Reciprocal signals between microglia and neurons regulate alpha-synuclein secretion by exophagy through a neuronal cJU-N-Nterminal kinase-signaling axis

BACKGROUND: Secretion of proteopathic α-synuclein (α-SNC) species from neurons is a suspected driving force in the propagation of Parkinson’s disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of α-SNC secretion in differentiated PC12 or SH-SY5Y nerve cells. Here we have examined the regulation of exophagy associated with different forms of nerve cell stress relevant to PD. RESULTS: We identify cJUN-N-terminal kinase (JNK) activity as pivotal in the secretory fate of autophagosomes containing α-SNC. Pharmacological inhibition or genetic (shRNA) knockdown of JNK2 or JNK3 decreases α-SNC secretion in differentiated PC12 and SH-SY5Y cells, respectively. Conversely, expression of constitutively active mitogen-activated protein kinase kinase 7 (MKK7)-JNK2 and -JNK3 constructs augment secretion. The transcriptional activity of cJUN was not required for the observed effects. We establish a causal relationship between increased α-SNC release by exophagy and JNK activation subsequent to lysosomal fusion deficiency (overexpression of Lewy body-localized protein p25α or bafilomycin A1). JNK activation following neuronal ER or oxidative stress was not correlated with exophagy, but of note, we demonstrate that reciprocal signaling between microglia and neurons modulates α-SNC secretion. NADPH oxidase activity of microglia cell lines was upregulated by direct co-culture with α-SNC-expressing PC12 neurons or by passive transfer of nerve cell-conditioned medium. Conversely, inflammatory factors secreted from activated microglia increased JNK activation and α-SNC secretion several-fold in PC12 cells. While we do not identify these factors, we extend our observations by showing that exposure of neurons in monoculture to TNFα, a classical pro-inflammatory mediator of activated microglia, is sufficient to increase α-SNC secretion in a mechanism dependent on JNK2 or JNK3. In continuation hereof, we show that also IFNβ and TGFβ increase the release of α-SNC from PC12 neurons. CONCLUSIONS: We implicate stress kinases of the JNK family in the regulation of exophagy and release of α-SNC following endogenous or exogenous stimulation. In a wider scope, our results imply that microglia not only inflict bystander damage to neurons in late phases of inflammatory brain disease but may also be active mediators of disease propagation

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.ISSN:0092-8674ISSN:1097-417

JNK1 protects against glucolipotoxicity-mediated beta-cell apoptosis

Pancreatic β-cell dysfunction is central to type 2 diabetes pathogenesis. Prolonged elevated levels of circulating free-fatty acids and hyperglycemia, also termed glucolipotoxicity, mediate β-cell dysfunction and apoptosis associated with increased c-Jun N-terminal Kinase (JNK) activity. Endoplasmic reticulum (ER) and oxidative stress are elicited by palmitate and high glucose concentrations further potentiating JNK activity. Our aim was to determine the role of the JNK subtypes JNK1, JNK2 and JNK3 in palmitate and high glucose-induced β-cell apoptosis. We established insulin-producing INS1 cell lines stably expressing JNK subtype specific shRNAs to understand the differential roles of the individual JNK isoforms. JNK activity was increased after 3 h of palmitate and high glucose exposure associated with increased expression of ER and mitochondrial stress markers. JNK1 shRNA expressing INS1 cells showed increased apoptosis and cleaved caspase 9 and 3 compared to non-sense shRNA expressing control INS1 cells when exposed to palmitate and high glucose associated with increased CHOP expression, ROS formation and Puma mRNA expression. JNK2 shRNA expressing INS1 cells did not affect palmitate and high glucose induced apoptosis or ER stress markers, but increased Puma mRNA expression compared to non-sense shRNA expressing INS1 cells. Finally, JNK3 shRNA expressing INS1 cells did not induce apoptosis compared to non-sense shRNA expressing INS1 cells when exposed to palmitate and high glucose but showed increased caspase 9 and 3 cleavage associated with increased DP5 and Puma mRNA expression. These data suggest that JNK1 protects against palmitate and high glucose-induced β-cell apoptosis associated with reduced ER and mitochondrial stress

Altering β-cell number through stable alteration of miR-21 and miR-34a expression

Aim: An insufficient functional β-cell mass is a prerequisite to develop diabetes. Thus, means to protect or restore β-cell mass are important goals in diabetes research. Inflammation and proinflammatory cytokines play important roles in β-cell dysfunction and death, and recent data show that 2 miRNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed miR-21 and knocked down miR-34a, and investigated essential cellular processes.   Materials and Methods: miRNA expression was manipulated using Lentiviral transduction of the β-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were monitored in the absence or presence of cytokines. Results: Overexpression of miR-21 decreased net β-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a increased net β-cell number in the absence of cytokines, and reduced apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was decreased upon miR-34a knockdown. Conclusion: As overexpression of miR-21 increased proliferation, but also apoptosis and NO synthesis, the potential of miR-21 as a therapeutic agent to increase β-cell survival is doubtful. Knockdown of miR-34a slightly decreased proliferation, but as apoptosis and NO synthesis were highly reduced, miR-34a may be further investigated as a therapeutic target to reduce β-cell death and dysfunction

Figure 2. JNK1 knockdown increases palmitate and high glucose-induced β-cell apoptosis.

<p>INS1 cell lines stably expressing shRNA for JNK1, JNK2, JNK3, non-sense shRNA (ns), empty vector controls (ev) or wildtype INS1 cells were exposed to 0.5 mM palmitate and 25 mM glucose (GLT) (+) or vehicle (−) for 24 h. A: JNK1, B: JNK2, C: JNK3 knockdown specificity and efficiency in JNK1, 2 and 3 shRNA expressing INS1 cell lines were assessed by Western blotting with actin as loading control. Blots are representative of knockdown efficiency in the shRNA expressing INS1 cell lines. D: The specificity of the JNK antibodies were verified against JNK1 (73 kDa with GST tag), JNK2 (72 kDa with GST tag) and JNK3 (61 kDa with GST tag) recombinant proteins. INS1 cell lines stably expressing shRNA for JNK1, JNK2, JNK3, non-sense shRNA (ns), empty vector controls (ev) or wildtype INS1 cells were exposed to 0.5 mM palmitate and 25 mM glucose (black bars) or vehicle (white bars) for 24 h. E: Apoptosis was measured as the relative levels of cytoplasmic nucleosomes in INS1 shRNA stable cell lines lysates compared to ns vehicle using the Roche Cell Death detection ELISA kit. Data are shown as means+SEM of five independent experiments. F, G: Cleaved caspase 9 or 3 was assessed by Western blotting and normalized to actin. Data are shown as means+SEM of five independent experiments; representative blots are shown. *P<0.05, ***P<0.001.</p

JNK knockdown increase <i>Puma</i> mRNA expression.

<p>INS1 cells stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense control were exposed to 0.5 mM palmitate and 25 mM glucose for 12 or16 h (+). Relative mRNA expression was measured using quantitative RT-PCR and normalized to <i>Hprt1</i>. Relative <i>DP5</i> mRNA expression at A: 12 h, B: 16 h. Relative <i>Puma</i> mRNA expression C: 12 h, D: 16 h. Data are shown with+SEM of five - six independent experiments. INS1 cells stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense control were exposed to 0.5 mM palmitate and 25 mM glucose (black bars) or vehicle (white bars) for 24 h. Relative <i>Ins1</i> mRNA expression E: 24 h, Relative <i>Ins2</i> mRNA expression, F: 24 h. G: Total insulin content (ng insulin/total DNA) after GSIS. Data are shown with+SEM of four independent experiments *P<0.05, **P<0.01, ***P<0.001.</p

Primer sequence.

<p>Primer sequence.</p

JNK knockdown does not affect <i>sXbp-1</i>, P-eIF2α, <i>ATF4</i> and <i>ATF3</i> expression.

<p>INS1 cell lines stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense (ns) control were exposed to 0.5 mM palmitate and 25 mM glucose for 4–16 h (+). Relative mRNA expression was measured using quantitative RT-PCR and normalized to <i>Hprt1</i>. Relative <i>sXBP1</i>mRNA expression at A: 4 h, B: 12 h. C: Time-course analysis of P-eIF2α protein expression analyzed by Western blotting. Protein was isolated from INS1 cells exposed to 0.5 mM palmitate and 25 mM glucose for 0.5–24 h. Actin was used as loading control, representative blots are shown. Data are shown with+SEM of three independent experiments. D: Protein was isolated from INS1 cell lines stably expressing JNK1, JNK2 or JNK3 shRNA or the non-sense (ns) shRNA control after 16 h of palmitate and high glucose exposure, and p-eIF2α levels were analyzed by Western blotting. Actin was used as the loading control, representative blots are shown. Relative <i>ATF-4</i> mRNA expression at E: 12 h, F: 16 h. Relative <i>ATF-3</i> mRNA expression at G: 12 h, H: 16 h. Data are shown with+SEM of five independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p