Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis

Abstract

BackgroundSecretion 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.ResultsWe 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.ConclusionsWe 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.

APP upregulation contributes to retinal ganglion cell degeneration via JNK3

“…MAB343, Millipore) [ 16 ], rabbit antibody against JNK3 (1:200, catalog no. 2305, Cell Signaling Technology) [ 54 ], or chicken antibody against Tuj1 (1:200, catalog no. NB100–1612, Novus Biologicals) [ 55 ] in primary antibody dilution buffer (1% v/v BSA, 0.3% v/v Triton × 100 and 0.01% w/v NaN 3 in phosphate-buffered saline (PBS)) at 4 °C.…”

Unconventional Secretion and Intercellular Transfer of Mutant Huntingtin

“…The unconventional secretion of α-Synuclein occurs via extracellular vesicles [ 82 , 83 ], with exosomes most likely being the main vehicle [ 84 , 85 ]. Such secretion is enhanced by mutation [ 86 ] and promoted by neuronal activity [ 84 , 87 , 88 ] as well as autophagy failure or inhibition [ 89 , 90 , 91 ] and stress signaling [ 92 , 93 ]. Secreted α-Synuclein could be taken up via endocytosis by neighboring cells [ 85 , 94 , 95 ] and could presumably transmit neurotoxicity in this manner [ 90 , 96 ].…”

Prelysosomal Compartments in the Unconventional Secretion of Amyloidogenic Seeds

“…Thus, we have shown in vitro that microglia activated by conditioned medium from synucleinopathic neurons in culture (or LPS) increase α-synuclein secretion from the neurons [ 327 ]. While the microglia factors that mediate this effect are unknown, the ensuing neuronal hyper-secretion of α-synuclein depends on c-Jun N-terminal kinase (JNK) activation in the nerve cells [ 327 ]. Additionally, microglia might potentially ‘qualitatively enrich’ amyloids for their ability to transmit misfolding disease.…”