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

Abstract

Introduction: Loss of proteasome function, proteinopathy, and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology.

Methods: Using pigs, a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex, we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes.

Results: By Western blotting, proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus, PSMB5 immunoreactivity was diffuse, particulate, and clustered, including perinucleolar decorations. By fluorogenic assay, proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex, proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15-20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay, 26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay, MG132-inhibitable proteasome CTL activity was localized to the neuropil, as a mosaic, and to cell bodies, nuclei, and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h, brain proteasome CTL activity was modestly increased.

Discussion: This study shows that the proteasome in the pig brain has relative regional uniformity, prominent nuclear and perinuclear presence with catalytic activity, a mitochondrial association with activity, 26S-single cap dominance, and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable.

Keywords: aging; chlorpromazine; encephalopathy; neonatal brain injury; proteasome nuclear satellite; protein aggregation; proteinopathy; pyrazolone.

Figure 1

Piglet brain regions that were microdissected and used for proteasome Western blotting and biochemical or histochemical activity assays. Piglets were euthanized with Somnasol and then quickly perfused intra-aortically with ice-cold PBS for complete exsanguination. The fresh brains were quickly removed from the skull and kept ice-cold during microdissection on a chilled plate. The left hemisphere is shown in slabs from anterior (far left, frontal lobe) to posterior (far right, the occipital lobe was inadvertently rotated). The brainstem and cerebellum are shown at the bottom. The regions removed are identified. The identifications of the different areas of the neocortex are described (Primiani et al., 2023). Column micropunches of different white matter regions were taken as described (Lee et al., 2021): (blue, parietal subcortical white matter; yellow, corpus callosum; red, internal capsule). Microdissected brain samples were placed in Eppendorf tubes and snap frozen in isopentane (70°C). An additional sample of parietal somatosensory cortex was snap frozen as a small tissue block for cryostat sectioning and in situ proteasome activity assay.

Figure 2

Proteasome 20S subunit PSMB5 in the piglet brain. (A) Western blots show that the brain regional levels of PSMB5 (detected at ~25 kDa) are relatively uniform among regions. The abundance of PSMB5 in the nasal mucosa biopsy tissue of piglets is like the different brain regions. Lanes represent regional samples from two different pigs. On the left, the 25 kDa molecular weight standard (MWS) is identified. (B–H) Immunohistochemical localization of PSMB5. Immunoreactivity is seen as brown staining with a blue Nissl substance (basophilic) counterstain. In primary somatosensory cortex (B) and motor cortex (C), PSMB5 is ubiquitously present in patterns that define the horizontal neuronal layering in cerebral cortex (1–6) and vertical translaminar arrangements of neurons in somatosensory cortex (B, yellow lines). Essentially, all cortical neurons were PSMB5-positive (B,C). A large layer 5 neuron is delineated (D, hatched yellow lines). PSMB5 is enriched in the nucleus (N), while the core of the nucleolus is negative (D, open arrow). Particulate immunoreactivity is also present in the cytoplasm and in the neuropil. (E) Some neurons have a discrete perinuclear inclusion (~2 μm in diameter) that is positive for PSMB5 (yellow arrows). (F) Essentially, all hippocampal pyramidal neurons were strongly positive for PSMB5. (G) Neurons in the caudate nucleus were essentially all PSMB5 positive, but capillary endothelial cells (asterisk) and subsets of glial cells (open arrows) were not positive for PSMB5. The neuropil is positive for PSMB5. (H) In the subcortical white matter of the parietal cortex, glial cells can be PSMB5-positive (black arrows) or -negative (open arrows). Scale bars (in μm): (B) (same for C), 40; (D) 7; (E) 4; (F) (same for G,H), 12. (I) Western blots show that the levels of PSMA3 (detected at ~25 kDa) are relatively uniform among regions of cerebral cortex and white matter in pig brains. Lanes represent regional samples from two different pigs.

Figure 3

Diagram of the experimental protocol used for intravenous (iv) drug treatment of piglets. Piglets (2–3 days old, 1.5–2.0 kg, male) were sedated, intubated, and anesthetized for cardiovascular and hemodynamic monitoring before, during, and after iv administration of vehicle or a cocktail of pyrazolone (PYR) and chlorpromazine (CPZ). The piglets received multiple iv drug doses and were then euthanized for brain harvesting 24 h after the first dose (the group sizes are shown at right). Brain regional microdissections were taken (see Figure 1) and used for proteasome assays. Seasonal- and age-matched naïve piglets (N = 9) did not receive surgical anesthesia or any iv treatments and were euthanized with SomnaSol for immediate brain harvesting and microdissection. Animals and protocols used for other experiments (e.g., immunohistochemistry) are described specifically in the Materials and Methods.

Figure 4

Subcellular localization of the proteasome seen by confocal microscopy and brain tissue fractionation. (A) Confocal microscope image shows PSMB5 (red) and voltage-dependent anion channel (VDAC, green) as a mitochondrial marker. Some mitochondria are green only, and some are yellow (corresponding to mitochondria-proteasome colocalization). Other mitochondria are seen with proteasome immunoreactivity associated with the outer surface (a discrete green profile with closely associated red particles on the surface). Colocalization of proteasomes with mitochondria (yellow) is seen within the neuropil, perhaps corresponding to synaptic mitochondria proteasomes. (B) Confocal microscope image shows PSMB5 (red) and cyclophilin D (CyPD, green) as a mitochondrial marker. (C) Colocalization graph for VDAC-positive mitochondria located in the neuropil and PSMB5. Box plots show mean values with IQR and 5–95th percentile whiskers. (D) Colocalization graph for VDAC-positive mitochondria located in the neuronal perikaryal cytoplasm and PSMB5. Box plots show mean values with IQR and 5–95th percentile whiskers. (E) A representative nitrocellulose membrane with transferred proteins after SDS-PAGE and Ponceau S staining shows the exquisitely resolved protein banding patterns in different subcellular fractions resulting from Percoll ultracentrifugation of piglet somatosensory cortex homogenates. Somatosensory cortex was homogenized (Hom) and then fractionated into the following subcellular compartments: nuclear (Nuc), cytosolic (Cyto), crude mitochondria (Mc), pure mitochondria (Mp), mitochondrial-associated membranes-mitochondrial pellet (MAM-m), and pure mitochondrial-associated membranes (MAM). At left, the molecular weight kDa standards (Precision Plus Protein Dual Color Standards, Bio-Rad) are identified. (F) Western botting was done to characterize the fractions with antibodies to inositol triphosphate receptor (IP3R), cytochrome P450 reductase, mitochondrial complex V (ATP synthase), glyceraldehyde phosphate dehydrogenase (GAPDH), and histone H3. PSMB5 was detected in all fractions, including the pure mitochondrial fraction. Purified human P20S was a positive control.

Figure 5

Proteasome CTL activity in subcellular fractions of the somatosensory cortex of naïve piglets. (A) Brain tissue from two different piglets was subjected to subcellular fractionation and assayed for proteasome CTL activity in the different fractions. The UBPBio fluorogenic assay was done with Suc-LLVY-AMC as the substrate. Each line (black or red) represents a single piglet brain sample. (B) Mean of the proteasome CTL activities in the different subcellular fractions (n = 2 different pigs).

Figure 6

Proteasome CTL activity in crude homogenates of different brain regions of naïve piglets. (A) Proteasome fluorogenic assay shows reaction progress curve raw fluorescence measurements of AMC released from Suc-LLVY-AMC by chymotrypsin-like activity over 14 min in the somatosensory cortex of a single naïve piglet. To isolate the specific fluorescent signal produced by AMC liberation due to proteasome activity, MG132 was used as an inhibitor in separate duplicate control wells. (B) Boxplots of femtomoles (fmol) AMC released per minute by proteasome CTL activity in each brain region in naïve piglets (N = 9). The individual brain regions are: FC, frontal cortex; M, motor cortex; SC, somatosensory cortex; VC, visual cortex; HC, hippocampus; WM, subcortical white matter; CN, caudate nucleus; CC = cerebellar cortex. Each box plot shows the minimum/Q1/median/Q3/maximum values.

Figure 7

Kinetic analysis of proteasome CTL activity in different gray and white regions of the brain of naïve piglets (N = 4 or 5 for each brain region). (A–C) Relative AMC fluorescence at initial reaction time identified as t0 (A), peak fluorescence (B), and slope of the progress reaction curve (C) in hippocampus (HC), caudate nucleus (CN), primary somatosensory cortex (SC), frontal cortex (FC), and cerebellar cortex (CC). (D–F) Relative AMC fluorescence at t₀ (D), peak fluorescence (E), and slope of the progress reaction curve (F) in the internal capsule (IC), corpus callosum (CpC), and parietal subcortical white matter (SCWM). Box plots show mean values with IQR and 5–95th percentile whiskers.

Figure 8

Proteasome CTL activity reaction progress curve properties (fluorescence t₀, peak, slope) in different white and gray regions of sham piglet brains (N = 4 or 5) versus proteasome activity in nasal mucosa biopsy samples of the same piglets. (A–C) Scatterplots show the white matter regions of the internal capsule (IC), corpus callosum (CpC), and parietal subcortical white matter (SCWM). (D–F) Scatterplots show the gray matter regions of the hippocampus (HC), caudate nucleus (CN), primary somatosensory cortex (SC), frontal cortex (FC), and cerebellar cortex (CC).

Figure 9

Brain proteasome CTL activity in drug-treated piglets. (A) Box plots of femtomoles (fmol) of AMC released per minute by proteasome chymotrypsin-like activity in each brain region in the different piglet treatment groups identified. Each box plot shows the minimum/Q1/median/Q3/maximum values. The individual brain regions are: FC, frontal cortex; M, motor cortex; SC, somatosensory cortex; VC, visual cortex; HC, hippocampus; WM, parietal subcortical white matter; CN, caudate nucleus; CC, cerebellar cortex. Each box plot shows the minimum/Q1/median/Q3/maximum values. (B) Proteasome activity in brain regions grouped by functional relationships. Each box plot shows the minimum/Q1/median/Q3/maximum values. (C) Combined brain region holistic scatterplots of fmol AMC released per minute by proteasome chymotrypsin-like activity in each treatment group. Lines are medians. The significance was determined by two-way ANOVA (α = 0.05) of multiple comparisons between treatment groups.

Figure 10

In-gel proteasome CTL activity in piglet cerebral cortex. (A) Protease activity gel shows the presence of the 26S (singly capped) and 30S (26S-doubly capped) proteasomes in the piglet motor cortex. Purified proteasome 20S from human erythrocytes was used as a standard. (B) 26S and 30S proteasome activities in piglet cerebral cortex were inhibited by the standard proteasome inhibitor MG132 (left gel). Representative gel loading verification by Coomassie staining (right gel). (C) In-gel proteasome CTL in piglet cerebral cortex showed enhanced activity with PYR/CPZ treatment compared to vehicle. Enhancement of activity occurred with the 26S proteasome and as a smear of activity (bracket) above the 26S-singly capped.

Figure 11

Novel in situ histochemical detection of proteasome activity in piglet brain cryostat sections reveals new findings on proteasome localization. (A) Proteasome CTL activity (green) is enriched (but not uniformly) in the neocortical neuropil and in subsets of cells in the somatosensory cortex of piglet. Blue is the DAPI counterstaining of the nucleus. The white hatched box is shown at higher magnification in panel (B). (B) Large cells (solid white arrows), likely neurons, are enriched in proteasome CTL activity, but other neurons (open arrows; blue is DAPI staining of the large nucleus) appear without detectable activity. Some small cells (thin white arrows) have perinuclear inclusions with high proteasome CTL activity. The white hatched box is shown as the inset to better illustrate the proteasome-active perinuclear satellite. (C) MG132 treatment of sections completely inhibited the histochemical detection of proteasome activity (specific green reaction product) in the neuropil and in cell bodies (blue DAPI staining shows cell nuclei). (D) Some cells in the neocortex have proteasome activity focally within the nucleus (solid white arrow) with limited to no activity in the cytoplasm, while other cells appear completely enriched in proteasome activity (open white arrow). (E) A cell in the cerebral cortex highly enriched in proteasome activity. (F) Graph of the number of cells in cortical gray matter and subcortical white matter with perinuclear proteasome-active inclusions. Box plots show mean values with IQR and 5–95th percentile whiskers. Scale bars (in μm): (A) 65; (B) 32.5; (B) (inset) 4; (C) 3; (D) 8; (E) 4.