Oleuropein Activates Neonatal Neocortical Proteasomes, but Proteasome Gene Targeting by AAV9 Is Variable in a Clinically Relevant Piglet Model of Brain Hypoxia-Ischemia and Hypothermia

Abstract

Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically relevant neonatal piglet model. Protecting and activating proteasomes could be an adjunct therapy to hypothermia. We investigated whether chymotrypsin-like proteasome activity differs regionally and developmentally in the neonatal brain. We also tested whether neonatal brain proteasomes can be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by targeting a proteasome subunit gene using recombinant adeno-associated virus-9 (AAV). During post-HI hypothermia, we treated piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ using AAV-PA28γ with green fluorescent protein (GFP). Neonatal neocortex and subcortical white matter had greater proteasome activity than did liver and kidney. Neonatal white matter had higher proteasome activity than did juvenile white matter. Lower arterial pH 1 h after HI correlated with greater subsequent cortical proteasome activity. With increasing brain homogenate protein input into the assay, the initial proteasome activity increased only among shams, whereas HI increased total kinetic proteasome activity. OLE increased the initial neocortical proteasome activity after hypothermia. AAV drove GFP expression, and white matter PA28γ levels correlated with proteasome activity and subunit levels. However, AAV proteasome modulation varied. Thus, neonatal neocortical proteasomes can be pharmacologically activated. HI slows the initial proteasome performance, but then augments ongoing catalytic activity. AAV-mediated genetic manipulation in the piglet brain holds promise, though proteasome gene targeting requires further development.

Proteasome localization and activity in pig brain and in vivo small molecule screening for activators

“…Western blotting was done to determine relative protein levels of the β5 proteasome subunit (PSMB5) in different regions of the neonatal pig brain ( Figure 2A ). Immunoreactivity for the β5 proteasome subunit was detected at ~25 kDa, consistent with expectations from the work of others ( Sutovsky et al, 2004 ) and us ( Santos et al, 2018 ; El Demerdash et al, 2021 ). In precisely microdissected white matter regions ( Figure 1 ) such as parietal cortex subcortical white matter, corpus callosum, and internal capsule β5 levels were similar ( Figure 2A ).…”

“…The proteasome garners considerable attention in the cell biology of aging and neurodegeneration ( Davidson and Pickering, 2023 ). With proteasome loss of functioning as a putative key mechanism of nervous system disease and injury throughout the lifelong spectrum, proteasome enhancement strategies are now a focus of research and development (Tripper et al, 2014; Jones et al, 2017 ; Trader et al, 2017 ; Santoro et al, 2020 ; El Demerdash et al, 2021 ; George and Tepe, 2021 ). Small molecule activators offer hope for clinically relevant proteasome enhancement (Tripper et al, 2014; Jones et al, 2017 ; Santoro et al, 2020 ; George and Tepe, 2021 ); however, much work needs completion.…”

“…Protein oxidation evolves rapidly in concert with α-synuclein oligomerization in a neonatal mouse model of traumatic brain injury ( Martin et al, 2019 ). Aberrant brain proteasome localization and activity, increased protein oxidative damage (carbonyl accumulation), and elevated protein ubiquitination were discovered in neonatal hypoxic–ischemic piglets ( Santos et al, 2018 ; El Demerdash et al, 2021 ). These studies seed the idea that proteinopathy as a pathological mechanism in the nervous system is not limited to aging and neurodegenerative disease in adulthood ( Martin et al, 2019 , 2022 ); thus, the clinical need and application of targeting the proteasome for activation may broaden.…”

“…Experiments on the proteasome in pigs can satisfy important preclinical needs to advance the field of nervous system therapeutics for injury and disease. Prior studies suggest that species differences exist in the localization of proteasome subunits ( Adori et al, 2006 ) and the biochemical activities ( Sesma et al, 2003 ; Pride et al, 2015 ; El Demerdash et al, 2021 ). Information on proteasome localization and activity in the swine brain is limited ( Santos et al, 2018 ; El Demerdash et al, 2021 ).…”

“…Prior studies suggest that species differences exist in the localization of proteasome subunits ( Adori et al, 2006 ) and the biochemical activities ( Sesma et al, 2003 ; Pride et al, 2015 ; El Demerdash et al, 2021 ). Information on proteasome localization and activity in the swine brain is limited ( Santos et al, 2018 ; El Demerdash et al, 2021 ). It is important to examine proteasome localizations and activities in animal alternatives to rodents to better appreciate the attributes and relevance of models used for brain injury and disease, particularly those involving proteinopathy, and to develop proteasome-targeted therapeutics.…”