Key features of the innate immune response is mediated by the immunoproteasome in microglia.

Microglia are the resident immune cells of the central nervous system (CNS). We and others have shown that the inflammatory response of microglia is partially regulated by the immunoproteasome, an inducible form of the proteasome responsible for the generation of major histocompatibility complex (MHC) class I epitopes. While the role of the proteasome in the adaptive immune system is well established, emerging evidence suggests the immunoproteasome may have discrete functions in the innate immune response. Here, we show that inhibiting the immunoproteasome reduces the IFNγ-dependent induction of complement activator C1q, suppresses phagocytosis, and alters the cytokine expression profile in a microglial cell line and microglia derived from human inducible pluripotent stem cells. Moreover, we show that the immunoproteasome regulates the degradation of IκBα, a modulator of NF-κB signaling. Finally, we demonstrate that NADH prevents induction of the immunoproteasome, representing a potential pathway to suppress immunoproteasome-dependent immune responses.

SMN-deficiency disrupts SERCA2 expression and intracellular Ca2+ signaling in cardiomyocytes from SMA mice and patient-derived iPSCs.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed, and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T50 for Ca2+ sequestration increased to 146 ± 4 ms in SMN-deficient cardiomyocytes from 126 ± 4 ms in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent-derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca2+ cycling dysregulation.

Neuroinflammatory astrocytes generated from cord blood-derived human induced pluripotent stem cells.

BACKGROUND: Astrocytes respond to central nervous system (CNS) injury and disease by transforming to a reactive astrogliosis cell state that can contribute to either CNS dysfunction or repair. Neuroinflammation is a powerful driver of a harmful A1 astrogliosis phenotype associated with in vitro neurotoxicity and histopathology in human neurodegenerative diseases. Here we report a protocol for the rapid development of a human cell culture model of neuroinflammatory astrogliosis using induced pluripotent stem cells (iPSCs). METHODS: Using RNA sequencing and in vitro cell assays, we measured transcriptional and cellular effects of chronic exposure of human iPSC-derived astrocytes to the cytokines TNFα (tumor necrosis factor alpha) or IL-1ß (interleukin-1 beta). RESULTS: We show TNFα and IL-1ß induce pro-inflammatory gene signatures but by widely different magnitudes. TNFα treatment results in 606 differential expressed genes, the suppression of glutamate-uptake, and increased phagocytic activity in astrocyte cultures. In contrast, IL-1ß effects are attenuated to 33 differential expressed genes and no significant effects on glutamate-uptake or increased phagocytic activity. CONCLUSION: Our approach demonstrates a rapid tool for modeling neuroinflammatory human astrocytic responses in nervous system trauma and disease. In particular, we reveal a model for robust TNFα-induced human astrogliosis suitable for the study of neurotoxic A1 astrocytes.

Iron accentuated reactive oxygen species release by NADPH oxidase in activated microglia contributes to oxidative stress in vitro.

BACKGROUND: Excessive iron contributes to oxidative stress after central nervous system injury. NADPH oxidase (NOX) enzymes are upregulated in microglia after pro-inflammatory activation and contribute to oxidative stress. The relationship between iron, microglia, NOX, and oxidative stress is currently unclear. METHODS: We evaluated the effects of iron on lipopolysaccharide (LPS)-activated microglia and its secondary effect within neuronal co-cultures. Further, NOX2 and four specific inhibitors were tested to evaluate the relationship with the reactive oxygen species (ROS)-producing enzymes. RESULTS: An iron dose-dependent increase in ROS production among microglia treated with LPS was identified. Interestingly, despite this increase in ROS, inflammatory polarization alterations were not detected among the microglia after exposure to iron and LPS. Co-culture experimentation between primary neurons and exposed microglia (iron and LPS) significantly reduced neuronal cell number at 24 h, suggesting a profound neurotoxic effect despite the lack of a change in polarization phenotype. NOX2 and NOX4 inhibition significantly reduced ROS production among microglia exposed to iron and LPS and reduced neuronal damage and death in response to microglial co-culture. CONCLUSIONS: In conclusion, iron significantly increased ROS production and neurotoxicity without exacerbating LP-activated microglia phenotype in vitro, suggesting that iron contributes to microglia-related oxidative stress, and this may be a viable therapeutic target for injury or neurodegeneration. Further, this study highlights both NOX2 and NOX4 as potential therapeutic targets in the treatment of iron-induced microglia-related inflammation and neurotoxicity.

Aging alters glucose uptake in the naïve and injured rodent spinal cord.

Aging results in increased activation of inflammatory glial cells and decreased neuronal viability following spinal cord injury (SCI). Metabolism and transport of glucose is also decreased with age, although the influence of age on glucose transporter (GLUT) expression or glucose uptake in SCI is currently unknown. We therefore performed [18F]Fluorodeoxyglucose (FDG) PET imaging of young (3 month) and middle-aged (12 month) rats. Glucose uptake in middle-aged rats was decreased compared to young rats at baseline, followed by increased uptake 14 days post contusion SCI. qRT-PCR and protein analysis revealed an association between 14 day glucose uptake and 14 day post-injury inflammation. Further, gene expression analysis of neuron-specific GLUT3 and non-specific GLUT4 (present on glial cells) revealed an inverse relationship between GLUT3/4 gene expression and glucose uptake patterns. Protein expression revealed increased GLUT3 in 3 month rats only, consistent with age related decreases in glucose uptake, and increased GLUT4 in 12 month rats only, consistent with age related increases in inflammatory activity and glucose uptake. Inconsistencies between gene and protein suggest an influence of age-related impairment of translation and/or protein degradation. Overall, our findings show that age alters glucose uptake and GLUT3/4 expression profiles before and after SCI, which may be dependent on level of inflammatory response, and may suggest a therapeutic avenue in addressing glucose uptake in the aging population.

Age exacerbates microglial activation, oxidative stress, inflammatory and NOX2 gene expression, and delays functional recovery in a middle-aged rodent model of spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) among people over age 40 has been steadily increasing since the 1980s and is associated with worsened outcome than injuries in young people. Age-related increases in reactive oxygen species (ROS) are suggested to lead to chronic inflammation. The NADPH oxidase 2 (NOX2) enzyme is expressed by microglia and is a primary source of ROS. This study aimed to determine the effect of age on inflammation, oxidative damage, NOX2 gene expression, and functional performance with and without SCI in young adult (3 months) and middle-aged (12 months) male rats. METHODS: Young adult and middle-aged rats were assessed in two groups-naïve and moderate contusion SCI. Functional recovery was determined by weekly assessment with the Basso, Beattie, and Breshnahan general motor score (analyzed two-way ANOVA) and footprint analysis (analyzed by Chi-square analysis). Tissue was analyzed for markers of oxidative damage (8-OHdG, Oxyblot, and 3-NT), microglial-related inflammation (Iba1), NOX2 component (p47PHOX, p22PHOX, and gp91PHOX), and inflammatory (CD86, CD206, TNFα, and NFκB) gene expression (all analyzed by unpaired Student's t test). RESULTS: In both naïve and injured aged rats, compared to young rats, tissue analysis revealed significant increases in 8-OHdG and Iba1, as well as inflammatory and NOX2 component gene expression. Further, injured aged rats showed greater lesion volume rostral and caudal to the injury epicenter. Finally, injured aged rats showed significantly reduced Basso-Beattie-Bresnahan (BBB) scores and stride length after SCI. CONCLUSIONS: These results show that middle-aged rats demonstrate increased microglial activation, oxidative stress, and inflammatory gene expression, which may be related to elevated NOX2 expression, and contribute to worsened functional outcome following injury. These findings are essential to elucidating the mechanisms of age-related differences in response to SCI and developing age-appropriate therapeutics.

The role of the immunoproteasome in interferon-γ-mediated microglial activation.

Microglia regulate the brain microenvironment by sensing damage and neutralizing potentially harmful insults. Disruption of central nervous system (CNS) homeostasis results in transition of microglia to a reactive state characterized by morphological changes and production of cytokines to prevent further damage to CNS tissue. Immunoproteasome levels are elevated in activated microglia in models of stroke, infection and traumatic brain injury, though the exact role of the immunoproteasome in neuropathology remains poorly defined. Using gene expression analysis and native gel electrophoresis we characterize the expression and assembly of the immunoproteasome in microglia following interferon-gamma exposure. Transcriptome analysis suggests that the immunoproteasome regulates multiple features of microglial activation including nitric oxide production and phagocytosis. We show that inhibiting the immunoproteasome attenuates expression of pro-inflammatory cytokines and suppresses interferon-gamma-dependent priming of microglia. These results imply that targeting immunoproteasome function following CNS injury may attenuate select microglial activity to improve the pathophysiology of neurodegenerative conditions or the progress of inflammation-mediated secondary injury following neurotrauma.

ML372 blocks SMN ubiquitination and improves spinal muscular atrophy pathology in mice.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. SMA results from insufficient levels of the survival motor neuron (SMN) protein, and studies in animal models of the disease have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient-derived cells. We show here that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice.

Combining enriched environment and induced pluripotent stem cell therapy results in improved cognitive and motor function following traumatic brain injury.

PURPOSE: Despite advances towards potential clinically viable therapies there has been only limited success in improving functional recovery following traumatic brain injury (TBI). In rats, exposure to an enriched environment (EE) improves learning and fosters motor skill development. Induced pluripotent stem cells (iPSC) have been shown to survive transplantation and influence the recovery process. The current study evaluated EE and iPSC as a polytherapy for remediating cognitive deficits following medial frontal cortex (mFC) controlled cortical impact (CCI) injury. METHODS: Sixty adult male rats received a midline mFC CCI or sham injury and were randomly placed in either EE or standard environment (SE). Seven days post-injury rats received bilateral transplantation of iPSCs or media. Behavioral measures were conducted throughout the remainder of the study. Following behavioral analysis, brains were extracted and prepared for histological analysis. RESULTS: Open-field data revealed that combined therapy resulted in typical Sham/EE activity rearing patterns by the conclusion of the study. On the Vermicelli Handling task, rats with EE/iPSC polytherapy performed better than media-treated rats. Furthermore, rats treated with polytherapy performed equivalently to Sham/EE rats on the Morris water maze. Proficiency on the Rotarod was consistently better in EE when compared to SE counterparts. Confocal microscopy confirmed that iPSCs survived and migrated away from the transplantation site. CONCLUSIONS: Overall, EE or iPSC therapy improved cognition and motor performance, however, full cognitive restoration was seen only with the EE/iPSC treatment. These data suggest that EE/iPSC therapy should be explored as a potential, clinically relevant, treatment for TBI.

Post-operative environmental enrichment improves spatial and motor deficits but may not ameliorate anxiety- or depression-like symptoms in rats following traumatic brain injury.

PURPOSE: Anxiety-like (ANX) and depression-like (DEP) symptoms are common consequences of traumatic brain injury (TBI). Environmental enrichment (EE) attenuates many deficits, though its impact on ANX and DEP symptoms has yet to be described. METHODS: Adult male Long-Evans rats were subject to a medial frontal cortex (mFC) cortical impact injury or sham preparation, then placed into EE or standard housing (SE). ANX symptoms were analyzed using the open field test (OFT) and elevated plus maze (EPM). The forced swim task (FST) and sucrose consumption task (SCT) were used to quantify DEP symptoms. In order to measure changes in spatial learning and motor performance, the Barnes maze (BM) and rotor rod (RR) were utilized. RESULTS: Damage to the mFC resulted in functional losses in motor and cognitive behavior and an increase in ANX and DEP symptoms. Placement of injured rats into the EE improves motor functioning after TBI and resulted in an decreased latency to locate the escape box in the BM. Though the application of an EE attenuated deficits in BM and RR performance, the ANX and DEP behavioral symptoms persisted. CONCLUSIONS: Additional therapeutic approaches paired with EE may be necessary to address all functional changes post-TBI. Additionally, no single behavioral assessment appears to clearly identify symptoms of ANX or DEP in rats following TBI, however utilizing multiple tests can be potentially confounding.