Irina Lonskaya

Nilotinib reverses loss of dopamine neurons and improves motor behavior via autophagic degradation of α-synuclein in Parkinson's disease models

Parkinsons disease is a movement disorder characterized by death of dopaminergic substantia nigra (SN) neurons and brain accumulation of α-synuclein. The tyrosine kinase Abl is activated in neurodegeneration. Here, we show that lentiviral expression of α-synuclein in the mouse SN leads to Abl activation (phosphorylation) and lentiviral Abl expression increases α-synuclein levels, in agreement with Abl activation in PD brains. Administration of the tyrosine kinase inhibitor nilotinib decreases Abl activity and ameliorates autophagic clearance of α-synuclein in transgenic and lentiviral gene transfer models. Subcellular fractionation shows accumulation of α-synuclein and hyper-phosphorylated Tau (p-Tau) in autophagic vacuoles in α-synuclein expressing brains, but nilotinib enhances protein deposition into the lysosomes. Nilotinib is used for adult leukemia treatment and it enters the brain within US Food and Drug Administration approved doses, leading to autophagic degradation of α-synuclein, protection of SN neurons and amelioration of motor performance. These data suggest that nilotinib may be a therapeutic strategy to degrade α-synuclein in PD and other α-synucleinopathies.

Parkin ubiquitinates Tar-DNA binding protein-43 (TDP-43) and promotes its cytosolic accumulation via interaction with histone deacetylase 6 (HDAC6).

Background: TDP-43 pathology and the role of E3 ubiquitin ligases are increasingly recognized in neurodegeneration. Results: Parkin ubiquitinates TDP-43 and forms a multiprotein complex with HDAC6 to sequester TDP-43 in cytosol. Conclusion: Parkin E3 ubiquitin ligase activity promotes TDP-43 inclusion formation and nuclear translocation. Significance: Parkin-TDP-43 interaction may be exploited as a therapeutic strategy in ALS/FTLD pathology. The importance of E3 ubiquitin ligases, involved in the degradation of misfolded proteins or promotion of protein-protein interaction, is increasingly recognized in neurodegeneration. TDP-43 is a predominantly nuclear protein, which regulates the transcription of thousands of genes and binds to mRNA of the E3 ubiquitin ligase Parkin to regulate its expression. Wild type and mutated TDP-43 are detected in ubiquitinated forms within the cytosol in several neurodegenerative diseases. We elucidated the mechanisms of TDP-43 interaction with Parkin using transgenic A315T mutant TDP-43 (TDP43-Tg) mice, lentiviral wild type TDP-43, and Parkin gene transfer rat models. TDP-43 expression increased Parkin mRNA and protein levels. Lentiviral TDP-43 increased the levels of nuclear and cytosolic protein, whereas Parkin co-expression mediated Lys-48 and Lys-63-linked ubiquitin to TDP-43 and led to cytosolic co-localization of Parkin with ubiquitinated TDP-43. Parkin and TDP-43 formed a multiprotein complex with HDAC6, perhaps to mediate TDP-43 translocation. In conclusion, Parkin ubiquitinates TDP-43 and facilitates its cytosolic accumulation through a multiprotein complex with HDAC6.

Tyrosine kinase inhibition increases functional parkin‐Beclin‐1 interaction and enhances amyloid clearance and cognitive performance

Tyrosine kinase inhibitors (TKIs) are effective therapies for leukaemia. Alzheimer is a neurodegenerative disease characterized by accumulation of β‐amyloid (plaques) and hyper‐phosphorylated Tau (tangles). Here we show that AD animals have high levels of insoluble parkin and decreased parkin‐Beclin‐1 interaction, while peripheral administration of TKIs, including Nilotinib and Bosutinib, increases soluble parkin leading to amyloid clearance and cognitive improvement. Blocking Beclin‐1 expression with shRNA or parkin deletion prevents tyrosine kinase (TK) inhibition‐induced amyloid clearance, suggesting that functional parkin‐Beclin‐1 interaction mediates amyloid degradation. Isolation of autophagic vacuoles (AVs) in AD mouse brain shows accumulation of parkin and amyloid, consistent with previous results in AD brains, while Bosutinib and Nilotinib increase parkin‐Beclin‐1 interaction and result in protein deposition in the lysosome. These data suggest that decreased parkin solubility impedes parkin‐Beclin‐1 interaction and amyloid clearance. We identified two FDA‐approved anti‐cancer drugs as potential treatment for AD.

Methamphetamine-associated cleavage of the synaptic adhesion molecule intercellular adhesion molecule-5.

J. Neurochem. (2011) 118, 521–532.

Diminished parkin solubility and co-localization with intraneuronal amyloid-β are associated with autophagic defects in Alzheimer's disease.

Alzheimers disease (AD) is an aging disorder characterized by amyloid-β (Aβ) accumulation in extracellular plaques and formation of intracellular tangles containing hyperphosphorylated tau (p-Tau). Autophagic defects, leading to accumulation of autophagosomes, are recognized in AD. Parkin is an E3 ubiquitin ligase involved in degradation of proteins via autophagy and the proteasome. We investigated the role of parkin in postmortem brain tissues from 21 AD patients and 15 control subjects. We detected decreased parkin solubility in AD cortex and parkin co-localization with intraneuronal Aβ(1-42) in the hippocampus and cortex of AD patients. Parkin accumulation with intraneuronal Aβ and p-Tau was detected in autophagosomes in AD brains. To determine the role of parkin in Aβ clearance, we generated gene transfer animals expressing lentiviral Aβ(1-42)with and without parkin and examined autophagic mechanisms. Lentiviral expression of Aβ(1-42) led to p-Tau accumulation and induced autophagic defects, leading to accumulation of autophagic vacuoles. However, co-expression of wild type parkin facilitated autophagic clearance and promoted deposition of Aβ(1-42) and p-Tau into the lysosome. Taken together, these data suggest that Aβ(1-42) alters normal autophagy and parkin enhances autophagic clearance. In conclusion, decreased parkin solubility may lead to co-localization with intraneuronal Aβ(1-42) and compromise the cell autophagic clearance ability. Parkin may clear autophagic defects via autophagosome degradation.

Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance

Alzheimers disease (AD) is a neurodegenerative disorder associated with amyloid accumulation and autophagic changes. Parkin is an E3 ubiquitin ligase involved in proteasomal and autophagic clearance. We previously demonstrated decreased parkin solubility and interaction with the key autophagy enzyme beclin-1 in AD, but tyrosine kinase inhibition restored parkin–beclin-1 interaction. In the current studies, we determined the mechanisms of nilotinib-induced parkin–beclin-1 interaction, which leads to amyloid clearance. Nilotinib increased endogenous parkin levels and ubiquitination, which may enhance parkin recycling via the proteasome, leading to increased activity and interaction with beclin-1. Parkin solubility was decreased and autophagy was altered in amyloid expressing mice, suggesting that amyloid stress affects parkin stability, leading to failure of protein clearance via the lysosome. Isolation of autophagic vacuoles revealed amyloid and parkin accumulation in autophagic compartments but nilotinib decreased insoluble parkin levels and facilitated amyloid deposition into lysosomes in wild type, but not parkin−/− mice, further underscoring an essential role for endogenous parkin in amyloid clearance. These results suggest that nilotinib boosts the autophagic machinery, leading to increased level of endogenous parkin that undergoes ubiquitination and interacts with beclin-1 to facilitate amyloid clearance. These data suggest that nilotinib-mediated autophagic changes may trigger parkin response via increased protein levels, providing a therapeutic strategy to reduce Aβ and Tau in AD.Key messageParkin solubility (stability) is decreased in AD and APP transgenic mice.Nilotinib-induced autophagic changes increase endogenous parkin level.Increased parkin level leads to ubiquitination and proteasomal recycling.Re-cycling decreases insoluble parkin and increases parkin–beclin-1 interaction.Beclin-1–parkin interaction enhances amyloid clearance.

Ubiquitination Increases Parkin Activity to Promote Autophagic α-Synuclein Clearance

Parkinson’s disease (PD) is a movement disorder associated with genetic and age related causes. Although autosomal recessive early onset PD linked to parkin mutations does not exhibit α-Synuclein accumulation, while autosomal dominant and sporadic PD manifest with α-Synuclein inclusions, loss of dopaminergic substantia nigra neurons is a common denominator in PD. Here we show that decreased parkin ubiquitination and loss of parkin stability impair interaction with Beclin-1 and alter α-Synuclein degradation, leading to death of dopaminergic neurons. Tyrosine kinase inhibition increases parkin ubiquitination and interaction with Beclin-1, promoting autophagic α-Synuclein clearance and nigral neuron survival. However, loss of parkin via deletion increases α-Synuclein in the blood compared to the brain, suggesting that functional parkin prevents α-Synuclein release into the blood. These studies demonstrate that parkin ubiquitination affects its protein stability and E3 ligase activity, possibly leading to α-Synuclein sequestration and subsequent clearance.

Decreased parkin solubility is associated with impairment of autophagy in the nigrostriatum of sporadic Parkinson’s disease

Parkinsons disease (PD) is a motor disorder that involves death of dopaminergic neurons in the substantia nigra pars compacta. Parkin is an autosomal recessive gene that is mutated in early onset PD. We investigated the role of parkin and autophagic clearance in postmortem nigrostriatal tissues from 22 non-familial sporadic PD patients and 15 control samples. Parkin was insoluble with altered cytosolic expression in the nigrostriatum of sporadic PD. Parkin insolubility was associated with lack of degradation of ubiquitinated proteins and accumulation of α-Synuclein and parkin in autophagosomes, suggesting autophagic defects in PD. To test parkins role in mediating autophagic clearance, we used lentiviral gene transfer to express human wild type or mutant parkin (T240R) with α-Synuclein in the rat striatum. Lentiviral expression of α-Synuclein led to accumulation of autophagic vacuoles, while co-expression of parkin with α-Synuclein facilitated autophagic clearance. Subcellular fractionation showed accumulation of α-Synuclein and tau hyper-phosphorylation (p-Tau) in autophagosomes in gene transfer models, similar to the effects observed in PD brains, but parkin expression led to protein deposition into lysosomes. However, parkin loss of function mutation did not affect autophagic clearance. Taken together, these data suggest that functional parkin regulates autophagosome clearance, while decreased parkin solubility may alter normal autophagy in sporadic PD.

Tyrosine kinase inhibition facilitates autophagic SNCA/α-synuclein clearance

The effects of ABL1/ABL inhibition on clearance of SNCA/α-synuclein were evaluated in animal models of α-synucleinopathies. Parkinson disease (PD) is a movement disorder characterized by death of dopaminergic substantia nigra (SN) neurons and brain accumulation of SNCA. The tyrosine kinase ABL1 is activated in several neurodegenerative diseases. An increase in ABL1 activity is detected in human postmortem PD brains. Lentiviral expression of SNCA in the mouse SN activates ABL1 via phosphorylation, while lentiviral Abl expression increases SNCA levels. Administration of the brain-penetrant tyrosine kinase inhibitor Nilotinib decreases Abl activity and facilitates autophagic clearance of SNCA in transgenic and lentiviral gene transfer models. Subcellular fractionation demonstrates accumulation of SNCA and hyperphosphorylated MAPT/Tau (p-MAPT) in autophagic vacuoles in SNCA-expressing brains, while Nilotinib treatment leads to protein deposition into the lysosomes, suggesting enhanced autophagic clearance. These data suggest that Nilotinib may be a therapeutic strategy to degrade SNCA in PD and other α-synucleinopathies.

Nilotinib and bosutinib modulate pre-plaque alterations of blood immune markers and neuro-inflammation in Alzheimer's disease models.

Alzheimers disease (AD) brains exhibit plaques and tangles in association with inflammation. The non-receptor tyrosine kinase Abl is linked to neuro-inflammation in AD. Abl inhibition by nilotinib or bosutinib facilitates amyloid clearance and may decrease inflammation. Transgenic mice that express Dutch, Iowa and Swedish APP mutations (TgAPP) and display progressive Aβ plaque deposition were treated with tyrosine kinase inhibitors (TKIs) to determine pre-plaque effects on systemic and CNS inflammation using milliplex® ELISA. Plaque Aβ was detected at 4months in TgAPP and pre-plaque intracellular Aβ accumulation (2.5months) was associated with changes of cytokines and chemokines prior to detection of glial changes. Plaque formation correlated with increased levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1α, IL-1β) and markers of immunosuppressive and adaptive immunity, including, IL-4, IL-10, IL-2, IL-3, Vascular Endothelial Growth Factor (VEGF) and IFN-γ. An inverse relationship of chemokines was observed as CCL2 and CCL5 were lower than WT mice at 2months and significantly increased after plaque appearance, while soluble CX3CL1 decreased. A change in glial profile was only robustly detected at 6months in Tg-APP mice and TKIs reduced astrocyte and dendritic cell number with no effects on microglia, suggesting alteration of brain immunity. Nilotinib decreased blood and brain cytokines and chemokines and increased CX3CL1. Bosutinib increased brain and blood IL-10 and CX3CL1, suggesting a protective role for soluble CX3CL1. Taken together these data suggest that TKIs regulate systemic and CNS immunity and may be useful treatments in early AD through dual effects on amyloid clearance and immune modulation.