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

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.

Hypothermic Protection in Neocortex Is Topographic and Laminar, Seizure Unmitigating, and Partially Rescues Neurons Depleted of RNA Splicing Protein Rbfox3/NeuN in Neonatal Hypoxic-Ischemic Male Piglets.

The effects of hypothermia on neonatal encephalopathy may vary topographically and cytopathologically in the neocortex with manifestations potentially influenced by seizures that alter the severity, distribution, and type of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia (HT) with continuous electroencephalography (cEEG) for seizures. Neonatal male piglets received HI-normothermia (NT), HI-HT, sham-NT, or sham-HT treatments. Randomized unmedicated sham and HI piglets underwent cEEG during recovery. Survival was 2-7 days. Normal and pathological neurons were counted in different neocortical areas, identified by cytoarchitecture and connectomics, using hematoxylin and eosin staining and immunohistochemistry for RNA-binding FOX-1 homolog 3 (Rbfox3/NeuN). Seizure burden was determined. HI-NT piglets had a reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to sham-NT and sham-HT piglets with differing severities in the anterior and posterior motor, somatosensory, and frontal cortices. Neocortical neuropathology was attenuated by HT. HT protection was prominent in layer III of the inferior parietal cortex. Rbfox3 immunoreactivity distinguished cortical neurons as: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had an increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT piglets. Neuronal Rbfox3 depletion was partly rescued by HT. Seizure burdens in HI-NT and HI-HT piglets were similar. We conclude that the neonatal HI piglet neocortex has: (1) suprasylvian vulnerability to HI and seizures; (2) a limited neuronal cytopathological repertoire in functionally different regions that engages protective mechanisms with HT; (3) higher seizure burden, insensitive to HT, that is correlated with more panlaminar ischemic-necrotic neurons in the somatosensory cortex; and (4) pathological RNA splicing protein nuclear depletion that is sensitive to HT. This work demonstrates that HT protection of the neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores neuronal depletion of RNA splicing factor.

Plasma Biomarkers of Evolving Encephalopathy and Brain Injury in Neonates with Hypoxic-Ischemic Encephalopathy.

OBJECTIVE: The objective of the study was to evaluate the relationship between a panel of candidate plasma biomarkers and (1) death or severe brain injury on magnetic resonance imaging (MRI) and (2) dysfunctional cerebral pressure autoregulation as a measure of evolving encephalopathy. STUDY DESIGN: Neonates with moderate-to-severe hypoxic-ischemic encephalopathy (HIE) at 2 level IV neonatal intensive care units were enrolled into this observational study. Patients were treated with therapeutic hypothermia (TH) and monitored with continuous blood pressure monitoring and near-infrared spectroscopy. Cerebral pressure autoregulation was measured by the hemoglobin volume phase (HVP) index; a higher HVP index indicates poorer autoregulation. Serial blood samples were collected during TH and assayed for Tau, glial fibrillary acidic protein, and neurogranin. MRIs were assessed using National Institutes of Child Health and Human Development scores. The relationships between the candidate biomarkers and (1) death or severe brain injury on MRI (defined as a National Institutes of Child Health and Human Development score of ≥ 2B) and (2) autoregulation were evaluated using bivariate and adjusted logistic regression models. RESULTS: Sixty-two patients were included. Elevated Tau levels on days 2-3 of TH were associated with death or severe injury on MRI (aOR: 1.06, 95% CI: 1.03-1.09; aOR: 1.04, 95% CI: 1.01-1.06, respectively). Higher Tau was also associated with poorer autoregulation (higher HVP index) on the same day (P = .022). CONCLUSIONS: Elevated plasma levels of Tau are associated with death or severe brain injury by MRI and dysfunctional cerebral autoregulation in neonates with HIE. Larger-scale validation of Tau as a biomarker of brain injury in neonates with HIE is warranted.

The Utility of Cerebral Autoregulation Indices in Detecting Severe Brain Injury Varies by Cooling Treatment Phase in Neonates with Hypoxic-Ischemic Encephalopathy.

Identifying the hemodynamic range that best supports cerebral perfusion using near infrared spectroscopy (NIRS) autoregulation monitoring is a potential physiologic marker for neonatal hypoxic-ischemic encephalopathy (HIE) during therapeutic hypothermia. However, an optimal autoregulation monitoring algorithm has not been identified for neonatal clinical medicine. We tested whether the hemoglobin volume phase (HVP), hemoglobin volume (HVx), and pressure passivity index (PPI) identify changes in autoregulation that are associated with brain injury on MRI or death. The HVP measures the phase difference between a NIRS metric of cerebral blood volume, the total hemoglobin (THb), and mean arterial blood pressure (MAP) at the frequency of maximum coherence. The HVx is the correlation coefficient between MAP and THb. The PPI is the percentage of coherent MAP-DHb (difference between oxygenated and deoxygenated hemoglobin, a marker of cerebral blood flow) epochs in a chosen time period. Neonates cooled for HIE were prospectively enrolled in an observational study in two neonatal intensive care units. In analyses adjusted for study site and encephalopathy level, all indices detected relationships between poor autoregulation in the first 6 h after rewarming with a higher injury score on MRI. Only HVx and PPI during hypothermia and the PPI during rewarming identified autoregulatory dysfunction associated with a poor outcome independent of study site and encephalopathy level. Our findings suggest that the accuracy of mathematical autoregulation algorithms in detecting the risk of brain injury or death may depend on temperature and postnatal age. Extending autoregulation monitoring beyond the standard 72 h of therapeutic hypothermia may serve as a method to provide personalized care by assessing the need for and efficacy of future therapies after the hypothermia treatment phase.

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.

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.

Wavelet Autoregulation Monitoring Identifies Blood Pressures Associated With Brain Injury in Neonatal Hypoxic-Ischemic Encephalopathy.

Dysfunctional cerebrovascular autoregulation may contribute to neurologic injury in neonatal hypoxic-ischemic encephalopathy (HIE). Identifying the optimal mean arterial blood pressure (MAPopt) that best supports autoregulation could help identify hemodynamic goals that support neurologic recovery. In neonates who received therapeutic hypothermia for HIE, we hypothesized that the wavelet hemoglobin volume index (wHVx) would identify MAPopt and that blood pressures closer to MAPopt would be associated with less brain injury on MRI. We also tested a correlation-derived hemoglobin volume index (HVx) and single- and multi-window data processing methodology. Autoregulation was monitored in consecutive 3-h periods using near infrared spectroscopy in an observational study. The neonates had a mean MAP of 54 mmHg (standard deviation: 9) during hypothermia. Greater blood pressure above the MAPopt from single-window wHVx was associated with less injury in the paracentral gyri (p = 0.044; n = 63), basal ganglia (p = 0.015), thalamus (p = 0.013), and brainstem (p = 0.041) after adjustments for sex, vasopressor use, seizures, arterial carbon dioxide level, and a perinatal insult score. Blood pressure exceeding MAPopt from the multi-window, correlation HVx was associated with less injury in the brainstem (p = 0.021) but not in other brain regions. We conclude that applying wavelet methodology to short autoregulation monitoring periods may improve the identification of MAPopt values that are associated with brain injury. Having blood pressure above MAPopt with an upper MAP of ~50-60 mmHg may reduce the risk of brain injury during therapeutic hypothermia. Though a cause-and-effect relationship cannot be inferred, the data support the need for randomized studies of autoregulation and brain injury in neonates with HIE.

Targeting the mitochondrial permeability transition pore for neuroprotection in a piglet model of neonatal hypoxic-ischemic encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes significant morbidity despite treatment with therapeutic hypothermia. Mitochondrial dysfunction may drive the mechanisms underlying neuronal cell death, thereby making mitochondria prime targets for neuroprotection. The mitochondrial permeability transition pore (mPTP) is one such target within mitochondria. In adult animal models, mPTP inhibition is neuroprotective. However, evidence for mPTP inhibition in neonatal models of neurologic disease is less certain. We tested the therapeutic efficacy of the mPTP small molecule inhibitor GNX-4728 and examined the developmental presence of brain mPTP proteins for drug targeting in a neonatal piglet model of hypoxic-ischemic brain injury. Male neonatal piglets were randomized to hypoxia-ischemia (HI) or sham procedure with GNX-4728 (15 mg/kg, IV) or vehicle (saline/cyclodextrin/DMSO, IV). GNX-4728 was administered as a single dose within 5 min after resuscitation from bradycardic arrest. Normal, ischemic, and injured neurons were counted in putamen and somatosensory cortex using hematoxylin and eosin staining. In separate neonatal and juvenile pigs, western blots of putamen mitochondrial-enriched fractions were used to evaluate mitochondrial integrity and the presence of mPTP proteins. We found that a single dose of GNX-4728 did not protect putamen and cortical neurons from cell death after HI. However, loss of mitochondrial matrix integrity occurred within 6h after HI, and while mPTP components are present in the neonatal brain their levels were significantly different compared to that of a mature juvenile brain. Thus, the neonatal brain mPTP may not be a good target for current neurotherapeutic drugs that are developed based on adult mitochondria.

Combining Hypothermia and Oleuropein Subacutely Protects Subcortical White Matter in a Swine Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxia-ischemia (HI) causes white matter injury that is not fully prevented by therapeutic hypothermia. Adjuvant treatments are needed. We compared myelination in different piglet white matter regions. We then tested whether oleuropein (OLE) improves neuroprotection in 2- to 4-day-old piglets randomized to undergo HI or sham procedure and OLE or vehicle administration beginning at 15 minutes. All groups received overnight hypothermia and rewarming. Injury in the subcortical white matter, corpus callosum, internal capsule, putamen, and motor cortex gray matter was assessed 1 day later. At baseline, piglets had greater subcortical myelination than in corpus callosum. Hypothermic HI piglets had scant injury in putamen and cerebral cortex. However, hypothermia alone did not prevent the loss of subcortical myelinating oligodendrocytes or the reduction in subcortical myelin density after HI. Combining OLE with hypothermia improved post-HI subcortical white matter protection by preserving myelinating oligodendrocytes, myelin density, and oligodendrocyte markers. Corpus callosum and internal capsule showed little HI injury after hypothermia, and OLE accordingly had minimal effect. OLE did not affect putamen or motor cortex neuron counts. Thus, OLE combined with hypothermia protected subcortical white matter after HI. As an adjuvant to hypothermia, OLE may subacutely improve regional white matter protection after HI.

Evaluating congenital syphilis in a reverse sequence testing environment.

OBJECTIVES: To examine the effect of maternal reverse-sequence (RS) syphilis screening on management of infants at risk for congenital syphilis (CS) using a standardized approach. STUDY DESIGN: A retrospective study from 2011 to 2014 at an academic medical center using RS testing, involving chemiluminescent immunoassay (CIA), rapid plasma  reagin (RPR), and fluorescent treponemal antibody-absorption (FTA-ABS) assays for syphilis. Clinical management and outcomes of infants born to mothers with discordant (CIA+/RPR-/FTA+) serology were compared with national or internal guidelines. RESULTS: Sixty-three infants were classified as discordant (n = 21), presumed false positive (CIA+/RPR-/FTA-; n = 16), or true positive (CIA+/RPR+; n = 26) based on maternal serology. Only 24% of cases in the discordant group underwent recommended full evaluation. None of the evaluated infants in the discordant group (n = 8) were diagnosed with CS. CONCLUSIONS: Management of infants with discordant maternal RS serology remained reliant on clinical judgment. In our high-risk population, RS testing did not identify additional cases of CS.

Proteasome Biology Is Compromised in White Matter After Asphyxic Cardiac Arrest in Neonatal Piglets.

Background Neurological deficits in hypoxic-ischemic encephalopathy, even with therapeutic hypothermia, are partially attributed to white matter injury. We theorized that proteasome insufficiency contributes to white matter injury. Methods and Results Neonatal piglets received hypoxia-ischemia ( HI ) or sham procedure with normothermia, hypothermia, or hypothermia+rewarming. Some received a proteasome activator drug (oleuropein) or white matter-targeted, virus-mediated proteasome knockdown. We measured myelin oligodendrocyte glycoprotein, proteasome subunit 20S (P20S), proteasome activity, and carbonylated and ubiquitinated protein levels in white matter and cerebral cortex. HI reduced myelin oligodendrocyte glycoprotein levels regardless of temperature, and myelin oligodendrocyte glycoprotein loss was associated with increased ubiquitinated and carbonylated protein levels. Ubiquitinated and carbonyl-damaged proteins increased in white matter 29 hours after HI during hypothermia to exceed levels at 6 to 20 hours. In cortex, ubiquitinated proteins decreased. Ubiquitinated and carbonylated protein accumulation coincided with lower P20S levels in white matter; P20S levels also decreased in cerebral cortex. However, proteasome activity in white matter lagged behind that in cortex 29 hours after HI during hypothermia. Systemic oleuropein enhanced white matter P20S and protected the myelin, whereas proteasome knockdown exacerbated myelin oligodendrocyte glycoprotein loss and ubiquitinated protein accumulation after HI . At the cellular level, temperature and HI interactively affected macroglial P20S enrichment in subcortical white matter. Rewarming alone increased macroglial P20S immunoreactivity, but this increase was blocked by HI . Conclusions Oxidized and ubiquitinated proteins accumulate with HI -induced white matter injury. Proteasome insufficiency may drive this injury. Hypothermia did not prevent myelin damage, protect the proteasome, or preserve oxidized and ubiquitinated protein clearance after HI .

Abdominal near-infrared spectroscopy in a piglet model of gastrointestinal hypoxia produced by graded hypoxia or superior mesenteric artery ligation.

BackgroundAbdominal near-infrared spectroscopy (aNIRS) may detect gastrointestinal hypoxia before necrotizing enterocolitis develops. We sought to validate aNIRS during splanchnic hypoxia and hypoperfusion in neonatal piglets.MethodsAnesthetized piglets underwent systemic hypoxia or 3 h superior mesenteric artery (SMA) ligation with aNIRS monitoring.ResultsDuring progressive hypoxia, gastrointestinal tissue oxyhemoglobin saturation measured by aNIRS decreased linearly with oxyhemoglobin saturation measured directly in the portal vein. Correlation coefficients were 0.94-0.99 in each of 10 piglets, the average regression slope of 0.73 (95% confidence interval: 0.57, 0.89) differed from one (P<0.004), and the intercept on the aNIRS axis of 9.5% (4.4, 14.6) differed from zero (P<0.0025). Umbilical venous oxyhemoglobin saturation also correlated strongly with the portal vein oxyhemoglobin saturation (r=0.83-0.99), with a slope not different from one. SMA ligation caused ileal blood flow to decrease by ~50%, and produced a sustained decrease in aNIRS oximetry from approximately 60 to 30%.ConclusionaNIRS can detect abrupt and sustained gastrointestinal hypoperfusion associated with arterial occlusion in a neonatal model. The highly linear relationship of portal venous oxyhemoglobin saturation with aNIRS and umbilical vein saturation during graded hypoxia implies that these measures can accurately track tissue oxygenation trends over a wide range in individual subjects.