Huntington’s disease (HD) is a neurodegenerative progressive genetic disorder characterized by motor deficits, neuropsychiatric disturbances, and cognitive decline. The beginning of HD is generally in midlife and death occurs 15-20 years from the symptoms’ onset. HD is an autosomal dominant genetic disorder affecting the gene codifying for the huntingtin protein (Htt) on chromosome 4. An expansion of a CAG triplet repeat stretch results in a mutant form of the Htt. The mutant Htt (mHtt) induces cellular toxicity and causes a diffuse loss of neurons in the whole brain, but particularly in cortex and striatum. Moreover, mHtt causes progressive alterations in morphology, excitability, and synaptic properties of cortical pyramidal neurons (CPNs) and striatal medium-sized spiny neurons (MSNs). The striatum is one of the subcortical gray matter nuclei, called basal ganglia. They are strongly interconnected and form a large cortical-subcortical-cortical circuit that allows to regulate and refine movements, and furthermore to control specific cognitive activities. The cellular impairments and the diffuse loss of neurons during HD progression result in a cortical and striatal atrophy and lead to functional alterations of the basal ganglia circuit, that determine the HD symptoms. The striatal MSNs – that are the principal and the most numerous neurons of the striatum and project to the basal ganglia output nuclei – receive numerous cortical projections from most of the cortical areas. Alterations in CPNs/MSNs connectivity may exacerbate the striatal deficits in HD. A variety of other pathophysiological mechanisms are implicated in Huntington’s disease. Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of cholesterol in the HD brain, we evaluate the effects of cholesterol administration in the striatum of HD mice (R6/2 models) through two different approaches: the exogenous approach involved the direct infusion of cholesterol at the striatal level through the use of osmotic mini-pumps, while the endogenous approach was implemented by stimulating the activation of glial cholesterol synthesis by means of the transcription factor hSERBP2 administered with viral vectors. To verify the effect of the increase in cholesterol levels, electrophysiological experiments were carried out using the patch-clamp technique applied to coronal brain slices obtained at the level of the striatum. The main objective of these experiments was to investigate whether and how cholesterol could correct the impairments in excitatory and inhibitory synaptic transmission directed to striatal MSNs that occur in HD. We found morphological and functional recovery of synaptic transmission involving either excitatory and inhibitory synapses onto the striatal medium spiny neurons in HD mice, with both the exogenous and the endogenous approach. These results indicate that the increase of cholesterol levels into the striatum can exert a disease-modifying effect and may be therapeutically relevant in Huntington’s disease. Since HD can be considered a synaptopathy, the confirmation that cholesterol, regardless of the modality of supplementation, can represent an effective pharmacological treatment for resolving the synaptic impairments typical of HD could constitute an important starting point for the development of further pharmacological therapies against this devastating disease.
La malattia di Huntington (HD) è una malattia genetica neurodegenerativa caratterizzata da deficit motori, disturbi neuropsichiatrici e cognitivi. L’HD insorge generalmente nella mezza età con un’irreversibile progressione dei sintomi in 10/15 anni. L’HD è una malattia genetica autosomica dominante che colpisce il gene che codifica per la proteina huntingtina (Htt) sul cromosoma 4. Un’anomala espansione della tripletta CAG risulta in una forma mutata dell’Htt. L’Htt mutata (mHtt) induce tossicità cellulare e provoca una perdita diffusa di neuroni in tutto il cervello, in particolare nella corteccia e nello striato. Inoltre, l’mHtt causa alterazioni progressive nella morfologia, nell’eccitabilità e nelle proprietà sinaptiche dei neuroni piramidali corticali (CPNs) e dei neuroni spinosi di medie dimensioni (MSNs) striatali. Lo striato è uno dei nuclei di sostanza grigia sottocorticale, detti gangli della base. Sono fortemente interconnessi e formano un grande circuito corticale-subcorticale-corticale che permette di regolare e affinare i movimenti, oltre che controllare specifiche attività cognitive. Le alterazioni cellulari e la perdita diffusa di neuroni durante la progressione della malattia provocano un'atrofia corticale e striatale e portano ad un'alterazione funzionale del circuito dei gangli della base, che determina i sintomi dell’HD. I MSNs striatali - che sono i principali e i più numerosi neuroni dello striato e proiettano verso i nuclei di uscita dei gangli della base - ricevono numerose proiezioni corticali dalla maggior parte delle aree corticali. Le alterazioni nella connettività CPNs / MSNs possono esacerbare i deficit striatali nell’HD. Altri meccanismi fisiopatologici sono però implicati nella malattia di Huntington; tra questi, una ridotta biosintesi del colesterolo è stata rilevata nel cervello di topi HD in stadi pre-sintomatici, portando a una ridotta sintesi del colesterolo, in particolare nello striato. Per identificare un possibile trattamento e verificare se e in che modo il colesterolo potrebbe dare beneficio in un cervello affetto da HD, abbiamo valutato gli effetti della somministrazione di colesterolo nello striato di topi HD attraverso due diversi approcci: uno esogeno e uno endogeno. L’approccio esogeno ha previsto l’infusione diretta a livello striatale di colesterolo attraverso l’impiego di minipompe osmotiche, mentre quello endogeno è stato messo in atto stimolando l’attivazione della sintesi gliale di colesterolo mediante il fattore di trascrizione hSERBP2 somministrato con vettori virali. Per verificare l’effetto dell’aumento dei livelli del colesterolo sono stati effettuati esperimenti di elettrofisiologia mediante la tecnica del patch-clamp applicata a brain slices coronali ottenute a livello del corpo striato. L’obiettivo principale di tali esperimenti era di indagare se e in che modo il colesterolo potesse correggere le compromissioni nella trasmissione sinaptica eccitatoria e inibitoria diretta ai MSNs striatali che si verificano nell’HD. I risultati mostrano un recupero morfologico e funzionale della trasmissione sinaptica che coinvolge sinapsi eccitatorie e inibitorie di neuroni spinosi medi striatali in topi HD sia con l'approccio esogeno che con quello endogeno. Dal momento che l’HD può essere considerato una sinaptopatia– le compromissioni nella connettività e nella trasmissione sinaptica hanno infatti un ruolo chiave nella patogenesi dell’HD– la conferma che il colesterolo, indipendentemente dalla modalità di supplementazione, possa rappresentare un bersaglio farmacologico efficace per risolvere o mitigare le compromissioni sinaptiche tipiche dell’HD potrebbe costituire un importante punto di partenza per la messa a punto di terapie farmacologiche contro questa difficile malattia
Ripristino delle alterazioni sinaptiche striatali in topi Huntington attraverso l’aumento dei livelli di colesterolo nello striato
ZAMBONI, MATTIA
2019/2020
Abstract
Huntington’s disease (HD) is a neurodegenerative progressive genetic disorder characterized by motor deficits, neuropsychiatric disturbances, and cognitive decline. The beginning of HD is generally in midlife and death occurs 15-20 years from the symptoms’ onset. HD is an autosomal dominant genetic disorder affecting the gene codifying for the huntingtin protein (Htt) on chromosome 4. An expansion of a CAG triplet repeat stretch results in a mutant form of the Htt. The mutant Htt (mHtt) induces cellular toxicity and causes a diffuse loss of neurons in the whole brain, but particularly in cortex and striatum. Moreover, mHtt causes progressive alterations in morphology, excitability, and synaptic properties of cortical pyramidal neurons (CPNs) and striatal medium-sized spiny neurons (MSNs). The striatum is one of the subcortical gray matter nuclei, called basal ganglia. They are strongly interconnected and form a large cortical-subcortical-cortical circuit that allows to regulate and refine movements, and furthermore to control specific cognitive activities. The cellular impairments and the diffuse loss of neurons during HD progression result in a cortical and striatal atrophy and lead to functional alterations of the basal ganglia circuit, that determine the HD symptoms. The striatal MSNs – that are the principal and the most numerous neurons of the striatum and project to the basal ganglia output nuclei – receive numerous cortical projections from most of the cortical areas. Alterations in CPNs/MSNs connectivity may exacerbate the striatal deficits in HD. A variety of other pathophysiological mechanisms are implicated in Huntington’s disease. Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of cholesterol in the HD brain, we evaluate the effects of cholesterol administration in the striatum of HD mice (R6/2 models) through two different approaches: the exogenous approach involved the direct infusion of cholesterol at the striatal level through the use of osmotic mini-pumps, while the endogenous approach was implemented by stimulating the activation of glial cholesterol synthesis by means of the transcription factor hSERBP2 administered with viral vectors. To verify the effect of the increase in cholesterol levels, electrophysiological experiments were carried out using the patch-clamp technique applied to coronal brain slices obtained at the level of the striatum. The main objective of these experiments was to investigate whether and how cholesterol could correct the impairments in excitatory and inhibitory synaptic transmission directed to striatal MSNs that occur in HD. We found morphological and functional recovery of synaptic transmission involving either excitatory and inhibitory synapses onto the striatal medium spiny neurons in HD mice, with both the exogenous and the endogenous approach. These results indicate that the increase of cholesterol levels into the striatum can exert a disease-modifying effect and may be therapeutically relevant in Huntington’s disease. Since HD can be considered a synaptopathy, the confirmation that cholesterol, regardless of the modality of supplementation, can represent an effective pharmacological treatment for resolving the synaptic impairments typical of HD could constitute an important starting point for the development of further pharmacological therapies against this devastating disease.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/12538