Original Research ArticleBDNF and Netrin-1 repression by C/EBPβ in the gut triggers Parkinson’s disease pathologies, associated with constipation and motor dysfunctions
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.
Section snippets
C/EBPβ is inversely coupled with BDNF and netrin-1 in PD patients
To explore whether C/EBPβ is involved in PD pathogenesis and its pathological relationship with BDNF and netrin-1, we conducted immunofluorescent (IF) staining on PD patient brain and gut sections. Noticeably, both BDNF and netrin-1 were evidently reduced in dopaminergic neurons in the SN regions of PD versus health control (Fig. 1A). C/EBPβ was highly phosphorylated (p-T235), a biomarker for its activation, in PD brains (Supplementary Fig. 1A). Moreover, BDNF and netrin-1 gene expression
Netrin-1 deprivation in BDNF gut conditional knockout mice elicits PD-like pathogenesis in the gut and brain
Next, we extended our study into BDNF gut specific KO mice by breeding BDNF flox/flox mice with SM22α-Cre mice. The BDNF gut specific KO mice were treated with His-Tat-DCC-4Fbn (2 mg/kg), a recombinant protein of netrin binding domain on DCC sequestrating netrins (Delloye-Bourgeois et al., 2009), via i.p. injection twice a week for consecutively for 3 months. IF study showed that BDNF was successfully eliminated from the gut, and netrin-1 in both the gut and SN tissues was substantially reduced
Netrin-1 deficiency induces constipation and motor disorders in BDNF gut conditional knockout mice
To assess the pathological effect of Tat-DCC-4Fbn treatment on BDNF gut specific KO mice, we monitored the constipation by examining GI transit time, stool moisture and colon length and found that Tat-DCC-4Fbn treatment alone elicited both GI transit time increase and stool dryness without changing the colon length; and these first two effects were further escalated in BDNF gut specific KO mice that also displayed shorted colon length (Fig. 7A–C), suggesting that Tat-DCC-4Fbn triggers
Discussion
In the current study, we show that both netrin-1 and BDNF are substantially decreased in PD patient’s gut and SN tissues, accompanied with noticeable upregulation of p-C/EBPβ and C/EBPβ. Our in vitro netrin-1 and BDNF promoter luciferase, EMSA and ChIP assays support that C/EBPβ acts as a repressor by directly associating with the promoters of both genes. Accordingly, depletion of C/EBPβ alleviates rotenone-induced suppression of netrin-1 and BDNF in dopaminergic SH-SY5Y cells, rat neurons and
Healthy control and PD post-mortem brain samples
Post-mortem brain samples were dissected from frozen brains of PD patients and age-matched non-demented controls (63–72 years old) from the Emory Alzheimer's Disease Research Center (ADRC). The study was approved by the Biospecimen Committee (The Goizueta ADRC, Emory University). PD was diagnosed according to the criteria of the Consortium to Establish a Registry for PD and the National Institute on Aging. Informed consent was obtained from all cases.
PD patients gut biopsy and immunofluorescent staining
The included subjects were recruited from
CRediT authorship contribution statement
Keqiang Ye: Conceptualization, Funding, and Writing-original draft. Eun Hee Ahn: Data curation, Formal analysis, Methodology, Validation, and Writing-original. Seong Su Kang: Validation, Resources, and Wrinting-review & editing. Xia Liu: Resources, and Project administration. Laura Musazzi: Wrinting-review & editing, and Resources. Soo Young Choi: Resources. Patrick Mehlen and Xuebing Cao: Wrinting-review & editing.
Declaration of Competing Interest
The authors declare no competing financial interests.
Acknowledgments
This work was supported by grants from NIH grant (RF1, AG051538) to K.Y. We thank ADRC at Emory University for human AD patients and healthy control samples. This study was supported in part by the Rodent Behavioral Core (RBC), which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities. Additional support was provided by the Viral Vector Core of the Emory Neuroscience NINDS Core Facilities (P30NS055077). Further support was provided by the
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