4R-cembranoid suppresses glial cells inflammatory phenotypes and prevents hippocampal neuronal loss in LPS-treated mice.

Chronic neuroinflammation has been implicated in neurodegenerative disease pathogenesis. A key feature of neuroinflammation is neuronal loss and glial activation, including microglia and astrocytes. 4R-cembranoid (4R) is a natural compound that inhibits hippocampal pro-inflammatory cytokines and increases memory function in mice. We used the lipopolysaccharide (LPS) injection model to study the effect of 4R on neuronal density and microglia and astrocyte activation. C57BL/6J wild-type mice were injected with LPS (5 mg/kg) and 2 h later received either 4R (6 mg/kg) or vehicle. Mice were sacrificed after 72 h for analysis of brain pathology. Confocal images of brain sections immunostained for microglial, astrocyte, and neuronal markers were used to quantify cellular hippocampal phenotypes and neurons. Hippocampal lysates were used to measure the expression levels of neuronal nuclear protein (NeuN), inducible nitrous oxide synthase (iNOS), arginase-1, thrombospondin-1 (THBS1), glial cell-derived neurotrophic factor (GDNF), and orosomucoid-2 (ORM2) by western blot. iNOS and arginase-1 are widely used protein markers of pro- and anti-inflammatory microglia, respectively. GDNF promotes neuronal survival, and ORM2 and THBS1 are astrocytic proteins that regulate synaptic plasticity and inhibit microglial activation. 4R administration significantly reduced neuronal loss and the number of pro-inflammatory microglia 72 h after LPS injection. It also decreased the expression of the pro-inflammatory protein iNOS while increasing arginase-1 expression, supporting its anti-inflammatory role. The protein expression of THBS1, GDNF, and ORM2 was increased by 4R. Our data show that 4R preserves the integrity of hippocampal neurons against LPS-induced neuroinflammation in mice.

Caudal DMN neurons innervate the spleen and release CART peptide to regulate neuroimmune function.

BACKGROUND: Inflammation is a fundamental biological response to injury and infection, which if unregulated can contribute to the pathophysiology of many diseases. The vagus nerve, which primarily originates from the dorsal motor nucleus (DMN), plays an important role in rapidly dampening inflammation by regulating splenic function. However, direct vagal innervation of the spleen, which houses the majority of immune and inflammatory cells, has not been established. As an alternative to direct innervation, an anti-inflammatory reflex pathway has been proposed which involves the vagus nerve, the sympathetic celiac ganglion, and the neurotransmitter norepinephrine. Although sympathetic regulation of inflammation has been shown, the interaction of the vagus nerve and the celiac ganglia requires a unique interaction of parasympathetic and sympathetic inputs, making this putative mechanism of brain-spleen interaction controversial. BODY: As neuropeptides can be expressed at relatively high levels in neurons, we reasoned that DMN neuropeptide immunoreactivity could be used to determine their target innervation. Employing immunohistochemistry, subdiaphragmatic vagotomy, viral tract tracing, CRISPR-mediated knock-down, and functional assays, we show that cocaine and amphetamine-regulated transcript (CART) peptide-expressing projection neurons in the caudal DMN directly innervate the spleen. In response to lipopolysaccharide (LPS) stimulation, CART acts to reduce inflammation, an effect that can be augmented by intrasplenic administration of a synthetic CART peptide. These in vivo effects could be recapitulated in cultured splenocytes, suggesting that these cells express the as yet unidentified CART receptor(s). CONCLUSION: Our results provide evidence for direct connections between the caudal DMN and spleen. In addition to acetylcholine, these neurons express the neuropeptide CART that, once released, acts to suppress inflammation by acting directly upon splenocytes.

Selective Endothelial Hyperactivation of Oncogenic KRAS Induces Brain Arteriovenous Malformations in Mice.

OBJECTIVE: Brain arteriovenous malformations (bAVMs) are a leading cause of hemorrhagic stroke and neurological deficits in children and young adults, however, no pharmacological intervention is available to treat these patients. Although more than 95% of bAVMs are sporadic without family history, the pathogenesis of sporadic bAVMs is largely unknown, which may account for the lack of therapeutic options. KRAS mutations are frequently observed in cancer, and a recent unprecedented finding of these mutations in human sporadic bAVMs offers a new direction in the bAVM research. Using a novel adeno-associated virus targeting brain endothelium (AAV-BR1), the current study tested if endothelial KRASG12V mutation induces sporadic bAVMs in mice. METHODS: Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRASG12V . At 8 weeks after the AAV injection, bAVM formation and characteristics were addressed by histological and molecular analyses. The effect of MEK/ERK inhibition on KRASG12V -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. RESULTS: The viral-mediated KRASG12V overexpression induced bAVMs, which were composed of a tangled nidus mirroring the distinctive morphology of human bAVMs. The bAVMs were accompanied by focal angiogenesis, intracerebral hemorrhages, altered vascular constituents, neuroinflammation, and impaired sensory/cognitive/motor functions. Finally, we confirmed that bAVM growth was inhibited by trametinib treatment. INTERPRETATION: Our innovative approach using AAV-BR1 confirms that KRAS mutations promote bAVM development via the MEK/ERK pathway, and provides a novel preclinical mouse model of bAVMs which will be useful to develop a therapeutic strategy for patients with bAVM. ANN NEUROL 2021;89:926-941.

Hypothermia for Patients Requiring Evacuation of Subdural Hematoma: A Multicenter Randomized Clinical Trial.

BACKGROUND: Hypothermia is neuroprotective in some ischemia-reperfusion injuries. Ischemia-reperfusion injury may occur with traumatic subdural hematoma (SDH). This study aimed to determine whether early induction and maintenance of hypothermia in patients with acute SDH would lead to decreased ischemia-reperfusion injury and improve global neurologic outcome. METHODS: This international, multicenter randomized controlled trial enrolled adult patients with SDH requiring evacuation of hematoma within 6 h of injury. The intervention was controlled temperature management of hypothermia to 35 °C prior to dura opening followed by 33 °C for 48 h compared with normothermia (37 °C). Investigators randomly assigned patients at a 1:1 ratio between hypothermia and normothermia. Blinded evaluators assessed outcome using a 6-month Glasgow Outcome Scale Extended score. Investigators measured circulating glial fibrillary acidic protein and ubiquitin C-terminal hydrolase L1 levels. RESULTS: Independent statisticians performed an interim analysis of 31 patients to assess the predictive probability of success and the Data and Safety Monitoring Board recommended the early termination of the study because of futility. Thirty-two patients, 16 per arm, were analyzed. Favorable 6-month Glasgow Outcome Scale Extended outcomes were not statistically significantly different between hypothermia vs. normothermia groups (6 of 16, 38% vs. 4 of 16, 25%; odds ratio 1.8 [95% confidence interval 0.39 to ∞], p = .35). Plasma levels of glial fibrillary acidic protein (p = .036), but not ubiquitin C-terminal hydrolase L1 (p = .26), were lower in the patients with favorable outcome compared with those with unfavorable outcome, but differences were not identified by temperature group. Adverse events were similar between groups. CONCLUSIONS: This trial of hypothermia after acute SDH evacuation was terminated because of a low predictive probability of meeting the study objectives. There was no statistically significant difference in functional outcome identified between temperature groups.

4R-cembranoid confers neuroprotection against LPS-induced hippocampal inflammation in mice.

BACKGROUND: Chronic brain inflammation has been implicated in the pathogenesis of various neurodegenerative diseases and disorders. For example, overexpression of pro-inflammatory cytokines has been associated with impairments in hippocampal-dependent memory. Lipopolysaccharide (LPS) injection is a widely used model to explore the pathobiology of inflammation. LPS injection into mice causes systemic inflammation, neuronal damage, and poor memory outcomes if the inflammation is not controlled. Activation of the alpha-7 nicotinic receptor (α7) plays an anti-inflammatory role in the brain through vagal efferent nerve signaling. 4R-cembranoid (4R) is a natural compound that crosses the blood-brain barrier, induces neuronal survival, and has been shown to modulate the activity of nicotinic receptors. The purpose of this study is to determine whether 4R reduces the deleterious effects of LPS-induced neuroinflammation and whether the α7 receptor plays a role in mediating these beneficial effects. METHODS: Ex vivo population spike recordings were performed in C57BL/6J wild-type (WT) and alpha-7-knockout (α7KO) mouse hippocampal slices in the presence of 4R and nicotinic receptor inhibitors. For in vivo studies, WT and α7KO mice were injected with LPS for 2 h, followed by 4R or vehicle for 22 h. Analyses of IL-1ß, TNF-α, STAT3, CREB, Akt1, and the long-term novel object recognition test (NORT) were performed for both genotypes. In addition, RNA sequencing and RT-qPCR analyses were carried out for 12 mRNAs related to neuroinflammation and their modification by 4R. RESULTS: 4R confers neuroprotection after NMDA-induced neurotoxicity in both WT and α7KO mice. Moreover, hippocampal TNF-α and IL-1ß levels were decreased with 4R treatment following LPS exposure in both strains of mice. 4R restored LPS-induced cognitive decline in NORT. There was a significant increase in the phosphorylation of STAT3, CREB, and Akt1 with 4R treatment in the WT mouse hippocampus following LPS exposure. In α7KO mice, only pAkt levels were significantly elevated in the cortex. 4R significantly upregulated mRNA levels of ORM2, GDNF, and C3 following LPS exposure. These proteins are known to play a role in modulating microglial activation, neuronal survival, and memory. CONCLUSION: Our results indicate that 4R decreases the levels of pro-inflammatory cytokines; improves memory function; activates STAT3, Akt1, and CREB phosphorylation; and upregulates the mRNA levels of ORM2, GDNF, and C3. These effects are independent of the α7 nicotinic receptor.

Morphology of mitochondria in spatially restricted axons revealed by cryo-electron tomography.

Neurons project axons to local and distal sites and can display heterogeneous morphologies with limited physical dimensions that may influence the structure of large organelles such as mitochondria. Using cryo-electron tomography (cryo-ET), we characterized native environments within axons and presynaptic varicosities to examine whether spatial restrictions within these compartments influence the morphology of mitochondria. Segmented tomographic reconstructions revealed distinctive morphological characteristics of mitochondria residing at the narrowed boundary between presynaptic varicosities and axons with limited physical dimensions (approximately 80 nm), compared to mitochondria in nonspatially restricted environments. Furthermore, segmentation of the tomograms revealed discrete organizations between the inner and outer membranes, suggesting possible independent remodeling of each membrane in mitochondria at spatially restricted axonal/varicosity boundaries. Thus, cryo-ET of mitochondria within axonal subcompartments reveals that spatial restrictions do not obstruct mitochondria from residing within them, but limited available space can influence their gross morphology and the organization of the inner and outer membranes. These findings offer new perspectives on the influence of physical and spatial characteristics of cellular environments on mitochondrial morphology and highlight the potential for remarkable structural plasticity of mitochondria to adapt to spatial restrictions within axons.

Endoplasmic Reticulum Stress Contributes to the Loss of Newborn Hippocampal Neurons after Traumatic Brain Injury.

Adult hippocampal neurogenesis has been shown to be required for certain types of cognitive function. For example, studies have shown that these neurons are critical for pattern separation, the ability to store similar experiences as distinct memories. Although traumatic brain injury (TBI) has been shown to cause the loss of newborn hippocampal neurons, the signaling pathway(s) that triggers their death is unknown. Endoplasmic reticulum (ER) stress activates the PERK-eIF2α pathway that acts to restore ER function and improve cell survival. However, unresolved/intense ER stress activates C/EBP homologous protein (CHOP), leading to cell death. We show that TBI causes the death of hippocampal newborn neurons via CHOP. Using CHOP KO mice, we show that loss of CHOP markedly reduces newborn neuron loss after TBI. Injured CHOP mice performed significantly better in a context fear discrimination task compared with injured wild-type mice. In contrast, the PERK inhibitor GSK2606414 exacerbated doublecortin cell loss and worsened contextual discrimination. Administration of guanabenz (which reduces ER stress) to injured male rats reduced the loss of newborn neurons and improved one-trial contextual fear memory. Interestingly, we also found that the surviving newborn neurons in brain-injured animals had dendritic loss, which was not observed in injured CHOP KO mice or in animals treated with guanabenz. These results indicate that ER stress plays a key role in the death of newborn neurons after TBI. Further, these findings indicate that ER stress can alter dendritic arbors, suggesting a role for ER stress in neuroplasticity and dendritic pathologies.SIGNIFICANCE STATEMENT The hippocampus, a structure in the temporal lobe, is critical for learning and memory. The hippocampus is one of only two areas in which neurons are generated in the adult brain. These newborn neurons are required for certain types of memory, and are particularly vulnerable to traumatic brain injury (TBI). However, the mechanism(s) that causes the loss of these cells after TBI is poorly understood. We show that endoplasmic reticulum (ER) stress pathways are activated in newborn neurons after TBI, and that manipulation of the CHOP cascade improves newborn neuron survival and cognitive outcome. These results suggest that treatments that prevent/resolve ER stress may be beneficial in treating TBI-triggered memory dysfunction.

A role for autophagy in long-term spatial memory formation in male rodents.

A hallmark of long-term memory formation is the requirement for protein synthesis. Administration of protein synthesis inhibitors impairs long-term memory formation without influencing short-term memory. Rapamycin is a specific inhibitor of target of rapamycin complex 1 (TORC1) that has been shown to block protein synthesis and impair long-term memory. In addition to regulating protein synthesis, TORC1 also phosphorylates Unc-51-like autophagy activating kinase-1 (Ulk-1) to suppress autophagy. As autophagy can be activated by rapamycin (and rapamycin inhibits long-term memory), our aim was to test the hypothesis that autophagy inhibitors would enhance long-term memory. To examine if learning alters autophagosome number, we used male reporter mice carrying the GFP-LC3 transgene. Using these mice, we observed that training in the Morris water maze task increases the number of autophagosomes, a finding contrary to our expectations. For learning and memory studies, male Long Evans rats were used due to their relatively larger size (compared to mice), making it easier to perform intrahippocampal infusions in awake, moving animals. When the autophagy inhibitors 3-methyladenine (3-MA) or Spautin-1 were administered bilaterally into the hippocampii prior to training in the Morris water maze task, the drugs did not alter learning. In contrast, when memory was tested 24 hours later by a probe trial, significant impairments were observed. In addition, intrahippocampal infusion of an autophagy activator peptide (TAT-Beclin-1) improved long-term memory. These results indicate that autophagy is not necessary for learning, but is required for long-term memory formation.

Treatment of Severe Adult Traumatic Brain Injury Using Bone Marrow Mononuclear Cells.

Preclinical studies using bone marrow derived cells to treat traumatic brain injury have demonstrated efficacy in terms of blood-brain barrier preservation, neurogenesis, and functional outcomes. Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe traumatic brain injury demonstrated safety and potentially a central nervous system structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Adults with severe traumatic brain injury (Glasgow Coma Scale 5-8) and without signs of irreversible brain injury were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6, 9, 12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re-infusion occurred within 48 hours after injury. Patients were monitored for harvest-related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included magnetic resonance imaging-based measurements of supratentorial and corpus callosal volumes as well as diffusion tensor imaging-based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months postinjury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pretreatment, posttreatment, and at 1 and 6 month follow-up. There were no serious adverse events. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were downregulated. Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by central nervous system structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are downregulated after cell infusion. Stem Cells 2016 Video Highlight: https://youtu.be/UiCCPIe-IaQ Stem Cells 2017;35:1065-1079.

Activation of Alpha 7 Cholinergic Nicotinic Receptors Reduce Blood-Brain Barrier Permeability following Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major human health concern that has the greatest impact on young men and women. The breakdown of the blood-brain barrier (BBB) is an important pathological consequence of TBI that initiates secondary processes, including infiltration of inflammatory cells, which can exacerbate brain inflammation and contribute to poor outcome. While the role of inflammation within the injured brain has been examined in some detail, the contribution of peripheral/systemic inflammation to TBI pathophysiology is largely unknown. Recent studies have implicated vagus nerve regulation of splenic cholinergic nicotinic acetylcholine receptor α7 (nAChRa7) signaling in the regulation of systemic inflammation. However, it is not known whether this mechanism plays a role in TBI-triggered inflammation and BBB breakdown. Following TBI, we observed that plasma TNF-α and IL-1ß levels, as well as BBB permeability, were significantly increased in nAChRa7 null mice (Chrna7(-/-)) relative to wild-type mice. The administration of exogenous IL-1ß and TNF-α to brain-injured animals worsened Evans Blue dye extravasation, suggesting that systemic inflammation contributes to TBI-triggered BBB permeability. Systemic administration of the nAChRa7 agonist PNU-282987 or the positive allosteric modulator PNU-120596 significantly attenuated TBI-triggered BBB compromise. Supporting a role for splenic nAChRa7 receptors, we demonstrate that splenic injection of the nicotinic receptor blocker α-bungarotoxin increased BBB permeability in brain-injured rats, while PNU-282987 injection decreased such permeability. These effects were not seen when α-bungarotoxin or PNU-282987 were administered to splenectomized, brain-injured rats. Together, these findings support the short-term use of nAChRa7-activating agents as a strategy to reduce TBI-triggered BBB permeability. SIGNIFICANCE STATEMENT: Breakdown of the blood-brain barrier (BBB) in response to traumatic brain injury (TBI) allows for the accumulation of circulating fluids and proinflammatory cells in the injured brain. These processes can exacerbate TBI pathology and outcome. While the role of inflammation in the injured tissue has been examined in some detail, the contribution of peripheral inflammation in BBB breakdown and ensuing pathology has not been well defined. We present experimental evidence to indicate that the stimulation of nicotinic acetylcholine α7 receptors (nAChRa7s) can reduce peripheral inflammation and BBB breakdown after TBI. These results suggest that activators of nAChRa7 may have therapeutic utility for the treatment of TBI.

Wnt3a, a Protein Secreted by Mesenchymal Stem Cells Is Neuroprotective and Promotes Neurocognitive Recovery Following Traumatic Brain Injury.

Intravenous administration of bone marrow derived mesenchymal stem cells (MSCs) has been shown to reduce blood brain barrier compromise and improve neurocognition following traumatic brain injury (TBI). These effects occur in the absence of engraftment and differentiation of these cells in the injured brain. Recent studies have shown that soluble factors produced by MSCs mediate a number of the therapeutic effects. In this study, we sought to determine if intravenous administration of MSCs (IV-MSCs) could enhance hippocampal neurogenesis following TBI. Our results demonstrate that IV-MSC treatment attenuates loss of neural stem cells and promotes hippocampal neurogenesis in TBI injured mice. As Wnt signaling has been implicated in neurogenesis, we measured circulating Wnt3a levels in serum following IV-MSC administration and found a significant increase in Wnt3a. Concurrent with this increase, we detected increased activation of the Wnt/ß-catenin signaling pathway in hippocampal neurons. Furthermore, IV recombinant Wnt3a treatment provided neuroprotection, promoted neurogenesis, and improved neurocognitive function in TBI injured mice. Taken together, our results demonstrate a role for Wnt3a in the therapeutic potential of MSCs and identify Wnt3a as a potential stand-alone therapy or as part of a combination therapeutic strategy for the treatment of TBI. Stem Cells 2016;34:1263-1272.

Traumatic brain injury decreases AMP-activated protein kinase activity and pharmacological enhancement of its activity improves cognitive outcome.

Prolonged metabolic suppression in the brain is a well-characterized secondary pathology of both experimental and clinical traumatic brain injury (TBI). AMP-activated kinase (AMPK) acts as a cellular energy sensor that, when activated, regulates various metabolic and catabolic pathways to decrease ATP consumption and increase ATP synthesis. As energy availability after TBI is suppressed, we questioned if increasing AMPK activity after TBI would improve cognitive outcome. TBI was delivered using the electromagnetic controlled cortical impact model on male Sprague-Dawley rats (275-300 g) and C57BL/6 mice (20-25 g). AMPK activity within the injured parietal cortex and ipsilateral hippocampus was inferred by western blots using phospho-specific antibodies. The consequences of acute manipulation of AMPK signaling on cognitive function were assessed using the Morris water maze task. We found that AMPK activity is decreased as a result of injury, as indicated by reduced AMPK phosphorylation and corresponding changes in the phosphorylation of its downstream targets: ribosomal protein S6 and Akt Substrate of 160 kDa (AS160). Increasing AMPK activity after injury using the drugs 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide or metformin did not affect spatial learning, but significantly improved spatial memory. Taken together, our results suggest that decreased AMPK activity after TBI may contribute to the cellular energy crisis in the injured brain, and that AMPK activators may have therapeutic utility. Increased phosphorylation of Thr172 activates AMP-activated protein kinase (AMPK) under conditions of low cellular energy availability. This leads to inhibition of energy consuming, while activating energy generating, processes. Hill et al., present data to indicate that TBI decreases Thr172 phosphorylation and that its stimulation by pharmacological agents offers neuroprotection and improves memory. These results suggest that decreased AMPK phosphorylation after TBI incorrectly signals the injured brain that excess energy is available, thereby contributing to the cellular energy crisis and memory impairments.

TIMP3 Attenuates the Loss of Neural Stem Cells, Mature Neurons and Neurocognitive Dysfunction in Traumatic Brain Injury.

Mesenchymal stem cells (MSCs) have been shown to have potent therapeutic effects in a number of disorders including traumatic brain injury (TBI). However, the molecular mechanism(s) underlying these protective effects are largely unknown. Herein we demonstrate that tissue inhibitor of matrix metalloproteinase-3 (TIMP3), a soluble protein released by MSCs, is neuroprotective and enhances neuronal survival and neurite outgrowth in vitro. In vivo in a murine model of TBI, intravenous recombinant TIMP3 enhances dendritic outgrowth and abrogates loss of hippocampal neural stem cells and mature neurons. Mechanistically we demonstrate in vitro and in vivo that TIMP3-mediated neuroprotection is critically dependent on activation of the Akt-mTORC1 pathway. In support of the neuroprotective effect of TIMP3, we find that intravenous delivery of recombinant TIMP3 attenuates deficits in hippocampal-dependent neurocognition. Taken together, our data strongly suggest that TIMP3 has direct neuroprotective effects that can mitigate the deleterious effects associated with TBI, an area with few if any therapeutic options.

Intrahippocampal glutamine administration inhibits mTORC1 signaling and impairs long-term memory.

The mechanistic Target of Rapamycin Complex 1 (mTORC1), a key regulator of protein synthesis and cellular growth, is also required for long-term memory formation. Stimulation of mTORC1 signaling is known to be dependent on the availability of energy and growth factors, as well as the presence of amino acids. In vitro studies using serum- and amino acid-starved cells have reported that glutamine addition can either stimulate or repress mTORC1 activity, depending on the particular experimental system that was used. However, these experiments do not directly address the effect of glutamine on mTORC1 activity under physiological conditions in nondeprived cells in vivo. We present experimental results indicating that intrahippocampal administration of glutamine to rats reduces mTORC1 activity. Moreover, post-training administration of glutamine impairs long-term spatial memory formation, while coadministration of glutamine with leucine had no influence on memory. Intracellular recordings in hippocampal slices showed that glutamine did not alter either excitatory or inhibitory synaptic activity, suggesting that the observed memory impairments may not result from conversion of glutamine to either glutamate or GABA. Taken together, these findings indicate that glutamine can decrease mTORC1 activity in the brain and may have implications for treatments of neurological diseases associated with high mTORC1 signaling.

Prolonging the survival of Tsc2 conditional knockout mice by glutamine supplementation.

The genetic disease tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by loss of function mutations in either TSC1 (hamartin) or TSC2 (tuberin), which serve as negative regulators of mechanistic target of rapamycin complex 1 (mTORC1) activity. TSC patients exhibit developmental brain abnormalities and tuber formations that are associated with neuropsychological and neurocognitive impairments, seizures and premature death. Mechanistically, TSC1 and TSC2 loss of function mutations result in abnormally high mTORC1 activity. Thus, the development of a strategy to inhibit abnormally high mTORC1 activity may have therapeutic value in the treatment of TSC. mTORC1 is a master regulator of growth processes, and its activity can be reduced by withdrawal of growth factors, decreased energy availability, and by the immunosuppressant rapamycin. Recently, glutamine has been shown to alter mTORC1 activity in a TSC1-TSC2 independent manner in cells cultured under amino acid- and serum-deprived conditions. Since starvation culture conditions are not physiologically relevant, we examined if glutamine can regulate mTORC1 in non-deprived cells and in a murine model of TSC. Our results show that glutamine can reduce phosphorylation of S6 and S6 kinase, surrogate indicators of mTORC1 activity, in both deprived and non-deprived cells, although higher concentrations were required for non-deprived cultures. When administered orally to TSC2 knockout mice, glutamine reduced S6 phosphorylation in the brain and significantly prolonged their lifespan. Taken together, these results suggest that glutamine supplementation can be used as a potential treatment for TSC.

The differential effects of prenatal and/or postnatal rapamycin on neurodevelopmental defects and cognition in a neuroglial mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is caused by heterozygous mutations in either the TSC1 (hamartin) or the TSC2 (tuberin) gene. Among the multisystemic manifestations of TSC, the neurodevelopmental features cause the most morbidity and mortality, presenting a considerable clinical challenge. Hamartin and tuberin form a heterodimer that inhibits the mammalian target of rapamycin complex 1 (mTORC1) kinase, a major cellular regulator of protein translation, cell growth and proliferation. Hyperactivated mTORC1 signaling, an important feature of TSC, has prompted a number of preclinical and clinical studies with the mTORC1 inhibitor rapamycin. Equally exciting is the prospect of treating TSC in the perinatal period to block the progression of brain pathologies and allow normal brain development to proceed. We hypothesized that low-dose rapamycin given prenatally and/or postnatally in a well-established neuroglial (Tsc2-hGFAP) model of TSC would rescue brain developmental defects. We developed three treatment regimens with low-dose intraperitoneal rapamycin (0.1 mg/kg): prenatal, postnatal and pre/postnatal (combined). Combined rapamycin treatment resulted in almost complete histologic rescue, with a well-organized cortex and hippocampus almost identical to control animals. Other treatment regimens yielded less complete, but significant improvements in brain histology. To assess how treatment regimens affected cognitive function, we continued rapamycin treatment after weaning and performed behavioral testing. Surprisingly, the animals treated with the combined therapy did not perform as well as postnatally-treated animals in learning and memory tasks. These results have important translational implications in the optimization of the timing and dosage of rapamycin treatment in TSC affected children.

Disruption of the perineuronal net in the hippocampus or medial prefrontal cortex impairs fear conditioning.

The perineuronal net (PNN) surrounds neurons in the central nervous system and is thought to regulate developmental plasticity. A few studies have shown an involvement of the PNN in hippocampal plasticity and memory storage in adult animals. In addition to the hippocampus, plasticity in the medial prefrontal cortex (mPFC) has been demonstrated to be critical for the storage of long-term memory, particularly memories for temporally separated events. In the present study, we examined the role of PNN in the acquisition and retention of memories for trace (in which the conditioned and unconditioned stimuli are temporally separated) and delayed (in which the conditioned and unconditioned stimuli overlap) fear conditioning in both the hippocampus and the mPFC. Consistent with a role for the hippocampus in memory storage in both delayed and trace fear conditioning, removal of hippocampal PNN disrupted contextual and trace fear memory. Disruption of the PNN in the mPFC impaired long-term trace and conditioned stimulus (CS)-elicited fear memory in the trace fear conditioning task. Interestingly, CS-elicited fear memory was also impaired when a delayed fear conditioning paradigm was used. These findings further support a role for the PNN in neural plasticity and implicate PNN-regulated plasticity in neocortical memory storage.

Lysophospholipids Are Associated With Outcomes in Hospitalized Patients With Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) accounts for 70-90% of all TBI cases. Lipid metabolites have important roles in plasma membrane biogenesis, function, and cell signaling. As TBI can compromise plasma membrane integrity and alter brain cell function, we sought to identify circulating phospholipid alterations after mTBI, and determine if these changes were associated with clinical outcomes. Patients with mTBI (Glasgow Coma Score [GCS] ≥13 and loss of consciousness <30 min) were recruited. A total of 84 mTBI subjects were enrolled after admission to a level I trauma center, with the majority having evidence of traumatic intracranial hemorrhage on brain computed tomography (CT). Plasma samples were collected within 24 h of injury with 32 mTBI subjects returning at 3 months after injury for a second plasma sample to be collected. Thirty-five healthy volunteers were enrolled as controls and had a one-time blood draw. Lipid metabolomics was performed on plasma samples from each subject. Fold change of selected lipid metabolites was determined. Multivariable regression models were created to test associations between lipid metabolites and discharge and 6-month Glasgow Outcomes Scale-Extended (GOSE) outcomes (dichotomized between "good" [GOSE ≥7] and "bad" [GOSE ≤6] functional outcomes). Plasma levels of 31 lipid metabolites were significantly associated with discharge GOSE using univariate models; three of these metabolites were significantly increased, while 14 were significantly decreased in subjects with good outcomes compared with subjects with poor outcomes. In multivariable logistic regression models, higher circulating levels of the lysophospholipids (LPL) 1-linoleoyl-glycerophosphocholine (GPC) (18:2), 1-linoleoyl-GPE (18:2), and 1-linolenoyl-GPC (18:3) were associated with both good discharge GOSE (odds ratio [OR] 12.2 [95% CI 3.35, 58.3], p = 5.23 × 10-4; OR 9.43 [95% CI 2.87, 39.6], p = 7.26 × 10-4; and OR 5.26 [95% CI 1.99, 16.7], p = 2.04 × 10-3, respectively) and 6-month (OR 4.67 [95% CI 1.49, 17.7], p = 0.013; OR 2.93 [95% CI 1.11, 8.87], p = 0.039; and OR 2.57 [95% CI 1.08, 7.11], p = 0.046, respectively). Compared with healthy volunteers, circulating levels of these three LPLs were decreased early after injury and had normalized by 3 months after injury. Logistic regression models to predict functional outcomes were created by adding each of the described three LPLs to a baseline model that included age and sex. Including 1-linoleoyl-GPC (18:2) (8.20% improvement, p = 0.009), 1-linoleoyl-GPE (18:2) (8.85% improvement, p = 0.021), or 1-linolenoyl-GPC (18:3) (7.68% improvement, p = 0.012), significantly improved the area under the curve (AUC) for predicting discharge outcomes compared with the baseline model. Models including 1-linoleoyl-GPC (18:2) significantly improved AUC for predicting 6-month outcomes (9.35% improvement, p = 0.034). Models including principal components derived from 25 LPLs significantly improved AUC for prediction of 6-month outcomes (16.0% improvement, p = 0.020). Our results demonstrate that higher plasma levels of LPLs (1-linoleoyl-GPC, 1-linoleoyl-GPE, and 1-linolenoyl-GPC) after mTBI are associated with better functional outcomes at discharge and 6 months after injury. This class of phospholipids may represent a potential therapeutic target.

Loss of Tsc2 in Purkinje cells is associated with autistic-like behavior in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a dominant tumor suppressor disorder caused by mutations in either TSC1 or TSC2. TSC causes substantial neuropathology, often leading to autism spectrum disorders (ASDs) in up to 60% of patients. The anatomic and neurophysiologic links between these two disorders are not well understood. We have generated and characterized a novel TSC mouse model with Purkinje cell specific Tsc2 loss. These Tsc2f/-;Cre mice exhibit progressive Purkinje cell degeneration. Since loss of Purkinje cells is a well reported postmortem finding in patients with ASD, we conducted a series of behavior tests to asses if Tsc2f/-;Cre mice displayed autistic-like deficits. Tsc2f/-;Cre mice demonstrated increased repetitive behavior as assessed with marble burying activity. Using the three chambered apparatus to asses social behavior, we found that Tsc2f/-;Cre mice showed behavioral deficits, exhibiting no preference between a stranger mouse and an inanimate object, or between a novel and a familiar mouse. We also detected social deficits in Tsc2f/f;Cre mice, suggesting that Purkinje cell pathology is sufficient to induce ASD-like behavior. Importantly, social behavior deficits were prevented with rapamycin treatment. Altogether, these results demonstrate that loss of Tsc2 in Purkinje cells in a Tsc2-haploinsufficient background leads to autistic-like behavioral deficits. These studies provide compelling evidence that Purkinje cell loss and/or dysfunction may be an important link between TSC and ASD as well as a general anatomic phenomenon that contributes to the ASD phenotype.