Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases

The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair

A TLR/AKT/FoxO3 immune tolerance–like pathway disrupts the repair capacity of oligodendrocyte progenitors

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance–like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation

A TLR/AKT/FoxO3 immune tolerance–like pathway disrupts the repair capacity of oligodendrocyte progenitors

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance–like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation

Role of Recurrent Hypoxia-Ischemia in Preterm White Matter Injury Severity

<div><p>Objective</p><p>Although the spectrum of white matter injury (WMI) in preterm infants is shifting from cystic necrotic lesions to milder forms, the factors that contribute to this changing spectrum are unclear. We hypothesized that recurrent hypoxia-ischemia (rHI) will exacerbate the spectrum of WMI defined by markers of inflammation and molecules related to the extracellular matrix (hyaluronan (HA) and the PH20 hyaluronidase) that regulate maturation of the oligodendrocyte (OL) lineage after WMI.</p><p>Methods</p><p>We employed a preterm fetal sheep model of <i>in utero</i> moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. The response to rHI was compared against corresponding early or later single episodes of HI. An ordinal rating scale of WMI was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microglial activation. Late oligodendrocyte progenitors (preOLs) were quantified by stereology. Analysis of hyaluronan and the hyaluronidase PH20 defined the progressive response of the extracellular matrix to WMI.</p><p>Results</p><p>rHI resulted in a more severe spectrum of WMI with a greater burden of necrosis, but an expanded population of preOLs that displayed reduced susceptibility to cell death. WMI from single episodes of HI or rHI was accompanied by elevated HA levels and increased labeling for PH20. Expression of PH20 in fetal ovine WMI was confirmed by RT-PCR and RNA-sequencing.</p><p>Conclusions</p><p>rHI is associated with an increased risk for more severe WMI with necrosis, but reduced risk for preOL degeneration compared to single episodes of HI. Expansion of the preOL pool may be linked to elevated hyaluronan and PH20.</p></div

Spectrum of WMI in the four experimental conditions, as assessed by analysis of GFAP and Iba-1 staining using quantitation of area fractions stained for each marker.

<p>(A) Astrogliosis, measured by GFAP area fraction (AF), is increased following rHI (Kruskal-Wallis H = 13.96, p = 0.003, on 3df; mean ± SD Control: 0.20±0.06; Late HI: 0.25±0.05; Early HI: 0.33±0.05; rHI: 0.37±0.04; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **rHI vs. Control, p = 0.002; *Early HI vs. Control, p = 0.018; *rHI vs. Late HI, p = 0.021). (B) Iba-1 AF revealed similar patterns to Iba-1 pathology in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112800#pone-0112800-g003" target="_blank">Fig. 3</a> (Kruskal-Wallis H = 11.7, p = 0.008; mean ± SD Control: 0.06±0.05; Late HI: 0.08±0.06; Early HI: 0.18±0.13; rHI: 0.33±0.170; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **rHI vs. Control p = 0.01; *rHI vs. Late HI p = 0.020). (C) Iba-1 and GFAP AFs are significantly associated over a broad spectrum of WMI (***Spearman’s Rank Correlation: ρ = 0.91, p&lt;0.0001).</p

Spectrum of WMI in the four experimental conditions, as assessed by analysis of H &amp; E staining using ordinal rating scores.

<p>(A) Neuropathological scoring of H &amp; E staining in frontal white matter (Kruskal-Wallis H = 8.57, p = 0.036 on 3df; mean ± SD Control: 0.20±0.45; Late HI: 0.28±0.44; Early HI: 0.80±0.84; rHI: 2.0±1.3). (B) Neuropathological scoring of H &amp; E stained parietal white matter (Kruskal-Wallis H = 10.5, p = 0.015; mean ± SD Control: 0.60±0.89; Late HI: 1.00±0.00; Early HI: 1.4±0.89; rHI: 2.3±0.82; Bonferroni-corrected post-hoc Mann-Whitney U-test: *rHI vs. Late HI, p = 0.036). (C–F) Representative images of H &amp; E staining from (C) Control, (D) Early HI, (E) Late HI, (F) rHI frontal white matter. Panel scale bars: 200 µm; inset scale bars: 20 µm.</p

The pool of premyelinating OL lineage cells is significantly increased following rHI; PreOLs are less susceptible to acute degeneration following rHI than following late HI.

<p>(A–B) Representative confocal images of premyelinating OL lineage cells in the PVWM labeled with the O4 monoclonal antibody (arrowheads) in controls (A) and after rHI (B). Scale bars 20 µm. (C) Unbiased stereological counts of O4-labeled cells show an expansion of the preOL pool in the rHI group relative to control and early HI (Kruskal-Wallis: H = 7.7, p = 0.053, on 3 df; mean ± SD Control: 16,854±4,752 cells/mm³; Early HI: 16,430±7,182 cells/mm³; Late HI: 22,855±3,210 cells/mm³; rHI: 29,917±11,254 cells/mm³; CE range: 0.048–0.154). (D) Stereology further shows a decreased rate of preOL death following rHI relative to late HI (Kruskal-Wallis: H = 7.5, p = 0.057, on 3 df; mean ± SD Control: 0.9±0.4%; Early HI: 1.2±0.9%; Late HI: 5.0±2.4%; rHI: 1.7±1.8%; Bonferroni-corrected post-hoc Mann-Whitney U-tests: *Late HI vs. Control, p = 0.037; CE range for pyknotic counts: 0.21–0.95). (E) There is a greater density of activated caspase 3-labeled cells following a single late HI episode (Kruskal-Wallis H = 10.5, p = 0.015, on 3df; mean ± SD, Control: 1.62±0.76 Cells/mm²; Late HI: 6.30±5.40; Early HI: 0.92±0.21 cells/mm²; rHI: 1.73±1.33 cells/mm²; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **Early HI vs. Late HI, p = 0.001). (F) Representative confocal images of a degenerating O4-positive cell with a fragmented pyknotic nucleus in a late HI case. (F1) O4, (F2) Hoechst 33342 nuclear stain, (F3) Merge. Scale bar: 5 µm.</p

HA is present in fetal ovine white matter and displays a persistent increase for several weeks after HI.

<p>Representative confocal image of staining for HA with an HA binding protein (HABP) in frontal white matter (pseudocolors: green: HABP; red: Hoechst 33342-labeled nuclei). (A) Control: One-week post-insult. (B) Early HI. (C) Late HI. (D) rHI. (E) Control: 24-hours post-insult. (F) HI: 24-hours post-insult. (G) Control: Two weeks post-insult. (H) HI: Two weeks post-insult. (I) Control: Four weeks post-insult. (J) HI: Four weeks post-insult. Scale bars 20 µm.</p