to divide the studied patients into two groups, i.e. those above and those below the median "Work difficulties" subscale score. Independent sample t test was used to compare these two groups as far as all the other studied variables. A total of 296 patients (102 with EM and 194 with CM-MO) were enrolled. Patients with higher work difficulties score also displayed higher scores in the other WHODAS 2.0 subscales; for those with CM-MO, the differences were significant. The results of this study indicate that having more and more severe workplace problems is associated to a higher disability level in migraineurs. Further studies are needed to better understand workplace disability in different migraine forms, particularly in a qualitative way.

Electromyography data in chronic migraine patients by using neurostimulation with the Cefaly® device.

The objective of this observational study is to report clinical and instrumental results obtained in 23 chronic migraine sufferers treated with transcutaneous neurostimulation with the Cefaly(®) device. The electrom yography (EMG) parameters of the patients monitored before and during neurostimulation with the Cefaly(®) device showed a significant increase in the EMG amplitude and frequency values in the frontalis, anterior temporalis, auricularis posterior and middle trapezius muscles. The Cefaly(®) device could act on the inhibitory circuit in the spinal cord thus causing a neuromuscular facilitation and may help reduce contraction of frontalis muscles.

Study of parafunctions in patients with chronic migraine.

The purpose of this paper is to present the results of a questionnaire investigating parafunctions (particularly clenching and grinding) in patients with chronic migraine presenting sign of temporomandibular disorder. The questionnaire was elaborated by the Dental Clinic of the University of Milano and completed by 125 patients experiencing chronic migraine and attending the Neurological Institute Carlo Besta for an inpatient withdrawal protocol to treat medication overuse. Our results showed high percentages of parafunctions, which were present in 80 % of patients. We note that patient information on possible behaviours and coexisting conditions which may be involved in the mechanisms of chronic headaches, as well as education about these factors, are crucial aspects in the management of chronic headache patients. We suggest that patients suffering from chronic migraine with medication overuse headache should be evaluated in relation to the possible presence of parafunctions, and as far as the need for interocclusal devices, in order to limit the role of temporomandibular dysfunctions as trigger factors or coexisting conditions favouring the development/maintaining of headache chronification.

Occipital neurostimulation in primary headaches: update.

A subset of headache patients are chronic and results refractory to standard medical treatments, they are unsatisfied or unable to tolerate the side effects of medications. In the lack of more effective prophylactic treatment, there is need of alternative approach. Migraine is conceptualized as a chronic and potentially progressive disorder. It is conceivable that more aggressive therapeutic efforts could be warranted in drug-refractory chronic migraine. In this prospective, the new, device-based therapies that allow to affect brain function in less invasive ways may represent a therapeutic opportunity. Peripheral occipital neurostimulation resulted in several trials and case reports to be beneficial in a large variety of headache and craniofacial pain disorders, with chronic primary headache the most studied. We comment on our experience in the application of ONS in drug-refractory chronic cluster headache and chronic migraine patients.

A 14-month study of change in disability and mood state in patients with chronic migraine associated to medication overuse.

This paper aims to evaluate changes in disease severity, disability and mood state in patients with chronic migraine associated to medication overuse (CM-MO). MIDAS was used for assessing disease activity, WHO-DAS-2 for disability, DBI-2 for mood state. ANOVA was used to test change over time; t-test to assess follow-up differences in WHO-DAS-2 and BDI-2 between patients with MIDAS ≤20 and ≥21. Change in MIDAS, WHO-DAS-2 and BDI-2 scores were computed: Pearson's index was used to assess correlation between them; linear regression to assess change in WHO-DAS-2, using MIDAS and BDI-2 change as predictors. Mean MIDAS decreased significantly (from 101.9 to 52.0). In 26.1 % of the sample, MIDAS fell below 21 at follow-up: these patients had lower WHO-DAS-2 score. WHO-DAS-2 change was little correlated to MIDAS change and strongly correlated to changes in BDI-2 scores. 57.1 % of WHO-DAS-2 change variance is explained by change in BDI-2 and MIDAS scores. There was a clear clinical improvement 14 months after detoxification, and a modest reduction in disability which is explained by reduced disease activity and improved mood state. An appropriate treatment of CM-MO, based on detoxification and prophylaxis, is likely to reduce disease burden: recognition and treatment of mood problems may be a key factor to reduce disability.

Positive outcome of occlusal freeway space reestablishment in patients with medication overuse due to chronic migraine.

Considering the great chapter of migraines, it is important to note the signs and symptoms caused by an alteration of the relationship of the facial musculature and the occlusal freeway space (FWS) that is the distance from maximal intercuspation to the habitual rest position (measurable in 1.4-2.5 mm). To any mandible position changing (detected by periodontal, muscle and joint proprioceptors), there is an influence on the neuromuscular system and then an alteration of the FWS. A group of 60 patients with chronic migraine (CM) underwent a withdrawal of overused medication and were subjected to electromyographic and kinesiographic evaluation. All those who presented an alteration of the FWS at rest position have been subjected to treatment with orthosis device for about 12 months. The aim of our work is to obtain and define a correct mandibular position, physiological, in agreement with the neuromuscular structures of the patient. Clinical results obtained on this pool of patients, in terms of reduction of the VAS, let us understand the importance of the inviolability of the FWS and to eliminate any type of mandibular deviation with respect to the closure trajectory induced by TENS in patients suffering from CM.

CDK12 promotes tumorigenesis but induces vulnerability to therapies inhibiting folate one-carbon metabolism in breast cancer.

Cyclin-dependent kinase 12 (CDK12) overexpression is implicated in breast cancer, but whether it has a primary or only a cooperative tumorigenic role is unclear. Here, we show that transgenic CDK12 overexpression in the mouse mammary gland per se is sufficient to drive the emergence of multiple and multifocal tumors, while, in cooperation with known oncogenes, it promotes earlier tumor onset and metastasis. Integrative transcriptomic, metabolomic and functional data reveal that hyperactivation of the serine-glycine-one-carbon network is a metabolic hallmark inherent to CDK12-induced tumorigenesis. Consistently, in retrospective patient cohort studies and in patient-derived xenografts, CDK12-overexpressing breast tumors show positive response to methotrexate-based chemotherapy targeting CDK12-induced metabolic alterations, while being intrinsically refractory to other types of chemotherapy. In a retrospective analysis of hormone receptor-negative and lymph node-positive breast cancer patients randomized in an adjuvant phase III trial to 1-year low-dose metronomic methotrexate-based chemotherapy or no maintenance chemotherapy, a high CDK12 status predicts a dramatic reduction in distant metastasis rate in the chemotherapy-treated vs. not-treated arm. Thus, by coupling tumor progression with metabolic reprogramming, CDK12 creates an actionable vulnerability for breast cancer therapy and might represent a suitable companion biomarker for targeted antimetabolite therapies in human breast cancers.

Endocytosis and mitogenic signaling.

Is there mitogenic signaling during endocytosis or is receptor internalization mainly an attenuator of signals? Recent data indicate that the answer appears to be yes to both questions. Signal transduction occurs physiologically from the cell surface and endocytosis downregulates signaling by removing receptors from the plasma membrane. In cancer, the involvement of endocytic/sorting proteins points to dysregulation of apparently unrelated pathways, which might account for an important causative role in neoplasia.

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling.

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

Understanding biological dynamics: following cells and molecules to track functions and mechanisms.

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.

Deliberative ethics in a biomedical institution: an example of integration between science and ethics.

The deliberative ethics guidelines elaborated and implemented by members of the IFOM-IEO Campus (Firc Institute of Molecular Oncology (IFOM) and the European Institute of Oncology (IEO)). These should serve the dual purpose of establishing a minimal set of standard rules for bioethical debate and any ensuing decision-making process, especially for the perspective of providing real instruments to foster public engagement and public awareness on the ethical issues involved in biomedical research. It is shown that these guidelines instantiate the scheme of one of the correct ways of debating formalised by the western thought.

Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF).

Epsin (Eps15 interactor) is a cytosolic protein involved in clathrin-mediated endocytosis via its direct interactions with clathrin, the clathrin adaptor AP-2, and Eps15. The NH(2)-terminal portion of epsin contains a phylogenetically conserved module of unknown function, known as the ENTH domain (epsin NH(2)-terminal homology domain). We have now solved the crystal structure of rat epsin 1 ENTH domain to 1.8 A resolution. This domain is structurally similar to armadillo and Heat repeats of beta-catenin and karyopherin-beta, respectively. We have also identified and characterized the interaction of epsin 1, via the ENTH domain, with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). Leptomycin B, an antifungal antibiotic, which inhibits the Crm1- dependent nuclear export pathway, induces an accumulation of epsin 1 in the nucleus. These findings suggest that epsin 1 may function in a signaling pathway connecting the endocytic machinery to the regulation of nuclear function.

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis.

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport.

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.

Numb is an endocytic protein.

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.

Nucleocytoplasmic shuttling of endocytic proteins.

Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and alpha-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.

An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.