Extracellular uridine diphosphate-mediated microglial inflammation in a mouse model of Sandhoff disease

Eri Kawashita1, Daisuke Tsuji2, Kohji Itoh2*

1Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Kyoto, Kyoto, Japan
2Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima, Tokushima, Japan

 Sandhoff disease (SD) is an inherited lysosomal storage disease caused by a β-hexosaminidase deficiency involving excessive accumulation of undegraded substrates, including GM2 ganglioside, which leads to neurological symptoms, such as mental retardation, spasms and quadriplegia. Macrophage inflammatory protein-1α (MIP-1α) is a crucial factor for microglia-mediated neuroinflammation in the onset or progression of SD. However, there was no therapeutic approach to control the abnormal production of MIP-1α in the brain of SD, and the mechanisms underlying the MIP-1α production by microglia, especially the transmitter-mediated production, remains unclear.

Extracellular nucleotides, including uridine diphosphate (UDP), are leaked by injured or damaged neurons. It has been shown that the nucleotide leakage activates microglia to trigger chemotaxis, phagocytosis, macropinocytosis and cytokine production, suggesting that extracellular nucleotides may be important neurotransmitters for microglia to regulate their functions physiologically and pathologically.

In the present study, we review the essential roles of extracellular nucleotides in the microglial functions and the UDP-enhanced MIP-1α production by microglia in SD model mice, providing a potential therapeutic approach for SD.


Sandhoff disease (SD) is a progressive neurodegenerative disorder caused by deficiencies of β-hexosaminidase (Hex), HexA (αβ) and HexB (ββ), associated with a defect in the Hex β-subunit gene1,2. Both HexA and HexB can degrade the terminal β-linked N-acetylglucosamine residues of oligosaccharides, but only HexA degrades GM2 ganglioside containing an N-acetylgalactosamine residue. In SD patients, an excessive accumulation of undegraded substrates, including GM2 ganglioside, is observed, particularly within lysosomes in the neuronal cells, due to the deficiencies of HexA and HexB, which leads to neurological symptoms in the central nervous system (CNS), such as mental retardation, spasms and quadriplegia. SD model mice (SD mice), established by means of Hex β-subunit gene disruption, exhibit the accumulation of GM2 gangliosides throughout the CNS and the abnormalities in motor functions, which are quite similar to those observed in SD patients3. Several therapeutic approaches for SD have been investigated for decades, including substrate reduction therapy4-6, bone marrow transplantation7,8, stem cell therapy9,10, enzyme replacement therapy10-14, gene therapy15-17 and chaperon therapy18, where the aim is to reduce the accumulated substrates. However, the disease remains incurable.

A progressive increase in microglial activation/expansion and subsequent neuronal apoptosis have been observed in the brains of SD patients and mice, suggesting that microglial inflammation is likely involved in the neurodegenerative mechanism in SD8,19-21. We demonstrated that macrophage inflammatory protein-1α (MIP-1α) is upregulated in the brains of SD mice from the age of 1 week, and in microglial cells derived from neonatal SD mice22,23. Wu and Proia also demonstrated that the deletion of MIP-1α expression results in not only a substantial decrease in macrophage/microglial-associated pathology together with neuronal apoptosis in SD mice, but also an increase in the life span of SD mice24. These studies suggest that MIP-1α is a crucial factor for microglia-mediated neuroinflammation during the pathogenesis of SD, and the downregulation of the abnormal production of MIP-1α by microglia may be another approach to delay the onset or progression of SD. However, the mechanisms underlying the abnormal production of MIP-1α by microglia, especially the transmitter-mediated production, is still poorly understood.

Microglia exists in their resting ramified form in the CNS under normal conditions; however, they are transformed into the activated ameboid form when they recognize a pathological state in the brain25. Extracellular nucleotides, adenosine triphosphate and uridine diphosphate (ATP and UDP, respectively), are leaked from injured or damaged neuronal cells and activate microglia to trigger cytokine production, chemotaxis, phagocytosis and macropinocytosis. Extracellular ATP and UDP induce the expression or release of cytokines and chemokines, including TNF-α, MCP-1 and MIP-1α, in microglia26,27. ATP regulates the microglial branch dynamics in the intact brain, and the ATP leakage from the damaged tissue mediates a rapid microglial response towards injury28. UDP is an “eat-me” signal from the dying cells: microglia recognize the extracellular UDP leakage from damaged neuronal cells, leading to the removal of the dying cells or their debris29,30. The extracellular nucleotides modulate the cellular functions by activating P2 receptors, which are classified into ionotropic P2X receptors and metabotropic P2Y receptors. Microglia have been shown to express functional P2X4, P2X7, P2Y6 and P2Y12 receptors29. These studies suggest that extracellular nucleotide signaling may regulate microglia-mediated physiological or pathological events in the brain.

We demonstrated that MIP-1α is prominently upregulated in the brain of SD mice22, and that the basal production of MIP-1α is higher in microglia derived from SD mice (SD-Mg) than in that from wild-type mice (WT-Mg)23, suggesting that the higher MIP-1α production is due to the abnormal signal transduction caused by the deficiencies of HexA and HexB in SD-Mg as well as the effects of other neuronal cells, including neurons and astrocytes. We furthermore investigated whether or not extracellular nucleotides enhance the production of MIP-1α by SD-Mg. We found that UDP induces the production of MIP-1α in SD-Mg but not WT-Mg, while ATP has no effect on the production of MIP-1α by WT- or SD-Mg31. The UDP leakage from the damaged neurons might enhance the MIP-1α production in microglia of SD mice to recruit other microglia to the damaged area, thereby resulting in a rapid microglial inflammation in the progression of SD. We also showed that SD-Mg is more strongly activated than WT-Mg due to the excessive accumulation of undegraded substrates, based on the observed increase in the IL-1β and TNF-α expression in SD-Mg compared with WT-Mg. The different activation states of WT- and SD-Mg may lead to differences in the response to UDP.

UDP is a known ligand of the P2Y2 and P2Y6 receptors, in addition to the CysLT1 and CysLT2 receptors, which are receptors for cysteinyl leukotrienes; UTP that is converted from UDP by ecto-nucleoside diphosphokinase binds to P2Y2, P2Y4 and P2Y632. We demonstrated that UDP and potentially UTP converted from UDP induce the production of MIP-1α by SD-Mg via the P2Y6 receptor but not via the P2Y2, P2Y4, CysLT1 or CysLT2 receptors31. A recent study reported that P2Y6 receptors are present in neuronal cells as monomeric and dimeric forms33. The protein expression of the dimeric P2Y6 receptor as well as the mRNA expression of P2Y6 receptor were found to be increased in SD-Mg in comparison to WT-Mg, suggesting that the increase in the expression of dimeric P2Y6 receptor may cause the enhanced response of SD-Mg to UDP in MIP-1α production compared with that of WT-Mg. We also confirmed that the activation of ERK and JNK was involved in the UDP-induced MIP-1α production in SD-Mg. Our previous study indicated that the activation of PLC, PKC, ERK and JNK mediates the enhanced production of MIP-1α in SD-Mg23. P2Y6 receptor couples to Gq protein to activate PLCβ and mobilize intracellular Ca2+ 34 and also modulates several cellular functions through the activation of ERK, JNK or PKC35,36. These findings suggest that the activation of PLC, PKC, ERK and JNK may be critical signaling events in the transmitter-induced abnormal production of MIP-1α in an autocrine or paracrine manner in SD-Mg.

The dimeric P2Y6 receptors are distributed in a non-raft microdomain and are thought to regulate the uracil nucleotide signaling33. Microdomains, such as lipid rafts, are known to be rich in cholesterol and glycosphingolipids including GM1 and GM3 gangliosides, where the gangliosides associate with receptors or signal transducers to modulate their functions. Glycosylation and deglycosylation have been reported to be responsible for the ganglioside composition of the plasma membrane37. GD3 synthase and GM2/GD2 synthase double knockout mice have disordered lipid rafts and subsequent inflammation, suggesting that the ganglioside composition is critical in the maintenance of lipid rafts38. Our previous studies by immunoblotting and immunocytochemical analyses have demonstrated little difference in the distribution of flotillin-1, a raft marker, between WT- and SD-Mg, but the intensity of flotillin-1 was decreased in SD-Mg compared with WT-Mg, suggesting the disordered maintenance of the lipid rafts in SD-Mg31. Previous studies have shown the level of cholesterol in fibroblasts from Tay-Sachs variant GM2 gangliosidosis to be similar to that of control fibroblasts39, and that the amounts of cholesterol and GM1 ganglioside in the brain did not differ significantly between the Hexb−/− and the Hexb+/− mice40. Thus, the disordered maintenance of the lipid rafts in SD-Mg might be caused by the altered ganglioside composition due to the failure to catalyze GM2 to GM3 ganglioside, with no marked changes in the amounts of cholesterol or GM1 ganglioside. We found that the disruption of the lipid rafts by pretreatment with methyl-β-cyclodextrin enhanced UDP-induced MIP-1α production in both WT- and SD-Mg, suggesting that lipid raft formation plays an important role in regulating the UDP-P2Y6 receptor signaling31. The disordered maintenance of the lipid rafts in SD-Mg is likely not involved in the enhanced dimeric formation of P2Y6 receptors31, and therefore both the increase in the expression of the dimeric P2Y6 receptor and the disruption of the lipid rafts may independently cause the enhanced response of SD-Mg to UDP in MIP-1α production.

A recent study demonstrated that UDP and UTP, as well as uridine, are detectable in the brain extracellular fluid obtained from freely moving rats, and the exposure of striatum to depolarizing concentrations of potassium chloride increases the level of the extracellular uracil nucleotides41. The extracellular uracil nucleotides have been shown to play roles in neural precursor cell proliferation and differentiation, and kainic acid-induced neuronal damage29,42. Furthermore, the uracil nucleotides are possibly involved in several neurological disorders, including epilepsy, cerebral ischemia, Alzheimer’s disease and amyotrophic lateral sclerosis43-46; however, the detailed roles of the uracil nucleotides in those diseases remain unclear and thus require further investigation.

MIP-1α is a crucial factor for microglia-mediated neuroinflammation in the brain of SD mice, and the downregulation of the abnormal production of MIP-1α by microglia may delay the onset or progression of SD22-24. The activation of EP2 and 4/cAMP/PKA signaling has been shown as a potential target to control the abnormal production of MIP-1α in SD-Mg47. Our findings additionally provide a new therapeutic approach for SD; that is, the P2Y6 receptor antagonist is thought to be a potential therapeutic target for reducing the UDP-enhanced MIP-1α production in SD. This approach can be used for the other lysosomal storage disease, including Tay-Sachs disease and Gaucher disease, although further investigation of the involvement of the uracil nucleotide signaling is required.

This work was supported by MEXT/JSPS KAKENHI (Grant No. 26293120).

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Article Info

Article Notes

  • Published on: October 19, 2016


  • Sandhoff disease

  • Lysosomal storage disease
  • Microglia
  • UDP
  • MIP-1α
  • Inflammation
  • P2Y6 receptor
  • Lipid raft
  • Microdomain
  • Nucleotides


Dr. Kohji Itoh PhD
Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, Tokushima University, 1-78 Sho-machi, Tokushima, Tokushima 770-8505, Japan, Telephone: +81-88-633-7290
Email: kitoh@tokushima-u.ac.jp