Senthilkumar S. Karuppagounder

Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkins ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD.

Sulfhydration mediates neuroprotective actions of parkin

Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson’s Disease (PD) are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic PD. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide (H2S), termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and investigated by site directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with PD, suggesting that this loss may be pathologic. This implies that H2S donors may be therapeutic.

Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3

INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder and leads to slowness of movement, tremor, rigidity, and, in the later stages of PD, cognitive impairment. Pathologically, PD is characterized by the accumulation of α-synuclein in Lewy bodies and neurites. There is degeneration of neurons throughout the nervous system, with the degeneration of dopamine neurons in the substantia nigra pars compacta leading to the major symptoms of PD. RATIONALE In the brains of PD patients, pathologic α-synuclein seems to spread from cell to cell via self-amplification, propagation, and transmission in a stereotypical and topographical pattern among neighboring cells and/or anatomically connected brain regions. The spread or transmission of pathologic α-synuclein is emerging as a potentially important driver of PD pathogenesis. The underlying mechanisms and molecular entities responsible for the transmission of pathologic α-synuclein from cell to cell are not known, but the entry of pathologic α-synuclein into neurons is thought to occur, in part, through an active clathrin-dependent endocytic process. RESULTS Using recombinant α-synuclein preformed fibrils (PFF) as a model system with which to study the transmission of misfolded α-synuclein from neuron to neuron, we screened a library encoding transmembrane proteins for α-synuclein-biotin PFF–binding candidates via detection with streptavidin-AP (alkaline phosphatase) staining. Three positive clones were identified that bind α-synuclein PFF and include lymphocyte-activation gene 3 (LAG3), neurexin 1β, and amyloid β precursor-like protein 1 (APLP1). Of these three transmembrane proteins, LAG3 demonstrated the highest ratio of selectivity for α-synuclein PFF over the α-synuclein monomer. α-Synuclein PFF bind to LAG3 in a saturable manner (dissociation constant = 77 nM), whereas the α-synuclein monomer does not bind to LAG3. Co-immunoprecipitation also suggests that pathological α-synuclein PFF specifically bind to LAG3. Tau PFF, β-amyloid oligomer, and β-amyloid PFF do not bind to LAG3, indicating that LAG3 is specific for α-synuclein PFF. The internalization of α-synuclein PFF involves LAG3 because deletion of LAG3 reduces the endocytosis of α-synuclein PFF. LAG3 colocalizes with the endosomal guanosine triphosphatases Rab5 and Rab7 and coendocytoses with pathologic α-synuclein. Neuron-to-neuron transmission of pathologic α-synuclein and the accompanying pathology and neurotoxicity is substantially attenuated by deletion of LAG3 or by antibodies to LAG3. The lack of LAG3 also substantially delayed α-synuclein PFF–induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. CONCLUSION We discovered that pathologic α-synuclein transmission and toxicity is initiated by binding to LAG3 and that neuron-to-neuron transmission of pathological α-synuclein involves the endocytosis of exogenous α-synuclein PFF by the engagement of LAG3 on neurons. Depletion of LAG3 or antibodies to LAG3 substantially reduces the pathology set in motion by the transmission of pathologic α-synuclein. The identification of LAG3 as an α-synuclein PFF–binding protein provides a new target for developing therapeutics designed to slow the progression of PD and related α-synucleinopathies. LAG3 deletion or antibodies to LAG3 delay α-synuclein PFF transmission. Compared with wild-type neurons, binding and endocytosis of α-synuclein PFF is dramatically reduced with antibodies to LAG3 or when LAG3 is deleted, resulting in delayed pathologic α-synuclein transmission and toxicity. Illustration credit: I-Hsun Wu Emerging evidence indicates that the pathogenesis of Parkinson’s disease (PD) may be due to cell-to-cell transmission of misfolded preformed fibrils (PFF) of α-synuclein (α-syn). The mechanism by which α-syn PFF spreads from neuron to neuron is not known. Here, we show that LAG3 (lymphocyte-activation gene 3) binds α-syn PFF with high affinity (dissociation constant = 77 nanomolar), whereas the α-syn monomer exhibited minimal binding. α-Syn-biotin PFF binding to LAG3 initiated α-syn PFF endocytosis, transmission, and toxicity. Lack of LAG3 substantially delayed α-syn PFF–induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. The identification of LAG3 as a receptor that binds α-syn PFF provides a target for developing therapeutics designed to slow the progression of PD and related α-synucleinopathies.

Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis

Significance Excessive activation of poly(ADP-ribose) (PAR) polymerase (PARP) is intimately linked to cell death in a variety of organ systems. It has long been thought that the cell death caused by excessive activation of PARP occurs through the catalytic consumption of NAD+ followed by reduction of ATP and bioenergetic collapse. This study shows that the bioenergetic collapse is caused not by the consumption of NAD+ but by PAR-dependent inhibition of hexokinase activity leading to defects in glycolysis. These results are consistent with the notion that cell death induced by excessive PARP activity (parthanatos) is an active cell-death program that is initiated by PAR signaling. Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated “parthanatos” in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD+ and energetic collapse, which have been thought to be caused by the consumption of cellular NAD+ by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD+ depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD+ depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1–mediated mitochondrial dysfunction. Depleting neurons of NAD+ with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss

The defining pathogenic feature of Parkinsons disease is the age-dependent loss of dopaminergic neurons. Mutations and inactivation of parkin, an ubiquitin E3 ligase, induce Parkinsons disease through accumulation of pathogenic substrates. We found that transgenic overexpression of a parkin substrate, aminoacyl-tRNA synthetase complex interacting multifunctional protein-2 (AIMP2), led to a selective, age-dependent, progressive loss of dopaminergic neurons via activation of poly(ADP-ribose) polymerase-1 (PARP1). AIMP2 accumulation in vitro and in vivo resulted in PARP1 overactivation and dopaminergic cell toxicity via direct association of these proteins in the nucleus, providing a path to PARP1 activation other than DNA damage. Inhibition of PARP1 through gene deletion or drug inhibition reversed behavioral deficits and protected against dopamine neuron death in AIMP2 transgenic mice. These data indicate that brain-permeable PARP inhibitors could effectively delay or prevent disease progression in Parkinsons disease.

The c-Abl inhibitor, Nilotinib, protects dopaminergic neurons in a preclinical animal model of Parkinson's disease

c-Abl is activated in the brain of Parkinsons disease (PD) patients and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice where it inhibits parkin through tyrosine phosphorylation leading to the accumulation of parkin substrates, and neuronal cell death. In the present study, we evaluated the in vivo efficacy of nilotinib, a brain penetrant c-Abl inhibitor, in the acute MPTP-induced model of PD. Our results show that administration of nilotinib reduces c-Abl activation and the levels of the parkin substrate, PARIS, resulting in prevention of dopamine (DA) neuron loss and behavioral deficits following MPTP intoxication. On the other hand, we observe no reduction in the tyrosine phosphorylation of parkin and the parkin substrate, AIMP2 suggesting that the protective effect of nilotinib may, in part, be parkin-independent or to the pharmacodynamics properties of nilotinib. This study provides a strong rationale for testing other brain permeable c-Abl inhibitors as potential therapeutic agents for the treatment of PD.

MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway

Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma-infected mice 5days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin (5-HT) and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and 5-HT metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.

Genetic deficiency of the mitochondrial protein PGAM5 causes a Parkinson’s-like movement disorder

Mitophagy is a specialized form of autophagy that selectively disposes of dysfunctional mitochondria. Delineating the molecular regulation of mitophagy is of great importance because defects in this process lead to a variety of mitochondrial diseases. Here we report that mice deficient for the mitochondrial protein, phosphoglycerate mutase family member 5 (PGAM5), displayed a Parkinsons-like movement phenotype. We determined biochemically that PGAM5 is required for the stabilization of the mitophagy-inducing protein PINK1 on damaged mitochondria. Loss of PGAM5 disables PINK1-mediated mitophagy in vitro and leads to dopaminergic neurodegeneration and mild dopamine loss in vivo. Our data indicate that PGAM5 is a regulator of mitophagy essential for mitochondrial turnover and serves a cytoprotective function in dopaminergic neurons in vivo. Moreover, PGAM5 may provide a molecular link to study mitochondrial homeostasis and the pathogenesis of a movement disorder similar to Parkinsons disease.

Activation of tyrosine kinase c-Abl contributes to α -synuclein–induced neurodegeneration

Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein-induced neuropathology. In mice expressing a human α-synucleinopathy-associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein-induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies.

GBA1 deficiency negatively affects physiological α-synuclein tetramers and related multimers

Significance Recent studies have identified a helically folded tetramer as the major normal structure of α-synuclein (α-syn) and that the tetramer resists aggregation. However, the underlying mechanisms that regulate the formation of α-syn tetramers remain elusive. Our study shows that mutations in glucocerebrosidase 1 (GBA1) and depletion-induced GBA1 deficiency leading to accumulation of glycosphingolipids (GSLs) are sufficient to cause destabilization of α-syn tetramers and increase the susceptibility of human dopaminergic neurons to cytotoxicity due to exposure to pathologic α-syn fibrils. Therefore, maintaining GBA1 activity and reducing GSLs are potentially important in reducing misfolding and pathogenic aggregation of α-syn in Parkinson’s disease. Accumulating evidence suggests that α-synuclein (α-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of α-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of α-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of α-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of α-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases α-syn tetramers and related multimers and increases α-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, α-syn tetramers and related multimers are decreased in N370S GBA1 Parkinson’s disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P GBA1 mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of α-syn tetramers and protect against α-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric α-syn to α-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.