Digestion products of the PH20 hyaluronidase inhibit remyelination

Abstract

OBJECTIVE Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. METHODS Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. RESULTS OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA-digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. INTERPRETATION We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions.

Comment on: PH20 is not expressed in murine CNS and oligodendrocyte precursor cells

“…This finding is in contrast to work in our laboratory showing that OPCs digest hyaluronan (HA), and that PH20, but not other hyaluronidases, as well as PH20‐generated digestion products of HA potently block OPC maturation and remyelination 2. The basis for the claim that PH20 does not influence OPC maturation is that in the Marella et al paper, the authors report that the effects of a preparation of hyaluronidase, bovine testicular hyaluronidase (whose hyaluronidase activity is mostly PH20), are due entirely to contaminating fibroblast growth factor‐2 (FGF‐2; also called basic fibroblast growth factor).…”

“…We also reported, using size exclusion chromatography with multi‐angle laser light scattering, that distinct hyaluronidases generated HA digestion products that varied in their size ranges 2. We found that PH20‐digested HA but not HA digested by another hyaluronidase blocked OPC maturation.…”

“…We2, 4 and others5 also previously reported that the PH20 hyaluronidase is elevated in demyelinating white matter lesions as well as oligodendrocyte progenitor cells. PH20 was also reported to be present, albeit at low levels, in the rat brain in a study on traumatic brain injury 6.…”

“…While it remains unclear if our preparation of bovine testicular hyaluronidase was contaminated with sufficient FGF‐2 to impact OPC maturation, Marella et al failed to acknowledge that we also reported that transducing the PH20 cDNA into OPCs potently inhibited OPC maturation to a far greater degree than other hyaluronidases 2. This experiment demonstrates that elevated PH20 directly blocks OPC maturation, given that contaminating factors did not exist in these experimental conditions.…”

The Activity of Hyaluronan and Hyaluronidase PH20 in Inflammation-A Role by Reagent Contaminants?

“…A recent report suggested that HA fragments generated by PH20 in vitro inhibited differentiation of oligodendrocyte progenitor cells (OPC) and subsequent remyelination of brain lesions in a mouse model [17]. However, the key experiments characterizing the effect of HA fragments in this remyelination model used BTH-digested HA rather than rHuPH20-digested HA or purified LMW HA fragments, again raising the possibility that the observed effect might have been influenced by reagent contamination.…”