Pimozide reduces toxic forms of tau in TauC3 mice via 5' adenosine monophosphate-activated protein kinase-mediated autophagy.

In neurodegenerative diseases like Alzheimer's disease (AD), tau is hyperphosphorylated and forms aggregates and neurofibrillary tangles in affected neurons. Autophagy is critical to clear the aggregates of disease-associated proteins and is often altered in patients and animal models of AD. Because mechanistic target of rapamycin (mTOR) negatively regulates autophagy and is hyperactive in the brains of patients with AD, mTOR is an attractive therapeutic target for AD. However, pharmacological strategies to increase autophagy by targeting mTOR inhibition cause various side effects. Therefore, autophagy activation mediated by non-mTOR pathways is a new option for autophagy-based AD therapy. Here, we report that pimozide activates autophagy to rescue tau pathology in an AD model. Pimozide increased autophagic flux through the activation of the AMPK-Unc-51 like autophagy activating kinase 1 (ULK1) axis, but not of mTOR, in neuronal cells, and this function was independent of dopamine D2 receptor inhibition. Pimozide reduced levels of abnormally phosphorylated tau aggregates in neuronal cells. Further, daily intraperitoneal (i.p.) treatment of pimozide led to a recovery from memory deficits of TauC3 mice expressing a caspase-cleaved form of tau. In the brains of these mice, we found increased phosphorylation of AMPK1 and ULK1, and reduced levels of the soluble oligomers and NP40-insoluble aggregates of abnormally phosphorylated tau. Together, these results suggest that pimozide rescues memory impairments in TauC3 mice and reduces tau aggregates by increasing autophagic flux through the mTOR-independent AMPK-ULK1 axis.

AK2 is an AMP-sensing negative regulator of BRAF in tumorigenesis.

The RAS-BRAF signaling is a major pathway of cell proliferation and their mutations are frequently found in human cancers. Adenylate kinase 2 (AK2), which modulates balance of adenine nucleotide pool, has been implicated in cell death and cell proliferation independently of its enzyme activity. Recently, the role of AK2 in tumorigenesis was in part elucidated in some cancer types including lung adenocarcinoma and breast cancer, but the underlying mechanism is not clear. Here, we show that AK2 is a BRAF-suppressor. In in vitro assays and cell model, AK2 interacted with BRAF and inhibited BRAF activity and downstream ERK phosphorylation. Energy-deprived conditions in cell model and the addition of AMP to cell lysates strengthened the AK2-BRAF interaction, suggesting that AK2 is involved in the regulation of BRAF activity in response to cell metabolic state. AMP facilitated the AK2-BRAF complex formation through binding to AK2. In a panel of HCC cell lines, AK2 expression was inversely correlated with ERK/MAPK activation, and AK2-knockdown or -knockout increased BRAF activity and promoted cell proliferation. Tumors from HCC patients showed low-AK2 protein expression and increased ERK activation compared to non-tumor tissues and the downregulation of AK2 was also verified by two microarray datasets (TCGA-LIHC and GSE14520). Moreover, AK2/BRAF interaction was abrogated by RAS activation in in vitro assay and cell model and in a mouse model of HRASG12V-driven HCC, and AK2 ablation promoted tumor growth and BRAF activity. AK2 also bound to BRAF inhibitor-insensitive BRAF mutants and attenuated their activities. These findings indicate that AK2 monitoring cellular AMP levels is indeed a negative regulator of BRAF, linking the metabolic status to tumor growth.

Aberrant role of pyruvate kinase M2 in the regulation of gamma-secretase and memory deficits in Alzheimer's disease.

Toxic amyloid beta (Aß) species cause synaptic dysfunction and neurotoxicity in Alzheimer's disease (AD). As of yet, however, there are no reported regulators for gamma-secretase, which links a risky environment to amyloid accumulation in AD. Here, we report that pyruvate kinase M2 (PKM2) is a positive regulator of gamma-secretase under hypoxia. From a genome-wide functional screen, we identify PKM2 as a gamma-secretase activator that is highly expressed in the brains of both patients and murine models with AD. PKM2 regulates Aß production and the amount of active gamma-secretase complex by changing the gene expression of aph-1 homolog. Hypoxia induces PKM2 expression, thereby promoting gamma-secretase activity. Moreover, transgenic expression of PKM2 in 3xTg AD model mice enhances hippocampal production of Aß and exacerbates the impairment of spatial and recognition memory. Taken together, these findings indicate that PKM2 is an important gamma-secretase regulator that promotes Aß production and memory impairment under hypoxia.

Alpha-synuclein overexpression reduces gap junctional intercellular communication in dopaminergic neuroblastoma cells.

Alpha-synuclein has been implicated in the pathology of certain neurodegenerative diseases, including Parkinson disease (PD) and dementia with Lewy bodies (LBs). Overexpression of human alpha-synuclein in neuronal cells reduces cell viability, but the precise cellular and molecular mechanisms remain poorly understood. Gap junctional intercellular communication (GJIC) is thought to be essential for maintaining cellular homeostasis and growth control. In the present study, the effect of alpha-synuclein overexpression on GJIC in human dopaminergic neuroblastoma SH-SY5Y cells was investigated. Cells overexpressing wild-type alpha-synuclein were more vulnerable to hydrogen peroxide and 6-hydroxydopamine. GJIC was decreased in cells overexpressing alpha-synuclein. In addition, alpha-synuclein binds directly to connexin-32 (Cx32). As such, the post-translational modification of Cx32 was enhanced in cells overexpressing alpha-synuclein. These findings suggest that alpha-synuclein can modulate GJIC in a dopaminergic neuronal cell line through specific binding to Cx32.

Phosphorylated CAV1 activates autophagy through an interaction with BECN1 under oxidative stress.

CAV1/Caveolin1, an integral membrane protein, is involved in caveolae function and cellular signaling pathways. Here, we report that CAV1 is a positive regulator of autophagy under oxidative stress and cerebral ischemic injury. Treatment with hydrogen peroxide enhanced autophagy flux and caused the localization of BECN1 to the mitochondria, whereas these changes were impaired in the absence of CAV1. Among many autophagy signals, only LC3 foci formation in response to hydrogen peroxide was abolished by CAV1 deficiency. Under oxidative stress, CAV1 interacted with a complex of BECN1/VPS34 through its scaffolding domain, and this interaction facilitated autophagosome formation. Interestingly, the phosphorylation of CAV1 at tyrosine-14 was essential for the interaction with BECN1 and their localization to the mitochondria, and the activation of autophagy in response to hydrogen peroxide. In addition, the expression of a phosphatase PTPN1 reduced the phosphorylation of CAV1 and inhibited autophagy. Further, compared to that in wild-type mice, autophagy was impaired and cerebral infarct damage was aggravated in the brain of Cav1 knockout mice. These results suggest that the phosphorylated CAV1 functions to activate autophagy through binding to the BECN1/VPS34 complex under oxidative stress and to protect against ischemic damage.

SUMO-Modified FADD Recruits Cytosolic Drp1 and Caspase-10 to Mitochondria for Regulated Necrosis.

Fas-associated protein with death domain (FADD) plays a key role in extrinsic apoptosis. Here, we show that FADD is SUMOylated as an essential step during intrinsic necrosis. FADD was modified at multiple lysine residues (K120/125/149) by small ubiquitin-related modifier 2 (SUMO2) during necrosis caused by calcium ionophore A23187 and by ischemic damage. SUMOylated FADD bound to dynamin-related protein 1 (Drp1) in cells both in vitro and in ischemic tissue damage cores, thus promoting Drp1 recruitment by mitochondrial fission factor (Mff) to accomplish mitochondrial fragmentation. Mitochondrial-fragmentation-associated necrosis was blocked by FADD or Drp1 deficiency and SUMO-defective FADD expression. Interestingly, caspase-10, but not caspase-8, formed a ternary protein complex with SUMO-FADD/Drp1 on the mitochondria upon exposure to A23187 and potentiated Drp1 oligomerization for necrosis. Moreover, the caspase-10 L285F and A414V mutants, found in autoimmune lymphoproliferative syndrome and non-Hodgkin lymphoma, respectively, regulated this necrosis. Our study reveals an essential role of SUMOylated FADD in Drp1- and caspase-10-dependent necrosis, providing insights into the mechanism of regulated necrosis by calcium overload and ischemic injury.

E2-25K SUMOylation inhibits proteasome for cell death during cerebral ischemia/reperfusion.

Cerebral ischemia/reperfusion (I/R) causes brain damage accompanied by ubiquitin accumulation and impairment of proteasome activity. In this study, we report that E2-25K, an E2-conjugating enzyme, is SUMOylated during oxidative stress and regulates cerebral I/R-induced damage. Knockdown of E2-25K expression protects against oxygen/glucose deprivation and reoxygenation (OGD/R)-induced neuronal cell death, whereas ectopic expression of E2-25K stimulates it. Compared with the control mice, cerebral infarction lesions and behavioral/neurological disorders are ameliorated in E2-25K knockout mice during middle cerebral artery occlusion and reperfusion. In particular, E2-25K is SUMOylated at Lys14 under oxidative stress, OGD/R and I/R to prompt cell death. Further, E2-25K downregulates the proteasome subunit S5a to impair proteasome complex and thus restrain proteasome activity under oxidative stress. This proteasome inhibitory activity of E2-25K is dependent on its SUMOylation. These results suggest that E2-25K has a crucial role in oxidative stress and cerebral I/R-induced damage through inhibiting proteasome via its SUMOylation.