br Materials and methods br Results br Discussion In

Materials and methods
Results
Discussion In this study, we administrated BDE-153 to newborn rat pups at PND 10 and cultured primary neurons, measured neuronal apoptosis and LDH activity in vivo and in vitro, then explored the possible predominant pathway underlying the neuronal apoptosis by examining p53, caspase-3, 8, 9, and calpain-1, 2 at mRNA and protein levels in the cerebral cortices in adult rats, and further measured five neurotrophins (at mRNA and protein levels) and the ChaT and AchE activities in adult rats’ cerebral cortices and primary neurons. The neuronal apoptosis was demonstrated to be dependent on p53, and dependent on more calpain-2 than caspase-3 in the cerebral cortices in rats following the BDE-153 treatment. Moreover, the BDE-153 treatment had significantly decreased the neurotrophins levels (at mRNA and protein) and the activities of ChaT and AchE in the Creatinine and primary neurons, and which were reverted by the pretreatment with calpain inhibitor PD150606 or calpain’s downstream cdk5/p25 inhibitor Roscovintine. The findings suggested that the neuronal apoptosis was dependent on more calpain-2 than caspase-3, and the neurotrophins and the cholinergic enzymes regulated by calpain-2 activation were likely involved in the neuronal apoptosis induced by the BDE-153 treatment. Caspases, a family of aspartate-specific cysteine proteases, are activated through proteolytic cleavage and well characterized for their roles in apoptosis (Wyllie, 1997). Caspases are categorized into initiator caspases (8, 9, and 10) and their downstream targets, executioner caspases (3, 6, and 7). The initiator caspases serve to initiate apoptosis and can be triggered and activated either through extracellular stimulation of the Fas receptor and tumor necrosis factor receptor or through the intracellular cytochrome c release from mitochondria (Wyllie, 1997). Caspase-3 or another executioner caspase is activated by the initiator caspases, and then executes apoptosis (Wyllie, 1997). Calpains is a ubiquitous family of Ca2+-activated intracellular neutral proteases, are believed to play critical roles in apoptosis under certain conditions O’Donovan et al., 2001). Calpain is maintained as an inactive proenzyme until a sharp increase in cytosolic free Ca2+ concentration triggers its activation. Calpain-1 and calpain-2 are the two highly expressed isoforms in the central nervous system, their activation requires different intracellular Ca2+ concentrations (1–20 μM for calpain-1, and 0.25–0.75 for calpain-2) ex vivo (Croall and Ersfeld, 2007; Sorimachi and Suzuki, 2001). It is regarded that calpain-1 activation induces cell survival, and calpain-2 induces cell death (Wang et al., 2013; Wang et al., 2014). Activated calpain cleaves p35 into p25 in the central nervous system in rodents, results in the downstream p25/Cdk5 complex formation and activation, which phosphorylates the cytoplasmic substrates including many enzymatic, signaling, and cytoskeletal proteins (tau, tubulin and other neurofilament proteins) (Croall and Ersfeld, 2007; Sorimachi and Suzuki, 2001). Furthermore, caspases-7, 8, 9 can be directly cleaved by calpain (Chua et al., 2000). The involvement of calpain-2 mediated cdk5/p25 pathway was demonstrated in the neuronal apoptosis induced by the BDE-153 treatment in our previous publication (Zhang et al., 2017). Neurotrophins, a family of small secreted proteins including BDNF, GDNF, NGF, NT-3 and NT-4, are essential for neural growth, survival, cell differentiation, and synaptic plasticity in normal brain development and adult brain. The neurotrophins protein and mRNA levels were significantly decreased following the BDE-153 treatment in this study, which is consistent with the decreased BDNF mRNA levels in primary culture cerebellar granular neurons and rat hippocampus following the BDE-99 treatment (Blanco et al., 2013; Blanco et al., 2011). Decreased BDNF contents in brain worsen the symptoms of Alzheimer’s disease (AD) (Hachisu et al., 2015). Intra-hippocampal BDNF administration rescues learning deficits induced by amyloid beta-42 peptide in rats (Zhang et al., 2015). GDNF enhances the dopamine levels, release and re-uptake in corpus striatum, increases adult dopamine neuron numbers and dopaminergic terminals (Kumar et al., 2015), and improves the spatial learning in mice via inducing the serotonin key genes in brain (Naumenko et al., 2013; Naumenko et al., 2014).