Interorganelle communication, aging, and neurodegeneration.

Our cells are comprised of billions of proteins, lipids, and other small molecules packed into their respective subcellular organelles, with the daunting task of maintaining cellular homeostasis over a lifetime. However, it is becoming increasingly evident that organelles do not act as autonomous discrete units but rather as interconnected hubs that engage in extensive communication through membrane contacts. In the last few years, our understanding of how these contacts coordinate organelle function has redefined our view of the cell. This review aims to present novel findings on the cellular interorganelle communication network and how its dysfunction may contribute to aging and neurodegeneration. The consequences of disturbed interorganellar communication are intimately linked with age-related pathologies. Given that both aging and neurodegenerative diseases are characterized by the concomitant failure of multiple cellular pathways, coordination of organelle communication and function could represent an emerging regulatory mechanism critical for long-term cellular homeostasis. We anticipate that defining the relationships between interorganelle communication, aging, and neurodegeneration will open new avenues for therapeutics.

Ventilator Weaning and Terminal Extubation: Withdrawal of Life-Sustaining Therapy in Children. Secondary Analysis of the Death One Hour After Terminal Extubation Study.

OBJECTIVE: Terminal extubation (TE) and terminal weaning (TW) during withdrawal of life-sustaining therapies (WLSTs) have been described and defined in adults. The recent Death One Hour After Terminal Extubation study aimed to validate a model developed to predict whether a child would die within 1 hour after discontinuation of mechanical ventilation for WLST. Although TW has not been described in children, pre-extubation weaning has been known to occur before WLST, though to what extent is unknown. In this preplanned secondary analysis, we aim to describe/define TE and pre-extubation weaning (PW) in children and compare characteristics of patients who had ventilatory support decreased before WLST with those who did not. DESIGN: Secondary analysis of multicenter retrospective cohort study. SETTING: Ten PICUs in the United States between 2009 and 2021. PATIENTS: Nine hundred thirteen patients 0-21 years old who died after WLST. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: 71.4% ( n = 652) had TE without decrease in ventilatory support in the 6 hours prior. TE without decrease in ventilatory support in the 6 hours prior = 71.4% ( n = 652) of our sample. Clinically relevant decrease in ventilatory support before WLST = 11% ( n = 100), and 17.6% ( n = 161) had likely incidental decrease in ventilatory support before WLST. Relevant ventilator parameters decreased were F io2 and/or ventilator set rates. There were no significant differences in any of the other evaluated patient characteristics between groups (weight, body mass index, unit type, primary diagnostic category, presence of coma, time to death after WLST, analgosedative requirements, postextubation respiratory support modality). CONCLUSIONS: Decreasing ventilatory support before WLST with extubation in children does occur. This practice was not associated with significant differences in palliative analgosedation doses or time to death after extubation.

Neurologic effects of short-term treatment with a soluble epoxide hydrolase inhibitor after cardiac arrest in pediatric swine.

BACKGROUND: Cardiac arrest (CA) is the most common cause of acute neurologic insult in children. Many survivors have significant neurocognitive deficits at 1 year of recovery. Epoxyeicosatrienoic acids (EETs) are multifunctional endogenous lipid signaling molecules that are involved in brain pathobiology and may be therapeutically relevant. However, EETs are rapidly metabolized to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH), limiting their bioavailability. We hypothesized that sEH inhibition would improve outcomes after CA in an infant swine model. Male piglets (3-4 kg, 2 weeks old) underwent hypoxic-asphyxic CA. After resuscitation, they were randomized to intravenous treatment with an sEH inhibitor (TPPU, 1 mg/kg; n = 8) or vehicle (10% poly(ethylene glycol); n = 9) administered at 30 min and 24 h after return of spontaneous circulation. Two sham-operated groups received either TPPU (n = 9) or vehicle (n = 8). Neurons were counted in hematoxylin- and eosin-stained sections from putamen and motor cortex in 4-day survivors. RESULTS: Piglets in the CA + vehicle groups had fewer neurons than sham animals in both putamen and motor cortex. However, the number of neurons after CA did not differ between vehicle- and TPPU-treated groups in either anatomic area. Further, 20% of putamen neurons in the Sham + TPPU group had abnormal morphology, with cell body attrition and nuclear condensation. TPPU treatment also did not reduce neurologic deficits. CONCLUSION: Treatment with an sEH inhibitor at 30 min and 24 h after resuscitation from asphyxic CA does not protect neurons or improve acute neurologic outcomes in piglets.

The use of pressure-controlled mechanical ventilation in a swine model of intraoperative pediatric cardiac arrest.

BACKGROUND: Current pediatric resuscitation guidelines suggest that resuscitators using an advanced airway deliver 8-10 breaths per minute while carefully avoiding excessive ventilation. In the intraoperative setting, having a dedicated ventilation rescuer may be difficult because of limited personnel. Continuing pressure-controlled mechanical ventilation during resuscitation for intraoperative cardiac arrest reduces personnel needed and the risk of hyperventilation but might risk hypoventilation during chest compression delivery. AIMS: To determine whether the use of pressure-controlled mechanical ventilation at prearrest settings provides normoxia and normocarbia during resuscitation from cardiac arrest. METHODS: We retrospectively analyzed combined data from preclinical randomized controlled trials. Two-week-old swine (3-4 kg) underwent asphyxia-induced cardiac arrest. Animals were resuscitated with periods of basic and advanced life support. During resuscitation, pressure-controlled mechanical ventilation was delivered at the prearrest respiratory rate, peak inspiratory pressure, and positive end-expiratory pressure. Arterial blood gases were measured prearrest, at 11 minutes of asphyxia, and at 8 and 20 minutes of cardiopulmonary resuscitation. RESULTS: Piglets (n = 154) received pressure-controlled mechanical ventilation before and during cardiopulmonary resuscitation with a peak inspiratory pressure of 14-15 cm H2 O, positive end-expiratory pressure of 4 cm H2 O, 20 breaths/minute, and an inspiratory:expiratory ratio of 1:2. During asphyxia, the arterial blood gas showed the expected severe hypercarbia and hypoxia. Continuing pressure-controlled mechanical ventilation using prearrest parameters and increasing the FiO2 to 1.0 returned the PaCO2 to prearrest levels and slightly increased the partial pressure of arterial oxygen at 8 and 20 minutes of cardiopulmonary resuscitation. CONCLUSION: In this piglet model of resuscitation from asphyxial arrest, pressure-controlled mechanical ventilation during cardiopulmonary resuscitation at the prearrest ventilator settings with an FiO2 of 1.0 provides adequate oxygenation and restores normocarbia. Clinical investigation is warranted to determine the benefits of continuing pressure-controlled mechanical ventilation at prearrest parameters during pediatric cardiopulmonary resuscitation.

Hypoxia-Ischemia and Hypothermia Independently and Interactively Affect Neuronal Pathology in Neonatal Piglets with Short-Term Recovery.

Therapeutic hypothermia is the standard of clinical care for moderate neonatal hypoxic-ischemic encephalopathy. We investigated the independent and interactive effects of hypoxia-ischemia (HI) and temperature on neuronal survival and injury in basal ganglia and cerebral cortex in neonatal piglets. Male piglets were randomized to receive HI injury or sham procedure followed by 29 h of normothermia, sustained hypothermia induced at 2 h, or hypothermia with rewarming during fentanyl-nitrous oxide anesthesia. Viable and injured neurons and apoptotic profiles were counted in the anterior putamen, posterior putamen, and motor cortex at 29 h after HI injury or sham procedure. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) identified genomic DNA fragmentation to confirm cell death. Though hypothermia after HI preserved viable neurons in the anterior and posterior putamen, hypothermia prevented neuronal injury in only the anterior putamen. Hypothermia initiated 2 h after injury did not protect against apoptotic cell death in either the putamen or motor cortex, and rewarming from hypothermia was associated with increased apoptosis in the motor cortex. In non-HI shams, sustained hypothermia during anesthesia was associated with neuronal injury and corresponding viable neuron loss in the anterior putamen and motor cortex. TUNEL confirmed increased neurodegeneration in the putamen of hypothermic shams. Anesthetized, normothermic shams did not show abnormal neuronal cytopathology in the putamen or motor cortex, thereby demonstrating minimal contribution of the anesthetic regimen to neuronal injury during normothermia. We conclude that the efficacy of hypothermic protection after HI is region specific and that hypothermia during anesthesia in the absence of HI may be associated with neuronal injury in the developing brain. Studies examining the potential interactions between hypothermia and anesthesia, as well as with longer durations of hypothermia, are needed.

The Effect of Asphyxia Arrest Duration on a Pediatric End-Tidal CO2-Guided Chest Compression Delivery Model.

OBJECTIVES: To determine the effect of the duration of asphyxial arrest on the survival benefit previously seen with end-tidal CO2-guided chest compression delivery. DESIGN: Preclinical randomized controlled study. SETTING: University animal research laboratory. SUBJECTS: Two-week-old swine. INTERVENTIONS: After either 17 or 23 minutes of asphyxial arrest, animals were randomized to standard cardiopulmonary resuscitation or end-tidal CO2-guided chest compression delivery. Standard cardiopulmonary resuscitation was optimized by marker, monitor, and verbal feedback about compression rate, depth, and release. End-tidal CO2-guided delivery used adjustments to chest compression rate and depth to maximize end-tidal CO2 level without other feedback. Cardiopulmonary resuscitation for both groups proceeded from 10 minutes of basic life support to 10 minutes of advanced life support or return of spontaneous circulation. MEASUREMENTS AND MAIN RESULTS: After 17 minutes of asphyxial arrest, mean end-tidal CO2 during 10 minutes of cardiopulmonary resuscitation was 18 ± 9 torr in the standard group and 33 ± 15 torr in the end-tidal CO2 group (p = 0.004). The rate of return of spontaneous circulation was three of 14 (21%) in the standard group rate and nine of 14 (64%) in the end-tidal CO2 group (p = 0.05). After a 23-minute asphyxial arrest, neither end-tidal CO2 values (20 vs 26) nor return of spontaneous circulation rate (3/14 vs 1/14) differed between the standard and end-tidal CO2-guided groups. CONCLUSIONS: Our previously observed survival benefit of end-tidal CO2-guided chest compression delivery after 20 minutes of asphyxial arrest was confirmed after 17 minutes of asphyxial arrest. The poor survival after 23 minutes of asphyxia shows that the benefit of end-tidal CO2-guided chest compression delivery is limited by severe asphyxia duration.

Non-rheumatic streptococcal myocarditis mimicking acute myocardial infarction in an adolescent male.

An adolescent male with a recent history of streptococcal pharyngitis presented with severe substernal chest pain, troponin leak, and ST-segment elevation, which are suggestive of acute inferolateral myocardial infarction. The coronary angiogram was normal. The patient was subsequently diagnosed with non-rheumatic streptococcal myocarditis. He was treated with amoxicillin and had excellent recovery. Non-rheumatic streptococcal myocarditis is an important mimic of acute myocardial infarction in young adults.

Behavioral development in embryonic and early juvenile cuttlefish (Sepia officinalis).

Though a mollusc, the cuttlefish Sepia officinalis possesses a sophisticated brain, advanced sensory systems, and a large behavioral repertoire. Cuttlefish provide a unique perspective on animal behavior due to their phylogenic distance from more traditional (vertebrate) models. S. officinalis is well-suited to addressing questions of behavioral ontogeny. As embryos, they can perceive and learn from their environment and experience no direct parental care. A marked progression in learning and behavior is observed during late embryonic and early juvenile development. This improvement is concomitant with expansion and maturation of the vertical lobe, the cephalopod analog of the mammalian hippocampus. This review synthesizes existing knowledge regarding embryonic and juvenile development in this species in an effort to better understand cuttlefish behavior and animal behavior in general. It will serve as a guide to future researchers and encourage greater awareness of the utility of this species to behavioral science.

The GARP complex prevents sterol accumulation at the trans-Golgi network during dendrite remodeling.

Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, we find that both complexes are required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, we investigated genes that regulate sterols and related lipids at the TGN. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.

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.

Association of end-tidal carbon dioxide levels during cardiopulmonary resuscitation with survival in a large paediatric cohort.

AIM: To examine the associations between ETCO2, ROSC, and chest compression quality markers in paediatric patients during active resuscitation. METHODS: This was a single-centre cohort study of data collected as part of an institutional prospective quality initiative improvement program that included all paediatric patients who received chest compressions of any duration from January 1, 2013, through July 10, 2018, in the Johns Hopkins Children's Center. Data was collected from Zoll R Series® defibrillators. Events were included if Zoll data files contained both chest compression and ETCO2 data. 2,746 minutes corresponding to 143 events were included in the analyses. RESULTS: The median event ETCO2 for all 143 events was 16.8 [9.3-26.3] mmHg. There was a significant difference in median event ETCO2 between events that achieved ROSC and those that did not (ROSC: 19.3 [14.4-26.6] vs. NO ROSC: 13.9 [6.6-25.5] mmHg; p < 0.05). When the events were based on patient age, this relationship held in adolescents (ROSC: 18.8 [15.5-22.3] vs. NO ROSC: 9.6 [4.4-15.9] mmHg; p < 0.05), but not in children or infants. Median event ETCO2 was significantly associated with chest compression rate less than 140 (p < 0.0001) and chest compression fraction 90-100 (p < 0.0001). CONCLUSIONS: This represents the largest collection of ETCO2 and chest compression data in paediatric patients to date and unadjusted analyses suggests an association between ETCO2 and ROSC in some paediatric patients.

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.

Use of an end-tidal carbon dioxide-guided algorithm during cardiopulmonary resuscitation improves short-term survival in paediatric swine.

AIM: To evaluate an algorithm that uses an end-tidal carbon dioxide (ETCO2) target of ≥ 30 torr to guide specific changes in chest compression rate and epinephrine administration during cardiopulmonary resuscitation (CPR) in paediatric swine. METHODS: Swine underwent asphyxial cardiac arrest followed by resuscitation with either standard or ETCO2-guided algorithm CPR. The standard group received chest compressions at a rate of 100/min and epinephrine every 4 min during advanced life support consistent with the American Heart Association paediatric resuscitation guidelines. In the ETCO2-guided algorithm group, chest compression rate was increased by 10 compressions/min for every minute that the ETCO2 was < 30 torr, and the epinephrine administration interval was decreased to every 2 min if the ETCO2 remained < 30 torr. Short-term survival and physiologic data during active resuscitation were compared. RESULTS: Short-term survival was significantly greater in the ETCO2-guided algorithm CPR group than in the standard CPR group (16/28 [57.1%] versus 4/28 [14.3%]; p = 0.002). Additionally, the algorithm group had higher predicted mean ETCO2, chest compression rate, diastolic and mean arterial pressure, and myocardial perfusion pressure throughout resuscitation. Swine in the algorithm group also exhibited significantly greater improvement in diastolic and mean arterial pressure and cerebral perfusion pressure after the first dose of epinephrine than did those in the standard group. Incidence of resuscitation-related injuries was similar in the two groups. CONCLUSIONS: Use of a resuscitation algorithm with stepwise guidance for changes in the chest compression rate and epinephrine administration interval based on a goal ETCO2 level improved survival and intra-arrest hemodynamics in this porcine cardiac arrest model.

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.

Association of diastolic blood pressure with survival during paediatric cardiopulmonary resuscitation.

AIM: To examine the relationship between survival and diastolic blood pressure (DBP) throughout resuscitation from paediatric asphyxial cardiac arrest. METHODS: Retrospective, secondary analysis of 200 swine resuscitations. Swine underwent asphyxial cardiac arrest and were resuscitated with predefined periods of basic and advanced life support (BLS and ALS, respectively). DBP was recorded every 30 s. Survival was defined as 20-min sustained return of spontaneous circulation (ROSC). RESULTS: During BLS, DBP peaked between 1-3 min and was greater in survivors (20.0 [11.3, 33.3] mmHg) than in non-survivors (5.0 [1.0, 10.0] mmHg; p < 0.001). After this transient increase, the DBP in survivors progressively decreased but remained greater than that of non-survivors after 10 min of resuscitation (9.0 [6.0, 13.8] versus 3.0 [1.0, 6.8] mmHg; p < 0.001). During ALS, the magnitude of DBP change after the first adrenaline (epinephrine) administration was greater in survivors (22.0 [16.5, 36.5] mmHg) than in non-survivors (6.0 [2.0, 11.0] mmHg; p < 0.001). Survival rate was greater when DBP improved by ≥26 mmHg after the first dose of adrenaline (20/21; 95%) than when DBP improved by ≤10 mmHg (1/99; 1%). The magnitude of DBP change after the first adrenaline administration correlated with the timetoROSC (r = -0.54; p < 0.001). CONCLUSIONS: Survival after asphyxial cardiac arrest is associated with a higher DBP throughout resuscitation, but the difference between survivors and non-survivors was reduced after prolonged BLS. During ALS, response to adrenaline administration correlates with survival and time to ROSC. If confirmed clinically, these findings may be useful for titrating adrenaline during resuscitation and prognosticating likelihood of ROSC. Institutional Protocol Numbers: SW14M223 and SW17M101.

The Current State of Cephalopod Science and Perspectives on the Most Critical Challenges Ahead From Three Early-Career Researchers.

Here, three researchers who have recently embarked on careers in cephalopod biology discuss the current state of the field and offer their hopes for the future. Seven major topics are explored: genetics, aquaculture, climate change, welfare, behavior, cognition, and neurobiology. Recent developments in each of these fields are reviewed and the potential of emerging technologies to address specific gaps in knowledge about cephalopods are discussed. Throughout, the authors highlight specific challenges that merit particular focus in the near-term. This review and prospectus is also intended to suggest some concrete near-term goals to cephalopod researchers and inspire those working outside the field to consider the revelatory potential of these remarkable creatures.

Proteolytic cleavage of Beclin 1 exacerbates neurodegeneration.

BACKGROUND: Neuronal cell loss contributes to the pathology of acute and chronic neurodegenerative diseases, including Alzheimer's disease (AD). It remains crucial to identify molecular mechanisms sensitizing neurons to various insults and cell death. To date, the multifunctional, autophagy-related protein Beclin 1 has been shown to be both necessary and sufficient for neuronal integrity in neurodegenerative models associated with protein aggregation. Interestingly, besides its role in cellular homeostasis, Beclin 1 has also been ascribed a role in apoptosis. This makes it critical to elucidate whether Beclin 1 regulates neuronal death and survival across neurodegenerative conditions independent of protein clearance. Here, we provide experimental evidence for a direct functional link between proteolytic cleavage of Beclin 1 and apoptotic neuronal cell loss in two independent models of neurodegeneration in vivo. METHODS: Proteolytic cleavage of Beclin 1 was characterized in lysates of human AD brain samples. We developed viral tools allowing for the selective neuronal expression of the various Beclin 1 forms, including Beclin 1 cleavage products as well as a cleavage-resistant form. The effect of these Beclin 1 forms on survival and integrity of neurons was examined in models of acute and chronic neurodegeneration in vitro and in vivo. Markers of neuronal integrity, neurodegeneration and inflammation were further assessed in a Kainic acid-based mouse model of acute excitotoxic neurodegeneration and in a hAPP-transgenic mouse model of AD following perturbation of Beclin 1 in the susceptible CA1 region of the hippocampus. RESULTS: We find a significant increase in caspase-mediated Beclin 1 cleavage fragments in brain lysates of human AD patients and mimic this phenotype in vivo using both an excitotoxic and hAPP-transgenic mouse model of neurodegeneration. Surprisingly, overexpression of the C-terminal cleavage-fragment exacerbated neurodegeneration in two distinct models of degeneration. Local inhibition of caspase activity ameliorated neurodegeneration after excitotoxic insult and prevented Beclin 1 cleavage. Furthermore, overexpression of a cleavage-resistant form of Beclin 1 in hippocampal neurons conferred neuroprotection against excitotoxic and Amyloid beta-associated insults in vivo. CONCLUSIONS: Together, these findings indicate that the cleavage state of Beclin 1 determines its functional involvement in both neurodegeneration and neuroprotection. Hence, manipulating the cleavage state of Beclin 1 may represent a therapeutic strategy for preventing neuronal cell loss across multiple forms of neurodegeneration.

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 .

Pilot Study to Compare the Use of End-Tidal Carbon Dioxide-Guided and Diastolic Blood Pressure-Guided Chest Compression Delivery in a Swine Model of Neonatal Asphyxial Cardiac Arrest.

Background The American Heart Association recommends use of physiologic feedback when available to optimize chest compression delivery. We compared hemodynamic parameters during cardiopulmonary resuscitation in which either end-tidal carbon dioxide ( ETCO 2) or diastolic blood pressure ( DBP ) levels were used to guide chest compression delivery after asphyxial cardiac arrest. Methods and Results One- to 2-week-old swine underwent a 17-minute asphyxial-fibrillatory cardiac arrest followed by alternating 2-minute periods of ETCO 2-guided and DBP -guided chest compressions during 10 minutes of basic life support and 10 minutes of advanced life support. Ten animals underwent resuscitation. We found significant changes to ETCO 2 and DBP levels within 30 s of switching chest compression delivery methods. The overall mean ETCO 2 level was greater during ETCO 2-guided cardiopulmonary resuscitation (26.4±5.6 versus 22.5±5.2 mm Hg; P=0.003), whereas the overall mean DBP was greater during DBP -guided cardiopulmonary resuscitation (13.9±2.3 versus 9.4±2.6 mm Hg; P=0.003). ETCO 2-guided chest compressions resulted in a faster compression rate (149±3 versus 120±5 compressions/min; P=0.0001) and a higher intracranial pressure (21.7±2.3 versus 16.0±1.1 mm Hg; P=0.002). DBP -guided chest compressions were associated with a higher myocardial perfusion pressure (6.0±2.8 versus 2.4±3.2; P=0.02) and cerebral perfusion pressure (9.0±3.0 versus 5.5±4.3; P=0.047). Conclusions Using the ETCO 2 or DBP level to optimize chest compression delivery results in physiologic changes that are method-specific and occur within 30 s. Additional studies are needed to develop protocols for the use of these potentially conflicting physiologic targets to improve outcomes of prolonged cardiopulmonary resuscitation.