Scarisbrick, Isobel A.

ABSTRACT Disruptions in the metabolism of cholesterol and other lipids are strongly implicated in the pathogenesis of neurological disease. The CNS is highly enriched in cholesterol, which is primarily synthesized de novo . Cholesterol synthesis is also rate limiting for myelin regeneration. Given that knockout of the thrombin receptor (Protease Activated Receptor 1 (PAR1)) accelerates myelin regeneration, here we sought to determine the potential regulatory actions of PAR1 in CNS cholesterol and lipid metabolism in the intact adult CNS and during myelin regeneration. We present quantitative PCR and RNAseq evidence from murine spinal cords at the peak of myelination and in adulthood showing PAR1 knockout is associated with increased gene expression for cholesterol biosynthesis (Hmgcs1, Hmgcr, Sqle, and Dhcr7), lipid transport (ApoE, Abca1, and Ldlr), and intracellular processing (Lcat, Npc1, and Npc2) at one or more time points examined. An upregulation of genes involved in the synthesis of other lipids enriched in the myelin membrane, specifically Fa2h, Ugt8a, and Gal3st1, was also observed in PAR1 knockouts. Transcription factors essential for lipid and cholesterol production (Srebf1 and Srebf2) were also increased in PAR1 knockout spinal cords at the postnatal day 21 peak of myelination and at day 45. GC–MS and LC–MS quantification of lipids demonstrated coordinate increases in the abundance of select cholesterol and lipid species in the spinal cords of PAR1 knockout mice, including enrichment of esterified cholesterol, together with sphingomyelins and sphingolipids. Co‐localization of the SREBP1 and SREBP2 transcription factors, as well as HMGCS1, a rate‐limiting enzyme in cholesterol biosynthesis, to glia during remyelination post‐lysolecithin or cuprizone‐mediated demyelination showed a prominent regulatory role for PAR1 in Olig2+ oligodendrocytes. PAR1 knockouts also demonstrated elevated levels of SREBP2 in more mature GST3+ oligodendrocytes and SREBP1 in GFAP+ astrocytes during remyelination post‐lysolecithin. These findings demonstrate novel roles for PAR1 as a regulator of CNS cholesterol and lipid metabolism and its potential as a therapeutic target to increase cholesterol availability to improve myelin regeneration. image

Abstract Objectives Neurofilament light (NfL) chain is a marker of neuroaxonal damage in various neurological diseases. Here we quantitated NfL levels in the cerebrospinal fluid (CSF) and serum from patients with multiple sclerosis (MS) and controls, using the R-PLEX NfL assay, which employs advanced Meso Scale Discovery ® (MSD) electrochemiluminescence (ECL)-based detection technology. Methods NfL was quantitated in samples from 116 individuals from two sites (Ottawa Hospital Research Institute and Mayo Clinic), consisting of patients with MS (n=71) and age- and sex-matched inflammatory neurological controls (n=13) and non-inflammatory controls (n=32). Correlation of NfL levels between CSF and serum was assessed in paired samples in a subset of MS patients and controls (n=61). Additionally, we assessed the correlation between NfL levels obtained with MSD’s R-PLEX ® and Quanterix’s single molecule array (Simoa ® ) assays in CSF and serum (n=32). Results Using the R-PLEX, NfL was quantitated in 99% of the samples tested, and showed a broad range in the CSF (82–500,000 ng/L) and serum (8.84–2,014 ng/L). Nf-L levels in both biofluids correlated strongly (r=0.81, p<0.0001). Lastly, Nf-L measured by MSD’s R-PLEX and Quanterix’s Simoa assays were highly correlated for both biofluids (CSF: r=0.94, p<0.0001; serum: r=0.95, p<0.0001). Conclusions We show that MSD’s R-PLEX NfL assay can reliably quantitate levels of NfL in the CSF and serum from patients with MS and controls, where levels correlate strongly with Simoa.

AbstractKallikrein related peptidase 6 (Klk6) is a secreted serine protease highly expressed in oligodendrocytes and implicated in demyelinating conditions. To gain insights into the significance of Klk6 to oligodendrocyte biology, we investigated the impact of global Klk6 gene knockout on CNS developmental myelination using the spinal cord of male and female mice as a model. Results demonstrate that constitutive loss of Klk6 expression accelerates oligodendrocyte differentiation developmentally, including increases in the expression of myelin proteins such as MBP, PLP and CNPase, in the number of CC‐1+ mature oligodendrocytes, and myelin thickness by the end of the first postnatal week. Co‐ordinate elevations in the pro‐myelinating signaling pathways ERK and AKT, expression of fatty acid 2‐hydroxylase, and myelin regulatory transcription factor were also observed in the spinal cord of 7d Klk6 knockouts. LC/MS/MS quantification of spinal cord lipids showed sphingosine and sphingomyelins to be elevated in Klk6 knockouts at the peak of myelination. Oligodendrocyte progenitor cells (OPCs)‐derived from Klk6 knockouts, or wild type OPCs‐treated with a Klk6 inhibitor (DFKZ‐251), also showed increased MBP and PLP. Moreover, inhibition of Klk6 in OPC cultures enhanced brain derived neurotrophic factor‐driven differentiation. Altogether, these findings suggest that oligodendrocyte‐derived Klk6 may operate as an autocrine or paracrine rheostat, or brake, on pro‐myelinating signaling serving to regulate myelin homeostasis developmentally and in the adult. These findings document for the first time that inhibition of Klk6 globally, or specifically in oligodendrocyte progenitors, is a strategy to increase early stages of oligodendrocyte differentiation and myelin production in the CNS.

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGFR-2 to control vasculogenesis and angiogenesis. Dysregulation of these physiological processes contributes to the pathologies of heart disease, stroke, and cancer. Protein kinase D (PKD) plays a crucial role in the regulation of angiogenesis by modulating endothelial cell proliferation and migration. In human umbilical vein endothelial cells (HUVEC) and human blood outgrowth endothelial cells (BOEC), knockdown of PKD-1 or PKD-2 downregulates VEGFR-2 and significantly inhibits VEGF-induced endothelial cell proliferation and migration. We sought to determine the molecular mechanism through which PKD modulates VEGFR-2 expression. Based on bioinformatics data, activating enhancer binding protein 2 (AP2) binding sites exist within the VEGFR-2 promoter. Thus, we hypothesized PKD may downregulate VEGFR-2 through AP2-mediated transcriptional repression of the VEGFR-2 promoter. Indeed, AP2β binds the VEGFR-2 promoter upon PKD knockdown in HUVEC as evident by chromatin immunoprecipitation assay. Luciferase reporter assays using serial deletions of AP2β binding sites within the VEGFR-2 promoter revealed transcriptional activity negatively correlated with the number of AP2β binding sites, thus confirming negative regulation of VEGFR-2 transcription by AP2β. Next, using siRNA, we demonstrated that upregulation of AP2β decreased VEGFR-2 expression and loss of AP2β enhanced VEGFR-2 expression. In vivo studies confirmed this finding as we observed increased VEGFR-2 immunostaining in the dorsal horn of the spinal cord of embryonic day 13 AP2β knockout mice. We hypothesize that PKD directly regulates AP2β function by serine phosphorylation and ongoing studies are being conducted to determine phosphorylation sites in AP2β directly regulated by PKD. Taken together, we demonstrate AP2β negatively regulates VEGFR-2 transcription and VEGFR-2 is a major downstream target of PKD. Our findings describing how PKD regulates angiogenesis may contribute to the development of therapies to improve the clinical outcome of patients afflicted by heart disease, stroke, and cancer.

The cardinal signs of systemic metabolic dysfunction, including insulin resistance, hypertension and a systemic pro‐inflammatory state are risk factors for the development of cardiovascular disease, type 2 diabetes, and neurodegenerative conditions, but there is limited information regarding the impact on recovery after spinal cord injury (SCI). To address this gap, we investigated the impact of systemic insulin resistance generated by consumption of a diet high in fat and sucrose, often referred to as a Western Diet, on neuropathophysiological outcomes in a murine compression model of incomplete SCI. Ten‐week‐old female C57BL6 mice were provided a regular diet (RD) or a diet high in fat and sucrose (HFHS) for 7 weeks prior to experimental compression SCI. Mice consuming a Western Diet showed signs of systemic insulin resistance such as evaluated blood glucose. In addition, signs of insulin resistance within the substance of the spinal cord were also observed, including reductions in the abundance of insulin‐like growth factor 1 receptor (IGF‐1R). Impairments in motor recovery evaluated by BMS score and subscore were present in mice consuming HFHS at 14 and 30 days post injury (dpi). Consumption of a diet rich in fat and sucrose also impaired the mean maximal angle achieved on the inclined plane test and resulted in significant impairments in bladder release. Immunochemical analysis of the spinal cord revealed a number of differences in markers of neural injury between the two groups. First, mice consuming HFHS exhibited significant increases in astrogliosis measured by glial fibrillary acidic protein (GFAP) prior to injury, compared to mice consuming a RD. In fact, the prominent increases in GFAP observed in the intact spinal cord of mice consuming HFHS were equivalent to those observed after SCI in mice consuming a regular chow. In the subacute period, markers of microglial/monocyte activation were elevated in all mice with SCI; however, the increase was substantially greater in the spinal cords of mice consuming HFHS. The spinal cord white matter of mice consuming HFHS also showed reductions in the number of oligodendrocyte progenitor and mature myelinating cells prior to SCI and HFHS‐associated reductions in myelin producing cells persisted at 14 and 30 dpi. In addition, loss of serotonergic axons in the spinal cord after SCI was exacerbated in mice consuming HFHS. Growth associated protein 43 (GAP43), a marker of axonal growth cones was elevated after SCI in the spinal cords of mice consuming a RD or a HFHS diet; however, the magnitude of the increase in growth cones was severely impaired in mice consuming HFHS. These findings suggest that consumption of a Western Diet increases the risk of insulin resistance within the central nervous system and impairs neurobehavioral recovery after SCI. These studies highlight insulin resistance as a new mechanism that limits nervous system repair after SCI and as a potential target for intervention to improve functional outcomes. Support or Funding Information NIH R01 NS052741 and The Minnesota State SCI and TBI Research Grant Program. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

We recently demonstrated that high dietary fat (HFD) consumption results in a dramatic loss of myelinating cells and their progenitors in the adult CNS (Yoon et al., 2016). However, the mechanism(s) by which HFD drives dysmyelination are unknown. Since epidemiological studies indicate that metabolic syndrome is a key risk factor for multiple sclerosis (MS), a better understanding of how HFD contributes to oligodendrocyte loss and the capacity for myelin regeneration has the potential to highlight new treatment targets. In mice fed HFD, we found significant decreases in vertical activity by 4 wk and significant changes in ambulatory and stereotypy movements by 12 wk in the Open Field Test. HFD‐fed mice also spent less time in the center of the field as opposed to the perimeter, indicating an anxious phenotype. Mitochondrial isolated from the spinal cord of mice consuming HFD for 4 wk had significantly higher state 3 and state 4 respiration as determined by high‐resolution respirometry. However, by 12 wk, state 4 mitochondrial respiration was significantly less in the HFD group. Targeted LC‐LC‐mass spectroscopy approaches demonstrated that Citric Acid Cycle intermediates are also depleted in the spinal cord of mice consuming HFD for 12 wk compared to controls. Changes in genes related to mitochondrial morphology, quality control processes, and oxidative stress response were also found. In addition, there were significantly fewer Olig2+ cells and increased signs of lipid peroxidation products (4‐Hydroxynonenal‐immunoreactivity) in the corpus callosum of HFD consuming mice compared to those fed a regular diet. Supporting in vivo findings that oligodendrocytes are reduced in the spinal cord of mice consuming HFD, we observed increased mitochondrial fragmentation and a reduced oxygen consumption rate in oligodendrocyte precursors cultured with palmitic acid. Astrocytes however, significantly increased oxygen consumption following exposure to palmitic acid. Together, these findings suggest that changes in the adult spinal cord in response to HFD consumption creates an environment that is less conducive to neural repair by impacting mitochondria function and morphology and impairing oligodendrocyte function. To determine any impact of HFD on myelin regeneration, studies are ongoing to examine myelin regeneration using a murine model of Cuprizone‐mediated demyelination. Neurobehavioral assessments to date reveal significant interactions between HFD and myelin injury and repair. Understanding the cellular environment in response to dietary factors such as HFD consumption can help to guide care for individuals with MS and other conditions in which myelin loss plays a pathological role and has potential to identify new targets for intervention. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Trauma to the CNS generates a proteolytic imbalance that contributes to secondary injury, including axonopathy and neuron degeneration; however the mechanism by which this occurs is not well understood. Kallikrein 6 (KLK6) is a serine protease implicated in neurotoxicity and in these studies we investigate the role of protease activated receptors (PARs) in mediating these effects. We demonstrate KLK6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and ‐2, are each elevated in a murine experimental spinal cord injury at acute or subacute time points. KLK6 and thrombin each promoted cerebellar granule neuron toxicity and potentiated glutamate excitotoxicity in vitro. Importantly, neural injury in each case was reduced or blocked in neurons derived from PAR1 deficient mice. KLK6 was shown to activate ERK in cerebellar neurons in a PI3K and MEK‐dependent fashion. Novel lipopeptide inhibitors specific for PAR1 or PAR2, in addition to neurons derived from PAR1 deficient mice were utilized to demonstrate both receptors play an important role in KLK6‐elicited ERK signaling and in the activation of the proapoptotic protein Bim. Taken together these data point to novel KLK6‐PAR1 and ‐PAR2 mediated signaling cascades in CNS neurons that are positioned to participate in neuron degeneration and glutamate‐mediated excitotoxicity in cases of CNS injury. Supported by NIH R01 NS052741

A remarkable feature of the expression of kallikrein 6 (KLK6) in the CNS is its abundance in oligodendroglia and neurons, but relative lack of expression in astrocytes. We have shown in cases of CNS injury however, that KLK6 is induced in reactive astrocytes and exhibits prolonged expression in astroglial scar tissue in cases of spinal cord injury, multiple sclerosis and glioblastoma multiforme. In this study, we address the hypothesis that KLK6 plays a role in the promotion of astrogliosis and that this occurs by selective activation of protease activated receptors (PARs). Using primary murine astrocytes we show KLK6 promotes rapid transformation from an epithelioid to a stellate morphology and secretion of interleukin 6, each a hallmark of astrogliosis. The ability of KLK6 to promote stellation was shown to occur as early as 4 hours after a single pulse of KLK6, to be dose‐dependent, and to require activation of PKC and AKT signaling cascades. The stellation promoting activities of KLK6 were further shown to depend at least in part on activation of the thrombin receptor PAR1, since these effects were reduced in astrocytes derived from PAR1‐deficient mice and by a PAR1 specific inhibitor, SCH79797. These results point to novel roles for KLK6‐mediated PAR signaling in the spectrum of events that culminate in astrogliosis. This work was supported by NIH R01 NS052741.

AbstractExcessive activation of the thrombin receptor, protease activated receptor 1 (PAR1) is implicated in diverse neuropathologies from neurodegenerative conditions to neurotrauma. PAR1 knockout mice show improved outcomes after experimental spinal cord injury (SCI), however information regarding the underpinning cellular and molecular mechanisms is lacking. Here we demonstrate that genetic blockade of PAR1 in female mice results in improvements in sensorimotor co‐ordination after thoracic spinal cord lateral compression injury. We document improved neuron preservation with increases in Synapsin‐1 presynaptic proteins and GAP43, a growth cone marker, after a 30 days recovery period. These improvements were coupled to signs of enhanced myelin resiliency and repair, including increases in the number of mature oligodendrocytes, their progenitors and the abundance of myelin basic protein. These significant increases in substrates for neural recovery were accompanied by reduced astrocyte (Serp1) and microglial/monocyte (CD68 and iNOS) pro‐inflammatory markers, with coordinate increases in astrocyte (S100A10 and Emp1) and microglial (Arg1) markers reflective of pro‐repair activities. Complementary astrocyte‐neuron co‐culture bioassays suggest astrocytes with PAR1 loss‐of‐function promote both neuron survival and neurite outgrowth. Additionally, the pro‐neurite outgrowth effects of switching off astrocyte PAR1 were blocked by inhibiting TrkB, the high affinity receptor for brain derived neurotrophic factor. Altogether, these studies demonstrate unique modulatory roles for PAR1 in regulating glial‐neuron interactions, including the capacity for neurotrophic factor signaling, and underscore its position at neurobiological intersections critical for the response of the CNS to injury and the capacity for regenerative repair and restoration of function.