The Absence of Parkin Does Not Promote Dopamine or Mitochondrial Dysfunction in PolgAD257A/D257A Mitochondrial Mutator Mice.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). In this study, we generated a transgenic model by crossing germline Parkin-/- mice with PolgAD257A mice, an established model of premature aging and mitochondrial stress. We hypothesized that loss of Parkin-/- in PolgAD257A/D257A mice would exacerbate mitochondrial dysfunction, leading to loss of dopamine neurons and nigral-striatal specific neurobehavioral motor dysfunction. We found that aged Parkin-/-/PolgAD257A/D257A male and female mice exhibited severe behavioral deficits, nonspecific to the nigral-striatal pathway, with neither dopaminergic neurodegeneration nor reductions in striatal dopamine. We saw no difference in expression levels of nuclear-encoded subunits of mitochondrial markers and mitochondrial Complex I and IV activities, although we did observe substantial reductions in mitochondrial-encoded COX41I, indicating mitochondrial dysfunction as a result of PolgAD257A/D257A mtDNA mutations. Expression levels of mitophagy markers LC3I/LC3II remained unchanged between cohorts, suggesting no overt mitophagy defects. Expression levels of the parkin substrates, VDAC, NLRP3, and AIMP2 remained unchanged, suggesting no parkin dysfunction. In summary, we were unable to observe dopaminergic neurodegeneration with corresponding nigral-striatal neurobehavioral deficits, nor Parkin or mitochondrial dysfunction in Parkin-/-/PolgAD257A/D257A mice. These findings support a lack of synergism of Parkin loss on mitochondrial dysfunction in mouse models of mitochondrial deficits.SIGNIFICANCE STATEMENT Producing a mouse model of Parkinson's disease (PD) that is etiologically relevant, recapitulates clinical hallmarks, and exhibits reproducible results is crucial to understanding the underlying pathology and in developing disease-modifying therapies. Here, we show that Parkin-/-/PolgAD257A/D257A mice, a previously reported PD mouse model, fails to reproduce a Parkinsonian phenotype. We show that these mice do not display dopaminergic neurodegeneration nor nigral-striatal-dependent motor deficits. Furthermore, we report that Parkin loss does not synergize with mitochondrial dysfunction. Our results demonstrate that Parkin-/-/PolgAD257A/D257A mice are not a reliable model for PD and adds to a growing body of work demonstrating that Parkin loss does not synergize with mitochondrial dysfunction in mouse models of mitochondrial deficits.

Robust kinase- and age-dependent dopaminergic and norepinephrine neurodegeneration in LRRK2 G2019S transgenic mice.

Mutations in LRRK2 are known to be the most common genetic cause of sporadic and familial Parkinson's disease (PD). Multiple lines of LRRK2 transgenic or knockin mice have been developed, yet none exhibit substantial dopamine (DA)-neuron degeneration. Here we develop human tyrosine hydroxylase (TH) promoter-controlled tetracycline-sensitive LRRK2 G2019S (GS) and LRRK2 G2019S kinase-dead (GS/DA) transgenic mice and show that LRRK2 GS expression leads to an age- and kinase-dependent cell-autonomous neurodegeneration of DA and norepinephrine (NE) neurons. Accompanying the loss of DA neurons are DA-dependent behavioral deficits and α-synuclein pathology that are also LRRK2 GS kinase-dependent. Transmission EM reveals that that there is an LRRK2 GS kinase-dependent significant reduction in synaptic vesicle number and a greater abundance of clathrin-coated vesicles in DA neurons. These transgenic mice indicate that LRRK2-induced DA and NE neurodegeneration is kinase-dependent and can occur in a cell-autonomous manner. Moreover, these mice provide a substantial advance in animal model development for LRRK2-associated PD and an important platform to investigate molecular mechanisms for how DA neurons degenerate as a result of expression of mutant LRRK2.

Deubiquitinase CYLD acts as a negative regulator of dopamine neuron survival in Parkinson's disease.

Mutations in PINK1 and parkin highlight the mitochondrial axis of Parkinson's disease (PD) pathogenesis. PINK1/parkin regulation of the transcriptional repressor PARIS bears direct relevance to dopamine neuron survival through augmentation of PGC-1α-dependent mitochondrial biogenesis. Notably, knockout of PARIS attenuates dopaminergic neurodegeneration in mouse models, indicating that interventions that prevent dopaminergic accumulation of PARIS could have therapeutic potential in PD. To this end, we have identified the deubiquitinase cylindromatosis (CYLD) to be a regulator of PARIS protein stability and proteasomal degradation via the PINK1/parkin pathway. Knockdown of CYLD in multiple models of PINK1 or parkin inactivation attenuates PARIS accumulation by modulating its ubiquitination levels and relieving its repressive effect on PGC-1α to promote mitochondrial biogenesis. Together, our studies identify CYLD as a negative regulator of dopamine neuron survival, and inhibition of CYLD may potentially be beneficial in PD by lowering PARIS levels and promoting mitochondrial biogenesis.

Neuronal NLRP3 is a parkin substrate that drives neurodegeneration in Parkinson's disease.

Parkinson's disease (PD) is mediated, in part, by intraneuronal accumulation of α-synuclein aggregates andsubsequent death of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). Microglial hyperactivation of the NOD-like receptor protein 3 (NLRP3) inflammasome has been well-documented in various neurodegenerative diseases, including PD. We show here that loss of parkin activity in mouse and human DA neurons results in spontaneous neuronal NLRP3 inflammasome assembly, leading to DA neuron death. Parkin normally inhibits inflammasome priming by ubiquitinating and targeting NLRP3 for proteasomal degradation. Loss of parkin activity also contributes to the assembly of an active NLRP3 inflammasome complex via mitochondrial-derived reactive oxygen species (mitoROS) generation through the accumulation of another parkin ubiquitination substrate, ZNF746/PARIS. Inhibition of neuronal NLRP3 inflammasome assembly prevents degeneration of DA neurons in familial and sporadic PD models. Strategies aimed at limiting neuronal NLRP3 inflammasome activation hold promise as a disease-modifying therapy for PD.

PARIS farnesylation prevents neurodegeneration in models of Parkinson's disease.

Accumulation of the parkin-interacting substrate (PARIS; ZNF746), due to inactivation of parkin, contributes to Parkinson's disease (PD) through repression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α; PPARGC1A) activity. Here, we identify farnesol as an inhibitor of PARIS. Farnesol promoted the farnesylation of PARIS, preventing its repression of PGC-1α via decreasing PARIS occupancy on the PPARGC1A promoter. Farnesol prevented dopaminergic neuronal loss and behavioral deficits via farnesylation of PARIS in PARIS transgenic mice, ventral midbrain transduction of AAV-PARIS, adult conditional parkin KO mice, and the α-synuclein preformed fibril model of sporadic PD. PARIS farnesylation is decreased in the substantia nigra of patients with PD, suggesting that reduced farnesylation of PARIS may play a role in PD. Thus, farnesol may be beneficial in the treatment of PD by enhancing the farnesylation of PARIS and restoring PGC-1α activity.

Overexpression of Parkinson's Disease-Associated Mutation LRRK2 G2019S in Mouse Forebrain Induces Behavioral Deficits and α-Synuclein Pathology.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an unambiguous cause of late-onset, autosomal-dominant familial Parkinson's disease (PD) and LRRK2 mutations are the strongest genetic risk factor for sporadic PD known to date. A number of transgenic mice expressing wild-type or mutant LRRK2 have been described with varying degrees of LRRK2-related abnormalities and modest pathologies. None of these studies directly addressed the role of the kinase domain in the changes observed and none of the mice present with robust features of the human disease. In an attempt to address these issues, we created a conditional LRRK2 G2019S (LRRK2 GS) mutant and a functionally negative control, LRRK2 G2019S/D1994A (LRRK2 GS/DA). Expression of LRRK2 GS or LRRK2 GS/DA was conditionally controlled using the tet-off system in which the presence of tetracycline-transactivator protein (tTA) with a CAMKIIα promoter (CAMKIIα-tTA) induced expression of TetP-LRRK2 GS or TetP-LRRK2 GS/DA in the mouse forebrain. Overexpression of LRRK2 GS in mouse forebrain induced behavioral deficits and α-synuclein pathology in a kinase-dependent manner. Similar to other genetically engineered LRRK2 GS mice, there was no significant loss of dopaminergic neurons. These mice provide an important new tool to study neurobiological changes associated with the increased kinase activity from the LRRK2 G2019S mutation, which may ultimately lead to a better understanding of not only the physiologic actions of LRRK2, but also potential pathologic actions that underlie LRRK2 GS-associated PD.

Thorase variants are associated with defects in glutamatergic neurotransmission that can be rescued by Perampanel.

The AAA+ adenosine triphosphatase (ATPase) Thorase plays a critical role in controlling synaptic plasticity by regulating the expression of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Bidirectional sequencing of exons of ATAD1, the gene encoding Thorase, in a cohort of patients with schizophrenia and healthy controls revealed rare Thorase variants. These variants caused defects in glutamatergic signaling by impairing AMPAR internalization and recycling in mouse primary cortical neurons. This contributed to increased surface expression of the AMPAR subunit GluA2 and enhanced synaptic transmission. Heterozygous Thorase-deficient mice engineered to express these Thorase variants showed altered synaptic transmission and several behavioral deficits compared to heterozygous Thorase-deficient mice expressing wild-type Thorase. These behavioral impairments were rescued by the competitive AMPAR antagonist Perampanel, a U.S. Food and Drug Administration-approved drug. These findings suggest that Perampanel may be useful for treating disorders involving compromised AMPAR-mediated glutamatergic neurotransmission.

PINK1 Primes Parkin-Mediated Ubiquitination of PARIS in Dopaminergic Neuronal Survival.

Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin cause autosomal-recessive Parkinson's disease through a common pathway involving mitochondrial quality control. Parkin inactivation leads to accumulation of the parkin interacting substrate (PARIS, ZNF746) that plays an important role in dopamine cell loss through repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α) promoter activity. Here, we show that PARIS links PINK1 and parkin in a common pathway that regulates dopaminergic neuron survival. PINK1 interacts with and phosphorylates serines 322 and 613 of PARIS to control its ubiquitination and clearance by parkin. PINK1 phosphorylation of PARIS alleviates PARIS toxicity, as well as repression of PGC-1α promoter activity. Conditional knockdown of PINK1 in adult mouse brains leads to a progressive loss of dopaminergic neurons in the substantia nigra that is dependent on PARIS. Altogether, these results uncover a function of PINK1 to direct parkin-PARIS-regulated PGC-1α expression and dopaminergic neuronal survival.

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1.

Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1-dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1-dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.