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1.
ACS Chem Neurosci ; 9(11): 2849-2858, 2018 11 21.
Article in English | MEDLINE | ID: mdl-29906101

ABSTRACT

The preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta is responsible for the motor impairment associated with Parkinson's disease. Dopamine is a highly reactive molecule, which is usually stored inside synaptic vesicles where it is stabilized by the ambient low pH. However, free cytosolic dopamine can auto-oxidize, generating reactive oxygen species, and lead to the formation of toxic quinones. In the present work, we have analyzed the mechanisms through which the dysfunction of dopamine homeostasis could induce cell toxicity, by focusing in particular on the damage induced by dopamine oxidation products at the mitochondrial level. Our results indicate that dopamine derivatives affect mitochondrial morphology and induce mitochondrial membrane depolarization, leading to a reduction of ATP synthesis. Moreover, our results suggest that opening of the mitochondrial transition pore induced by dopamine-derived quinones may contribute to the specific Parkinson's disease-associated vulnerability of dopamine containing neurons.


Subject(s)
Dopamine/metabolism , Mitochondria/drug effects , Parkinson Disease , Quinones/pharmacology , Reactive Oxygen Species/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Death/drug effects , Cell Line, Tumor , Dopaminergic Neurons , Endotoxins/metabolism , Endotoxins/pharmacology , Mice , Mitochondria/metabolism , Mitochondria, Liver/drug effects , Mitochondrial Membrane Transport Proteins/drug effects , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Mitochondrial Permeability Transition Pore , Oxidation-Reduction , Pars Compacta , Rats
2.
PLoS One ; 11(6): e0155747, 2016.
Article in English | MEDLINE | ID: mdl-27271685

ABSTRACT

Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile in the context of emerging cellular distress. The data provide some new candidates for the toxic species and some new reservations about more well-established candidates. Compared to other known markers of HTT toxicity, nuclear DNA damage appears to be a relatively early pathological event.


Subject(s)
Huntingtin Protein/chemistry , Huntingtin Protein/genetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Protein Multimerization , Amyloid/chemistry , Amyloid/genetics , Amyloid/metabolism , Animals , Cell Survival/genetics , DNA Damage/genetics , Huntingtin Protein/metabolism , Mutant Proteins/metabolism , Mutation/physiology , PC12 Cells , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Folding , Rats
3.
J Neurophysiol ; 116(2): 438-47, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27146984

ABSTRACT

The influence of hyperpolarization-activated cation current (h-current; Ih) upon synaptic integration in paravertebral sympathetic neurons was studied together with expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunit isoforms. All four HCN subunits were detected in homogenates of the rat superior cervical ganglion (SCG) using the PCR to amplify reverse-transcribed messenger RNAs (RT-PCR) and using quantitative PCR. Voltage clamp recordings from dissociated SCG neurons at 35°C detected Ih in all cells, with a maximum hyperpolarization-activated cation conductance of 1.2 ± 0.1 nS, half-maximal activation at -87.6 mV, and reversal potential of -31.6 mV. Interaction between Ih and synaptic potentials was tested with virtual fast nicotinic excitatory postsynaptic potentials (EPSPs) created with dynamic clamp. The blocking of Ih with 15 µM ZD7288 hyperpolarized cells by 4.7 mV and increased the virtual synaptic conductance required to stimulate an action potential from 7.0 ± 0.9 nS to 12.1 ± 0.9 nS. In response to stimulation with 40 s long trains of virtual EPSPs, ZD7288 reduced postsynaptic firing from 2.2 to 1.7 Hz and the associated synaptic amplification from 2.2 ± 0.1 to 1.7 ± 0.2. Cyclic nucleotide binding to HCN channels was simulated by blocking native Ih with ZD7288, followed by reconstitution with virtual Ih using a dynamic clamp model of the voltage clamp data. Over a 30-mV range, shifting the half-activation voltage for Ih in 10 mV depolarizing increments always increased synaptic gain. These results indicate that Ih, in sympathetic neurons, can strengthen nicotinic EPSPs and increase synaptic amplification, while also working as a substrate for cyclic nucleotide-dependent modulation.


Subject(s)
Excitatory Postsynaptic Potentials/drug effects , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Neurons/physiology , Nicotine/pharmacology , Superior Cervical Ganglion/cytology , Animals , Biophysical Phenomena/drug effects , Biophysical Phenomena/physiology , Biophysics , Electric Stimulation , Female , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Male , Neurons/drug effects , Neuropeptide Y/genetics , Neuropeptide Y/metabolism , Patch-Clamp Techniques , Pyrimidines/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Synapses/drug effects , User-Computer Interface
4.
J Mol Biol ; 426(4): 816-29, 2014 Feb 20.
Article in English | MEDLINE | ID: mdl-24291210

ABSTRACT

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either L- or D-polyQ peptides and found that D-fibrils are as cytotoxic as L-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced L-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized D- and L-polyQ in vitro. We found that, as expected, D-polyQ monomers are not recognized by proteins that recognize L-polyQ monomers. However, amyloid fibrils prepared from D-polyQ peptides can efficiently seed the aggregation of L-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a "rippled ß-sheet" interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.


Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Peptides/metabolism , Amino Acid Sequence , Animals , Huntington Disease/metabolism , Isomerism , Kinetics , Molecular Sequence Data , PC12 Cells/drug effects , PC12 Cells/metabolism , PC12 Cells/pathology , Peptides/chemical synthesis , Peptides/genetics , Peptides/pharmacology , Protein Conformation , Rats , Spectroscopy, Fourier Transform Infrared
5.
J Mol Biol ; 425(7): 1183-97, 2013 Apr 12.
Article in English | MEDLINE | ID: mdl-23353826

ABSTRACT

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington's disease. Here, we explore the response of various biophysical parameters to the introduction of ß-hairpin motifs within polyQ sequences. These motifs (tryptophan zipper, disulfide, d-Pro-Gly, Coulombic attraction, l-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well correlated with their known abilities to enhance ß-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n=4 for a simple, unbroken Q23 sequence to approximate unitary n values for similar length polyQs containing ß-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of solid-state NMR (13)C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a ß-hairpin polyQ. Importantly, while ß-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn values remain quite low (~10(-)(10)) and there is no evidence for significant enhancement of ß-structure within the monomer ensemble. The results indicate an important role for ß-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.


Subject(s)
Amyloid/chemistry , Glutamine/chemistry , Peptides/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Amyloid/metabolism , Amyloid/ultrastructure , Animals , Exons/genetics , Glutamine/genetics , Glutamine/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Huntingtin Protein , Kinetics , Magnetic Resonance Spectroscopy , Microscopy, Confocal , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , PC12 Cells , Peptides/genetics , Peptides/metabolism , Protein Conformation , Rats , Spectroscopy, Fourier Transform Infrared
6.
J Mol Biol ; 415(5): 900-17, 2012 Feb 03.
Article in English | MEDLINE | ID: mdl-22178478

ABSTRACT

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (htt(NT) segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the htt(NT) segment forms the α-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of htt(NT) self-assembly by describing inhibitory htt(NT)-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the htt(NT) domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to htt(NT). Studies with D-amino acid and scrambled sequence versions of htt(NT) suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. Htt(NT) derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The htt(NT)-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.


Subject(s)
Amyloid/chemical synthesis , Nerve Tissue Proteins/chemical synthesis , Nuclear Proteins/chemical synthesis , Polymers/chemical synthesis , Amino Acid Sequence , Amino Acids/chemistry , Amyloid/antagonists & inhibitors , Amyloid/genetics , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Humans , Huntingtin Protein , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Peptides/chemistry , Protein Structure, Secondary
7.
Biochim Biophys Acta ; 1802(9): 699-706, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20600874

ABSTRACT

Oxidative stress and mitochondrial dysfunction, especially at the level of complex I of the electronic transport chain, have been proposed to be involved in the pathogenesis of Parkinson disease (PD). A plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (DA) and produce various toxic molecules, i.e., free radicals and quinone species (DAQ). It has been shown that DA oxidation products can induce various forms of mitochondrial dysfunction, such as mitochondrial swelling and decreased electron transport chain activity. In the present work, we analyzed the potentially toxic effects of DAQ on mitochondria and, specifically, on the NADH and GSH pools. Our results demonstrate that the generation of DAQ in isolated respiring mitochondria triggers the opening of the permeability transition pore most probably by inducing oxidation of NADH, while GSH levels are not affected. We then characterized in vitro, by UV and NMR spectroscopy, the reactivity of different DA-derived quinones, i.e., dopamine-o-quinone (DQ), aminochrome (AC) and indole-quinone (IQ), toward NADH and GSH. Our results indicate a very diverse reactivity for the different DAQ studied that may contribute to unravel the complex molecular mechanisms underlying oxidative stress and mitochondria dysfunction in the context of PD.


Subject(s)
Dopamine/analogs & derivatives , Glutathione/metabolism , Mitochondria, Liver/drug effects , NAD/metabolism , Parkinson Disease/metabolism , Animals , Dopamine/analysis , Dopamine/metabolism , Dopamine/pharmacology , Humans , In Vitro Techniques , Magnetic Resonance Spectroscopy , Mice , Mitochondria, Liver/metabolism , Mitochondria, Liver/physiology , Mitochondrial Swelling/drug effects , Models, Biological , Oxidative Stress/drug effects , Parkinson Disease/physiopathology , Quinones/analysis , Quinones/metabolism , Quinones/pharmacology , Spectrophotometry, Ultraviolet
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