Ca 2+ -dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca 2+ influx through CaV1.3 Ca 2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca 2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca 2+ -dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinson's disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.

Neuron type-specific proteomics reveals distinct Shank3 proteoforms in iSPNs and dSPNs lead to striatal synaptopathy in Shank3B-/- mice.

Combinatorial expression of postsynaptic proteins underlies synapse diversity within and between neuron types. Thus, characterization of neuron-type-specific postsynaptic proteomes is key to obtaining a deeper understanding of discrete synaptic properties and how selective dysfunction manifests in synaptopathies. To overcome the limitations associated with bulk measures of synaptic protein abundance, we developed a biotin proximity protein tagging probe to characterize neuron-type-specific postsynaptic proteomes in vivo. We found Shank3 protein isoforms are differentially expressed by direct and indirect pathway spiny projection neurons (dSPNs and iSPNs). Investigation of Shank3B-/- mice lacking exons 13-16 within the Shank3 gene, reveal distinct Shank3 protein isoform expression in iSPNs and dSPNs. In Shank3B-/- striatum, Shank3E and Shank3NT are expressed by dSPNs but are undetectable in iSPNs. Proteomic analysis indicates significant and selective alterations in the postsynaptic proteome of Shank3B-/- iSPNs. Correspondingly, the deletion of exons 13-16 diminishes dendritic spine density, reduces spine head diameter, and hampers corticostriatal synaptic transmission in iSPNs. Remarkably, reintroducing Shank3E in adult Shank3B-/- iSPNs significantly rectifies the observed dendritic spine morphological and corticostriatal synaptic transmission deficits. We report unexpected cell-type specific synaptic protein isoform expression which could play a key causal role in specifying synapse diversity and selective synapse dysfunction in synaptopathies.

GABAergic regulation of striatal spiny projection neurons depends upon their activity state.

Synaptic transmission mediated by GABAA receptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at subthreshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical, and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. In perforated patch recordings, activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in both juvenile and adult SPNs. Transcriptomic analysis and pharmacological work suggested that this relatively positive GABAAR reversal potential was not attributable to NKCC1 expression, but rather to HCO3- permeability. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to dendritic iGluR signaling and promoted dendritic spikes. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation for the role of intrastriatal GABAergic circuits.

Mitochondrial dysfunction in Parkinson's disease - a key disease hallmark with therapeutic potential.

Mitochondrial dysfunction is strongly implicated in the etiology of idiopathic and genetic Parkinson's disease (PD). However, strategies aimed at ameliorating mitochondrial dysfunction, including antioxidants, antidiabetic drugs, and iron chelators, have failed in disease-modification clinical trials. In this review, we summarize the cellular determinants of mitochondrial dysfunction, including impairment of electron transport chain complex 1, increased oxidative stress, disturbed mitochondrial quality control mechanisms, and cellular bioenergetic deficiency. In addition, we outline mitochondrial pathways to neurodegeneration in the current context of PD pathogenesis, and review past and current treatment strategies in an attempt to better understand why translational efforts thus far have been unsuccessful.

Distributed dopaminergic signaling in the basal ganglia and its relationship to motor disability in Parkinson's disease.

The degeneration of mesencephalic dopaminergic neurons that innervate the basal ganglia is responsible for the cardinal motor symptoms of Parkinson's disease (PD). It has been thought that loss of dopaminergic signaling in one basal ganglia region - the striatum - was solely responsible for the network pathophysiology causing PD motor symptoms. While our understanding of dopamine (DA)'s role in modulating striatal circuitry has deepened in recent years, it also has become clear that it acts in other regions of the basal ganglia to influence movement. Underscoring this point, examination of a new progressive mouse model of PD shows that striatal dopamine DA depletion alone is not sufficient to induce parkinsonism and that restoration of extra-striatal DA signaling attenuates parkinsonian motor deficits once they appear. This review summarizes recent advances in the effort to understand basal ganglia circuitry, its modulation by DA, and how its dysfunction drives PD motor symptoms.

Parkinson's disease-linked parkin mutation disrupts recycling of synaptic vesicles in human dopaminergic neurons.

Parkin-mediated mitophagy has been studied extensively, but whether mutations in parkin contribute to Parkinson's disease pathogenesis through alternative mechanisms remains unexplored. Using patient-derived dopaminergic neurons, we found that phosphorylation of parkin by Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) at Ser9 leads to activation of parkin in a neuronal-activity-dependent manner. Activated parkin ubiquitinates synaptojanin-1, facilitating its interaction with endophilin A1 and synaptic vesicle recycling. Neurons from PD patients with mutant parkin displayed defective recycling of synaptic vesicles, leading to accumulation of toxic oxidized dopamine that was attenuated by boosting endophilin A1 expression. Notably, combined heterozygous parkin and homozygous PTEN-induced kinase 1 (PINK1) mutations led to earlier disease onset compared with homozygous mutant PINK1 alone, further underscoring a PINK1-independent role for parkin in contributing to disease. Thus, this study identifies a pathway for selective activation of parkin at human dopaminergic synapses and highlights the importance of this mechanism in the pathogenesis of Parkinson's disease.

State-dependent GABAergic regulation of striatal spiny projection neuron excitability.

Synaptic transmission mediated by GABA A receptors (GABA A Rs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABA A Rs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABA A Rs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.

Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington's disease model mice.

Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

Disease mechanisms as Subtypes: Mitochondrial and bioenergetic dysfunction.

Parkinson disease (PD) is the second most common neurodegenerative disease in the world. Despite its enormous human and societal cost, there is no disease-modifying therapy for PD. This unmet medical need reflects our limited understanding of PD pathogenesis. One of the most important clues comes from the recognition that PD motor symptoms arises from the dysfunction and degeneration of a very select group of neurons in the brain. These neurons have a distinctive set of anatomic and physiologic traits that reflect their role in brain function. These traits elevate mitochondrial stress, potentially making them particularly vulnerable to age, as well as to genetic mutations and environmental toxins linked to PD incidence. In this chapter, the literature supporting this model is outlined, along with gaps in our knowledge base. The translational implications of this hypothesis are then discussed, with a focus on why disease-modification trials have failed to date and what this means for the development of new strategies for altering disease course.

α-Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson's disease.

Background: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson's disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain. Methods: aSYN PPFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell- type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser- scanning microscopy of genetically encoded sensors for bioenergetic and redox status. Results: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure. Conclusions: Taken together, our findings suggest that disruption of mitochondrial function, and the subsequent bioenergetic deficit, is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.

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.

Palladium-Catalyzed α-Arylation of Cyclic ß-Dicarbonyl Compounds for the Synthesis of CaV1.3 Inhibitors.

Cyclic α-aryl ß-dicarbonyl derivatives are important scaffolds in medicinal chemistry. Palladium-catalyzed coupling reactions of haloarenes were conducted with diverse five- to seven-membered cyclic ß-dicarbonyl derivatives including barbiturate, pyrazolidine-3,5-dione, and 1,4-diazepane-5,7-dione. The coupling reactions of various para- or meta-substituted aryl halides occurred efficiently when Pd(t-Bu3P)2, Xphos, and Cs2CO3 were used under 1,4-dioxane reflux conditions. Although the couplings of ortho-substituted aryl halides with pyrazolidine-3,5-dione and 1,4-diazepane-5,7-dione were moderate, the coupling with barbiturate was limited. Using the optimized reaction conditions, we synthesized several 5-aryl barbiturates as new scaffolds of CaV1.3 Ca2+ channel inhibitors. Among the synthesized molecules, 14e was the most potent CaV1.3 inhibitor with an IC50 of 1.42 µM.

Pathway-Specific Remodeling of Thalamostriatal Synapses in a Mouse Model of Parkinson's Disease.

BACKGROUND: The network pathophysiology underlying the motor symptoms of Parkinson's disease (PD) is poorly understood. In models of late-stage PD, there is significant cell-specific remodeling of corticostriatal, axospinous glutamatergic synapses on principal spiny projection neurons (SPNs). Neurons in the centrolateral nucleus (CLN) of the thalamus that relay cerebellar activity to the striatum also make axospinous synapses on SPNs, but the extent to which they are affected in PD has not been definitively characterized. OBJECTIVE: To fill this gap, transgenic mice in which CLN neurons express Cre recombinase were used in conjunction with optogenetic and circuit mapping approaches to determine changes in the CLN projection to SPNs in a unilateral 6-hydroxydopamine (6-OHDA) model of late-stage PD. METHODS: Adeno-associated virus vectors carrying Cre-dependent opsin expression constructs were stereotaxically injected into the CLN of Grp-KH288 mice in which CLN, but not parafascicular nucleus neurons, expressed Cre recombinase. The properties of this projection to identify direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) were then studied in ex vivo brain slices of the dorsolateral striatum from control and 6-OHDA lesioned mice using anatomic, optogenetic, and electrophysiological approaches. RESULTS: Optogenetically evoked excitatory synaptic currents in both iSPNs and dSPNs were reduced in lesioned mice; however, the reduction was significantly greater in dSPNs. In iSPNs, the reduction in evoked responses was attributable to synaptic pruning, because synaptic channelrhodopsin assisted circuit mapping (sCRACm) revealed fewer synapses per cell after lesioning. In contrast, sCRACm mapping of CLN inputs to dSPNs failed to detect any change in synapse abundance in lesioned mice. However, the ratio of currents through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors to those through N-methyl-D-aspartate receptors was significantly reduced in dSPNs. Moreover, the distribution of currents evoked by optical stimulation of individual synapses shifted toward smaller amplitudes by lesioning, suggesting that they had undergone long-term depression. CONCLUSIONS: Taken together, our results demonstrate that the CLN projection to the striatum undergoes a pathway-specific remodeling that could contribute to the circuit imbalance thought to drive the hypokinetic features of PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Striatal synaptic adaptations in Parkinson's disease.

The striatum is densely innervated by mesencephalic dopaminergic neurons that modulate acquisition and vigor of goal-directed actions and habits. This innervation is progressively lost in Parkinson's disease (PD), contributing to the defining movement deficits of the disease. Although boosting dopaminergic signaling with levodopa early in the course of the disease alleviates these deficits, later this strategy leads to the emergence of debilitating dyskinesia. Here, recent advances in our understanding of how striatal cells and circuits adapt to this progressive de-innervation and to levodopa therapy are discussed. First, we discuss how dopamine (DA) depletion triggers cell type-specific, homeostatic changes in spiny projection neurons (SPNs) that tend to normalize striatal activity but also lead to disruption of the synaptic architecture sculpted by experience. Second, we discuss the roles played by cholinergic and nitric oxide-releasing interneurons in these adaptations. Third, we examine recent work in freely moving mice suggesting that alterations in the spatiotemporal dynamics of striatal ensembles contributes to PD movement deficits. Lastly, we discuss recently published evidence from a progressive model of PD suggesting that contrary to the classical model, striatal pathway imbalance is necessary but not sufficient to produce frank parkinsonism.

The roles of connectivity and neuronal phenotype in determining the pattern of α-synuclein pathology in Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and motor dysfunction has been attributed to loss of dopaminergic neurons. However, motor dysfunction is only one of many symptoms experienced by patients. A neuropathological hallmark of PD is intraneuronal protein aggregates called Lewy pathology (LP). Neuropathological staging studies have shown that dopaminergic neurons are only one of the many cell types prone to manifest LP. Progressive appearance of LP in multiple brain regions, as well as peripheral nerves, has led to the popular hypothesis that LP and misfolded forms of one of its major components - α-synuclein (aSYN) - can spread through synaptically connected circuits. However, not all brain regions or neurons within connected circuits develop LP, suggesting that cell autonomous factors modulate the development of pathology. Here, we review studies about how LP develops and progressively engages additional brain regions. We focus on how connectivity constrains progression and discuss cell autonomous factors that drive pathology development. We propose a mixed model of cell autonomous factors and trans-synaptic spread as mediators of pathology progression and put forward this model as a framework for future experiments exploring PD pathophysiology.

Re-Analysis of the STEADY-PD II Trial-Evidence for Slowing the Progression of Parkinson's Disease.

BACKGROUND: Recent examination of the STEADY-PD III isradipine clinical trial data concluded that early-stage Parkinson's disease (PD) participants who had longer exposure to isradipine had a significant delay in their need for symptomatic medication, as well as a lower medication burden at the end of the trial. These findings suggest that greater exposure to isradipine might slow disease progression. OBJECTIVES: To test this hypothesis, the data from the STEADY-PD II isradipine clinical trial, in which an extended-release (ER) formulation of the drug was used, was re-examined. METHODS: The re-analysis of the STEADY-PD II data was restricted to participants assigned placebo or tolerable isradipine treatment (10 mg isradipine/day or less). The effect of isradipine treatment was assessed by Unified Parkinson's Disease Rating Scale (UPDRS) at the end of the 52-week trial, rather than by last observation carried forward at the beginning of symptomatic therapy. RESULTS: Participant cohorts were well-matched for baseline disability, initial disease progression, and time to initiation of symptomatic therapy. Participants given 10 mg/day ER isradipine had significantly smaller total and part 3 UPDRS scores at the end of the trial than did the placebo cohort. Post hoc adjustment for symptomatic therapy diminished the statistical significance of these differences. In those participants not taking a monoamine oxidase B inhibitor, the progression in UPDRS scores also was significantly reduced. CONCLUSIONS: These results are consistent with the recent secondary analysis of the STEADY-PD III clinical trial-suggesting that clinically attainable brain exposure to isradipine may slow early-stage PD progression. © 2021 International Parkinson and Movement Disorder Society.

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.

CalDAG-GEFI mediates striatal cholinergic modulation of dendritic excitability, synaptic plasticity and psychomotor behaviors.

CalDAG-GEFI (CDGI) is a protein highly enriched in the striatum, particularly in the principal spiny projection neurons (SPNs). CDGI is strongly down-regulated in two hyperkinetic conditions related to striatal dysfunction: Huntington's disease and levodopa-induced dyskinesia in Parkinson's disease. We demonstrate that genetic deletion of CDGI in mice disrupts dendritic, but not somatic, M1 muscarinic receptors (M1Rs) signaling in indirect pathway SPNs. Loss of CDGI reduced temporal integration of excitatory postsynaptic potentials at dendritic glutamatergic synapses and impaired the induction of activity-dependent long-term potentiation. CDGI deletion selectively increased psychostimulant-induced repetitive behaviors, disrupted sequence learning, and eliminated M1R blockade of cocaine self-administration. These findings place CDGI as a major, but previously unrecognized, mediator of cholinergic signaling in the striatum. The effects of CDGI deletion on the self-administration of drugs of abuse and its marked alterations in hyperkinetic extrapyramidal disorders highlight CDGI's therapeutic potential.