BDNF and Netrin-1 repression by C/EBPβ in the gut triggers Parkinson’s disease pathologies, associated with constipation and motor dysfunctions

Highlights

• Parkinson's disease patients display decreased BDNF and netrin-1 in the gastrointestinal (GI) tract, associated with TH-positive neuronal loss and Lewy body-like inclusions.

• p-C/EBPβ inversely correlates with BDNF and netrin-1 levels in dopaminergic neurons in the gut and the brain.

• C/EBPβ binds the promoters and is a transcription repressor for both BDNF and netrin-1.

• Gut conditional knockout of BDNF and netrin-1 elicits PD non-motor, Lewy body-like pathologies and motor symptoms in SNCA PD mouse model.

Abstract

Chronic constipation is one of the most prominent prodromal symptoms in Parkinson’s disease (PD), and Lewy bodies, enriched with aggregated α-Synuclein (α-Syn), propagation from the gut into the brain has been proposed to play a key role in PD etiopathogenesis. BDNF (Brain-derived neurotrophic factor) and Netrin-1 promote both neuronal survival and regulate the gut functions. We hypothesize that C/EBPβ represses BDNF and Netrin-1 in peripheral nervous system and central nervous system, contributing to GI tract and brain malfunctions in PD. To test the hypothesis, we performed the studies in both human PD gut tissues and BDNF or Netrin-1 gut conditional KO mice models. Lewy bodies with α-Syn aggregation and neuro-inflammation were measured in the colon and brain samples from PD patients and healthy controls and rotenone or vehicle-treated WT and CEBPβ (+/-) mice. We show that both BDNF and Netrin-1 are strongly decreased in the brain and the gut of PD patients, and conditional KO of these trophic factors in the gut elicits dopaminergic neuronal loss, constipation and motor dysfunctions. Interestingly, the inflammation and oxidative stress-induced transcription factor C/EBPβ acts as a robust repressor for both BDNF and Netrin-1 and suppresses the expression of trophic factors, and its levels inversely correlate with BDNF and Netrin-1 in PD patients. Our findings support that gut inflammation induces C/EBPβ activation that leads to both BDNF and Netrin-1 reduction and triggers PD non-motor and motor symptoms. Possibly, C/EBPβ-mediated biological events might be early diagnostic biomarkers for PD.

Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by motor dysfunction. Its pathological hallmarks include loss of dopaminergic neurons in the substantia nigra (SN)-striatum system and presence of proteinaceous cytoplasmic inclusions, known as Lewy Bodies, in the remaining dopaminergic neurons (Savitt et al., 2006). The main component in Lewy bodies is aggregated alpha-synuclein (α-Syn) that is hyperphosphorylated and truncated. Lewy body-like aggregation first manifests in enteric neurons of the gut (pre-stage 1 or stage 0) (Braak et al., 2006) long before it is present in dopaminergic neurons of the dorsal motor nucleus of the vagus (dmX) (Kingsbury et al., 2010) and, to a lesser extent, the olfactory system (Stages 1 and 2), progressing to the midbrain (Stages 3 and 4) and finally, to the basal forebrain and neocortex (Stages 5 and 6). The gap between stages 0 and 4 may take many years (Braak et al., 2003), leading to the hypothesis that the intestine might be an early site of PD pathology in response to gut luminal -likely bacterial-products, metabolites or toxins. PD mouse models exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine such as LPS (Kelly et al., 2014), which increases the oxidative stress in the enteric neurons (Forsyth et al., 2011). Thus, gut leakiness in patients with a genetic susceptibility to PD may be a pivotal early step contributing to the initiation and progression of PD etiopathogenesis. α-Syn-related neurodegeneration in the enteric nervous system (ENS) is a frequent and premotor manifestation of PD (Braak et al., 2006), associated with chronic constipation and pathophysiological changes in the intestinal wall (Forsyth et al., 2011).

PD patients experience nonmotor symptoms, such as olfactory disturbance, REM sleep behavior disorders and constipation and depression (Reichmann et al., 2009), which may precede the onset of motor symptoms by over a decade. Brain-derived neurotrophic factor (BDNF), the neurotrophin ligand for TrkB receptors, is distributed abundantly in the intestinal tract, including ENS neurons and intestinal mucosa epithelia and interstitial (Hoehner et al., 1996; Lommatzsch et al., 1999; Lucini et al., 2002; Radaelli et al., 2001). BDNF plays an essential role in potentiating enteric neural network formation from murine embryonic stem cells (Takaki et al., 2006) and facilitating reflex nerve pathway regeneration (Katsui et al., 2008). BDNF is important for ENS neuron survival and development in regulating intestinal function. BDNF’s physiologic effects on gut motility have been investigated in several studies. In healthy humans, BDNF accelerates colonic emptying and increases stool frequency, without altering stool consistency (Coulie et al., 2000). Moreover, BDNF facilitates gut motility in slow-transit constipation (Chen et al., 2014). Interestingly, its gut knockout (KO) increases vagal sensory innervation of the intestine and enhances satiation (Biddinger and Fox, 2014).

Netrin-1, initially identified as a diffusible factor for axon guidance (Kennedy et al., 1994; Serafini et al., 1994), is a multifunctional secreted molecule implicated both during tissue patterning and in regulating neuronal activity (Lai Wing Sun et al., 2011). Netrin-1 regulates various biological processes, including axonal growth (Forcet et al., 2002; Lin et al., 2005), synaptic plasticity (Bayat et al., 2012; Horn et al., 2013) and inflammation (He et al., 2018; Podjaski et al., 2015). The expressions of Netrin-1 and its receptor DCC (deletion in colorectal cancer) genes are observed in embryonic as well as adult nigrostriatal pathway (Cord et al., 2010; Livesey and Hunt, 1997), mediating the topographic guidance of dopaminergic axons by differential netrin-1 expression in the striatum via DCC receptor (Li et al., 2014). In peripheral organs, netrin-1 modulates inflammation, and is also expressed in intestinal epithelium (Paradisi et al., 2009). Netrin-1 attenuates experimental gut colitis via limitation of neutrophil trafficking (Aherne et al., 2012), and netrin/DCC pathway mediates the attraction of vagal sensory axons to the fetal mouse gut (Ratcliffe et al., 2006). However, whether netrin-1 is implicated in constipation remains unknown.

The CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors, binding to the CCAAT box of promoters and enhancer regions (van der Krieken et al., 2015). C/EBPβ is expressed in various tissues including the CNS: in neurons they participate in memory formation and synaptic plasticity, whereas in glial cells they regulate the pro-inflammatory program (Pulido-Salgado et al., 2015; Poli, 1998). Promoters of many pro-inflammatory genes contain putative C/EBPβ consensus sequences (Caivano et al., 2001; Lowenstein et al., 1993). Recently, we identified that C/EBPβ acts as a major transcription factor for AEP (also called delta-secretase, gene name: LGMN) and mediates its augmentation in the brain in an age-dependent manner (Wang et al., 2018). Moreover, AEP cleaves α-Syn at N103 and promotes its aggregation and neurotoxicity in dopaminergic neurons in PD patients (Zhang et al., 2017). Interestingly, BDNF deprivation in primary neuronal cultures, as well as reduction in Alzheimer’s disease (AD), elicits C/EBPβ activation via elevating pro-inflammatory cytokines, correlating with δ-secretase upregulation and APP N585 and Tau N368 cleavage (Wang et al., 2018). Furthermore, C/EBPβ mediates both SNCA and MAO-B mRNA transcription, implicated in PD pathogenesis (Wu et al., 2020). Most recently, we reported α-Syn N103 and Tau N368 form compact fibrils in the gut, and AEP mediates PD pathogenesis by regulating the spread of α-Syn N103/Tau N368 fibrils from the gut into the brain via vagus nerve. Chronic oral low-dose rotenone treatment demonstrated the gut dysfunctions in SNCA but not SNCA/AEP KO mice, coupled to the motor deficits (Ahn et al., 2020). Discovering both BDNF and netrin-1 reduction and inverse C/EBPβ elevation in PD patient colon biopsy samples, we hypothesize that C/EBPβ represses BDNF and netrin-1 mRNA transcription in the gut, which might initiate the prodromal effects and PD pathologies that are spread into the brain later. We employed C/EBPβ heterozygote mice and BDNF or netrin-1 gut-specific conditional knockout mice to test whether gut inflammation-stimulated C/EBPβ suppresses these trophic expression in the gut, eliciting PD pathologies in the brain. Interestingly, rotenone strongly activated C/EBPβ that subsequently decreased both BDNF and netrin-1 expression by acting as a repressor in dopaminergic (DA) cell line SH-SY5Y and DA neurons. Noticeably, netrin-1 deprivation from BDNF gut-specific KO mice induced aggregated α-Syn formation displaying Lewy body-like pathologies that were validated by pS129 α-Syn/14-3-3 or ubiquitin antibodies co-staining in the gut and the brain, accompanied with constipation and motor symptoms.