La propagazione anterograda e retrograda degli effetti della tossina botulinica A (BoNT/A) nel sistema visivo del ratto

Le tossine botuliniche sono enzimi batterici che bloccano il rilascio di neurotrasmettitore a livello delle sinapsi centrali e periferiche; la loro azione a livello della giunzione neuromuscolare viene utilizzata in clinica per il trattamento di disturbi del movimento quali le distonie e la spasticità. Esistono sette serotipi diversi di tossine botuliniche, che vengono indicati con le lettere dalla A alla G (BoNT/A-BoNT/G). Il loro meccanismo d’azione consiste nel tagliare alcune proteine del complesso SNARE : VAMP, SNAP-25 o sintaxina, interferendo così con la fusione delle vescicole al terminale sinaptico. Comunemente si riteneva che le azioni delle BoNTs rimanessero ristrette al sito di iniezione; tuttavia, dati recenti del nostro laboratorio indicano una possibilità di trasporto assonale. Il trasporto assonale potrebbe spiegare alcuni degli effetti a livello di sistema nervoso centrale che sono stati riscontrati nei pazienti trattati intramuscolarmente con le BoNTs. In questa tesi ho quindi investigato la possibilità di trasporto assonale utilizzando la BoNT/A, che è il serotipo più usato a livello clinico. Gli studi sono stati condotti sul sistema visivo di Ratto adulto, in cui abbiamo valutato sia il trasporto anterogrado (iniettando la BoNT/A nell’umor vitreo dell’occhio) che il trasporto retrogrado (iniettando la tossina nel collicolo superiore). In questo modo è stato possibile valutare la propagazione anterograda e retrograda degli effetti della tossina andando a valutare il taglio proteolitico del substrato di BoNT/A: la proteina sinaptica SNAP-25. A seguito di iniezioni intraoculari, l’analisi biochimica ha evidenziato alti livelli di SNAP-25 tagliata nella retina trattata e quantità significative dello stesso substrato tagliato nel collicolo controlaterale all’iniezione. Al contrario, nessun segnale si ritrovava nel collicolo ipsilaterale, escludendo quindi l’ipotesi di una diffusione sistemica della BoNT/A. Inoltre, quando la propagazione anterograda era bloccata dall’iniezione di colchicina, non si ritrovava SNAP-25 tagliata nel collicolo controlaterale, dimostrando così il ruolo cruciale del trasporto mediato dai microtubuli. A livello immunoistochimico la marcatura di SNAP-25 tagliata nel collicolo è associata a fibre e terminali sinaptici. In esperimenti preliminari non abbiamo riscontrato una colocalizzazione significativa tra SNAP-25 tagliata e marcatori delle terminazioni retiniche (il trasportatore vescicolare per il glutammato vGlut2 e il tracciante anterogrado tossina del colera – CTB – iniettato nell’occhio), suggerendo quindi che l’effetto della BoNT/A possa estendersi a sinapsi distinte da quelle delle cellule gangliari. Dopo iniezioni nel collicolo superiore, l’analisi immunoistochimica ha rivelato molte sinapsi positive per SNAP-25 tagliata nella retina (propagazione retrograda). In particolare, risultano marcate le cellule amacrine colinergiche e le cellule bipolari, due tipi cellulari presinaptici alle gangliari. Questo dato ci suggerisce la capacità della BoNT/A di eseguire transcitosi. Inoltre, anche alcuni fotorecettori sono risultati positivi per SNAP-25 tagliata e questo dato suggerisce che la BoNT/A possa compiere un secondo ciclo di transcitosi. Esperimenti in corso sono volti a determinare se questa propagazione anterograda e retrograda sia associata a effetti funzionali. In particolare stiamo valutando se la presenza di SNAP-25 tagliata in sinapsi lontane dal sito di iniezioni possa indurre un blocco del normale ciclo di esocitosi delle vescicole. In conclusione, i nostri dati dimostrano che gli effetti della BoNT/A si propagano sia anterogradamente che retrogradamente all’interno del sistema visivo del Ratto. Queste osservazioni potrebbero avere implicazioni importanti per l’uso clinico della BoNT/A

Disentangling the signaling complexity of nerve growth factor receptors by CRISPR/Cas9

The binding of nerve growth factor (NGF) to the tropomyosin-related kinase A (TrkA) and p75(NTR) receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA-p75(NTR) interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma-derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75(NTR), or both. We found that TrkA-null cells become unresponsive to NGF. Conversely, the absence of p75(NTR) enhances the phosphoiylation of TrkA and its effectors. Using a patch-clamp, we demonstrated that the individual activation of TrkA and p75(NTR) by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75(NTR) in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing

Longitudinal Bottom-Up Proteomics of Serum, Serum Extracellular Vesicles, and Cerebrospinal Fluid Reveals Candidate Biomarkers for Early Detection of Glioblastoma in a Murine Model

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset

The role of activity in synaptic degeneration in a protein misfolding disease, prion disease

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer's, Parkinson's and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration

Centrin 2: a novel marker of mature and neoplastic human astrocytes

As microtubule organizing centers, centrosomes play a pivotal role in cell division as well as in neurodevelopment and neuronal maturation. Among centrosomal proteins, centrin-2 (CETN2) contributes also to DNA repair mechanisms which are fundamental to prevent genomic instability during neural stem cell pool expansion. Nevertheless, the expression profile of CETN2 in human neural stem cells and their progeny is currently unknown. To address this question, we interrogated a platform of human neuroepithelial stem (NES) cells derived from post-mortem developing brain or established from pluripotent cells, and demonstrated that while CETN2 retains its centrosomal location in proliferating NES cells, its expression pattern changes upon differentiation. In particular, we found that CETN2 is selectively expressed in mature astrocytes with a broad cytoplasmic distribution. We then extended our findings on human autoptic nervous tissue samples. We investigated CETN2 distribution in diverse anatomical areas along the rostro-caudal neuraxis and pointed out a peculiar topography of CETN2-labelled astrocytes in humans which was not appreciable in murine tissues, where CETN2 was mostly confined to ependymal cells. As prototypical condition with glial overproliferation, we also explored CETN2 expression in glioblastoma multiforme, reporting a focal concentration of CETN2 in neoplastic astrocytes. This study expands CETN2 localization beyond centrosomes and reveals a unique expression pattern which makes it eligible as a novel astrocytic molecular marker, thus opening new roads to glial biology and human neural conditions

Molecular changes underlying decay of sensory responses and enhanced seizure propensity in peritumoral neurons

Background: Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target. Methods: We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection we analysed layer II-III pyramidal neurons molecular profile and with Local Field Potentials (LFP) recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice. Results: We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP-25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (i.e., AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signalling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in two mouse models of tumor-bearing mice. Conclusions: These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients' quality of life

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Beta-Blocker Use in Older Hospitalized Patients Affected by Heart Failure and Chronic Obstructive Pulmonary Disease: An Italian Survey From the REPOSI Register

Beta (β)-blockers (BB) are useful in reducing morbidity and mortality in patients with heart failure (HF) and concomitant chronic obstructive pulmonary disease (COPD). Nevertheless, the use of BBs could induce bronchoconstriction due to β2-blockade. For this reason, both the ESC and GOLD guidelines strongly suggest the use of selective β1-BB in patients with HF and COPD. However, low adherence to guidelines was observed in multiple clinical settings. The aim of the study was to investigate the BBs use in older patients affected by HF and COPD, recorded in the REPOSI register. Of 942 patients affected by HF, 47.1% were treated with BBs. The use of BBs was significantly lower in patients with HF and COPD than in patients affected by HF alone, both at admission and at discharge (admission, 36.9% vs. 51.3%; discharge, 38.0% vs. 51.7%). In addition, no further BB users were found at discharge. The probability to being treated with a BB was significantly lower in patients with HF also affected by COPD (adj. OR, 95% CI: 0.50, 0.37-0.67), while the diagnosis of COPD was not associated with the choice of selective β1-BB (adj. OR, 95% CI: 1.33, 0.76-2.34). Despite clear recommendations by clinical guidelines, a significant underuse of BBs was also observed after hospital discharge. In COPD affected patients, physicians unreasonably reject BBs use, rather than choosing a β1-BB. The expected improvement of the BB prescriptions after hospitalization was not observed. A multidisciplinary approach among hospital physicians, general practitioners, and pharmacologists should be carried out for better drug management and adherence to guideline recommendations

The “Diabetes Comorbidome”: A Different Way for Health Professionals to Approach the Comorbidity Burden of Diabetes

(1) Background: The disease burden related to diabetes is increasing greatly, particularly in older subjects. A more comprehensive approach towards the assessment and management of diabetes’ comorbidities is necessary. The aim of this study was to implement our previous data identifying and representing the prevalence of the comorbidities, their association with mortality, and the strength of their relationship in hospitalized elderly patients with diabetes, developing, at the same time, a new graphic representation model of the comorbidome called “Diabetes Comorbidome”. (2) Methods: Data were collected from the RePoSi register. Comorbidities, socio-demographic data, severity and comorbidity indexes (Cumulative Illness rating Scale CIRS-SI and CIRS-CI), and functional status (Barthel Index), were recorded. Mortality rates were assessed in hospital and 3 and 12 months after discharge. (3) Results: Of the 4714 hospitalized elderly patients, 1378 had diabetes. The comorbidities distribution showed that arterial hypertension (57.1%), ischemic heart disease (31.4%), chronic renal failure (28.8%), atrial fibrillation (25.6%), and COPD (22.7%), were the more frequent in subjects with diabetes. The graphic comorbidome showed that the strongest predictors of death at in hospital and at the 3-month follow-up were dementia and cancer. At the 1-year follow-up, cancer was the first comorbidity independently associated with mortality. (4) Conclusions: The “Diabetes Comorbidome” represents the perfect instrument for determining the prevalence of comorbidities and the strength of their relationship with risk of death, as well as the need for an effective treatment for improving clinical outcomes

Antidiabetic Drug Prescription Pattern in Hospitalized Older Patients with Diabetes

Objective: To describe the prescription pattern of antidiabetic and cardiovascular drugs in a cohort of hospitalized older patients with diabetes. Methods: Patients with diabetes aged 65 years or older hospitalized in internal medicine and/or geriatric wards throughout Italy and enrolled in the REPOSI (REgistro POliterapuie SIMI—Società Italiana di Medicina Interna) registry from 2010 to 2019 and discharged alive were included. Results: Among 1703 patients with diabetes, 1433 (84.2%) were on treatment with at least one antidiabetic drug at hospital admission, mainly prescribed as monotherapy with insulin (28.3%) or metformin (19.2%). The proportion of treated patients decreased at discharge (N = 1309, 76.9%), with a significant reduction over time. Among those prescribed, the proportion of those with insulin alone increased over time (p = 0.0066), while the proportion of those prescribed sulfonylureas decreased (p < 0.0001). Among patients receiving antidiabetic therapy at discharge, 1063 (81.2%) were also prescribed cardiovascular drugs, mainly with an antihypertensive drug alone or in combination (N = 777, 73.1%). Conclusion: The management of older patients with diabetes in a hospital setting is often sub-optimal, as shown by the increasing trend in insulin at discharge, even if an overall improvement has been highlighted by the prevalent decrease in sulfonylureas prescription