TGFβ1-Smad3 signaling mediates the formation of a stable serine racemase dimer in microglia.

Highlights

• Low activity stable serine racemase dimers are induced by TGFβ1-Smad3 in microglia.

• Inhibition of the Smad-dependent TGFβ1 signaling reduces total serine racemase.

• Lipopolysaccharide and SIS3 increase the high activity monomeric Serine Racemase.

• Smad-dependent TGFβ1 signaling reduces IL1β but not TNFα in adult mice microglia.

• TGFβ1 contributes to avoid d-serine and NMDAR mediated glutamate excitotoxicity.

Abstract

d-serine is synthesized by serine racemase (SR), a fold type II class of pyridoxal-5′-phosphate (PLP)-dependent enzyme. Whereas X-ray crystallography reveals that SR can be monomeric, reversible dimers having the highest racemase activity, or stable SR dimers resistant to both denaturation and reductive treatment, showing reduced racemase activity have been detected in microglia and astrocytes; the latter especially in oxidative or inflammatory environments. The microglial inflammatory environment depends largely on the TGFβ1-mediated regulation of inflammatory cytokines such as TNFα and IL1β. Here we evaluated the participation of TGFβ1 in the regulation of SR, and whether that regulation is associated with the induction of stable SR dimers in the microglia from adult mice. In contrast to the effect of lipopolysaccharide (LPS), TGFβ1 increased the formation of stable SR dimers and reduced the detection of monomers in microglia in culture. LPS or TGFβ1 did not change the amount of total SR. The increase of stable SR dimer was abolished when TGFβ1 treatment was done in the presence of the Smad inhibitor SIS3, showing that Smad3 has a role in the induction of stable dimers. Treatment with TGFβ1 + SIS3 also reduced total SR, indicating that the canonical TGFβ1 pathway participates in the regulation of the synthesis or degradation of SR. In addition, the decrease of IL1β, but not the decrease of TNFα induced by TGFβ1, was mediated by Smad3. Our results reveal a mechanism for the regulation of d-serine through the induction of stable SR dimers mediated by TGFβ1-Smad3 signaling in microglia.

Introduction

d-serine is an endogenous dextro amino acid synthesized by serine racemase (SR), a member of the fold type II class of pyridoxal-5′-phosphate (PLP)-dependent enzyme [1]. SR catalyzes the isomerization of l-serine into d-serine [2,3]. It is expressed by neurons, astrocytes and, in a lesser extent by activated and quiescent microglia [[4], [5], [6], [7], [8], [9]]. In addition, SR has the potential to degrade d-serine because it is also able to catalyze an α, β-elimination reaction that converts d-serine in pyruvate and ammonium [10].

d-serine regulates glutamatergic transmission mediated by the glutamate N-methyl-d-aspartate receptor (NMDAR) [11,12] acting as an agonist on the allosteric strychnine-insensitive glycine site of the NMDAR [13]. Thus, d-serine contributes for the regulation of synaptic plasticity and in the induction of long-term potentiation (LTP) [14] or synaptogenesis [15], process where glutamatergic transmission and NMDAR activities are pivotals. In addition to its regulatory functions, the excess of d-serine has the potential to generate neurotoxicity mediated by the overactivation of NMDAR resulting in neuronal impairment [[16], [17], [18]] and could participate in the induction or progression of neurodegenerative diseases as Alzheimer's disease [[19], [20], [21]].

The regulation of SR activity is complex and includes allosteric modulation exerted by ATP, malonate [22] or divalent cations as calcium or magnesium [23]. In fact, to know how allosteric modulators affect the structure and activity of SR contributes on the design of drugs for the regulation of d-serine levels [24]. In addition to allosteric modulation, several species of SR, with different quaternary conformation and activity coexist in cells [25,26]. The X-ray crystallography and gel-filtration chromatography confirm that functional SR is formed by symmetric dimers [23,25], although multiples dimeric structures have been reported [26]. A noncovalent SR dimer containing one or more free thiol groups in the enzyme's active center holds the highest activity [26]. In addition, a SR dimer with covalent disulfide bonds, resistant to SDS and reducing reagents as β-mercaptoethanol, and with reduced racemase activity is detected in astrocytes [26] and in microglia [8,9,27]. The stable dimer appears to be elevated during inflammatory stimulation, or when cells are exposed to an oxidative environment [28]. Thus, cytokines modulating the microglia activity and its oxidative state as transforming growth factor β1 (TGFβ1) [29,30], or proinflammatory cytokines, as tumor necrosis alfa (TNFα) or interleukin 1β (IL1β), could participate in the regulation of SR species in microglia.

TGFβ1 is a cytokine released from neurons and astrocytes [31,32] and plays a neuroprotective role during brain injury or infection, because it attenuates and restricts the neuroinflammation, avoiding the development of a neurotoxic environment [29,33]. TGFβ1 activates Smad-dependent (or canonical), and Smad-independent (non-canonical) pathways [34]. The activation of the TGFβ1-Smad pathway is mostly neuroprotective because it decreases the release of proinflammatory cytokines, ROS and NO· [29,34], and promotes Aβ clearance [35]. In contrast, the Smad-independent pathway involves the activation of MAPKs, including ERK, p38 and JNK, and PI3K [36,37], and if activated by itself, it could be associated with the potentiation of neuroinflammation.

There are reports that TGFβ1 promotes the release of d-serine without changes in SR expression in astrocytes and neurons [15], and that the synaptogenesis of cortical neurons mediated by astrocytes' secreted TGFβ1 requires of d-serine [15]. However, the role of TGFβ1 in the regulation of SR in microglia has not been studied.

On the other hand, the inflammatory cytokines TNFα and IL1β, both upregulated early during the inflammatory activation of microglia [38,39], are regulated by TGFβ1-Smad3 [40]. In addition, TNFα and IL1β can inhibit TGFβ1 signaling by upregulating the expression of the inhibitory Smad7 [41,42]. d-serine increases the expression of TNFα mRNA in the forebrain [43], whereas the induction of IL1β mRNA in macrophages requires of l-serine [44].

Because TGFβ1-Smad3 signaling mainly potentiate a neuroprotective activation of microglia, modifying the release of inflammatory cytokines TNFα or IL1β and regulating the microglial cell environment, here we assessed if the regulation by TGFβ1- Smad3 signaling includes the SR expression and the induction of the stable SR dimer in microglia.