Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

A Single Amino Acid Determines the Selectivity and Efficacy of Selective Negative Allosteric Modulators of CaV1.3 L-Type Calcium Channels.

Ca2+ channels with a CaV1.3 pore-forming α1 subunit have been implicated in both neurodegenerative and neuropsychiatric disorders, motivating the development of selective and potent inhibitors of CaV1.3 versus CaV1.2 channels, the calcium channels implicated in hypertensive disorders. We have previously identified pyrimidine-2,4,6-triones (PYTs) that preferentially inhibit CaV1.3 channels, but the structural determinants of their interaction with the channel have not been identified, impeding their development into drugs. By a combination of biochemical, computational, and molecular biological approaches, it was found that PYTs bind to the dihydropyridine (DHP) binding pocket of the CaV1.3 subunit, establishing them as negative allosteric modulators of channel gating. Site-directed mutagenesis, based on homology models of CaV1.3 and CaV1.2 channels, revealed that a single amino acid residue within the DHP binding pocket (M1078) is responsible for the selectivity of PYTs for CaV1.3 over CaV1.2. In addition to providing direction for chemical optimization, these results suggest that, like dihydropyridines, PYTs have pharmacological features that could make them of broad clinical utility.

Mutant huntingtin enhances activation of dendritic Kv4 K+ channels in striatal spiny projection neurons.

Huntington's disease (HD) is initially characterized by an inability to suppress unwanted movements, a deficit attributable to impaired synaptic activation of striatal indirect pathway spiny projection neurons (iSPNs). To better understand the mechanisms underlying this deficit, striatal neurons in ex vivo brain slices from mouse genetic models of HD were studied using electrophysiological, optical and biochemical approaches. Distal dendrites of iSPNs from symptomatic HD mice were hypoexcitable, a change that was attributable to increased association of dendritic Kv4 potassium channels with auxiliary KChIP subunits. This association was negatively modulated by TrkB receptor signaling. Dendritic excitability of HD iSPNs was rescued by knocking-down expression of Kv4 channels, by disrupting KChIP binding, by restoring TrkB receptor signaling or by lowering mutant-Htt (mHtt) levels with a zinc finger protein. Collectively, these studies demonstrate that mHtt induces reversible alterations in the dendritic excitability of iSPNs that could contribute to the motor symptoms of HD.

Systemic isradipine treatment diminishes calcium-dependent mitochondrial oxidant stress.

The ability of the Cav1 channel inhibitor isradipine to slow the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons and the progression of Parkinson's disease (PD) is being tested in a phase 3 human clinical trial. But it is unclear whether and how chronic isradipine treatment will benefit SNc DA neurons in vivo. To pursue this question, isradipine was given systemically to mice at doses that achieved low nanomolar concentrations in plasma, near those achieved in patients. This treatment diminished cytosolic Ca2+ oscillations in SNc DA neurons without altering autonomous spiking or expression of Ca2+ channels, an effect mimicked by selectively knocking down expression of Cav1.3 channel subunits. Treatment also lowered mitochondrial oxidant stress, reduced a high basal rate of mitophagy, and normalized mitochondrial mass - demonstrating that Cav1 channels drive mitochondrial oxidant stress and turnover in vivo. Thus, chronic isradipine treatment remodeled SNc DA neurons in a way that should not only diminish their vulnerability to mitochondrial challenges, but to autophagic stress as well.

Cryopreservation Maintains Functionality of Human iPSC Dopamine Neurons and Rescues Parkinsonian Phenotypes In Vivo.

A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

Mitochondrial oxidant stress in locus coeruleus is regulated by activity and nitric oxide synthase.

Loss of noradrenergic locus coeruleus (LC) neurons is a prominent feature of aging-related neurodegenerative diseases, such as Parkinson's disease (PD). The basis of this vulnerability is not understood. To explore possible physiological determinants, we studied LC neurons using electrophysiological and optical approaches in ex vivo mouse brain slices. We found that autonomous activity in LC neurons was accompanied by oscillations in dendritic Ca(2+) concentration that were attributable to the opening of L-type Ca(2+) channels. This oscillation elevated mitochondrial oxidant stress and was attenuated by inhibition of nitric oxide synthase. The relationship between activity and stress was malleable, as arousal and carbon dioxide increased the spike rate but differentially affected mitochondrial oxidant stress. Oxidant stress was also increased in an animal model of PD. Thus, our results point to activity-dependent Ca(2+) entry and a resulting mitochondrial oxidant stress as factors contributing to the vulnerability of LC neurons.

Calcium entry and α-synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons.

The core motor symptoms of Parkinson's disease (PD) are attributable to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed a major factor in PD pathogenesis. Previous work has shown that activity-dependent calcium entry through L-type channels elevates perinuclear mitochondrial oxidant stress in SNc dopaminergic neurons, providing a potential basis for their selective vulnerability. What is less clear is whether this physiological stress is present in dendrites and if Lewy bodies, the major neuropathological lesion found in PD brains, exacerbate it. To pursue these questions, mesencephalic dopaminergic neurons derived from C57BL/6 transgenic mice were studied in primary cultures, allowing for visualization of soma and dendrites simultaneously. Many of the key features of in vivo adult dopaminergic neurons were recapitulated in vitro. Activity-dependent calcium entry through L-type channels increased mitochondrial oxidant stress in dendrites. This stress progressively increased with distance from the soma. Examination of SNc dopaminergic neurons ex vivo in brain slices verified this pattern. Moreover, the formation of intracellular α-synuclein Lewy-body-like aggregates increased mitochondrial oxidant stress in perinuclear and dendritic compartments. This stress appeared to be extramitochondrial in origin, because scavengers of cytosolic reactive oxygen species or inhibition of NADPH oxidase attenuated it. These results show that physiological and proteostatic stress can be additive in the soma and dendrites of vulnerable dopaminergic neurons, providing new insight into the factors underlying PD pathogenesis.

Calcium entry induces mitochondrial oxidant stress in vagal neurons at risk in Parkinson's disease.

Mitochondrial oxidant stress is widely viewed as being critical to pathogenesis in Parkinson's disease. But the origins of this stress are poorly defined. One possibility is that it arises from the metabolic demands associated with regenerative activity. To test this hypothesis, we characterized neurons in the dorsal motor nucleus of the vagus (DMV), a population of cholinergic neurons that show signs of pathology in the early stages of Parkinson's disease, in mouse brain slices. DMV neurons were slow, autonomous pacemakers with broad spikes, leading to calcium entry that was weakly buffered. Using a transgenic mouse expressing a redox-sensitive optical probe targeted to the mitochondrial matrix, we found that calcium entry during pacemaking created a basal mitochondrial oxidant stress. Knocking out DJ-1 (also known as PARK7), a gene associated with early-onset Parkinson's disease, exacerbated this stress. These results point to a common mechanism underlying mitochondrial oxidant stress in Parkinson's disease and a therapeutic strategy to ameliorate it.

Physiological phenotype and vulnerability in Parkinson's disease.

This review will focus on the principles underlying the hypothesis that neuronal physiological phenotype-how a neuron generates and regulates action potentials-makes a significant contribution to its vulnerability in Parkinson's disease (PD) and aging. A cornerstone of this hypothesis is that the maintenance of ionic gradients underlying excitability can pose a significant energetic burden for neurons, particularly those that have sustained residence times at depolarized membrane potentials, broad action potentials, prominent Ca(2+) entry, and modest intrinsic Ca(2+) buffering capacity. This energetic burden is shouldered in neurons primarily by mitochondria, the sites of cellular respiration. Mitochondrial respiration increases the production of damaging superoxide and other reactive oxygen species (ROS) that have widely been postulated to contribute to cellular aging and PD. Many of the genetic mutations and toxins associated with PD compromise mitochondrial function, providing a mechanistic linkage between known risk factors and cellular physiology that could explain the pattern of pathology in PD. Because much of the mitochondrial burden created by this at-risk phenotype is created by Ca(2+) entry through L-type voltage-dependent channels for which there are antagonists approved for human use, a neuroprotective strategy to reduce this burden is feasible.

The origins of oxidant stress in Parkinson's disease and therapeutic strategies.

Parkinson's disease (PD) is a major world-wide health problem afflicting millions of the aged population. Factors that act on most or all cell types (pan-cellular factors), particularly genetic mutations and environmental toxins, have dominated public discussions of disease etiology. Although there is compelling evidence supporting an association between disease risk and these factors, the pattern of neuronal pathology and cell loss is difficult to explain without cell-specific factors. This article focuses on recent studies showing that the neurons at greatest risk in PD-substantia nigra pars compacta dopamine neurons-have a distinctive physiological phenotype that could contribute to their vulnerability. The opening of L-type calcium channels during autonomous pacemaking results in sustained calcium entry into the cytoplasm of substantia nigra pars compacta dopamine neurons, resulting in elevated mitochondrial oxidant stress and susceptibility to toxins used to create animal models of PD. This cell-specific stress could increase the negative consequences of pan-cellular factors that broadly challenge either mitochondrial or proteostatic competence. The availability of well-tolerated, orally deliverable antagonists for L-type calcium channels points to a novel neuroprotective strategy that could complement current attempts to boost mitochondrial function in the early stages of the disease.

HCN channelopathy in external globus pallidus neurons in models of Parkinson's disease.

Parkinson's disease is a common neurodegenerative disorder characterized by a profound motor disability that is traceable to the emergence of synchronous, rhythmic spiking in neurons of the external segment of the globus pallidus (GPe). The origins of this pathophysiology are poorly defined for the generation of pacemaking. After the induction of a parkinsonian state in mice, there was a progressive decline in autonomous GPe pacemaking, which normally serves to desynchronize activity. The loss was attributable to the downregulation of an ion channel that is essential in pacemaking, the hyperpolarization and cyclic nucleotide-gated (HCN) channel. Viral delivery of HCN2 subunits restored pacemaking and reduced burst spiking in GPe neurons. However, the motor disability induced by dopamine (DA) depletion was not reversed, suggesting that the loss of pacemaking was a consequence, rather than a cause, of key network pathophysiology, a conclusion that is consistent with the ability of L-type channel antagonists to attenuate silencing after DA depletion.

Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1.

Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.

What causes the death of dopaminergic neurons in Parkinson's disease?

The factors governing neuronal loss in Parkinson's disease (PD) are the subject of continuing speculation and experimental study. In recent years, factors that act on most or all cell types (pan-cellular factors), particularly genetic mutations and environmental toxins, have dominated public discussions of disease aetiology. Although there is compelling evidence supporting an association between disease risk and these factors, the pattern of neuronal pathology and cell loss is difficult to explain without cell-specific factors. This chapter focuses on recent studies showing that the neurons at greatest risk in PD--substantia nigra pars compacta (SNc) dopamine (DA) neurons--have a distinctive physiological phenotype that could contribute to their vulnerability. The opening of L-type calcium channels during autonomous pacemaking results in sustained calcium entry into the cytoplasm of SNc DA neurons, resulting in elevated mitochondrial oxidant stress and susceptibility to toxins used to create animal models of PD. This cell-specific stress could increase the negative consequences of pan-cellular factors that broadly challenge either mitochondrial or proteostatic competence. The availability of well-tolerated, orally deliverable antagonists for L-type calcium channels points to a novel neuroprotective strategy that could complement current attempts to boost mitochondrial function in the early stages of the disease.

Thalamic gating of corticostriatal signaling by cholinergic interneurons.

Salient stimuli redirect attention and suppress ongoing motor activity. This attentional shift is thought to rely upon thalamic signals to the striatum to shift cortically driven action selection, but the network mechanisms underlying this interaction are unclear. Using a brain slice preparation that preserved cortico- and thalamostriatal connectivity, it was found that activation of thalamostriatal axons in a way that mimicked the response to salient stimuli induced a burst of spikes in striatal cholinergic interneurons that was followed by a pause lasting more than half a second. This patterned interneuron activity triggered a transient, presynaptic suppression of cortical input to both major classes of principal medium spiny neuron (MSN) that gave way to a prolonged enhancement of postsynaptic responsiveness in striatopallidal MSNs controlling motor suppression. This differential regulation of the corticostriatal circuitry provides a neural substrate for attentional shifts and cessation of ongoing motor activity with the appearance of salient environmental stimuli.

Calcium, cellular aging, and selective neuronal vulnerability in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease in developed countries. The core motor symptoms are attributable to the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Why these neurons, and other restricted sets of non-dopamine neuron, succumb in PD is not clear. One potential clue has come from the observation that the engagement of L-type Ca2+ channels during autonomous pacemaking elevates the sensitivity of SNc DA neurons to mitochondrial toxins used to create animal models of PD, suggesting that Ca2+ entry is a factor in their selective vulnerability. Epidemiological data also supports a linkage between L-type Ca2+ channels and the risk of developing PD. This review examines the hypothesis that the primary factor driving neurodegenerative changes in PD is the metabolic stress created by sustained Ca2+ entry, particularly in the face of genetic or environmental factors that compromise oxidative defenses or proteostatic competence.

Robust pacemaking in substantia nigra dopaminergic neurons.

Dopaminergic neurons of the substantia nigra pars compacta are autonomous pacemakers. This activity is responsible for the sustained release of dopamine necessary for the proper functioning of target structures, such as the striatum. Somatodendritic L-type Ca2+ channels have long been viewed as important, if not necessary, for this activity. The studies reported here challenge this viewpoint. Using a combination of optical and electrophysiological approaches in brain slices, it was found that antagonism of L-type Ca2+ channel effectively stopped dendritic Ca2+ oscillations but left autonomous pacemaking unchanged. Moreover, damping intracellular Ca2+ oscillations with exogenous buffer had little effect on pacemaking rate. Although not necessary for pacemaking, L-type channels helped support pacemaking when challenged with cationic channel blockers. Simulations suggested that the insensitivity to antagonism of L-type channels reflected the multichannel nature of the pacemaking process. The robustness of pacemaking underscores its biological importance and provides a framework for understanding how therapeutics targeting L-type Ca2+ channels might protect dopaminergic neurons in Parkinson's disease without compromising their function.