Targeting Proteasome Complex in the CNS: Subunit-Specific Inhibition as a Promising Strategy for Novel Neuropathic Pain Therapies

Background and Purpose: Proteasomes are essential regulators of proteostasis and immune signaling under inflammatory conditions, including chronic pain. However, the contribution of individual catalytic subunits to neuroinflammation and microglial adaptation remains poorly understood. Here we investigated the abundance and activity of constitutive proteasome (c20s) and immunoproteasome (i20s) in neuroinflammatory conditions. Experimental Approach: We examined how selective inhibition of proteasome catalytic subunits influences the expression and activity of both constitutive and immunoproteasome subunits, or cellular viability, in LPS-stimulated human microglial cells. Transcript levels were assessed via qPCR, protein expression via western blotting and proteasomal activity was profiled using activity-based probes (ABP). Using a chronic constriction injury (CCI) mice model, we conducted transcript and protein level analyses of β-subunits in distinct brain regions associated with chronic pain conditions. Key Results: CCI induced region‑specific remodeling of proteasomal subunits, with the ipsilateral spinal cord showing the most pronounced shift toward immunoproteasome subunits at both mRNA and protein levels. A similar i20s‑biased transcript pattern was observed in the prefrontal cortex. In microglia, LPS robustly upregulated i20s subunit transcripts without proportional changes in protein levels, whereas inhibition of β1 selectively drove broad reorganization of catalytic subunit expression. ABP profiling revealed subunit‑specific changes in catalytic activity that did not consistently mirror mRNA or protein abundance. Dual targeting of constitutive and immunoproteasome isoforms reduced microglial viability, with β2/MECL‑1 and β5/LMP7 inhibition being more cytotoxic than β1 inhibition under inflammatory conditions and associated with a trend toward reduced pyroptosis‑related signaling. Conclusion and Implications: Our data show that chronic nerve injury and inflammatory activation of microglia engage a subunit-specific reorganization of c20s/i20s composition and activity that is not captured by transcriptomics alone. These findings support integrated, activity-centric analysis of proteasome subunits and suggest that subunit- and isoform-selective modulation of the immunoproteasome, rather than non-selective proteasome inhibition, may offer a more refined strategy for targeting neuroinflammatory mechanisms in chronic pain.