Research

Learn about the research in the lab from recent publications.

Impact of the brain metabolism-excitability relation, from mice to persons

Why is hyperexcitability or seizures, rather than hypoexcitability, the hallmark of decreased brain metabolism? Does this paradox offer an avenue for treatment?  

Individuals with glucose transporter type I deficiency (G1D) habitually experience nutrient-responsive epilepsy associated with decreased brain glucose. However, the mechanistic association between blood glucose concentration and brain excitability in the context of G1D remains to be elucidated. Electroencephalography (EEG) in G1D individuals revealed nutrition time-dependent seizure oscillations often associated with preserved volition despite electrographic generalization and uniform average oscillation duration and periodicity, suggesting increased facilitation of an underlying neural loop circuit. Nonlinear EEG ictal source localization analysis and simultaneous EEG/functional magnetic resonance imaging converged on the thalamus-sensorimotor cortex as one potential circuit, and 18F-deoxyglucose positron emission tomography (18F-DG-PET) illustrated decreased glucose accumulation in this circuit. This pattern, reflected in a decreased thalamic to striatal 18F signal ratio, can aid with the PET imaging diagnosis of the disorder, whereas the absence of noticeable ictal behavioral changes challenges the postulated requirement for normal thalamocortical activity during consciousness. In G1D mice, 18F-DG-PET and mass spectrometry also revealed decreased brain glucose and glycogen, but preserved tricarboxylic acid cycle intermediates, indicating no overall energy metabolism failure. In brain slices from these animals, synaptic inhibition of cortical pyramidal neurons and thalamic relay neurons was decreased, and neuronal disinhibition was mitigated by metabolic sources of carbon; tonic-clonic seizures were also suppressed by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibition. These results pose G1D as a thalamocortical synaptic disinhibition disease associated with increased glucose-dependent neuronal excitability, possibly in relation to reduced glycogen. Together with findings in other metabolic defects, inhibitory neuron dysfunction is emerging as a modulable mechanism of hyperexcitability.

Development of new experimental models. What information can be acquired from a larger complexity brain?

Rodents are not persons. Pigs are not either. But their brains are almost person-like. Or at least more so than rodent brains.

Gyriform mammals display neurophysiological and neural network activity that other species exhibit only in rudimentary or dissimilar form. However, neural recordings from large mammals such as the pig can be anatomically hindered and pharmacologically suppressed by anesthetics. This curtails comparative inferences. To mitigate these limitations, we set out to modify electrocorticography, intracerebral depth and intracortical recording methods to study the anesthetized pig. We aimed to safeguard neural network integrity and recording fidelity by developing surgical, anesthesia and noise reduction methods and by working inside a newly designed Faraday cage, and evaluated this from the point of view of neurophysiological power spectral density and coherence analyses. We also utilized novel silicon carbide electrodes to minimize mechanical disruption of single-neuron activity. These methods allowed for the preservation of native neurophysiological activity for several hours. Pig electrocorticography recordings were essentially indistinguishable from awake human recordings except for the small segment of electrical activity associated with vision in conscious persons. In addition, single-neuron and paired-pulse stimulation recordings were feasible simultaneously with electrocorticography and depth electrode recordings. The spontaneous and stimulus-elicited neuronal activities thus surveyed can be recorded with a degree of precision similar to that achievable in rodent or any other animal studies and prove as informative as unperturbed human electrocorticography.

Do individual genes really determine biology or disease in every case?

This was an unusual question to ask. After all, how else could biology or disease manifestations follow a clear, recognizable pattern?

Localization within the nervous system provides context for neurological disease manifestations and treatment, with numerous disease mechanisms exhibiting predilect locations. In contrast, the molecular function of most disease-causing genes is generally considered dissociated from such brain regional correlations because most genes are expressed throughout the brain. We tested the factual basis for this dissociation by discerning between two distinct genetic disease mechanism possibilities: One, gene-specific, in which genetic disorders are poorly localizable because they are multiform at the molecular level, with each mutant gene acting more widely or complexly than via mere loss or gain of one function. The other, more general, where aspects shared by groups of genes such as membership in a gene set that sustains a concerted biological process accounts for a common or localizable phenotype. We analyzed mitochondrial substrate disorders as a paradigm of apparently heterogeneous diseases when considered from the point of view of their manifestations and individual function of their causal genes. We used publicly available transcriptomes, disease phenotypes published in peer-reviewed journals and Human Ontology classifications for 27 mitochondrial substrate metabolism diseases and analyzed if these disorders manifest common phenotypes and if this relates to common brain regions or cells as demarcated by their transcriptome. The most frequent phenotypic manifestations and brain structures involved were almost stereotypic regardless of the individual gene affected, correlating with the regional abundance of the transcriptome that served mitochondrial substrate metabolism. This also applied to the transcriptome of inhibitory neurons, which are dysfunctional in some mitochondrial diseases. The results suggest that brain region or cell type dysfunction stemming from a broad process such as mitochondrial substrate metabolism is more relevant for disease manifestations than individual gene participation in specific molecular function.

Hidden (or nor so hidden) features of behavior that predict future performance

This was a first result from the study of over 32,000 mutant mice, one at a time.

Time-to-fall off an accelerating rotating rod (rotarod) is widely utilized to evaluate rodent motor performance. We reasoned that this simple outcome could be refined with additional measures explicit in the task (however inconspicuously) to examine what we call movement sub-structure. Our goal was to characterize normal variation or motor impairment more robustly than by using time-to-fall. We also hypothesized that measures (or features) early in the sub-structure could anticipate the learning expected of a mouse undergoing serial trials. Using normal untreated and baclofen-treated movement-impaired mice, we defined these features and automated their analysis using paw video-tracking in three consecutive trials, including paw location, speed, acceleration, variance and approximate entropy. Spectral arc length yielded speed and acceleration uniformity. We found that, in normal mice, paw movement smoothness inversely correlated with rotarod time-to-fall for the three trials. Greater approximate entropy in vertical movements, and opposite changes in horizontal movements, correlated with greater first-trial time-to-fall. First-trial horizontal approximate entropy in the first few seconds predicted subsequent time-to-fall. This allowed for the separation, after only one rotarod trial, of different-weight, untreated mouse groups, and for the detection of mice otherwise unimpaired after baclofen, which displayed a time-to-fall similar to control. A machine-learning support vector machine classifier corroborated these findings. In conclusion, time-to-fall off a rotarod correlated well with several measures, including some obtained during the first few seconds of a trial, and some responsive to learning over the first two trials, allowing for predictions or preemptive experimental manipulations before learning completion.

Observation of human brain and muscle metabolism by ultra-high field MRI

Better methods for the study of human metabolism in situ are needed. Even better, flux, rather than metabolite abundance, is what governs cellular function. Therefore, there is great interest in techniques that measure biochemical interconversions over time.

We examined an alternative approach to monitor glucose metabolism in the human brain. 13 C-enriched glucose was infused in healthy subjects outside the magnet to a steady-state level of 13 C enrichment. Subsequently, the subjects were scanned at 7T for 60 min without 1 H decoupling. Metabolic modeling was used to calculate anaplerosis. Biomarkers of energy metabolism and anaplerosis were detected. The glutamate C5 doublet provided information about glucose-derived acetyl-coenzyme A flux into the tricarboxylic acid (TCA) cycle via pyruvate dehydrogenase, and the bicarbonate signal reflected overall TCA cycle activity. The glutamate C1/C5 ratio is sensitive to anaplerosis. Brain 13 C MRS at 7T provides information about glucose oxidation and anaplerosis without the need of prolonged 13 C infusions inside the scanner and without technical challenges of 1 H decoupling, making it a feasible approach for clinical research.

We also explored intramyocellular lipid (IMCL) accumulation noninvasively by 7T magnetic resonance spectroscopy (MRS) of muscle as a reporter of metabolic dysfunction in MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). We reasoned that mitochondrial dysfunction may impair muscle fat metabolism, resulting in lipid deposition (as is sometimes observed in biopsies), and that MRS is well suited to quantify these lipids. In 10 MELAS participants and relatives, IMCL abundance correlates with percent mitochondrial DNA mutation abundance and with disease severity. These results indicate that IMCL accumulation is a novel potential disease hallmark in MELAS.

Developing a treatment derived from laboratory research

Often, we put to trial potential therapies for those afflicted by disorders that we consider treatable based on a body of work from the lab. The primary goal is not so much any of the so-called biomarkers, but aspects that impact personal autonomy such as cognition. For too many interesting biological phenomena mean too little to persons. We are grateful to a loyal following and to a worldwide network of patients willing to enable this research with their advice, patience and participation.

Glucose transporter type I deficiency (G1D) is commonly associated with seizures and with electrographic spike-waves. The G1D syndrome has long been attributed to energy (ie, adenosine triphosphate synthetic) failure such as that consequent to tricarboxylic acid (TCA) cycle intermediate depletion. Indeed, glucose and other substrates generate TCAs via anaplerosis. However, TCAs are preserved in murine G1D, rendering energy-failure inferences premature and suggesting a different hypothesis, also grounded on our work, that consumption of alternate TCA precursors is stimulated and may be detrimental. Second, common ketogenic diets lead to a therapeutically counterintuitive reduction in blood glucose available to the G1D brain and prove ineffective in one-third of patients. We aimed to identify the most helpful outcomes for treatment evaluation and to uphold (rather than diminish) blood glucose concentration and stimulate the TCA cycle, including anaplerosis, in G1D using the medium-chain, food-grade triglyceride triheptanoin. First, we show that, regardless of electroencephalographic spike-waves, most seizures are rarely visible, such that perceptions by patients or others are inadequate for treatment evaluation. Thus, we used quantitative electroencephalographic, neuropsychological, blood analytical, and magnetic resonance imaging cerebral metabolic rate measurements. Triheptanoin can favorably influence cardinal aspects of neural function in G1D. In addition, our outcome measures constitute an important framework for the evaluation of therapies for encephalopathies associated with impaired intermediary metabolism.

Drug Discovery

Some drugs can be repurposed, but many more are waiting to be discovered. Some should probably be used in combination therapy to harness several mechanisms at once. This idea is slowly getting some traction, but the drugs to try or use are few and neurological drug discovery has been almost stagnant for years.

One approach to Glucose transporter I (Glut1) deficiency syndrome (G1D), dementia and cancer treatment is modulation of glucose transport. For decreased or increased Glut1 expression or activity is a central biological event in these disparate disorders. Using lung adenocarcinoma cells rich in Glut1, we identified, in high-throughput fashion, activators and inhibitors of fluorescent glucose analog transport. About 9,000 compounds were studied in this fashion. Since the identified drugs and compounds were many more than expected, it was impractical to study each of them from a mechanism perspective. Therefore, we started with impact on disease-relevant behavior. Thus, we also developed a gait testing platform for the deep learning neural network analysis of G1D mice that quantifies drug impact on 49 gait parameters, thus evaluating the potential preclinical efficacy of these drugs and other interventions via analysis of locomotion.

An unusual way to modulate brain function, even in adulthood

Dr. Richard Wang had a heterodox idea that a quick textbook check could have disproved but we nevertheless agreed to test with help from his lab.

Red blood cells circulating through the brain are briefly but closely apposed to the capillary endothelium. We hypothesized that this contact provides a nearly direct pathway for metabolic substrate transfer to neural cells that complements the better characterized plasma to endothelium transfer. While brain function is considered independent of normal fluctuations in blood glucose concentration, this is not borne out by persons with glucose transporter I (GLUT1) deficiency (G1D). In them, encephalopathy is often ameliorated by meal or carbohydrate administration, and this enabled us to test our hypothesis: Since red blood cells contain glucose, and since the red cells of G1D individuals are also deficient in GLUT1, replacing them with normal donor cells via exchange transfusion could augment erythrocyte to neural cell glucose transport via mass action in the setting of unaltered erythrocyte count or plasma glucose abundance. This motivated us to perform red blood cell exchange in 3 G1D persons. There were rapid, favorable and unprecedented changes in cognitive, electroencephalographic and quality-of-life measures. The hypothesized transfer mechanism was further substantiated by in vitro measurement of direct erythrocyte to endothelial cell glucose flux. The results also indicate that the adult intellect is capable of significant enhancement without deliberate practice.