Non-homogeneous degeneration within midbrain dopaminergic (DA) neurons is a histopathological hallmark of Parkinson’s disease (PD). Typically, DA neurons in the Substantia Nigra pars compacta (SNpc) are markedly more vulnerable than in the adjacent ventral tegmental area (VTA) (Brichta and Greengard 2014, Schapira and Jenner 2011). Numerous animal models, both toxin-based or transgenic, show non-uniform DA degeneration patterns, strongly suggesting that intrinsic cellular properties, rather than etiologic factors, underlie differential vulnerability between distinct subsets (Blesa and Przedborski 2014). For therapeutic prospects, understanding the molecular bases of this key pathogenic feature would dramatically improve our chances to develop neuroprotective, disease-modifying treatments. Comparative SNpc-VTA gene expression studies have revealed extensively overlapping signatures between the two DA populations (Grimm et al. 2004, Greene, Dingledine and Greenamyre 2005), suggesting that quantitative, rather than qualitative differences in the expression or function of a limited number of genes subtend selective vulnerability. Over the last decade, it has been suggested that intrinsic electrophysiological properties of specific DA subsets, such as the differential expression or function of selected ion channels, provide a physiological substrate for differential vulnerability (Liss et al. 2005, Guzman et al. 2009, Surmeier et al. 2012, Dragicevic, Schiemann and Liss 2015) In this regard, my colleagues previously demonstrated that MPP+, a neurotoxin able to cause selective nigrostriatal degeneration in animal rodents and primates, inhibits the Hyperpolarization-activated current (Ih) in SNpc DA neurons (Masi et al. 2013). The aim of this PhD project was to investigate the contribution of Ih loss of function (LOF) to the selective degeneration of SNpc DA neurons in PD. For first, we studied the impact of Ih inhibition at the cellular and molecular level, focusing on the electrical properties discriminating among differentially vulnerable subsets of midbrain DA neurons in TH-GFP mice. We showed that pharmacological suppression of Ih increases the amplitude and decay time of excitatory post-synaptic potentials (EPSPs), leading to temporal summation of multiple excitatory potentials at somatic level. Importantly, these effects was quantitatively more evident in SNpc DA neurons. Furthermore, we investigated the participation of VGCC-dependent calcium entry during evoked synaptic activity by combined electrophysiological and calcium fluorometry experiments in the SNpc and VTA DA neuron in wild-type rats. And, we showed that Ih block-induced synaptic potentiation leads to the amplification of somatic calcium responses (SCRs) in vitro. This effect was specific for the SNpc subfield and largely mediated by L-Type calcium channels, as indicated by sensitivity to the CaV 1 blocker isradipine. We showed that Ih is downregulated in presence of low intracellular ATP and that Ih suppression reduced the inhibitory effect of GABAergic transmission, suggesting the existence of a mechanistic link between disruption of mitochondrial homeostasis and abnormal synaptic excitability in SNpc DA neurons. Finally, we tested the effect of Ih suppression in vivo and found that intracerebral stereotaxic injection of the selective blockers ivabradine or ZD7288 causes a pattern of DA degeneration strikingly resembling that seen in MitoPark mice and MPP+-treated mice, two distinct PD models characterized by mitochondrial failure and SNpc-specific DAergic degeneration (Ekstrand et al. 2007, Blesa and Przedborski 2014) Overall, the present data support the hypothesis that Ih LOF may possibly be regarded as an acquired alteration, caused by disruption of mitochondrial metabolism, affecting specifically, or to a larger extent, DA neurons in the SNpc, where Ih is critical in the regulation of synaptic excitability. In vivo, Ih LOF may result from mitochondrial dysfunction, a key disease mechanism at the basis of extensively studied PD animal models, which is gaining increasing attention in the human pathology too. In this regard, there is increasing evidence linking mitochondrial damage to Ih function. Ih is suppressed by the mitochondrial toxin MPP+ in vitro (Masi et al. 2013) and lamotrigine (LTG), a commercial anticonvulsant agent reported to activate Ih (Poolos, Migliore and Johnston 2002, Friedman et al. 2014), is neuroprotective in MPTP-induced DA degeneration models (Archer and Fredriksson 2000, Lagrue et al. 2007). Furthermore, Ih current density is diminished in SNpc DA neurons of MitoPark mice at 6 weeks of age, well before the appearance of neurodegeneration (Good et al. 2011). This evidence supports the proposition that Ih LOF, which may result from mitochondrial failure during PD progression, leads to SNpc-specific DA degeneration through toxic calcium overload.Based on these premises, we tested the hypothesis that Ih LOF is a necessary pathogenic step in relevant PD animal models, and that reversion of this defect is neuroprotective. To test our hypothesis, we used The MitoPark mouse, a model based on a mitochondrial mutation expressed in DA neurons and featuring HCN LOF at an early disease stage (Ekstrand et al. 2007). This model shows late-onset, slow-progressing DA degeneration with differential vulnerability between SNc and VTA. furthermore, electrophysiological recordings in slices from mice at presymptomatic stage (6 weeks) have shown an aberrant activity pattern of SNc DA neurons, with a concomitant inactivation of the Ih (Branch et al. 2016) In this regard, we performed a pharmacological rescue of HCN channels using LTG in presymptomatic Mitopark mice (6 weeks). LTG, is generally considered as a voltage-gated sodium (Nav) channel blocker, as a anticonvulsivant that selectively reduced action potential firing from dendritic depolarization, while minimally affecting firing at the soma. Recent studies suggest that this regional and input-specific effect resulted from an increase in Ih, present predominantly in dendrites. These results demonstrate that neuronal excitability can be altered by drugs acting selectively on dendrites, and suggest an important role for Ih in controlling dendritic excitability (Poolos et al. 2002). LTG has already been used to functionally upregulate HCN function in DA neurons in vivo (Friedman et al. 2014). Based on the above hypothesis, we attempted a pharmacological rescue of Ih function by administering chronic LTG. LTG was administered daily starting from 6 weeks (presymptomatic stage), when Ih loss of function has been described, until 18 weeks (the beginning of the symptomatic stage). What we observed is a dramatic reduction of motor decay compared to MitoPark mice treated with vehicle. Pharmacological manipulation of Ih function in vivo suggests a possible link to DA vulnerability during disease progression. MitoPark mice has better face and construct validity compared with major toxin-induced models, the rat 6-OHDA model and the mouse MPTP-model (Terzioglu and Galter 2008). Indeed, MitoPark mice display a very slow progression of the symptoms, more similarly reflecting the disease progression in PD patients vs. the acute ablation of the DA system manifested in most lesion models. In addition, the slow progression of symptoms in MitoPark mice offers the opportunity to study the effects of chronic treatments for prolonged periods of time (i.e. several months) under conditions of a slow and gradual aggravation of symptoms in line with the situation in PD patients (Galter et al. 2010). To conclude, our research supports the hypothesis that Ih loss of function represents a bona fide pathogenic mechanism which, possibly in concert with SNpc-specific connectivity, may determine differential DAergic vulnerability during disease progression in relevant animal models and in human PD. Considering the total lack of neuroprotective medications in PD therapy and in consideration of the serious side effects associated with long-term levodopa therapy, the targets emerging from this research may be exploited to design protective therapies, in individuals with an early diagnosis of PD.

Ih loss of function as a pathogenic mechanism underlying the selective vulnerability of nigral dopamine neurons in Parkinson’s disease / Carmen Carbone. - (2019).

Ih loss of function as a pathogenic mechanism underlying the selective vulnerability of nigral dopamine neurons in Parkinson’s disease

CARBONE, CARMEN
2019

Abstract

Non-homogeneous degeneration within midbrain dopaminergic (DA) neurons is a histopathological hallmark of Parkinson’s disease (PD). Typically, DA neurons in the Substantia Nigra pars compacta (SNpc) are markedly more vulnerable than in the adjacent ventral tegmental area (VTA) (Brichta and Greengard 2014, Schapira and Jenner 2011). Numerous animal models, both toxin-based or transgenic, show non-uniform DA degeneration patterns, strongly suggesting that intrinsic cellular properties, rather than etiologic factors, underlie differential vulnerability between distinct subsets (Blesa and Przedborski 2014). For therapeutic prospects, understanding the molecular bases of this key pathogenic feature would dramatically improve our chances to develop neuroprotective, disease-modifying treatments. Comparative SNpc-VTA gene expression studies have revealed extensively overlapping signatures between the two DA populations (Grimm et al. 2004, Greene, Dingledine and Greenamyre 2005), suggesting that quantitative, rather than qualitative differences in the expression or function of a limited number of genes subtend selective vulnerability. Over the last decade, it has been suggested that intrinsic electrophysiological properties of specific DA subsets, such as the differential expression or function of selected ion channels, provide a physiological substrate for differential vulnerability (Liss et al. 2005, Guzman et al. 2009, Surmeier et al. 2012, Dragicevic, Schiemann and Liss 2015) In this regard, my colleagues previously demonstrated that MPP+, a neurotoxin able to cause selective nigrostriatal degeneration in animal rodents and primates, inhibits the Hyperpolarization-activated current (Ih) in SNpc DA neurons (Masi et al. 2013). The aim of this PhD project was to investigate the contribution of Ih loss of function (LOF) to the selective degeneration of SNpc DA neurons in PD. For first, we studied the impact of Ih inhibition at the cellular and molecular level, focusing on the electrical properties discriminating among differentially vulnerable subsets of midbrain DA neurons in TH-GFP mice. We showed that pharmacological suppression of Ih increases the amplitude and decay time of excitatory post-synaptic potentials (EPSPs), leading to temporal summation of multiple excitatory potentials at somatic level. Importantly, these effects was quantitatively more evident in SNpc DA neurons. Furthermore, we investigated the participation of VGCC-dependent calcium entry during evoked synaptic activity by combined electrophysiological and calcium fluorometry experiments in the SNpc and VTA DA neuron in wild-type rats. And, we showed that Ih block-induced synaptic potentiation leads to the amplification of somatic calcium responses (SCRs) in vitro. This effect was specific for the SNpc subfield and largely mediated by L-Type calcium channels, as indicated by sensitivity to the CaV 1 blocker isradipine. We showed that Ih is downregulated in presence of low intracellular ATP and that Ih suppression reduced the inhibitory effect of GABAergic transmission, suggesting the existence of a mechanistic link between disruption of mitochondrial homeostasis and abnormal synaptic excitability in SNpc DA neurons. Finally, we tested the effect of Ih suppression in vivo and found that intracerebral stereotaxic injection of the selective blockers ivabradine or ZD7288 causes a pattern of DA degeneration strikingly resembling that seen in MitoPark mice and MPP+-treated mice, two distinct PD models characterized by mitochondrial failure and SNpc-specific DAergic degeneration (Ekstrand et al. 2007, Blesa and Przedborski 2014) Overall, the present data support the hypothesis that Ih LOF may possibly be regarded as an acquired alteration, caused by disruption of mitochondrial metabolism, affecting specifically, or to a larger extent, DA neurons in the SNpc, where Ih is critical in the regulation of synaptic excitability. In vivo, Ih LOF may result from mitochondrial dysfunction, a key disease mechanism at the basis of extensively studied PD animal models, which is gaining increasing attention in the human pathology too. In this regard, there is increasing evidence linking mitochondrial damage to Ih function. Ih is suppressed by the mitochondrial toxin MPP+ in vitro (Masi et al. 2013) and lamotrigine (LTG), a commercial anticonvulsant agent reported to activate Ih (Poolos, Migliore and Johnston 2002, Friedman et al. 2014), is neuroprotective in MPTP-induced DA degeneration models (Archer and Fredriksson 2000, Lagrue et al. 2007). Furthermore, Ih current density is diminished in SNpc DA neurons of MitoPark mice at 6 weeks of age, well before the appearance of neurodegeneration (Good et al. 2011). This evidence supports the proposition that Ih LOF, which may result from mitochondrial failure during PD progression, leads to SNpc-specific DA degeneration through toxic calcium overload.Based on these premises, we tested the hypothesis that Ih LOF is a necessary pathogenic step in relevant PD animal models, and that reversion of this defect is neuroprotective. To test our hypothesis, we used The MitoPark mouse, a model based on a mitochondrial mutation expressed in DA neurons and featuring HCN LOF at an early disease stage (Ekstrand et al. 2007). This model shows late-onset, slow-progressing DA degeneration with differential vulnerability between SNc and VTA. furthermore, electrophysiological recordings in slices from mice at presymptomatic stage (6 weeks) have shown an aberrant activity pattern of SNc DA neurons, with a concomitant inactivation of the Ih (Branch et al. 2016) In this regard, we performed a pharmacological rescue of HCN channels using LTG in presymptomatic Mitopark mice (6 weeks). LTG, is generally considered as a voltage-gated sodium (Nav) channel blocker, as a anticonvulsivant that selectively reduced action potential firing from dendritic depolarization, while minimally affecting firing at the soma. Recent studies suggest that this regional and input-specific effect resulted from an increase in Ih, present predominantly in dendrites. These results demonstrate that neuronal excitability can be altered by drugs acting selectively on dendrites, and suggest an important role for Ih in controlling dendritic excitability (Poolos et al. 2002). LTG has already been used to functionally upregulate HCN function in DA neurons in vivo (Friedman et al. 2014). Based on the above hypothesis, we attempted a pharmacological rescue of Ih function by administering chronic LTG. LTG was administered daily starting from 6 weeks (presymptomatic stage), when Ih loss of function has been described, until 18 weeks (the beginning of the symptomatic stage). What we observed is a dramatic reduction of motor decay compared to MitoPark mice treated with vehicle. Pharmacological manipulation of Ih function in vivo suggests a possible link to DA vulnerability during disease progression. MitoPark mice has better face and construct validity compared with major toxin-induced models, the rat 6-OHDA model and the mouse MPTP-model (Terzioglu and Galter 2008). Indeed, MitoPark mice display a very slow progression of the symptoms, more similarly reflecting the disease progression in PD patients vs. the acute ablation of the DA system manifested in most lesion models. In addition, the slow progression of symptoms in MitoPark mice offers the opportunity to study the effects of chronic treatments for prolonged periods of time (i.e. several months) under conditions of a slow and gradual aggravation of symptoms in line with the situation in PD patients (Galter et al. 2010). To conclude, our research supports the hypothesis that Ih loss of function represents a bona fide pathogenic mechanism which, possibly in concert with SNpc-specific connectivity, may determine differential DAergic vulnerability during disease progression in relevant animal models and in human PD. Considering the total lack of neuroprotective medications in PD therapy and in consideration of the serious side effects associated with long-term levodopa therapy, the targets emerging from this research may be exploited to design protective therapies, in individuals with an early diagnosis of PD.
2019
Guido Mannaioni
ITALIA
Carmen Carbone
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1155812
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