Small RNA signatures of acute ischemic stroke in L1CAM positive extracellular vesicles.

L1CAM-positive extracellular vesicles (L1EV) are an emerging biomarker that may better reflect ongoing neuronal damage than other blood-based biomarkers. The physiological roles and regulation of L1EVs and their small RNA cargoes following stroke is unknown. We sought to characterize L1EV small RNAs following stroke and assess L1EV RNA signatures for diagnosing stroke using weighted gene co-expression network analysis and random forest (RF) machine learning algorithms. Interestingly, small RNA sequencing of plasma L1EVs from patients with stroke and control patients (n = 28) identified micro(mi)RNAs known to be enriched in the brain. Weighted gene co-expression network analysis (WGCNA) revealed small RNA transcript modules correlated to diagnosis, initial NIH stroke scale, and age. L1EV RNA signatures associated with the diagnosis of AIS were derived from WGCNA and RF classification. These small RNA signatures demonstrated a high degree of accuracy in the diagnosis of AIS with an area under the curve (AUC) of the signatures ranging from 0.833 to 0.932. Further work is necessary to understand the role of small RNA L1EV cargoes in the response to brain injury, however, this study supports the utility of L1EV small RNA signatures as a biomarker of stroke.

ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Suppressing APOE4-induced mortality and cellular damage by targeting VHL.

Mortality rate increases with age and can accelerate upon extrinsic or intrinsic damage to individuals. Identifying factors and mechanisms that curb population mortality rate has wide-ranging implications. Here, we show that targeting the VHL-1 (Von Hippel-Lindau) protein suppresses C. elegans mortality caused by distinct factors, including elevated reactive oxygen species, temperature, and APOE4, the genetic variant that confers high risks of neurodegeneration in Alzheimer's diseases and all-cause mortality in humans. These mortality factors are of different physical-chemical nature, yet result in similar cellular dysfunction and damage that are suppressed by deleting VHL-1. Stabilized HIF-1 (hypoxia inducible factor), a transcription factor normally targeted for degradation by VHL-1, recapitulates the protective effects of deleting VHL-1. HIF-1 orchestrates a genetic program that defends against mitochondrial abnormalities, excess oxidative stress, cellular proteostasis dysregulation, and endo-lysosomal rupture, key events that lead to mortality. Genetic Vhl inhibition also alleviates cerebral vascular injury and synaptic lesions in APOE4 mice, supporting an evolutionarily conserved mechanism. Collectively, we identify the VHL-HIF axis as a potent modifier of APOE4 and mortality and propose that targeting VHL-HIF in non-proliferative animal tissues may suppress tissue injuries and mortality by broadly curbing cellular damage.

Management of Status Epilepticus and Indications for Inpatient Electroencephalography Monitoring.

Status epilepticus (SE) is a neurologic emergency requiring immediate time-sensitive treatment to minimize neuronal injury and systemic complications. Minimizing time to administration of first- and second-line therapy is necessary to optimize the chances of successful seizure termination in generalized convulsive SE (GCSE). The approach to refractory and superrefractory GCSE is less well defined. Multiple agents with differing complementary actions that facilitate seizure termination are recommended. Nonconvulsive SE (NCSE) has a wide range of presentations and approaches to treatment. Continuous electroencephalography is critical to the management of both GCSE and NCSE, while its use for patients without seizure continues to expand.

Diabetes, stroke, and neuroresilience: looking beyond hyperglycemia.

Ischemic stroke is a leading cause of morbidity and mortality among type 2 diabetic patients. Preclinical and translational studies have identified critical pathophysiological mediators of stroke risk, recurrence, and poor outcome in diabetic patients, including endothelial dysfunction and inflammation. Most clinical trials of diabetes and stroke have focused on treating hyperglycemia alone. Pioglitazone has shown promise in secondary stroke prevention for insulin-resistant patients; however, its use is not yet widespread. Additional research into clinical therapies directed at diabetic pathophysiological processes to prevent stroke and improve outcome for diabetic stroke survivors is necessary. Resilience is the process of active adaptation to a stressor. In patients with diabetes, stroke recovery is impaired by insulin resistance, endothelial dysfunction, and inflammation, which impair key neuroresilience pathways maintaining cerebrovascular integrity, resolving poststroke inflammation, stimulating neural plasticity, and preventing neurodegeneration. Our review summarizes the underpinnings of stroke risk in diabetes, the clinical consequences of stroke in diabetic patients, and proposes hypotheses and new avenues of research for therapeutics to stimulate neuroresilience pathways and improve stroke outcome in diabetic patients.

Cytoprotection by a naturally occurring variant of ATP5G1 in Arctic ground squirrel neural progenitor cells.

Many organisms in nature have evolved mechanisms to tolerate severe hypoxia or ischemia, including the hibernation-capable Arctic ground squirrel (AGS). Although hypoxic or ischemia tolerance in AGS involves physiological adaptations, little is known about the critical cellular mechanisms underlying intrinsic AGS cell resilience to metabolic stress. Through cell survival-based cDNA expression screens in neural progenitor cells, we identify a genetic variant of AGS Atp5g1 that confers cell resilience to metabolic stress. Atp5g1 encodes a subunit of the mitochondrial ATP synthase. Ectopic expression in mouse cells and CRISPR/Cas9 base editing of endogenous AGS loci revealed causal roles of one AGS-specific amino acid substitution in mediating cytoprotection by AGS ATP5G1. AGS ATP5G1 promotes metabolic stress resilience by modulating mitochondrial morphological change and metabolic functions. Our results identify a naturally occurring variant of ATP5G1 from a mammalian hibernator that critically contributes to intrinsic cytoprotection against metabolic stress.

When animals hibernate, they lower their body temperature and metabolism to conserve the energy they need to withstand cold harsh winters. One such animal is the Arctic ground squirrel, an extreme hibernator that can drop its body temperatures to below 0°C. This hibernation ability means the cells of Arctic ground squirrels can survive severe shortages of blood and oxygen. But, it is unclear how their cells are able to endure this metabolic stress. To answer this question, Singhal, Bai et al. studied the cells of Arctic ground squirrels for unique features that might make them more durable to stress. Examining the genetic code of these resilient cells revealed that Arctic ground squirrels may have a variant form of a protein called ATP5G1. This protein is found in a cellular compartment called the mitochondria, which is responsible for supplying energy to the rest of the cell and therefore plays an important role in metabolic processes. Singhal, Bai et al. found that when this variant form of ATP5G1 was introduced into the cells of mice, their mitochondria was better at coping with stress conditions, such as low oxygen, low temperature and poisoning. Using a gene editing tool to selectively substitute some of the building blocks, also known as amino acids, that make up the ATP5G1 protein revealed that improvements to the mitochondria were caused by switching specific amino acids. However, swapping these amino acids, which presumably affects the role of ATP5G1, did not completely remove the cells' resilience to stress. This suggests that variants of other genes and proteins may also be involved in providing protection. These findings provide the first evidence of a protein variant that is responsible for protecting cells during the metabolic stress conditions caused by hibernation. The approach taken by Singhal, Bai et al. could be used to identify and study other proteins that increase resilience to metabolic stress. These findings could help develop new treatments for diseases caused by a limited blood supply to human organs, such as a stroke or heart attack.

Resilience to Injury: A New Approach to Neuroprotection?

Despite thousands of neuroprotectants demonstrating promise in preclinical trials, a neuroprotective therapeutic has yet to be approved for the treatment of acute brain injuries such as stroke or traumatic brain injury. Developing a more detailed understanding of models and populations demonstrating "neurological resilience" in spite of brain injury can give us important insights into new translational therapies. Resilience is the process of active adaptation to a stressor. In the context of neuroprotection, models of preconditioning and unique animal models of extreme physiology (such as hibernating species) reliably demonstrate resilience in the laboratory setting. In the clinical setting, resilience is observed in young patients and can be found in those with specific genetic polymorphisms. These important examples of resilience can help transform and extend the current neuroprotective framework from simply countering the injurious cascade into one that anticipates, monitors, and optimizes patients' physiological responses from the time of injury throughout the process of recovery. This review summarizes the underpinnings of key adaptations common to models of resilience and how this understanding can be applied to new neuroprotective approaches.

Neuronal GDPGP1 and glycogen metabolism: friend or foe?

The adult brain consumes glucose for energy needs and stores glucose as glycogen mainly in astrocytes. Schulz et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201807127) identify the stress-regulated metabolic enzyme GDPGP1 that promotes neuronal survival likely through glycogen reserves in mouse and C. elegans neurons.

Diagnosing Balamuthia mandrillaris Encephalitis With Metagenomic Deep Sequencing.

OBJECTIVE: Identification of a particular cause of meningoencephalitis can be challenging owing to the myriad bacteria, viruses, fungi, and parasites that can produce overlapping clinical phenotypes, frequently delaying diagnosis and therapy. Metagenomic deep sequencing (MDS) approaches to infectious disease diagnostics are known for their ability to identify unusual or novel viruses and thus are well suited for investigating possible etiologies of meningoencephalitis. METHODS: We present the case of a 74-year-old woman with endophthalmitis followed by meningoencephalitis. MDS of her cerebrospinal fluid (CSF) was performed to identify an infectious agent. RESULTS: Sequences aligning to Balamuthia mandrillaris ribosomal RNA genes were identified in the CSF by MDS. Polymerase chain reaction subsequently confirmed the presence of B. mandrillaris in CSF, brain tissue, and vitreous fluid from the patient's infected eye. B. mandrillaris serology and immunohistochemistry for free-living amoebas on the brain biopsy tissue were positive. INTERPRETATION: The diagnosis was made using MDS after the patient had been hospitalized for several weeks and subjected to costly and invasive testing. MDS is a powerful diagnostic tool with the potential for rapid and unbiased pathogen identification leading to early therapeutic targeting.

Sarcoid polyneuropathy masquerading as chronic inflammatory demyelinating polyneuropathy.

INTRODUCTION: Sarcoid polyneuropathy is a rare and clinically heterogeneous disorder that may be the initial presentation of sarcoidosis. METHODS: We report the clinical, electrophysiological, and pathological findings of a patient who carried a diagnosis of sensory-predominant chronic inflammatory demyelinating polyneuropathy (CIDP) for over a decade but was ultimately found to have sarcoid polyneuropathy. RESULTS: A 36-year-old man presented with a several-week history of gait difficulty and muscle cramps. He had a diagnosis of CIDP but had not received lasting benefit from steroid-sparing immunosuppressive drugs. Electrodiagnostic studies were consistent with a chronic demyelinating polyradiculoneuropathy with conduction blocks. After he developed systemic symptoms, tissue biopsies revealed granulomatous disease. Symptoms improved with steroid therapy. CONCLUSIONS: Sarcoid polyneuropathy presents a diagnostic challenge, but, in patients with atypical neuropathy, characteristic systemic symptoms, or a poor response to standard treatment, nerve and muscle biopsies can help diagnose this treatable disorder.

Effect of rituximab in patients with leucine-rich, glioma-inactivated 1 antibody-associated encephalopathy.

IMPORTANCE: This observational study describes the efficacy and safety of rituximab in 5 patients with voltage-gated potassium channel (VGKC)-complex/leucine-rich, glioma-inactivated 1 (LGI1) antibody-associated encephalopathy. Rituximab is a monoclonal antibody that targets CD20 and is used to treat other neurologic and nonneurologic diseases. OBSERVATIONS: This case series reports sequential seizure frequencies, modified Rankin Scale scores, and VGKC-complex antibody titers in 5 adult patients (median age, 65 years; range, 48-73 years) treated with rituximab. Median time from symptom onset to rituximab initiation was 414 days (range, 312-851 days). One patient showed a rapid clinical improvement after treatment with rituximab alone and experienced a rituximab-responsive clinical relapse. Another showed possible improvement on neuropsychometric memory indexes after rituximab therapy. In contrast, all patients showed robust responses to treatment with glucocorticoids, intravenous immunoglobulins, and/or plasma exchange at some point in their illness. Treatment with glucocorticoids-less so with intravenous immunoglobulins and plasma exchange-was associated with the most marked reductions in VGKC-complex antibodies. The only patient who did not receive glucocorticoids showed the poorest clinical and serologic responses. CONCLUSIONS AND RELEVANCE: Rituximab was well tolerated in this predominantly older adult patient population and may be an effective option for some patients with LGI1 antibody-associated encephalopathy. Glucocorticoid therapy appears particularly efficacious. Earlier rituximab administration and randomized trials are required to formally assess efficacy.

A practical approach to neurologic evaluation in the intensive care unit.

Delirium and neurologic impairment are extremely common in the intensive care setting, and their delayed identification is an important contributor to patient morbidity. Even in comatose patients, the clinical neurologic examination remains the most accurate and effective tool in assessing nervous system function. Rapid identification of neurologic deficits with a practical and easily reproducible neurologic examination is a core skill for effectively caring for critically ill patients. The purpose of this tutorial is to discuss techniques of neurologic examination and localization with an emphasis on comatose patients. Commonly encountered cases of encephalopathy and coma along with clinical pearls are presented.

Unconscious processing of color and form in metacontrast masking.

Three experiments employed a metacontrast masking procedure to examine the extent and nature of priming effects from visual stimuli not consciously perceived. The results showed effects of unconscious stimuli on subsequent target responses that (1) were more consistent, reliable, and not subject to strategic control, as compared with consciously perceived stimuli (Experiment 1); (2) produced both facilitation and interference of subsequent processing (Experiment 2); and (3) did not influence indirect response-related levels of processing (Experiment 3). These results demonstrate that color and form attributes of unconscious stimuli are sufficiently registered within the visual system to influence behavior, and that some of these unconscious effects occur at early levels of stimulus encoding, prior to higher level perceptual and response-related processes.

Inhibition of ADRP prevents diet-induced insulin resistance.

Diets with high fat content induce steatosis, insulin resistance, and type 2 diabetes. The lipid droplet protein adipose differentiation-related protein (ADRP) mediates hepatic steatosis, but whether this affects insulin action in the liver or peripheral organs in diet-induced obesity is uncertain. We fed C57BL/6J mice a high-fat diet and simultaneously treated them with an antisense oligonucleotide (ASO) against ADRP for 4 wk. Glucose homeostasis was assessed with clamp and tracer techniques. ADRP ASO decreased the levels of triglycerides and diacylglycerol in the liver, but fatty acids, long-chain fatty acyl CoAs, ceramides, and cholesterol were unchanged. Insulin action in the liver was enhanced after ADRP ASO treatment, whereas muscle and adipose tissue were not affected. ADRP ASO increased the phosphorylation of insulin receptor substrate (IRS)1, IRS2, and Akt, and decreased gluconeogenic enzymes and PKCepsilon, consistent with its insulin-sensitizing action. These results demonstrate an important role for ADRP in the pathogenesis of diet-induced insulin resistance.

Loss of resistin ameliorates hyperlipidemia and hepatic steatosis in leptin-deficient mice.

Resistin has been linked to components of the metabolic syndrome, including obesity, insulin resistance, and hyperlipidemia. We hypothesized that resistin deficiency would reverse hyperlipidemia in genetic obesity. C57Bl/6J mice lacking resistin [resistin knockout (RKO)] had similar body weight and fat as wild-type mice when fed standard rodent chow or a high-fat diet. Nonetheless, hepatic steatosis, serum cholesterol, and very low-density lipoprotein (VLDL) secretion were decreased in diet-induced obese RKO mice. Resistin deficiency exacerbated obesity in ob/ob mice, but hepatic steatosis was drastically attenuated. Moreover, the levels of triglycerides, cholesterol, insulin, and glucose were reduced in ob/ob-RKO mice. The antisteatotic effect of resistin deficiency was related to reductions in the expression of genes involved in hepatic lipogenesis and VLDL export. Together, these results demonstrate a crucial role of resistin in promoting hepatic steatosis and hyperlipidemia in obese mice.

Central resistin induces hepatic insulin resistance via neuropeptide Y.

Sensing of peripheral hormones and nutrients by the hypothalamus plays an important role in maintaining peripheral glucose homeostasis. The hormone resistin impairs the response to insulin in liver and other peripheral tissues. Here we demonstrate that in normal mice resistin delivered in the lateral cerebral ventricle increased endogenous glucose production during hyperinsulinemic-euglycemic clamp, consistent with induction of hepatic insulin resistance. In agreement, central resistin inhibited Akt phosphorylation and increased the expression of glucose-6-phosphatase, the enzyme regulating glucose output in the liver. Central resistin induced expression of proinflammatory cytokines as well as suppressor of cytokine signaling-3, a negative regulator of insulin action in liver. Central infusion of resistin was associated with neuronal activation in the arcuate, paraventricular and dorsomedial nuclei, and increased neuropeptide Y (NPY) expression in the hypothalamus. The effects of central resistin on glucose production as well as hepatic expression of proinflammatory cytokines were abrogated in mice lacking NPY. Pretreatment of wild-type mice with antagonists of the NPY Y1 receptor, but not the Y5 receptor, also prevented the effects of central resistin. Together, these results suggest that resistin action on NPY neurons is an important regulator of hepatic insulin sensitivity.

Brain adipocytokine action and metabolic regulation.

Adipose tissue secretes factors that control various physiological systems. The fall in leptin during fasting mediates hyperphagia and suppresses thermogenesis, thyroid and reproductive hormones, and immune system. On the other hand, rising leptin levels in the fed state stimulate fatty acid oxidation, decrease appetite, and limit weight gain. These divergent effects of leptin occur through neuronal circuits in the hypothalamus and other brain areas. Leptin also regulates the activities of enzymes involved in lipid metabolism, e.g., AMP-activated protein kinase and stearoyl-CoA desaturase-1, and also interacts with insulin signaling in the brain. Adiponectin enhances fatty acid oxidation and insulin sensitivity, in part by stimulating AMP-activated protein kinase phosphorylation and activity in liver and muscle. Moreover, adiponectin decreases body fat by increasing energy expenditure and lipid catabolism. These effects involve peripheral and possibly central mechanisms. Adipose tissue mediates interconversion of steroid hormones and secretes proinflammatory cytokines, vasoactive peptides, and coagulation and complement proteins. Understanding the actions of these "adipocytokines" will provide insight into the pathogenesis and treatment of obesity and related diseases.

Loss of resistin improves glucose homeostasis in leptin deficiency.

Resistin levels are increased in obesity, and hyperresistinemia impairs glucose homeostasis in rodents. Here, we have determined the role of resistin in ob/ob mice that are obese and insulin resistant because of genetic deficiency of leptin. Loss of resistin increased obesity in ob/ob mice by further lowering the metabolic rate without affecting food intake. Nevertheless, resistin deficiency improved glucose tolerance and insulin sensitivity in these severely obese mice, largely by enhancing insulin-mediated glucose disposal in muscle and adipose tissue. In contrast, in C57BL/6J mice with diet-induced obesity but wild-type leptin alleles, resistin deficiency reduced hepatic glucose production and increased peripheral glucose uptake. Resistin deficiency enhanced Akt phosphorylation in muscle and liver and decreased suppressor of cytokine signaling-3 level in muscle, and these changes were reversed by resistin replacement. Together, these results provide strong support for an important role of resistin in insulin resistance and diabetes associated with genetic or diet-induced obesity.