Targeting neuronal populations by AAV-mediated gene transfer for studying the endocannabinoid system

The cannabinoid type 1 (CB1) receptor is involved in a plethora of physiological functions and heterogeneously expressed on different neuronal populations. Several conditional loss-of-function studies revealed distinct effects of CB1 receptor signaling on glutamatergic and GABAergic neurons, respectively. To gain a comprehensive picture of CB1 receptor-mediated effects, the present study aimed at developing a gain-of-function approach, which complements conditional loss-of-function studies. Therefore, adeno-associated virus (AAV)-mediated gene delivery and Cre-mediated recombination were combined to recreate an innovative method, which ensures region- and cell type-specific transgene expression in the brain. This method was used to overexpress the CB1 receptor in glutamatergic pyramidal neurons of the mouse hippocampus. Enhanced CB1 receptor activity at glutamatergic terminals caused impairment in hippocampus-dependent memory performance. On the other hand, elevated CB1 receptor levels provoked an increased protection against kainic acid-induced seizures and against excitotoxic neuronal cell death. This finding indicates the protective role of CB1 receptor on hippocampal glutamatergic terminals as a molecular stout guard in controlling excessive neuronal network activity. Hence, CB1 receptor on glutamatergic hippocampal neurons may represent a target for novel agents to restrain excitotoxic events and to treat neurodegenerative diseases. Endocannabinoid synthesizing and degrading enzymes tightly regulate endocannabinoid signaling, and thus, represent a promising therapeutic target. To further elucidate the precise function of the 2-AG degrading enzyme monoacylglycerol lipase (MAGL), MAGL was overexpressed specifically in hippocampal pyramidal neurons. This genetic modification resulted in highly increased MAGL activity accompanied by a 50 % decrease in 2-AG levels without affecting the content of arachidonic acid and anandamide. Elevated MAGL protein levels at glutamatergic terminals eliminated depolarization-induced suppression of excitation (DSE), while depolarization-induced suppression of inhibition (DSI) was unchanged. This result indicates that the on-demand availability of the endocannabinoid 2-AG is crucial for short-term plasticity at glutamatergic synapses in the hippocampus. Mice overexpressing MAGL exhibited elevated corticosterone levels under basal conditions and an increase in anxiety-like behavior, but surprisingly, showed no changes in aversive memory formation and in seizure susceptibility. This finding suggests that 2 AG-mediated hippocampal DSE is essential for adapting to aversive situations, but is not required to form aversive memory and to protect against kainic acid-induced seizures. Thus, specific inhibition of MAGL expressed in hippocampal pyramidal neurons may represent a potential treatment strategy for anxiety and stress disorders. Finally, the method of AAV-mediated cell type-specific transgene expression was advanced to allow drug-inducible and reversible transgene expression. Therefore, elements of the tetracycline-controlled gene expression system were incorporated in our “conditional” AAV vector. This approach showed that transgene expression is switched on after drug application and that background activity in the uninduced state was only detectable in scattered cells of the hippocampus. Thus, this AAV vector will proof useful for future research applications and gene therapy approaches.

Analisi delle caratteristiche di dispositivi per il brain computer interface

La brain computer interface sono state create per sopperire ai problemi motori e di linguistica nei pazienti che, a causa di malattie neurodegenerative o in seguito a traumi, sono paralizzate o non riescono a parlare. Vengono prese in questione le caratteristiche di un sistema per le BCI analizzando ogni blocco funzionale del sistema. Si fa visione delle applicazioni tra le quali la possibilità di far muovere un arto bionico a una donna tetraplegica e la possibilità di far scrivere una parola su uno schermo a un paziente non in grado di comunicare verbalmente. Si mettono in luce i problemi annessi a questo campo e i possibili sviluppi.

La spettroscopia di forza basata sull'AFM nello studio dello spazio conformazionale e dei processi aggregativi di proteine prioniche

Le malattie neurodegenerative sono caratterizzate da aggregazione proteica, dipendente dalla perdita della usuale struttura fisiologica funzionale delle proteine coinvolte, a favore di conformazioni tossiche (patologiche). Il modello corrente descrive questi cambiamenti conformazionali come eventi rari e ritiene che non esista una sola conformazione patogena, ma che tali possibili conformazioni siano piuttosto eterogenee. La caratterizzazione di queste strutture è, di conseguenza, difficile con le tradizionali tecniche in bulk che permettono di studiare solo la conformazione media e non rendono possibile il riconoscimento delle caratteristiche dei conformeri individuali. Lo sviluppo delle tecniche di singola molecola ha permesso di studiare in modo approfondito le conformazioni possibili. In questo lavoro la spettroscopia di forza di singola molecola basata sull'AFM viene applicata a PrP (proteina responsabile delle encefalopatie spongiformi trasmissibili). Si studiano gli equilibri conformazionali del monomero e quelli di costrutti oligomerici, allo scopo di caratterizzare gli step iniziali dei processi aggregativi. Nel corso di questo lavoro di tesi è stato, in particolare, sviluppato un sistema di analisi dati, al fine di studiare in modo quantitativo le distribuzioni di eventi ottenute. Grazie a tale strumento è stato possibile riconoscere i segnali di unfolding della conformazione nativa del monomero e notare come essa sia presente anche in costrutti oligomerici, ad indicare come questo ripiegamento sia stabile anche in presenza di più monomeri ravvicinati. Si è osservato l'effetto del pH sulla stabilità di tale struttura, notando come pH acidi destabilizzino il ripiegamento nativo. Inoltre si è studiato il ruolo dell'orientazione dei monomeri nella formazione di strutture dimeriche. Monomeri e oligomeri di PrP sono stati descritti come proteine parzialmente strutturate il cui panorama energetico contiene molti minimi locali, dando origine a parecchie conformazioni transienti.

The effect of diffusion gradient direction number on tractography of corticospinal tract in human brain: an along-tract analysis

Nel presente lavoro di tesi ho sviluppato un metodo di analisi di dati di DW-MRI (Diffusion-Weighted Magnetic Resonance Imaging)cerebrale, tramite un algoritmo di trattografia, per la ricostruzione del tratto corticospinale, in un campione di 25 volontari sani. Il diffusion tensor imaging (DTI) sfrutta la capacità del tensore di diffusione D di misurare il processo di diffusione dell’acqua, per stimare quantitativamente l’anisotropia dei tessuti. In particolare, nella sostanza bianca cerebrale la diffusione delle molecole di acqua è direzionata preferenzialmente lungo le fibre, mentre è ostacolata perpendicolarmente ad esse. La trattografia utilizza le informazioni ottenute tramite il DW imaging per fornire una misura della connettività strutturale fra diverse regioni del cervello. Nel lavoro si è concentrata l’attenzione sul fascio corticospinale, che è coinvolto nella motricità volontaria, trasmettendo gli impulsi dalla corteccia motoria ai motoneuroni del midollo spinale. Il lavoro si è articolato in 3 fasi. Nella prima ho sviluppato il pre-processing di immagini DW acquisite con un gradiente di diffusione sia 25 che a 64 direzioni in ognuno dei 25 volontari sani. Si è messo a punto un metodo originale ed innovativo, basato su “Regions of Interest” (ROIs), ottenute attraverso la segmentazione automatizzata della sostanza grigia e ROIs definite manualmente su un template comune a tutti i soggetti in esame. Per ricostruire il fascio si è usato un algoritmo di trattografia probabilistica che stima la direzione più probabile delle fibre e, con un numero elevato di direzioni del gradiente, riesce ad individuare, se presente, più di una direzione dominante (seconda fibra). Nella seconda parte del lavoro, ciascun fascio è stato suddiviso in 100 segmenti (percentili). Sono stati stimati anisotropia frazionaria (FA), diffusività media, probabilità di connettività, volume del fascio e della seconda fibra con un’analisi quantitativa “along-tract”, per ottenere un confronto accurato dei rispettivi percentili dei fasci nei diversi soggetti. Nella terza parte dello studio è stato fatto il confronto dei dati ottenuti a 25 e 64 direzioni del gradiente ed il confronto del fascio fra entrambi i lati. Dall’analisi statistica dei dati inter-subject e intra-subject è emersa un’elevata variabilità tra soggetti, dimostrando l’importanza di parametrizzare il tratto. I risultati ottenuti confermano che il metodo di analisi trattografica del fascio cortico-spinale messo a punto è risultato affidabile e riproducibile. Inoltre, è risultato che un’acquisizione con 25 direzioni di DTI, meglio tollerata dal paziente per la minore durata dello scan, assicura risultati attendibili. La principale applicazione clinica riguarda patologie neurodegenerative con sintomi motori sia acquisite, quali sindromi parkinsoniane sia su base genetica o la valutazione di masse endocraniche, per la definizione del grado di contiguità del fascio. Infine, sono state poste le basi per la standardizzazione dell’analisi quantitativa di altri fasci di interesse in ambito clinico o di studi di ricerca fisiopatogenetica.

Le interfacce neurali e le loro possibili applicazioni nell'assistive technology

Ogni anno si registra un crescente aumento delle persone affette da patologie neurodegenerative come la sclerosi laterale amiotrofica, la sclerosi multipla, la malattia di Parkinson e persone soggette a gravi disabilità motorie dovute ad ictus, paralisi cerebrale o lesioni al midollo spinale. Spesso tali condizioni comportano menomazioni molto invalidanti e permanenti delle vie nervose, deputate al controllo dei muscoli coinvolti nell’esecuzione volontaria delle azioni. Negli ultimi anni, molti gruppi di ricerca si sono interessati allo sviluppo di sistemi in grado di soddisfare le volontà dell’utente. Tali sistemi sono generalmente definiti interfacce neurali e non sono pensati per funzionare autonomamente ma per interagire con il soggetto. Tali tecnologie, note anche come Brain Computer Interface (BCI), consentono una comunicazione diretta tra il cervello ed un’apparecchiatura esterna, basata generalmente sull’elettroencefalografia (EEG), in grado di far comunicare il sistema nervoso centrale con una periferica esterna. Tali strumenti non impiegano le usuali vie efferenti coinvolte nella produzione di azioni quali nervi e muscoli, ma collegano l'attività cerebrale ad un computer che ne registra ed interpreta le variazioni, permettendo quindi di ripristinare in modo alternativo i collegamenti danneggiati e recuperare, almeno in parte, le funzioni perse. I risultati di numerosi studi dimostrano che i sistemi BCI possono consentire alle persone con gravi disabilità motorie di condividere le loro intenzioni con il mondo circostante e provano perciò il ruolo importante che esse sono in grado di svolgere in alcune fasi della loro vita.

Geometria frattale: analisi della lacunarita cerebrale in MRI

La geometria frattale descrive la complessità strutturale di oggetti che presentano, entro certi limiti, invarianza a fattori di scala. Obiettivo di questa tesi è l’analisi di indici frattali della morfologia cerebrale e cerebellare da immagini di risonanza magnetica (MRI) pesate T1 e della loro correlazione con l’età. A tale scopo sono state analizzate la dimensione frattale (D0) e la lacunarità (λs), indice di eterogeneità strutturale, della sostanza grigia (GM) e bianca (WM), calcolate mediante algoritmi di box counting e di differential gliding box, implementati in linguaggio C++, e regressione lineare con scelta automatica delle scale spaziali. Gli algoritmi sono stati validati su fantocci 3D ed è stato proposto un metodo per compensare la dipendenza di λs dalle dimensioni dell’immagine e dalla frazione di immagine occupata. L’analisi frattale è stata applicata ad immagini T1 a 3T del dataset ICBM (International Consortium for Brain Mapping) composto da 86 soggetti (età 19-85 anni). D0 e λs sono state rispettivamente 2.35±0.02 (media±deviazione standard) e 0.41±0.05 per la GM corticale, 2.34±0.03 e 0.35±0.05 per la WM cerebrale, 2.19±0.05 e 0.17±0.02 per la GM cerebellare, 1.95±0.06 e 0.30±0.04 per la WM cerebellare. Il coefficiente di correlazione lineare tra età e D0 della GM corticale è r=−0.38 (p=0.003); tra età e λs, r=0.72 (p<0.001) (mostrando che l’eterogeneità strutturale aumenta con l’invecchiamento) e tra età e λs compensata rispetto al volume della GM cerebrale (GMV), r=0.51 (p<0.001), superiore in valore assoluto a quello tra età e GMV (r=−0.45, p<0.001). In un modello di regressione lineare multipla, dove l’età è stata modellata da D0, λ e GMV della GM corticale, λs è risultato l’unico predittore significativo (r parziale=0.62, p<0.001). La lacunarità λs è un indice sensibile alle variazioni strutturali dovute all’invecchiamento cerebrale e si candida come biomarcatore nella valutazione della complessità cerebrale nelle malattie neurodegenerative.

The role of autophagy in anticancer therapy: promises and uncertainties

Autophagy (literally self-eating) is a catabolic mechanism involved in the recycling and turnover of cytoplasmic constituents. Although often referred to as type II programmed cell death, autophagy is primarily a survival rather than a cell death mechanism in response to different stress stimuli. Autophagy is a process in which part of the cytoplasm or entire organelles are sequestered into double-membrane vesicles, called autophagosomes, which ultimately fuse with lysosomes to degrade their contents. Studies show that autophagy is associated with a number of pathological conditions, including cancer, infectious diseases, myopathies and neurodegenerative disorders. With respect to cancer, it has been suggested that the early stages of tumourigenesis are associated with downregulation of autophagy-related (ATG) genes. Indeed, several ATG genes display tumour suppressor function, including Beclin1, which is frequently hemizygously deleted in breast cancer cells. Conversely, in advanced stages of tumourigenesis or during anticancer therapy, autophagy may promote survival of tumour cells in adverse environmental conditions. Therefore, a thorough understanding of autophagy in different cancer types and stages is a prerequisite to determine an autophagy-activating or autophagy-inhibiting treatment strategy.

Autophagy: in: Cell Death

Autophagy is a conserved proteolytic mechanism that degrades cytoplasmic material including cell organelles. Although the importance of autophagy for cell homeostasis and survival has long been appreciated, our understanding of how autophagy is regulated at a molecular level just recently evolved. The importance of autophagy for the quality control of proteins is underscored by the fact that many neurodegenerative and myodegenerative diseases are characterized by an increased but still insufficient autophagic activity. Similarly, if the cellular stress, leading to deoxyribonucleic acid (DNA) damage, mitochondrial damage and/or damaged proteins, does not result in sufficient autophagic repair mechanisms, cells seem to be prone to transform into tumour cells. Therefore, autophagy has multiple roles to play in the causation and prevention of human diseases.

The status of olfactory function and the striatal dopaminergic system in drug-induced parkinsonism

Olfactory impairment has been reported in drug-induced parkinsonism (DIP), but the relationship between dopaminergic dysfunction and smell deficits in DIP patients has not been characterized. To this end, we studied 16 DIP patients and 13 patients affected by Parkinson's disease (PD) using the "Sniffin' Sticks" test and [(123)I] FP-CIT SPECT (single-photon emission computed tomography). DIP patients were divided based on normal (n = 9) and abnormal (n = 7) putamen dopamine transporter binding. Nineteen healthy age- and sex-matched subjects served as controls of smell function. Patients with DIP and pathological putamen uptake had abnormal olfactory function. In this group of patients, olfactory TDI scores (odor threshold, discrimination and identification) correlated significantly with putamen uptake values, as observed in PD patients. By contrast, DIP patients with normal putamen uptake showed odor functions-with the exception of the threshold subtest-similar to control subjects. In this group of patients, no significant correlation was observed between olfactory TDI scores and putamen uptake values. The results of our study suggest that the presence of smell deficits in DIP patients might be more associated with dopaminergic loss rather than with a drug-mediated dopamine receptor blockade. These preliminary results might have prognostic and therapeutic implications, as abnormalities in these individuals may be suggestive of an underlying PD-like neurodegenerative process.

Cataract and cognitive impairment: a review of the literature

Acquired cataract and cognitive impairment are both common age-related problems, and ophthalmologists are increasingly likely to encounter patients who have both. Patients with dementia types who display early visuoperceptual impairment may present first to ophthalmology services. When these patients have coexisting cataract, it may be difficult to distinguish visual complaints due to cataract from those due to dementia. The interaction between visual impairment due to cataract and neurodegenerative disorders affecting the central visual pathways, is not fully understood. Visual impairment due to cataract may stress impaired attentional mechanisms and cataract extraction may improve cognitive performance in some patients with early cognitive impairment; however, the benefits of cataract surgery in established dementia are less clear. In this study, the literature on this subject was reviewed and the implications for practice were considered.

Republished review: cataract and cognitive impairment: a review of the literature

Acquired cataract and cognitive impairment are both common age-related problems, and ophthalmologists are increasingly likely to encounter patients who have both. Patients with dementia types who display early visuoperceptual impairment may present first to ophthalmology services. When these patients have coexisting cataract, it may be difficult to distinguish visual complaints due to cataract from those due to dementia. The interaction between visual impairment due to cataract and neurodegenerative disorders affecting the central visual pathways, is not fully understood. Visual impairment due to cataract may stress impaired attentional mechanisms and cataract extraction may improve cognitive performance in some patients with early cognitive impairment; however, the benefits of cataract surgery in established dementia are less clear. In this study, the literature on this subject was reviewed and the implications for practice were considered.

The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.

Nonmotor disturbances in Parkinson's disease

Nonmotor disturbances (NMDs) affect most patients with Parkinson's disease (PD) and often have a profound impact on their quality of life. NMDs such as depression, anxiety, fatigue, REM sleep behavior disorder, constipation, delayed gastric emptying, altered olfaction and pain can precede the onset of motor symptoms. Other NMDs, including hallucinations, dementia, excessive daytime sleepiness, insomnia, orthostatic hypotension and bladder disturbances, typically appear later in the course of PD. For most NMDs of PD, nondopaminergic and non-nigrostriatal mechanisms (e.g. neurodegeneration of other transmitter systems in the cortex and brainstem, side effects of medications, genetic and psychosocial factors) are considered more relevant than the 'classical' dopaminergic-nigrostriatal dysfunction. The recognition of NMDs requires a high degree of clinical suspicion, the use of specific questionnaires and ancillary tests. Pharmacological and nonpharmacological approaches can be effective, but for most forms of treatment of NMDs, the scientific evidence is limited.

Interleukin-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells

It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1β, TNFα and IL-6, in a validated rat model of experimental glaucoma. First, only weak evidence was found for increased expression of IL-1β and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.

Interferon γ-Dependent Migration of Microglial Cells in the Retina after Systemic Cytomegalovirus Infection

Microglial cells are the resident macrophages of the central nervous system and participate in both innate and adaptive immune responses but can also lead to exacerbation of neurodegenerative pathologies after viral infections. Microglia in the outer layers of the retina and the subretinal space are thought to be involved in retinal diseases where low-grade chronic inflammation and oxidative stress play a role. This study investigated the effect of systemic infection with murine cytomegalovirus on the distribution and dynamics of retinal microglia cells. Systemic infection with murine cytomegalovirus elicited a significant increase in the number of microglia in the subretinal space and an accumulation of iris macrophages, along with morphological signs of activation. Interferon γ (IFN-γ)-deficient mice failed to induce changes in microglia distribution. Bone marrow chimera experiments confirmed that microglial cells in the subretinal space were not recruited from the circulating monocyte pool, but rather represented an accumulation of resident microglial cells from within the retina. Our results demonstrate that a systemic viral infection can lead to IFN-γ-mediated accumulation of microglia into the outer retinal layers and offer proof of concept that systemic viral infections alter the ocular microenvironment and therefore, may influence the course of diseases such as macular degeneration, diabetic retinopathy, or autoimmune uveitis, where low-grade inflammation is implicated.