Nathaniel Hodgson

Early life stress (ELS) yields cognitive impairments of unknown molecular and physiological origin. We found that fragmented maternal care of mice during a neonatal critical period from postnatal days P2–9 elevated dopamine receptor D2R and suppressed D4R expression, specifically within the anterior cingulate cortex (ACC) in only the male offspring. This was associated with poor performance on a two-choice visual attention task, which was acutely rescued in adulthood by local or systemic pharmacological rebalancing of D2R/D4R activity. Furthermore, ELS male mice demonstrated heightened hypothalamic orexin and persistently disrupted sleep. Given that acute sleep deprivation in normally reared male mice mimicked the ACC dopamine receptor subtype modulation and disrupted attention of ELS mice, sleep loss likely underlies cognitive deficits in ELS mice. Likewise, sleep impairment mediated the attention deficits associated with early adversity in human children, as demonstrated by path analysis on data collected with multiple questionnaires for a large child cohort. A deeper understanding of the sex-specific cognitive consequences of ELS thus has the potential to reveal therapeutic strategies for overcoming them.

Traumatic brain injury (TBI) increases cerebral reactive oxygen species production, which leads to continuing secondary neuronal injury after the initial insult. Cortical parvalbumin-positive interneurons (PVIs; neurons responsible for maintaining cortical inhibitory tone) are particularly vulnerable to oxidative stress and are thus disproportionately affected by TBI. Systemic N-acetylcysteine (NAC) treatment may restore cerebral glutathione equilibrium, thus preventing post-traumatic cortical PVI loss. We therefore tested whether weeks-long post-traumatic NAC treatment mitigates cortical oxidative stress, and whether such treatment preserves PVI counts and related markers of PVI integrity and prevents pathologic electroencephalographic (EEG) changes, 3 and 6 weeks after fluid percussion injury in rats. We find that moderate TBI results in persistent oxidative stress for at least 6 weeks after injury and leads to the loss of PVIs and the perineuronal net (PNN) that surrounds them as well as of per-cell parvalbumin expression. Prolonged post-TBI NAC treatment normalizes the cortical redox state, mitigates PVI and PNN loss, and - in surviving PVIs - increases per-cell parvalbumin expression. NAC treatment also preserves normal spectral EEG measures after TBI. We cautiously conclude that weeks-long NAC treatment after TBI may be a practical and well-tolerated treatment strategy to preserve cortical inhibitory tone post-TBI.

Expression of the glutamate transporter GLT-1 in neurons has been shown to be important for synaptic mitochondrial function in the cerebral cortex. Here we determined whether neuronal GLT-1 plays a similar role in the hippocampus and striatum, using conditional GLT-1 knockout mice in which GLT-1 was inactivated in neurons by expression of synapsin-Cre (synGLT-1 KO). Ex vivo ¹³C-labelling using [1,2-¹³C]acetate, representing astrocytic metabolism, yielded increased [4,5-¹³C]glutamate levels, suggesting increased astrocyte-neuron glutamine transfer, in the striatum but not in the hippocampus of the synGLT-1 KO. Moreover, aspartate concentrations were reduced − 38% compared to controls in the hippocampus and the striatum of the synGLT-1 KO. Mitochondria isolated from the hippocampus of synGLT-1 KO mice exhibited a lower oxygen consumption rate in the presence of oligomycin A, indicative of a decreased proton leak across the mitochondrial membrane, whereas the ATP production rate was unchanged. Electron microscopy revealed reduced mitochondrial inter-cristae distance within excitatory synaptic terminals in the hippocampus and striatum of the synGLT-1 KO. Finally, dilution of ¹³C-labelling originating from [U-¹³C]glucose, caused by metabolism of unlabelled glutamate, was reduced in hippocampal synGLT-1 KO synaptosomes, suggesting that neuronal GLT-1 provides glutamate for synaptic tricarboxylic acid cycle metabolism. Collectively, these data demonstrate an important role of neuronal expression of GLT-1 in synaptic mitochondrial metabolism in the forebrain.

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. ¹³C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-¹³C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function. SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%–10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

The folate and cobalamin (Cbl‐) dependent enzyme methionine synthase (MS) is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient‐derived fibroblasts. Here we show that MS mRNA in SH‐SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl) or the combination of hydroxocobalamin (OHCbl) and S‐adenosylmethionine (SAM). OHCbl‐based activity was eliminated by depletion of the antioxidant glutathione (GSH) but could be rescued by provision of either glutathionylcobalamin (GSCbl) or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury‐containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate‐limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH‐SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.

Neuregulin 1 (NRG‐1) is a key neurotrophic factor involved in energy homeostasis and CNS development, and impaired NRG‐1 signaling is associated with neurological disorders. Cobalamin (Cbl), also known as vitamin B₁₂, is an essential micronutrient which mammals must acquire through diet, and neurologic dysfunction is a primary clinical manifestation of Cbl deficiency. Here we show that NRG‐1 stimulates synthesis of the two bioactive Cbl species adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) in human neuroblastoma cells by both promoting conversion of inactive to active Cbl species and increasing neuronal Cbl uptake. Formation of active Cbls is glutathione‐ (GSH‐) dependent and the NRG‐1‐initiated increase is dependent upon its stimulation of cysteine uptake by excitatory amino acid transporter 3 (EAAT3), leading to increased GSH. The stimulatory effect of NRG‐1 on cellular Cbl uptake is associated with increased expression of megalin, which is known to facilitate Cbl transport in ileum and kidney. MeCbl is a required cofactor for methionine synthase (MS) and we demonstrate the ability of NRG‐1 to increase MS activity, and affect levels of methionine methylation cycle metabolites. Our results identify novel neuroprotective roles of NRG‐1 including stimulating antioxidant synthesis and promoting active Cbl formation.

Casein-free, gluten-free diets have been reported to mitigate some of the inflammatory gastrointestinal and behavioral traits associated with autism, but the mechanism for this palliative effect has not been elucidated. We recently showed that the opioid peptide beta-casomorphin-7, derived from bovine (bBCM7) milk, decreases cysteine uptake, lowers levels of the antioxidant glutathione (GSH) and decreases the methyl donor S-adenosylmethionine (SAM) in both Caco-2 human GI epithelial cells and SH-SY5Y human neuroblastoma cells. While human breast milk can also release a similar peptide (hBCM-7), the bBCM7 and hBCM-7 vary greatly in potency; as the bBCM-7 is highly potent and similar to morphine in it's effects. Since SAM is required for DNA methylation, we wanted to further investigate the epigenetic effects of these food-derived opioid peptides. In the current study the main objective was to characterize functional pathways and key genes responding to DNA methylation effects of food-derived opioid peptides. SH-SY5Y neuroblastoma cells were treated with 1 μM hBCM7 and bBCM7 and RNA and DNA were isolated after 4 h with or without treatment. Transcriptional changes were assessed using a microarray approach and CpG methylation status was analyzed at 450,000 CpG sites. Functional implications from both endpoints were evaluated via Ingenuity Pathway Analysis 4.0 and KEGG pathway analysis was performed to identify biological interactions between transcripts that were significantly altered at DNA methylation or transcriptional levels (p < 0.05, FDR <0.1). Here we show that hBCM7 and bBCM7, as well as morphine, cause epigenetic changes affecting gene pathways related to gastrointestinal disease and inflammation. These epigenetic consequences exhibited the same potency order as opiate inhibition of cysteine uptake insofar as hBCM7 was less potent than bBCM7, which was less potent than morphine. Our findings indicate that epigenetic effects of milk-derived opiate peptides may contribute to GI dysfunction and inflammation in sensitive individuals. While the current study was performed using SH-SY5Y neuronal cellular models, similar actions on other cells types might combine to cause symptoms of intolerance. These actions may provide a potential contributing mechanism for the beneficial effects of a casein-free diet in alleviating gastrointestinal symptoms in neurological conditions including autism and other conditions. Lastly, our study also contributes to the evolving awareness of a “gut-brain connection”.

Oxidative stress, hyperhomocysteinemia, altered DNA methylation, and insulin resistance in the brain are associated with Alzheimer's disease (AD), but the role of amyloid-β (Aβ) in these events remains unclear. Intracellular cysteine is rate-limiting