Hypothesis

We propose that the age‑associated decline in both HAS2 synthesis and HYAL degradation creates a sustained pool of intermediate‑molecular‑weight hyaluronan (HA) fragments (50‑250 kDa) that act as a danger‑associated molecular pattern (DAMP) signal. This chronic DAMP signaling pushes fibroblasts into a proteostatic crisis where the usual chaperone‑mediated refolding and ubiquitin‑proteasome degradation pathways are overwhelmed. In response, the cell redirects misfolded proteins into highly ordered, amyloid‑like aggregates as a final ‘last resort’ to sequester toxic species, mirroring the idea that aggregation is not random failure but a protective, thermodynamically stable endpoint.

Rationale

• Coordinate HAS2/HYAL down‑regulation reduces both new HA production and complete catabolism, favoring accumulation of immunogenic HA fragments【https://pmc.ncbi.nlm.nih.gov/articles/PMC4474994/】【https://pmc.ncbi.nlm.nih.gov/articles/PMC3570623/】.
• HA fragments activate CD44/TLR2/4 and rapidly down‑regulate the anti‑inflammatory A2a adenosine receptor via PKC‑driven internalization, amplifying NF‑κB‑driven inflammation and ROS production【https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1125899/full】【https://pmc.ncbi.nlm.nih.gov/articles/PMC3208614/】.
• Inflammatory ROS and ER stress impair proteasome activity and chaperone capacity, pushing the proteostasis network toward collapse【https://www.tandfonline.com/doi/full/10.4161/derm.21923】 (collagen fragment feedback on HAS2).
• When refolding and degradation fail, cells may favor the formation of β‑sheet‑rich aggregates that are less reactive and can be sequestered in insoluble deposits, reducing proteotoxic load—a strategy observed in yeast and mammalian stress granules that transition to amyloid‑like states under prolonged stress.

Testable Predictions

1. Aged dermal fibroblasts will show higher levels of insoluble, SDS‑resistant protein aggregates that co‑localize with HA fragment deposits compared with young cells.
2. Exogenous addition of LMW‑HA (100 kDa) to young fibroblasts will recapitulate the aggregate phenotype, while enzymatic removal of HA fragments with hyaluronidase will reduce aggregates in aged cells.
3. Blocking CD44 or TLR4 signaling (using antibodies or small‑molecule inhibitors) will attenuate both the inflammatory response and the formation of amyloid‑like aggregates.
4. Enhancing proteasome activity (e.g., with sulforaphane) or overexpressing HSP70 will suppress aggregate formation despite persistent HA fragment presence.

Experimental Approach

• Cell model: Primary human dermal fibroblasts from donors <30 y (young) and >65 y (aged). Culture under standard conditions.
• HA fragment modulation: Treat cells with recombinant HYAL2 to degrade HA, or with purified LMW‑HA (50‑250 kDa) to increase fragments.
• Read‑outs:
  • Soluble vs. insoluble protein fractionation followed by SDS‑PAGE and western blot for ubiquitin, p62, and known aggregation‑prone proteins (e.g., SOD1, α‑synuclein).
  • Filter‑trap assay and Thioflavin‑T fluorescence to detect amyloid‑like aggregates.
  • Immunofluorescence for co‑localization of HA (using HA‑binding protein probe) and aggregate markers.
  • ELISA for IL‑6, IL‑8, ROS (DCFDA), and surface A2aR levels (flow cytometry).
  • Proteasome activity assay (LLVY‑AMC).
• Interventions: CD44 blocking antibody, TLR4 antagonist (TAK‑242), A2aR agonist (CGS‑21680), proteasome activator (sulforamide), HSP70 overexpression via lentivirus.

Potential Implications

If validated, this hypothesis reframes HA‑driven inflammation not merely as a parallel age‑related process but as a upstream trigger that forces the proteostasis network into a protective, aggregation‑based ‘last resort’. Therapeutically, targeting HA fragment signaling (e.g., with hyaluronidase mimetics or CD44/TLR4 blockers) could prevent the shift toward pathological aggregation in skin and possibly other tissues where HA turnover is altered, opening a new avenue for ameliorating age‑associated proteotoxic diseases.