Lycorine, a natural alkaloid, promotes the degradation of alpha-synuclein via PKA-mediated UPS activation in transgenic Parkinson's disease models.

Parkinson's disease (PD) is one of the most common neurodegenerative motor disorders, and is characterized by the presence of Lewy bodies containing misfolded α-synuclein (α-syn) and by selective degeneration of midbrain dopamine neurons. Studies have shown that upregulation of ubiquitin-proteasome system (UPS) activity promotes the clearance of aggregation-prone proteins such as α-syn and Tau, so as to alleviate the neuropathology of neurodegenerative diseases. To identify and investigate lycorine as a UPS enhancer able to decrease α-syn in transgenic PD models. Dot blot was used to screen α-syn-lowering compounds in an inducible α-syn overexpression cell model. Inducible wild-type (WT) and mutant α-syn-overexpressing PC12 cells, WT α-syn-overexpressing N2a cells and primary cultured neurons from A53T transgenic mice were used to evaluate the effects of lycorine on α-syn degradation in vitro. Heterozygous A53T transgenic mice were used to evaluate the effects of lycorine on α-syn degradation in vivo. mCherry-GFP-LC3 reporter was used to detect autophagy-dependent degradation. Ub-R-GFP and Ub-G76V-GFP reporters were used to detect UPS-dependent degradation. Proteasome activity was detected by fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC). Lycorine significantly promoted clearance of over-expressed WT and mutant α-syn in neuronal cell lines and primary cultured neurons. More importantly, 15 days' intraperitoneal administration of lycorine effectively promoted the degradation of α-syn in the brains of A53T transgenic mice. Mechanistically, lycorine accelerated α-syn degradation by activating cAMP-dependent protein kinase (PKA) to promote proteasome activity. Lycorine is a novel α-syn-lowering compound that works through PKA-mediated UPS activation. This ability to lower α-syn implies that lycorine has the potential to be developed as a pharmaceutical for the treatment of neurodegenerative diseases, such as PD, associated with UPS impairment and protein aggregations.

Zhu Q ，Zhuang XX ，Chen JY ，Yuan NN ，Chen Y ，Cai CZ ，Tan JQ ，Su HX ，Lu JH ... -  《-》

α-mangostin derivative 4e as a PDE4 inhibitor promote proteasomal degradation of alpha-synuclein in Parkinson's disease models through PKA activation.

Parkinson's disease (PD) is a multi-factorial neurodegenerative disease affecting motor function of patients. The hall markers of PD are dopaminergic neuron loss in the midbrain and the presence of intra-neuronal inclusion bodies mainly composed of aggregation-prone protein alpha-synuclein (α-syn). Ubiquitin-proteasome system (UPS) is a multi-step reaction process responsible for more than 80% intracellular protein degradation. Impairment of UPS function has been observed in the brain tissue of PD patients. PDE4 inhibitors have been shown to activate cAMP-PKA pathway and promote UPS activity in Alzheimer's disease model. α-mangostin is a natural xanthonoid with broad biological activities, such as antioxidant, antimicrobial and antitumour activities. Structure-based optimizations based on α-mangostin produced a potent PDE4 inhibitor, 4e. Herein, we studied whether 4e could promote proteasomal degradation of α-syn in Parkinson's disease models through PKA activation. cAMP Assay was conducted to quantify cAMP levels in samples. Model UPS substrates (Ub-G76V-GFP and Ub-R-GFP) were used to monitor UPS-dependent activity. Proteasome activity was investigated by short peptide substrate, Suc-LLVY-AMC, cleavage of which by the proteasome increases fluorescence sensitivity. Tet-on WT, A30P, and A53T α-syn-inducible PC12 cells and primary mouse cortical neurons from A53T transgenic mice were used to evaluate the effect of 4e against α-syn in vitro. Heterozygous A53T transgenic mice were employed to assess the effect of 4e on the clearance of α-syn in vivo, and further validations were applied by western blotting and immunohistochemistry. Taken together, α-mangostin derivative 4e, a PDE4 inhibitor, efficiently activated the cAMP/PKA pathway in neuronal cells, and promoted UPS activity as evidenced by enhanced degradation of UPS substrate Ub-G76V-GFP and Ub-R-GFP, as well as elevated proteasomal enzyme activity. Interestingly, 4e dramatically accelerated degradation of inducibly-expressed WT and mutant α-syn in PC12 cells, in a UPS dependent manner. Besides, 4e consistently activated PKA in primary neuron and A53T mice brain, restored UPS inhibition and alleviated α-syn accumulation in the A53T mice brain. 4e is a natural compound derived highly potent PDE4 inhibitor. We revealed its potential effect in promoting UPS activity to degrade pathogenic proteins associated with PD.

Chen JY ，Zhu Q ，Cai CZ ，Luo HB ，Lu JH ... -  《-》

Natural alkaloid harmine promotes degradation of alpha-synuclein via PKA-mediated ubiquitin-proteasome system activation.

Parkinson's disease (PD) is an age-dependent progressive movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Accumulation of <alpha>-synuclein (<alpha>-syn) positive protein aggregates in the substantia nigra is a pathological hallmark of PD, indicating that protein turnover defect is implicated in PD pathogenesis. This study aims to identify neuroprotective compounds which can alleviate the accumulation of <alpha>-syn in neuronal cells and dissect the underlying mechanisms. High throughput screening was performed by dot blot assay. The degradation of different forms of <alpha>-syn by candidate compounds were assessed by western blot. The autophagy lysosome pathway and ubiquitin-proteasome system were examined to dissect the degradation pathway. The UPS activity was assessed by cellular UPS substrates degradation assay and biochemical proteasome activity assay. Q-PCR was performed to test the mRNA level of different proteasome subunits. Furthermore, Neuroprotective effect of candidate compound was tested by LDH assay and PI staining. Through the high throughput screening, harmine was identified as a potent <alpha>-syn lowering compound. The time-dependent and dose-dependent effects of harmine on the degradation of different forms of <alpha>-syn were further confirmed. Harmine could dramatically promote the degradation of UPS substrates GFP-CL1, Ub-R-GFP and Ub-G76V-GFP, and activate cellular proteasome activity. Mechanistically, harmine dramatically enhanced PKA phosphorylation to enhance proteasome subunit PSMD1 expression. PKA inhibitor blocked the effects of harmine in activating UPS, up regulating PSMD1 and promoting <alpha>-syn degradation, indicating that harmine enhances UPS function via PKA activation. Moreover, harmine efficiently rescued cell death induced by over-expression of <alpha>-syn, via UPS-dependent manner. Harmine, as a new proteasome enhancer, may have potential to be developed into therapeutic agent against neurodegenerative diseases associated with UPS dysfunction and aberrant proteins accumulation.

Cai CZ ，Zhou HF ，Yuan NN ，Wu MY ，Lee SM ，Ren JY ，Su HX ，Lu JJ ，Chen XP ，Li M ，Tan JQ ，Lu JH ... -  《-》

Tetramethylpyrazine Analogue T-006 Promotes the Clearance of Alpha-synuclein by Enhancing Proteasome Activity in Parkinson's Disease Models.

Zhou H ，Shao M ，Guo B ，Li C ，Lu Y ，Yang X ，ShengnanLi ，Li H ，Zhu Q ，Zhong H ，Wang Y ，Zhang Z ，Lu J ，Lee SM ... -  《-》

Alpha-synuclein interferes with cAMP/PKA-dependent upregulation of dopamine β-hydroxylase and is associated with abnormal adaptive responses to immobilization stress.

Parkinson's disease (PD) is clinically characterized not only by motor symptoms but also by non-motor symptoms, such as anxiety and mood changes. Based on our previous study showing that overexpression of wild-type or mutant α-synuclein (α-SYN) interferes with cAMP/PKA-dependent transcriptional activation in norepinephrine (NE)-producing cells, the effect of wild-type and mutant α-SYN on cAMP response element (CRE)-mediated regulation of the NE-synthesizing enzyme dopamine β-hydroxylase (DBH) was evaluated in this study. Overexpression of wild-type or mutant α-SYN interfered with CRE-mediated regulation of DBH transcription in NE-producing SK-N-BE(2) cells. Upon entering the nucleus, α-SYN interacted with the DBH promoter region encompassing the CRE, which interfered with forskolin-induced CREB binding to the CRE region. Interestingly, mutant A53T α-SYN showed much higher tendency to nuclear translocation and interaction with the DBH promoter region encompassing the CRE than wild type. In addition, A53T α-SYN expressing transgenic mice exhibited increased anxiety-like behaviors under normal conditions and abnormal regulation of DBH expression in response to immobilization stress with abnormal adaptive responses. These data provide an insight into the physiological function of α-SYN in NErgic neuronal cells, which further indicates that the α-SYN mutation may play a causative role in the generation of non-motor symptoms in PD.

Kim S ，Park JM ，Moon J ，Choi HJ ... -  《-》