Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies. Key features • The protocol applies to small- or large-scale manufacturing of cell therapy products. • It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs). • Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures. • This protocol employs the Finia® Fill and Finish System and a controlled-rate freezer. Graphical overview.

Elevated plasma levels of TIMP-3 are associated with a higher risk of acute respiratory distress syndrome and death following severe isolated traumatic brain injury.

BACKGROUND: Complications after injury, such as acute respiratory distress syndrome (ARDS), are common after traumatic brain injury (TBI) and associated with poor clinical outcomes. The mechanisms driving non-neurologic organ dysfunction after TBI are not well understood. Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) is a regulator of matrix metalloproteinase activity, inflammation, and vascular permeability, and hence has plausibility as a biomarker for the systemic response to TBI. METHODS: In a retrospective study of 182 patients with severe isolated TBI, we measured TIMP-3 in plasma obtained on emergency department arrival. We used non-parametric tests and logistic regression analyses to test the association of TIMP-3 with the incidence of ARDS within 8 days of admission and in-hospital mortality. RESULTS: TIMP-3 was significantly higher among subjects who developed ARDS compared with those who did not (median 2810 pg/mL vs. 2260 pg/mL, p=0.008), and significantly higher among subjects who died than among those who survived to discharge (median 2960 pg/mL vs. 2080 pg/mL, p<0.001). In an unadjusted logistic regression model, for each SD increase in plasma TIMP-3, the odds of ARDS increased significantly, OR 1.5 (95% CI 1.1 to 2.1). This association was only attenuated in multivariate models, OR 1.4 (95% CI 1.0 to 2.0). In an unadjusted logistic regression model, for each SD increase in plasma TIMP-3, the odds of death increased significantly, OR 1.7 (95% CI 1.2 to 2.3). The magnitude of this association was greater in a multivariate model adjusted for markers of injury severity, OR 1.9 (95% CI 1.2 to 2.8). DISCUSSION: TIMP-3 may play an important role in the biology of the systemic response to brain injury in humans. Along with clinical and demographic data, early measurements of plasma biomarkers such as TIMP-3 may help identify patients at higher risk of ARDS and death after severe isolated TBI. LEVEL OF EVIDENCE: III.

Mesenchymal stem cell-derived extracellular vesicles attenuate pulmonary vascular permeability and lung injury induced by hemorrhagic shock and trauma.

BACKGROUND: Mesenchymal stem cells (MSCs) have been shown to mitigate vascular permeability in hemorrhagic shock (HS) and trauma-induced brain and lung injury. Mechanistically, paracrine factors secreted from MSCs have been identified that can recapitulate many of the potent biologic effects of MSCs in animal models of disease. Interestingly, MSC-derived extracellular vesicles (EVs), contain many of these key soluble factors, and have therapeutic potential independent of the parent cells. In this study we sought to determine whether MSC-derived EVs (MSC EVs) could recapitulate the beneficial therapeutic effects of MSCs on lung vascular permeability induced by HS in mice. METHODS: Mesenchymal stem cell EVs were isolated from human bone marrow-derived MSCs by ultracentrifugation. A mouse model of fixed pressure HS was used to study the effects of shock, shock + MSCs and shock + MSC EVs on lung vascular endothelial permeability. Mice were administered MSCs, MSC EVs, or saline IV. Lung tissue was harvested and assayed for permeability, RhoA/Rac1 activation, and for differential phosphoprotein expression. In vitro, human lung microvascular cells junctional integrity was evaluated by immunocytochemistry and endothelial cell impedance assays. RESULTS: Hemorrhagic shock-induced lung vascular permeability was significantly decreased by both MSC and MSC EV infusion. Phosphoprotein profiling of lung tissue revealed differential activation of proteins and pathways related to cytoskeletal rearrangement and regulation of vascular permeability by MSCs and MSC EVs. Lung tissue from treatment groups demonstrated decreased activation of the cytoskeletal GTPase RhoA. In vitro, human lung microvascular cells, MSC CM but not MSC-EVs prevented thrombin-induced endothelial cell permeability as measured by electrical cell-substrate impedance sensing system and immunocytochemistry of VE-cadherin and actin. CONCLUSION: Mesenchymal stem cells and MSC EVs modulate cytoskeletal signaling and attenuate lung vascular permeability after HS. Mesenchymal stem cell EVs may potentially be used as a novel "stem cell free" therapeutic to treat HS-induced lung injury.

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression.

A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1ß, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1ß, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies.IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells.

The effects of 22°C and 4°C storage of platelets on vascular endothelial integrity and function.

BACKGROUND: Although a majority of the studies conducted to date on platelet (PLT) storage have been focused on PLT hemostatic function, the effects of 4°C PLTs on regulation of endothelial barrier permeability are still not known. In this study, we compared the effects of room temperature (22°C) stored and (4°C) stored PLTs on the regulation of vascular endothelial cell (EC) permeability in vitro and in vivo. STUDY DESIGN AND METHODS: Day 1, Day 5, and Day 7 leukoreduced apheresis PLTs stored at 4 or 22°C were studied in vitro and in vivo. In vitro, PLT effects on EC permeability and barrier function, adhesion, and impedance aggregometry were investigated. In vivo, using a mouse model of vascular leak, attenuation of vascular leak and circulating PLT numbers were measured. RESULTS: Treatment of EC monolayers with Day 5 or Day 7 PLTs, stored at both 22°C and 4°C, resulted in similar decreases in EC permeability on average. However, analysis of individual samples revealed significant variation that was donor dependent. Additional in vitro measurements revealed a decrease in inflammatory mediators, nonspecific PLT-endothelial aggregation and attenuated loss of aggregation over time to TRAP, ASPI, ADP, and collagen with 4°C storage. In mice, while 22°C and 4°C PLTs both demonstrated significant protection against vascular endothelial growth factor A (VEGF-A)-induced vascular leak 22°C PLTs exhibited increased protection compared to 4°C PLTs. Systemic circulating levels of 4°C PLTs were decreased compared to 22°C PLTs. CONCLUSIONS: In vitro, 4°C-stored PLTs exhibit a greater capacity to inhibit EC permeability than 22°C-stored PLTs. In vivo, 22°C PLTs provide superior control of vascular leak induced by VEGF-A. This discrepancy may be due to increased clearance of 4°C PLTs from the systemic circulation.

Platelets regulate vascular endothelial stability: assessing the storage lesion and donor variability of apheresis platelets.

BACKGROUND: In current blood banking practices, platelets (PLTs) are stored in plasma at 22°C, with gentle agitation for up to 5 days. To date, the effects of storage and donor variability on PLT regulation of vascular integrity are not known. STUDY DESIGN AND METHODS: In this study, we examined the donor variability of leukoreduced fresh (Day 1) or stored (Day 5) PLTs on vascular endothelial barrier function in vitro and in vivo. In vitro, PLT effects on endothelial cell (EC) monolayer permeability were assessed by analyzing transendothelial electrical resistances (TEER). PLT aggregation, a measure of hemostatic potential, was analyzed by impedance aggregometry. In vivo, PLTs were investigated in a vascular endothelial growth factor A (VEGF-A)-induced vascular permeability model in NSG mice, and PLT circulation was measured by flow cytometry. RESULTS: Treatment of endothelial monolayers with fresh Day 1 PLTs resulted in an increase in EC barrier resistance and decreased permeability in a dose-dependent manner. Subsequent treatment of EC monolayers with Day 5 PLTs demonstrated diminished vasculoprotective effects. Donor variability was noted in all measures of PLT function. Day 1 PLT donors were more variable in their effects on TEER than Day 5 PLTs. In mice, while all PLTs regardless of storage time demonstrated significant protection against VEGF-A-induced vascular leakage, Day 5 PLTs exhibited reduced protection when compared to Day 1 PLTs. Day 1 PLTs demonstrated significant donor variability against VEGF-A-challenged vascular leakage in vivo. Systemic circulating levels of Day 1 PLTs were higher than those of Day 5 PLTs CONCLUSIONS: In vitro and in vivo, Day 1 PLTs are protective in measures of vascular endothelial permeability. Donor variability is most prominent in Day 1 PLTs. A decrease in the protective effects is found with storage of the PLT units between Day 1 and Day 5 at 22°C, thereby suggesting that Day 5 PLTs are diminished in their ability to attenuate vascular endothelial permeability.

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.

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.

Fresh frozen plasma and spray-dried plasma mitigate pulmonary vascular permeability and inflammation in hemorrhagic shock.

BACKGROUND: In retrospective and prospective observational studies, fresh frozen plasma (FFP) has been associated with a survival benefit in massively transfused trauma patients. A dry plasma product, such as spray-dried plasma (SDP), offers logistical advantages over FFP. Recent studies on FFP have demonstrated that FFP modulates systemic vascular stability and inflammation. The effect of SDP on these measures has not been previously examined. This study compares SDP with FFP using in vitro assays of endothelial function and in vivo assays of lung injury using a mouse model of hemorrhagic shock (HS) and trauma. METHODS: FFP, SDP, and lactated Ringer's (LR) solution were compared in vitro using assays of endothelial cell (EC) permeability, cytokine production and content, gene expression, as well as tight and adherens junction stability. All resuscitation products were also compared in a murine model of HS. Mean arterial pressures and physiologic measures were assessed. Pulmonary vascular permeability was measured using tagged dextran. Lung tissues were stained for CD68, VE-cadherin, and occludin. RESULTS: Treatment of ECs with FFP and SDP, but not LR, preserved the integrity of EC monolayers in vitro and resulted in similar EC gene expression patterns and cytokine/growth factor production. FFP and SDP also reduced HS-induced pulmonary vascular permeability in vivo to the same extent. In mice with HS, mean arterial pressures and base excess were corrected by both FFP and SDP to levels observed in sham-treated mice. Treatment after HS with FFP and SDP but not LR solution reduce alveolar wall thickening, leukocyte infiltration, and the breakdown of EC junctions, as measured by staining for VE-cadherin, and occludin. CONCLUSION: Both FFP and SDP similarly modulate pulmonary vascular integrity, permeability, and inflammation in vitro and in vivo in a murine model of HS and trauma.

Cortical activation of accumbens hyperpolarization-active NMDARs mediates aversion-resistant alcohol intake.

Compulsive drinking despite serious adverse medical, social and economic consequences is a characteristic of alcohol use disorders in humans. Although frontal cortical areas have been implicated in alcohol use disorders, little is known about the molecular mechanisms and pathways that sustain aversion-resistant intake. Here, we show that nucleus accumbens core (NAcore) NMDA-type glutamate receptors and medial prefrontal (mPFC) and insula glutamatergic inputs to the NAcore are necessary for aversion-resistant alcohol consumption in rats. Aversion-resistant intake was associated with a new type of NMDA receptor adaptation, in which hyperpolarization-active NMDA receptors were present at mPFC and insula but not amygdalar inputs in the NAcore. Accordingly, inhibition of Grin2c NMDA receptor subunits in the NAcore reduced aversion-resistant alcohol intake. None of these manipulations altered intake when alcohol was not paired with an aversive consequence. Our results identify a mechanism by which hyperpolarization-active NMDA receptors under mPFC- and insula-to-NAcore inputs sustain aversion-resistant alcohol intake.

Binge ethanol-drinking potentiates corticotropin releasing factor R1 receptor activity in the ventral tegmental area.

BACKGROUND: Corticotropin releasing factor (CRF) and urocortin play an important role in many stress responses and also can regulate ethanol (EtOH) intake. Adaptations in CRF signaling in the central amygdala promote EtOH consumption after long-term EtOH intake in dependent animals and also after brief periods of binge EtOH intake. Thus, even brief episodes of EtOH consumption can alter the function of the CRF system, allowing CRF to regulate EtOH intake. Here, we examined whether brief binge EtOH consumption leads to CRF receptor adaptations within the ventral tegmental area (VTA), a structure involved in signaling rewarding and aversive events and important in the development and expression of drug and alcohol addiction. METHODS: We utilized a mouse model of binge drinking known as drinking in the dark (DID), where C57BL/6J mice drink approximately 6 g/kg in 4 hours and achieve blood EtOH concentrations of approximately 100 mg/dl, which is equivalent to binge drinking in humans. We used ex vivo whole-cell recordings from putative VTA dopamine (DA) neurons to examine CRF regulation of NMDA receptor (NMDAR) currents. We also examined the impact of CRF receptor antagonist injection in the VTA on binge EtOH intake. RESULTS: Ex vivo whole-cell recordings from putative VTA DA neurons showed enhanced CRF-mediated potentiation of NMDAR currents in juvenile mice that consumed EtOH in the DID procedure. CRF-induced potentiation of NMDAR currents in EtOH-drinking mice was blocked by administration of CP-154,526 (3 µM), a selective CRF1 receptor antagonist. Furthermore, intra-VTA infusion of CP-154,526 (1 µg) significantly reduced binge EtOH consumption in adult mice. These results were not due to alterations of VTA NMDAR number or function, suggesting that binge drinking may enhance signaling through VTA CRF1 receptors onto NMDARs. CONCLUSIONS: Altered CRF1 receptor-mediated signaling in the VTA promotes binge-like EtOH consumption in mice, which supports the idea that CRF1 receptors may therefore be a promising pharmacological target for reducing binge drinking in humans.

Ethanol-mediated facilitation of AMPA receptor function in the dorsomedial striatum: implications for alcohol drinking behavior.

We found previously that acute ex vivo as well as repeated cycles of in vivo ethanol exposure and withdrawal, including excessive voluntary consumption of ethanol, produces a long-lasting increase in the activity of NR2B-containing NMDA receptors (NR2B-NMDARs) in the dorsomedial striatum (DMS) of rats (Wang et al., 2010a). Activation of NMDARs is required for the induction of long-term potentiation (LTP) of AMPA receptor (AMPAR)-mediated synaptic response. We therefore examined whether the ethanol-mediated upregulation of NMDAR activity alters the induction of LTP in the DMS. We found that ex vivo acute exposure of striatal slices to, and withdrawal from, ethanol facilitates the induction of LTP in DMS neurons, which is abolished by the inhibition of NR2B-NMDARs. We also report that repeated systemic administration of ethanol causes an NR2B-NMDAR-dependent facilitation of LTP in the DMS. LTP is mediated by the insertion of AMPAR subunits into the synaptic membrane, and we found that repeated systemic administration of ethanol, as well as cycles of excessive ethanol consumption and withdrawal, produced a long-lasting increase in synaptic localization of the GluR1 and GluR2 subunits of AMPARs in the DMS. Importantly, we report that inhibition of AMPARs in the DMS attenuates operant self-administration of ethanol, but not of sucrose. Together, our data suggest that aberrant synaptic plasticity in the DMS induced by repeated cycles of ethanol exposure and withdrawal contributes to the molecular mechanisms underlying the development and/or maintenance of excessive ethanol consumption.

Ethanol-induced increase in Fyn kinase activity in the dorsomedial striatum is associated with subcellular redistribution of protein tyrosine phosphatase α.

In vivo exposure of rodents to ethanol leads to a long-lasting increase in Fyn kinase activity in the dorsomedial striatum (DMS). In this study, we set out to identify a molecular mechanism that contributes to the enhancement of Fyn activity in response to ethanol in the DMS. Protein tyrosine phosphatase α (PTPα) positively regulates the activity of Fyn, and we found that repeated systemic administration or binge drinking of ethanol results in an increase in the synaptic localization of PTPα in the DMS, the same site where Fyn resides. We also demonstrate that binge drinking of ethanol leads to an increase in Fyn activity and to the co-localization of Fyn and PTPα in lipid rafts in the DMS. Finally, we show that the level of tyrosine phosphorylated (and thus active) PTPα in the synaptic fractions is increased in response to contingent or non-contingent exposure of rats to ethanol. Together, our results suggest that the redistribution of PTPα in the DMS into compartments where Fyn resides is a potential mechanism by which the activity of the kinase is increased upon ethanol exposure. Such neuroadaptations could be part of a mechanism that leads to the development of excessive ethanol consumption.

Ethanol-mediated long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum.

We recently found that ethanol-induced long-term facilitation (LTF) of NMDAR activity is mediated by NR2B-NMDARs and is observed in the dorsomedial striatum (DMS) but not in the dorsolateral striatum (DLS). We also showed that repeated administration of ethanol causes a long-lasting increase in NMDAR activity in the DMS, resulting from ethanol-mediated Fyn phosphorylation of NR2B subunits. In this addendum, we report that the different sensitivity of NMDARs to ethanol between the DMS and DLS is not attributed to the abundance of synaptic NR2B-NMDARs or differences in Fyn levels. We further show that LTF is specific for NR2B-, but not NR2A-NMDARs, and that the duration of the in vivo ethanol-mediated increase in NMDAR activity is associated with the period of ethanol exposure, but not with alteration in NR1 or NR2A protein levels. Together, these results suggest that upregulation of NR2B-NMDAR activity by ethanol is selective and that ethanol's effect on NMDAR activity is gradual and cumulative.

Lyn kinase regulates mesolimbic dopamine release: implication for alcohol reward.

We report here that the Src family tyrosine kinase Lyn negatively regulates the release of dopamine (DA) in the mesolimbic system, as well as the rewarding properties of alcohol. Specifically, we show that RNA interference-mediated knockdown of Lyn expression results in an increase in KCl-induced DA release in DAergic-like SH-SY5Y cells, whereas overexpression of a constitutively active form of Lyn (CA-Lyn) leads to a decrease of DA release. Activation of ventral tegmental area (VTA) DAergic neurons results in DA overflow in the nucleus accumbens (NAc), and we found that the evoked release of DA was higher in the NAc of Lyn knock-out (Lyn KO) mice compared with wild-type littermate (Lyn WT) controls. Acute exposure of rodents to alcohol causes a rapid increase in DA release in the NAc, and we show that overexpression of CA-Lyn in the VTA of mice blocked alcohol-induced (2 g/kg) DA release in the NAc. Increase in DA levels in the NAc is closely associated with reward-related behaviors, and overexpression of CA-Lyn in the VTA of mice led to an attenuation of alcohol reward, measured in a conditioned place preference paradigm. Conversely, alcohol place preference was increased in Lyn KO mice compared with Lyn WT controls. Together, our results suggest a novel role for Lyn kinase in the regulation of DA release in the mesolimbic system, which leads to the control of alcohol reward.

Long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum play a crucial role in alcohol consumption and relapse.

A growing number of studies suggest that the development of compulsive drug seeking and taking depends on dorsostriatal mechanisms. We previously observed that ex vivo acute exposure of the dorsal striatum to, and withdrawal from, alcohol induces long-term facilitation (LTF) of the activity of NR2B-containing NMDA receptors (NR2B-NMDARs) in a mechanism that requires the Src family protein tyrosine kinase (PTK), Fyn (Wang et al., 2007). In the present study, we first compared alcohol's actions in rat dorsomedial (DMS) and the dorsolateral (DLS) subregions of the striatum, which differ in their anatomical connectivity and function. We found that alcohol-mediated induction of LTF of NR2B-NMDAR activity is centered in the DMS. Next, we tested whether in vivo exposure of rats to alcohol leads to long-term adaptations of the NMDAR system in the DMS. We observed that repeated daily administration of alcohol results in a long-lasting increase in the activity of the NR2B-NMDARs in the DMS. The same procedure leads to a prolonged activation of Fyn, increased NR2B phosphorylation, and membrane localization of the subunit. Importantly, similar electrophysiological and biochemical modifications were observed in the DMS of rats that consumed large quantities of alcohol. Finally, we show that inhibition of NR2B-NMDARs or Src family PTKs in the DMS, but not in the DLS, significantly decreases operant self-administration of alcohol and reduces alcohol-priming-induced reinstatement of alcohol seeking. Our results suggest that the upregulation of NR2B-NMDAR activity within the DMS by alcohol contributes to the maladaptive synaptic changes that lead to excessive alcohol intake and relapse.

Mind bomb-2 is an E3 ligase that ubiquitinates the N-methyl-D-aspartate receptor NR2B subunit in a phosphorylation-dependent manner.

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity. Post-translational modifications of NMDARs, such as phosphorylation, alter both the activity and trafficking properties of NMDARs. Ubiquitination is increasingly being recognized as another post-translational modification that can alter synaptic protein composition and function. We identified Mind bomb-2 as an E3 ubiquitin ligase that interacts with and ubiquitinates the NR2B subunit of the NMDAR in mammalian cells. The protein-protein interaction and the ubiquitination of the NR2B subunit were found to be enhanced in a Fyn phosphorylation-dependent manner. Immunocytochemical studies reveal that Mind bomb-2 is localized to postsynaptic sites and colocalizes with the NMDAR in apical dendrites of hippocampal neurons. Furthermore, we show that NMDAR activity is down-regulated by Mind bomb-2. These results identify a specific E3 ubiquitin ligase as a novel interactant with the NR2B subunit and suggest a possible mechanism for the regulation of NMDAR function involving both phosphorylation and ubiquitination.

A caspase-3-cleaved fragment of the glial glutamate transporter EAAT2 is sumoylated and targeted to promyelocytic leukemia nuclear bodies in mutant SOD1-linked amyotrophic lateral sclerosis.

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.

Caspase-3 cleaves and inactivates the glutamate transporter EAAT2.

EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS.