Meccanismi molecolari coinvolti nell'epilettogenesi: espressione dell'importina beta 1 nell'ippocampo di Mus musculus e Rattus norvegicus dopo trattamento con acido kainico

Il termine “epilessia” si riferisce a un gruppo di patologie neurologiche che si manifestano con la ripetuta comparsa di crisi provocate da una scarica elettrica anomala, sincronizzata e prolungata di cellule nervose della corteccia o del tronco cerebrale. Una delle forme più diffuse è l’epilessia dei lobi temporali, che colpisce le strutture del sistema limbico. Essa compare per lo più in seguito ad un evento scatenante (trauma cranico, ischemia cerebrale, tumori, convulsioni febbrili prolungate) che, dopo un lungo periodo di latenza, porta all’insorgenza della malattia vera e propria, caratterizzata da crisi spontanee, sempre più gravi e frequenti, e spesso resistenti al trattamento farmacologico. Esperimenti condotti su modelli animali della patologia suggeriscono che durante il periodo di latenza, definito epilettogenesi, avvengano modificazioni molecolari, citologiche ed elettrofisiologiche a carico dei neuroni di ippocampo e corteccia entorinale, ed è stato ipotizzato che tali cambiamenti siano dovuti ad alterazione dell’espressione genica. Studi recenti hanno indicato le importine come possibili candidati nella modulazione dell’espressione genica nell’ippocampo, in riferimento al loro ruolo di trasportatori di fattori di trascrizione importanti per la regolazione dei geni neuronali. Esperimenti condotti nel nostro laboratorio su ratti trattati con 12 mg/kg di acido kainico (KA) suggeriscono inoltre una regolazione dell’espressione dell’importina β1 (Imp β1) durante l’epilettogenesi, nei neuroni piramidali delle regioni CA1 e CA3, nei granuli del giro dentato e negli astrociti. Sulla base di queste considerazioni, durante il mio lavoro di Tesi ho condotto esperimenti atti a caratterizzare meglio il profilo di espressione dell’Imp β1 nei ratti epilettici: ratti maschi adulti sono stati trattati con 12 mg/kg di KA e sacrificati a tempi diversi, in modo da valutare la regolazione dell’espressione dell’Imp β1 nelle varie fasi dell’epilettogenesi. In un primo blocco di esperimenti sono stati valutati i ratti acuti (30 minuti e 2 ore; rispettivamente, prima e durante l’insorgenza dello stato epilettico). I dati ottenuti mediante RT-PCR su RNA di ippocampo non mostrano differenze significative a livello trascrizionale tra i due gruppi di acuti e gli animali di controllo. Per valutare inoltre l’espressione della proteina, ho effettuato esperimenti di immunoistochimica su fettine di ippocampo, misurando poi l’intensità della marcatura per Impβ1 nei neuroni piramidali della regione CA1. Questi esperimenti hanno dimostrato la presenza di un aumento significativo della proteina dopo 30 minuti e 2 ore dal trattamento con KA. Da questi risultati si evince che l’espressione dell’Imp β1 è rapidamente indotta dall’attività epilettica. In un secondo blocco di esperimenti sono stati valutati i ratti cronici (sacrificati 5 settimane dopo la somministrazione di KA). Dall’analisi di RT-PCR risulta un aumento, ma non significativo, dell’mRNA dell’ Imp β1, e l’intensità di marcatura per cellula nei neuroni piramidali della CA1 indica che il livello di espressione della proteina è simile nei due trattamenti; tuttavia, la conta delle cellule Imp β1 positive nello strato radiato della CA1 mostra un aumento significativo negli animali cronici rispetto ai controlli. Ho quindi condotto esperimenti di doppia marcatura immunoistochimica per valutare i tipi cellulari coinvolti in questo aumento. L’analisi quantitativa dimostra che il numero di neuroni (NeuN positivi) e di astrociti (GFAP positivi) che esprimono l’Imp β1 non varia significativamente nei due gruppi di trattamento. In un altro blocco di esperimenti topi adulti sono stati trattati con 30 mg/kg di KA e con MPEP (un farmaco antagonista dei recettori metabotropici del glutammato mGluR5, specifici degli astrociti), quindi sono stati sacrificati dopo 1 settimana dalla somministrazione (a uno stadio equivalente al ratto cronico). I risultati degli esperimenti di doppia marcatura immunoistochimica per Imp β1 e GFAP non indicano però differenze significative tra i due tipi di trattamento (KA e KA/MPEP): in entrambi i casi si osserva una forte gliosi, con la presenza di molti astrociti Imp β1 positivi; tra i vari campioni si osserva una certa variabilità, che però non è possibile ricondurre al tipo di trattamento. Questi risultati dimostrano la presenza di una regolazione postrascrizionale sull’espressione della Imp β1 durante l’epilettogenesi, mentre non sembra che gli astrociti siano coinvolti in questo processo; esperimenti futuri saranno rivolti a caratterizzare il ruolo di atri tipi cellulari gliali (microglia, oligodendrociti) nella regolazione dell’Imp β1 nell’epilettogenesi

The antidepressant fluoxetine acts on energy balance and leptin sensitivity via BDNF

Leptin and Brain Derived Neurotrophic Factor (BDNF) pathways are critical players in body weight homeostasis. Noninvasive treatments like environmental stimulation are able to increase response to leptin and induce BDNF expression in the brain. Emerging evidences point to the antidepressant selective serotonin reuptake inhibitor Fluoxetine (FLX) as a drug with effects similar to environmental stimulation. FLX is known to impact on body weight, with mechanisms yet to be elucidated. We herein asked whether FLX affects energy balance, the leptin system and BDNF function. Adult lean male mice chronically treated with FLX showed reduced weight gain, higher energy expenditure, increased sensitivity to acute leptin, increased hypothalamic BDNF expression, associated to changes in white adipose tissue expression typical of "brownization". In the Ntrk2tm1Ddg/J model, carrying a mutation in the BDNF receptor Tyrosine kinase B (TrkB), these effects are partially or totally reversed. Wild type obese mice treated with FLX showed reduced weight gain, increased energy output, and differently from untreated obese mice, a preserved acute response to leptin in terms of activation of the intracellular leptin transducer STAT3. In conclusion, FLX impacts on energy balance and induces leptin sensitivity and an intact TrkB function is required for these effects to take place

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

GH Dysfunction in Engrailed-2 Knockout Mice, a Model for Autism Spectrum Disorders

Insulin-like growth factor 1 (IGF-1) signaling promotes brain development and plasticity. Altered IGF-1 expression has been associated to autism spectrum disorders (ASD). IGF-1 levels were found increased in the blood and decreased in the cerebrospinal fluid of ASD children. Accordingly, IGF-1 treatment can rescue behavioral deficits in mouse models of ASD, and IGF-1 trials have been proposed for ASD children. IGF-1 is mainly synthesized in the liver, and its synthesis is dependent on growth hormone (GH) produced in the pituitary gland. GH also modulates cognitive functions, and altered levels of GH have been detected in ASD patients. Here, we analyzed the expression of GH, IGF-1, their receptors, and regulatory hormones in the neuroendocrine system of adult male mice lacking the homeobox transcription factor Engrailed-2 (En2(−/−) mice). En2(−/−) mice display ASD-like behaviors (social interactions, defective spatial learning, increased seizure susceptibility) accompanied by relevant neuropathological changes (loss of cerebellar and forebrain inhibitory neurons). Recent studies showed that En2 modulates IGF-1 activity during postnatal cerebellar development. We found that GH mRNA expression was markedly deregulated throughout the neuroendocrine axis in En2(−/−) mice, as compared to wild-type controls. In mutant mice, GH mRNA levels were significantly increased in the pituitary gland, blood, and liver, whereas decreased levels were detected in the hippocampus. These changes were paralleled by decreased levels of GH protein in the hippocampus but not other tissues of En2(−/−) mice. IGF-1 mRNA was significantly up-regulated in the liver and down-regulated in the En2(−/−) hippocampus, but no differences were detected in the levels of IGF-1 protein between the two genotypes. Our data strengthen the notion that altered GH levels in the hippocampus may be involved in learning disabilities associated to ASD

Fluoxetine Modulates the Activity of Hypothalamic POMC Neurons via mTOR Signaling

Hypothalamic proopiomelanocortin (POMC) neurons are important players in the regulation of energy homeostasis; we previously demonstrated that environmental stimulation excites arcuate nucleus circuits to undergo plastic remodeling, leading to altered ratio between excitatory and inhibitory synaptic contacts on these neurons. The widely used selective serotonin reuptake inhibitor fluoxetine (FLX) is known to affect body weight. On the other hand, FLX administration mimics the effects of environmental stimulation on synaptic plasticity in the hippocampus and cortex. The mammalian target of rapamycin (mTOR) pathway is instrumental in these phenomena. Thus, we aimed at investigating whether and how FLX affects POMC neurons activity and hypothalamic mTOR function. Adult mice expressing green fluorescent protein (GFP) under the POMC promoter were treated with FLX for 3 weeks resulting in diminished body weight. Patch clamp recordings performed on POMC neurons indicate that FLX increases their firing rate and the excitatory AMPA-mediated transmission, and reduces the inhibitory GABAergic currents at presynaptic level. Immunofluorescence studies indicate that FLX increases the ratio between excitatory and inhibitory synaptic contacts on POMC neurons. These changes are associated with an increased activity of the hypothalamic mTOR pathway. Use of the mTOR inhibitor rapamycin blunts the effects of FLX on body weight and on functional and structural plasticity of POMC neurons. Our findings indicate that FLX is able to remodel POMC neurons, and that this may be partly mediated by the mTOR signaling pathway