Roles of neuronal toll-like receptors in neuropathic pain and central nervous system injuries and diseases.

Toll-like receptors (TLRs) are innate immune receptors that are expressed in immune cells as well as glia and neurons of the central and peripheral nervous systems. They are best known for their role in the host defense in response to pathogens and for the induction of inflammation in infectious and non-infectious diseases. In the central nervous system (CNS), TLRs modulate glial and neuronal functions as well as innate immunity and neuroinflammation under physiological or pathophysiological conditions. The majority of the studies on TLRs in CNS pathologies investigated their overall contribution without focusing on a particular cell type, or they analyzed TLRs in glia and infiltrating immune cells in the context of neuroinflammation and cellular activation. The role of neuronal TLRs in CNS diseases and injuries has received little attention and remains underappreciated. The primary goal of this review is to summarize findings demonstrating the pivotal and unique roles of neuronal TLRs in neuropathic pain, Alzheimer's disease, Parkinson's disease and CNS injuries. We discuss how the current findings warrant future investigations to better define the specific contributions of neuronal TLRs to these pathologies. We underline the paucity of information regarding the role of neuronal TLRs in other neurodegenerative, demyelinating, and psychiatric diseases. We draw attention to the importance of broadening research on neuronal TLRs in view of emerging evidence demonstrating their distinctive functional properties.

Toll-like Receptor 4 Signaling in Neurons Enhances Calcium-Permeable α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor Currents and Drives Post-Traumatic Epileptogenesis.

OBJECTIVE: Traumatic brain injury is a major risk factor for acquired epilepsies, and understanding the mechanisms underlying the early pathophysiology could yield viable therapeutic targets. Growing evidence indicates a role for inflammatory signaling in modifying neuronal excitability and promoting epileptogenesis. Here we examined the effect of innate immune receptor Toll-like receptor 4 (TLR4) on excitability of the hippocampal dentate gyrus and epileptogenesis after brain injury. METHODS: Slice and in vivo electrophysiology and Western blots were conducted in rats subject to fluid percussion brain injury or sham injury. RESULTS: The studies identify that TLR4 signaling in neurons augments dentate granule cell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (CP-AMPAR) currents after brain injury. Blocking TLR4 signaling in vivo shortly after brain injury reduced dentate network excitability and seizure susceptibility. When blocking of TLR4 signaling after injury was delayed, however, this treatment failed to reduce postinjury seizure susceptibility. Furthermore, TLR4 signal blocking was less efficacious in limiting seizure susceptibility when AMPAR currents, downstream targets of TLR4 signaling, were transiently enhanced. Paradoxically, blocking TLR4 signaling augmented both network excitability and seizure susceptibility in uninjured controls. Despite the differential effect on seizure susceptibility, TLR4 antagonism suppressed cellular inflammatory responses after injury without impacting sham controls. INTERPRETATION: These findings demonstrate that independently of glia, the immune receptor TLR4 directly regulates post-traumatic neuronal excitability. Moreover, the TLR4-dependent early increase in dentate excitability is causally associated with epileptogenesis. Identification and selective targeting of the mechanisms underlying the aberrant TLR4-mediated increase in CP-AMPAR signaling after injury may prevent epileptogenesis after brain trauma. ANN NEUROL 2020;87:497-515.

Role of astroglial toll-like receptors (TLRs) in central nervous system infections, injury and neurodegenerative diseases.

Central nervous system (CNS) innate immunity plays essential roles in infections, neurodegenerative diseases, and brain or spinal cord injuries. Astrocytes and microglia are the principal cells that mediate innate immunity in the CNS. Pattern recognition receptors (PRRs), expressed by astrocytes and microglia, sense pathogen-derived or endogenous ligands released by damaged cells and initiate the innate immune response. Toll-like receptors (TLRs) are a well-characterized family of PRRs. The contribution of microglial TLR signaling to CNS pathology has been extensively investigated. Even though astrocytes assume a wide variety of key functions, information about the role of astroglial TLRs in CNS disease and injuries is limited. Because astrocytes display heterogeneity and exhibit phenotypic plasticity depending on the effectors present in the local milieu, they can exert both detrimental and beneficial effects. TLRs are modulators of these paradoxical astroglial properties. The goal of the current review is to highlight the essential roles played by astroglial TLRs in CNS infections, injuries and diseases. We discuss the contribution of astroglial TLRs to host defense as well as the dissemination of viral and bacterial infections in the CNS. We examine the link between astroglial TLRs and the pathogenesis of neurodegenerative diseases and present evidence showing the pivotal influence of astroglial TLR signaling on sterile inflammation in CNS injury. Finally, we define the research questions and areas that warrant further investigations in the context of astrocytes, TLRs, and CNS dysfunction.

Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury.

BACKGROUND: The recruitment of immune system cells into the central nervous system (CNS) has a profound effect on the outcomes of injury and disease. Glia-derived chemoattractants, including chemokines, play a pivotal role in this process. In addition, cytokines and chemokines influence the phenotype of infiltrating immune cells. Depending on the stimuli present in the local milieu, infiltrating macrophages acquire the classically activated M1 or alternatively activated M2 phenotypes. The polarization of macrophages into detrimental M1 versus beneficial M2 phenotypes significantly influences CNS pathophysiology. Earlier studies indicated that a toll-like receptor 9 (TLR9) antagonist modulates astrocyte-derived cytokine and chemokine release. However, it is not known whether these molecular changes affect astrocyte-induced chemotaxis and polarization of macrophages. The present studies were undertaken to address these issues. METHODS: The chemotaxis and polarization of mouse peritoneal macrophages by spinal cord astrocytes were evaluated in a Transwell co-culture system. Arrays and ELISA were utilized to quantify chemokines in the conditioned medium (CM) of pure astrocyte cultures. Immunostaining for M1- and M2-specific markers characterized the macrophage phenotype. The percentage of M2 macrophages at the glial scar was determined by stereological approaches in mice sustaining a mid-thoracic spinal cord contusion injury (SCI) and intrathecally treated with oligodeoxynucleotide 2088 (ODN 2088), the TLR9 antagonist. Statistical analyses used two-tailed independent-sample t-test and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p value < 0.05 was considered to be statistically significant. RESULTS: ODN 2088-treated astrocytes significantly increased the chemotaxis of peritoneal macrophages via release of chemokine (C-C motif) ligand 1 (CCL1). Vehicle-treated astrocytes polarized macrophages into the M2 phenotype and ODN 2088-treated astrocytes promoted further M2 polarization. Reduced CCL2 and CCL9 release by astrocytes in response to ODN 2088 facilitated the acquisition of the M2 phenotype, suggesting that CCL2 and CCL9 are negative regulators of M2 polarization. The percentage of M2 macrophages at the glial scar was higher in mice sustaining a SCI and receiving ODN 2088 treatment as compared to vehicle-treated injured controls. CONCLUSIONS: TLR9 antagonism could create a favorable environment during SCI by supporting M2 macrophage polarization and chemotaxis via modulation of astrocyte-to-macrophage signals.

Pathological pain processing in mouse models of multiple sclerosis and spinal cord injury: contribution of plasma membrane calcium ATPase 2 (PMCA2).

BACKGROUND: Neuropathic pain is often observed in individuals with multiple sclerosis (MS) and spinal cord injury (SCI) and is not adequately alleviated by current pharmacotherapies. A better understanding of underlying mechanisms could facilitate the discovery of novel targets for therapeutic interventions. We previously reported that decreased plasma membrane calcium ATPase 2 (PMCA2) expression in the dorsal horn (DH) of healthy PMCA2+/- mice is paralleled by increased sensitivity to evoked nociceptive pain. These studies suggested that PMCA2, a calcium extrusion pump expressed in spinal cord neurons, plays a role in pain mechanisms. However, the contribution of PMCA2 to neuropathic pain processing remains undefined. The present studies investigated the role of PMCA2 in neuropathic pain processing in the DH of wild-type mice affected by experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and following SCI. METHODS: EAE was induced in female and male C57Bl/6N mice via inoculation with myelin oligodendrocyte glycoprotein fragment 35-55 (MOG35-55) emulsified in Complete Freund's Adjuvant (CFA). CFA-inoculated mice were used as controls. A severe SC contusion injury was induced at thoracic (T8) level in female C57Bl/6N mice. Pain was evaluated by the Hargreaves and von Frey filament tests. PMCA2 levels in the lumbar DH were analyzed by Western blotting. The effectors that decrease PMCA2 expression were identified in SC neuronal cultures. RESULTS: Increased pain in EAE and SCI was paralleled by a significant decrease in PMCA2 levels in the DH. In contrast, PMCA2 levels remained unaltered in the DH of mice with EAE that manifested motor deficits but not increased pain. Interleukin-1ß (IL-1ß), tumor necrosis factor α (TNFα), and IL-6 expression were robustly increased in the DH of mice with EAE manifesting pain, whereas these cytokines showed a modest increase or no change in mice with EAE in the absence of pain. Only IL-1ß decreased PMCA2 levels in pure SC neuronal cultures through direct actions. CONCLUSIONS: PMCA2 is a contributor to neuropathic pain mechanisms in the DH. A decrease in PMCA2 in DH neurons is paralleled by increased pain sensitivity, most likely through perturbations in calcium signaling. Interleukin-1ß is one of the effectors that downregulates PMCA2 by acting directly on neurons.

Toll-like receptor 9 antagonism modulates astrocyte function and preserves proximal axons following spinal cord injury.

Increasing evidence indicates that innate immune receptors play important, yet controversial, roles in traumatic central nervous system (CNS) injury. Despite many advances, the contributions of toll-like receptors (TLRs) to spinal cord injury (SCI) remain inadequately defined. We previously reported that a toll-like receptor 9 (TLR9) antagonist, oligodeoxynucleotide 2088 (ODN 2088), administered intrathecally, improves the functional and histopathological outcomes of SCI. However, the molecular and cellular changes that occur at the injury epicenter following ODN 2088 treatment are not completely understood. Following traumatic SCI, a glial scar, consisting primarily of proliferating reactive astrocytes, forms at the injury epicenter and assumes both beneficial and detrimental roles. Increased production of chondroitin sulfate proteoglycans (CSPGs) by reactive astrocytes inhibits the regeneration of injured axons. Astrocytes express TLR9, which can be activated by endogenous ligands released by damaged cells. It is not yet known how TLR9 antagonism modifies astrocyte function at the glial scar and how this affects axonal preservation or re-growth following SCI. The present studies were undertaken to address these issues. We report that in female mice sustaining a severe mid-thoracic (T8) contusion injury, the number of proliferating astrocytes in regions rostral and caudal to the lesion border increased significantly by 30- and 24-fold, respectively, compared to uninjured controls. Intrathecal ODN 2088 treatment significantly reduced the number of proliferating astrocytes by 60% in both regions. This effect appeared to be, at least partly, mediated through the direct actions of ODN 2088 on astrocytes, since the antagonist decreased proliferation in pure SC astrocyte cultures by preventing the activation of the Erk/MAPK signaling pathway. In addition, CSPG immunoreactivity at the lesion border was more pronounced in vehicle-treated injured mice compared to uninjured controls and was significantly reduced following administration of ODN 2088 to injured mice. Moreover, ODN 2088 significantly decreased astrocyte migration in an in vitro scratch-wound assay. Anterograde tracing and quantification of corticospinal tract (CST) axons in injured mice, indicated that ODN 2088 preserves proximal axons. Taken together, these findings suggest that ODN 2088 modifies the glial scar and creates a milieu that fosters axonal protection at the injury site.

Impaired sensitivity to pain stimuli in plasma membrane calcium ATPase 2 (PMCA2) heterozygous mice: a possible modality- and sex-specific role for PMCA2 in nociception.

Plasma membrane calcium ATPase 2 (PMCA2) is a calcium pump that plays important roles in neuronal function. Although it is expressed in pain-associated regions of the CNS, including in the dorsal horn (DH), its contribution to pain remains undefined. The present study assessed the role of PMCA2 in pain responsiveness and the link between PMCA2 and glutamate receptors, GABA receptors (GABARs), and glutamate transporters that have been implicated in pain processing in the DH of adult female and male PMCA2+/+ and PMCA2+/- mice. Behavioral assays evaluated mechanical and thermal pain responsiveness. Mechanical sensitivity was significantly increased by 52% and heat sensitivity was reduced by 29% in female, but not male, PMCA2+/- mice compared with PMCA2+/+ controls. There were female-specific changes in metabotropic glutamate receptor 1, NMDA receptor 2A, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR1, GABABR1, and GABABR2 levels, whereas metabotropic glutamate receptor 5, NMDA receptor 2B, GluR2, and GABAARα2 levels were not altered. Glutamate aspartate transporter levels were higher and glial glutamate transporter 1 levels were lower in the DH of female, but not male, PMCA2+/- mice. These findings indicate a novel role for PMCA2 in modality- and sex-dependent pain responsiveness. Female-specific molecular changes potentially account for the altered pain responses.-Khariv, V., Ni, L., Ratnayake, A., Sampath, S., Lutz, B. M., Tao, X.-X., Heary, R. F., Elkabes, S. Impaired sensitivity to pain stimuli in plasma membrane calcium ATPase 2 (PMCA2) heterozygous mice: a possible modality- and sex-specific role for PMCA2 in nociception.

Toll like receptor 9 antagonism modulates spinal cord neuronal function and survival: Direct versus astrocyte-mediated mechanisms.

Toll like receptors (TLRs) are expressed by cells of the immune system and mediate the host innate immune responses to pathogens. However, increasing evidence indicates that they are important contributors to central nervous system (CNS) function in health and in pathological conditions involving sterile inflammation. In agreement with this idea, we have previously shown that intrathecal administration of a TLR9 antagonist, cytidine-phosphate-guanosine oligodeoxynucleotide 2088 (CpG ODN 2088), ameliorates the outcomes of spinal cord injury (SCI). Although these earlier studies showed a marked effect of CpG ODN 2088 on inflammatory cells, the expression of TLR9 in spinal cord (SC) neurons and astrocytes suggested that the antagonist exerts additional effects through direct actions on these cells. The current study was undertaken to assess the direct effects of CpG ODN 2088 on SC neurons, astrocytes and astrocyte-neuron interactions, in vitro. We report, for the first time, that inhibition of TLR9 in cultured SC neurons alters their function and confers protection against kainic acid (KA)-induced excitotoxic death. Moreover, the TLR9 antagonist attenuated the KA-elicited endoplasmic reticulum (ER) stress response in neurons, in vitro. CpG ODN 2088 also reduced the transcript levels and release of chemokine (C-X-C) motif ligand 1 (CXCL1) and monocyte chemotactic protein 1 (MCP-1) by astrocytes and it diminished interleukin-6 (IL-6) release without affecting transcript levels in vitro. Conditioned medium (CM) of CpG ODN 2088-treated astroglial cultures decreased the viability of SC neurons compared to CM of vehicle-treated astrocytes. However, this toxicity was not observed when astrocytes were co-cultured with neurons. Although CpG ODN 2088 limited the survival-promoting effects of astroglia, it did not reduce neuronal viability compared to controls grown in the absence of astrocytes. We conclude that the TLR9 antagonist acts directly on both SC neurons and astrocytes. Neuronal TLR9 antagonism confers protection against excitotoxic death. It is likely that this neuroprotection is partly due to the attenuation of the ER stress response provoked by excitotoxicity. Although CpG ODN 2088 limits the supportive effects of astrocytes on neurons, it could potentially exert beneficial effects by decreasing the release of pro-inflammatory cytokines and chemokines by astroglia. These findings highlight the multiple roles of TLR9 in the SC and have implications for pathological conditions including SCI where excitotoxicity and neuroinflammation play a prominent role in neuronal degeneration.

Toll-like receptor 4 enhancement of non-NMDA synaptic currents increases dentate excitability after brain injury.

Concussive brain injury results in neuronal degeneration, microglial activation and enhanced excitability in the hippocampal dentate gyrus, increasing the risk for epilepsy and memory dysfunction. Endogenous molecules released during injury can activate innate immune responses including toll-like receptor 4 (TLR4). Recent studies indicate that immune mediators can modulate neuronal excitability. Since non-specific agents that reduce TLR4 signaling can limit post-traumatic neuropathology, we examined whether TLR4 signaling contributes to early changes in dentate excitability after brain injury. Concussive brain injury caused a transient increase in hippocampal TLR4 expression within 4h, which peaked at 24h. Post-injury increase in TLR4 expression in the dentate gyrus was primarily neuronal and persisted for one week. Acute, in vitro treatment with TLR4 ligands caused bidirectional modulation of dentate excitability in control and brain-injured rats, with a reversal in the direction of modulation after brain injury. TLR4 antagonists decreased, and agonist increased, afferent-evoked dentate excitability one week after brain injury. NMDA receptor antagonist did not occlude the ability of LPS-RS, a TLR4 antagonist, to decrease post-traumatic dentate excitability. LPS-RS failed to modulate granule cell NMDA EPSCs but decreased perforant path-evoked non-NMDA EPSC peak amplitude and charge transfer in both granule cells and mossy cells. Our findings indicate an active role for TLR4 signaling in early post-traumatic dentate hyperexcitability. The novel TLR4 modulation of non-NMDA glutamatergic currents, identified herein, could represent a general mechanism by which immune activation influences neuronal excitability in neurological disorders that recruit sterile inflammatory responses.

Toll-like receptors in central nervous system injury and disease: a focus on the spinal cord.

Toll-like receptors (TLRs) are best known for recognizing pathogens and initiating an innate immune response to protect the host. However, they also detect tissue damage and induce sterile inflammation upon the binding of endogenous ligands released by stressed or injured cells. In addition to immune system-related cells, TLRs have been identified in central nervous system (CNS) neurons and glial subtypes including microglia, astrocytes and oligodendrocytes. Direct and indirect effects of TLR ligands on neurons and glial subtypes have been documented in vitro. Likewise, the effects of TLR ligands have been demonstrated in vivo using animal models of CNS trauma and disease including spinal cord injury (SCI), amyotrophic lateral sclerosis (ALS) and neuropathic pain. The indirect effects are most likely mediated via microglia or immune system cells that infiltrate the diseased or injured CNS. Despite considerable progress over the past decade, the role of TLRs in the physiological and pathological function of the spinal cord remains inadequately defined. Published reports collectively highlight TLRs as promising targets for therapeutic interventions in spinal cord pathology. The findings also underscore the complexity of TLR-mediated mechanisms and the necessity for further research in this field. The goals of the current review are to recapitulate the studies that investigated the role of TLRs in the spinal cord, to discuss potential future research directions, and to examine some of the challenges associated with pre-clinical studies pertinent to TLRs in the injured or diseased spinal cord.

Cypin Inhibition as a Therapeutic Approach to Treat Spinal Cord Injury-Induced Mechanical Pain.

Cypin (cytosolic postsynaptic density protein 95 interactor) is the primary guanine deaminase in the central nervous system (CNS), promoting the metabolism of guanine to xanthine, an important reaction in the purine salvage pathway. Activation of the purine salvage pathway leads to the production of uric acid (UA). UA has paradoxical effects, specifically in the context of CNS injury as it confers neuroprotection, but it also promotes pain. Since neuropathic pain is a comorbidity associated with spinal cord injury (SCI), we postulated that small molecule cypin inhibitor B9 treatment could attenuate SCI-induced neuropathic pain, potentially by interfering with UA production. However, we also considered that this treatment could hinder the neuroprotective effects of UA and, in doing so, exacerbate SCI outcomes. To address our hypothesis, we induced a moderate midthoracic contusion SCI in female mice and assessed whether transient intrathecal administration of B9, starting at 1 d postinjury (dpi) until 7 dpi, attenuates mechanical pain in hindlimbs at 3 weeks pi. We also evaluated the effects of B9 on the spontaneous recovery of locomotor function. We found that B9 alleviates mechanical pain but does not affect locomotor function. Importantly, B9 does not exacerbate lesion volume at the epicenter. In accordance with these findings, B9 does not aggravate glutamate-induced excitotoxic death of SC neurons in vitro. Moreover, SCI-induced increased astrocyte reactivity at the glial scar is not altered by B9 treatment. Our data suggest that B9 treatment reduces mechanical pain without exerting major detrimental effects following SCI.

Cross-hemicord spinal fiber reorganization associates with cortical sensory and motor network expansion in the rat model of hemicontusion cervical spinal cord injury.

Magnetic resonance imaging plays an important role in characterizing microstructural changes and reorganization after traumatic injuries to the nervous system. In this study, we tested the feasibility of ex-vivo spinal cord diffusion tensor imaging (DTI) in combination with in vivo brain functional MRI to characterize spinal reorganization and its supraspinal association after a hemicontusion cervical spinal cord injury (SCI). DTI parameters (fractional anisotropy [FA], mean diffusion [MD]) and fiber orientation changes related to reorganization in the contused cervical spinal cord were compared to sham specimens. Altered fiber density and fiber directions occurred across the ipsilateral and contralateral hemicords but with only ipsilateral FA and MD changes. The hemicontusion SCI resulted in ipsilateral fiber breaks, voids and vivid fiber reorientations along the injury epicenter. Fiber directional changes below the injury level were primarily inter-hemispheric, indicating prominent below-level cross-hemispheric reorganization. In vivo resting state functional connectivity of the brain from the respective rats before obtaining the spinal cord samples indicated spatial expansion and increased connectivity strength across both the sensory and motor networks after SCI. The consistency of the neuroplastic changes along the neuraxis (both brain and spinal cord) at the single-subject level, indicates that distinctive reorganizational relationships exist between the spinal cord and the brain post-SCI.

A toll-like receptor 9 antagonist reduces pain hypersensitivity and the inflammatory response in spinal cord injury.

Toll-like receptors (TLRs) are mediators of the innate immune response to exogenous pathogens. They have also been implicated in sterile inflammation associated with systemic injury and non-infectious diseases via binding of endogenous ligands, possibly released by damaged cells. Emerging evidence indicates that some TLRs play a role in nervous system injury and especially in injury-elicited pain and sterile inflammation. However, no information is available about the contribution of TLR9, a member of the TLR family, to traumatic spinal cord injury (SCI). Moreover, the therapeutic potential of TLR9 ligands in the functional outcomes of SCI, including pain, has not been explored. We report, for the first time, that the intrathecal administration of a TLR9 antagonist, cytidine-phosphate-guanosine oligodeoxynucleotide 2088 (CpG ODN 2088), to mice sustaining a severe contusion SCI, diminishes injury-induced heat hypersensitivity. Investigations on the potential mechanisms underlying the reduction in pain sensitivity indicated an attenuation of the inflammatory reaction manifested by a decrease in the number of CD11b-, CD45- and CD3-immunoreactive cells and a reduction in tumor necrosis factor-α (TNF-α) expression at the epicenter. Conversely, intrathecal delivery of a TLR9 agonist, CpG ODN 1826, increased inflammatory cell numbers and TNF-α expression in the epicenter. The CpG ODN 2088 treatment did not appear to induce systemic adverse effects as shown by spleen histology and serum cytokine levels. We propose that CpG ODN 2088 dampens injury-induced heat hypersensitivity by suppressing the inflammatory response and TNF-α expression. This investigation defines a previously unreported therapeutic role for CpG ODN 2088 in SCI-induced pain.

Maternal immune stimulation during pregnancy shapes the immunological phenotype of offspring.

Epidemiological studies have associated infection during pregnancy with increased risk of neurodevelopmental disorders in children, which is modeled in rodents by stimulating the immune system of pregnant dams with microorganisms or their mimics, such as poly(I:C) or LPS. In two prenatal mouse models, we show that in utero exposure of the fetus to cytokines/inflammatory mediators elicited by maternal immune stimulation with poly(I:C) yields offspring that exhibit a proinflammatory phenotype due to alterations in developmental programming of their immune system. Changes in the innate and adaptive immune elements of these pro-inflammatory offspring result in more robust responses following exposure to immune stimuli than those observed in control offspring from PBS-injected pregnant dams. In the first model, offspring from poly(I:C)-injected immunologically naïve dams showed heightened cellular and cytokine responses 4 h after injection of zymosan, a TLR2 agonist. In the second model, using dams with immunological memory, poly(I:C) injection during pregnancy produced offspring that showed preferential differentiation toward Th17 cell development, earlier onset of clinical symptoms of EAE, and more severe neurological deficits following immunization with MOG35-55. Such "fetal programming" in offspring from poly(I:C)-injected dams not only persists into neonatal and adult life, but also can have profound consequences on health and disease.

Toll-like receptor 9 deficiency impacts sensory and motor behaviors.

Toll-like receptors (TLRs) mediate the induction of the innate immune system in response to pathogens, injury and disease. However, they also play non-immune roles and are expressed in the central nervous system (CNS) during prenatal and postnatal stages including adulthood. Little is known about their roles in the CNS in the absence of pathology. Several members of the TLR family have been implicated in the development of neural and cognitive function although the contribution of TLR9 to these processes has not been well defined. The current studies were undertaken to determine whether developmental TLR9 deficiency affects motor, sensory or cognitive functions. We report that TLR9 deficient (TLR9(-/-)) mice show a hyper-responsive sensory and motor phenotype compared to wild type (TLR9(+/+)) controls. This is indicated by hypersensitivity to thermal stimuli in the hot plate paw withdrawal test, enhanced motor-responsivity under anxious conditions in the open field test and greater sensorimotor reactivity in the acoustic startle response. Prepulse inhibition (PPI) of the acoustic startle response was also enhanced, which indicates abnormal sensorimotor gating. In addition, subtle, but significant, gait abnormalities were noted in the TLR9(-/-) mice on the horizontal balance beam test with higher foot slip numbers than TLR9(+/+) controls. In contrast, spatial learning and memory, assessed by the Morris water maze, was similar in the TLR9(-/-) and TLR9(+/+) mice. These findings support the notion that TLR9 is important for the appropriate development of sensory and motor behaviors.

Purkinje cell dysfunction and delayed death in plasma membrane calcium ATPase 2-heterozygous mice.

Purkinje cell (PC) dysfunction or death has been implicated in a number of disorders including ataxia, autism and multiple sclerosis. Plasma membrane calcium ATPase 2 (PMCA2), an important calcium (Ca(2+)) extrusion pump that interacts with synaptic signaling complexes, is most abundantly expressed in PCs compared to other neurons. Using the PMCA2 heterozygous mouse as a model, we investigated whether a reduction in PMCA2 levels affects PC function. We focused on Ca(2+) signaling and the expression of glutamate receptors which play a key role in PC function including synaptic plasticity. We found that the amplitude of depolarization and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptor (AMPAR)-mediated Ca(2+) transients are significantly higher in cultured PMCA2(+/-) PCs than in PMCA2(+/+) PCs. This is due to increased Ca(2+) influx, since P/Q type voltage-gated Ca(2+) channel (VGCC) expression was more pronounced in PCs and cerebella of PMCA2(+/-) mice and VGCC blockade prevented the elevation in amplitude. Neuronal nitric oxide synthase (nNOS) activity was higher in PMCA2(+/-) cerebella and inhibition of nNOS or the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway, which mediates nitric oxide (NO) signaling, reduced the amplitude of Ca(2+) transients in PMCA2(+/-) PCs, in vitro. In addition, there was an age-dependent decrease in metabotropic glutamate receptor 1 (mGluR1) and AMPA receptor subunit GluR2/3 transcript and protein levels at 8 weeks of age. These changes were followed by PC loss in the 20-week-old PMCA2(+/-) mice. Our studies highlight the importance of PMCA2 in Ca(2+) signaling, glutamate receptor expression and survival of Purkinje cells.

Proteomic identification of immunoproteasome accumulation in formalin-fixed rodent spinal cords with experimental autoimmune encephalomyelitis.

Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the "shotgun" quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method, have made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis, which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ∼50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.

Lateralized Supraspinal Functional Connectivity Correlate with Pain and Motor Dysfunction in Rat Hemicontusion Cervical Spinal Cord Injury.

Afferent nociceptive activity in the reorganizing spinal cord after SCI influences supraspinal regions to establish pain. Clinical evidence of poor motor functional recovery in SCI patients with pain, led us to hypothesize that sensory-motor integration transforms into sensory-motor interference to manifest pain. This was tested by investigating supraspinal changes in a rat model of hemicontusion cervical SCI. Animals displayed ipsilateral forelimb motor dysfunction and pain, which persisted at 6 weeks after SCI. Using resting state fMRI at 8 weeks after SCI, RSFC across 14 ROIs involved in nociception, indicated lateral differences with a relatively weaker right-right connectivity (deafferented-contralateral) compared to left-left (unaffected-ipsilateral). However, the sensory (S1) and motor (M1/M2) networks showed greater RSFC using right hemisphere ROI seeds when compared to left. Voxel seeds from the somatosensory forelimb (S1FL) and M1/M2 representations reproduced the SCI-induced sensory and motor RSFC enhancements observed using the ROI seeds. Larger local connectivity occurred in the right sensory and motor networks amidst a decreasing overall local connectivity. This maladaptive reorganization of the right (deafferented) hemisphere localized the sensory component of pain emerging from the ipsilateral forepaw. A significant expansion of the sensory and motor network s overlap occurred globally after SCI when compared to sham, supporting the hypothesis that sensory and motor interference manifests pain. Voxel-seed based analysis revealed greater sensory and motor network overlap in the left hemisphere when compared to the right. This left predominance of the overlap suggested relatively larger pain processing in the unaffected hemisphere, when compared to the deafferented side.

Neuropathic Pain in Multiple Sclerosis and Its Animal Models: Focus on Mechanisms, Knowledge Gaps and Future Directions.

Multiple sclerosis (MS) is a multifaceted, complex and chronic neurological disease that leads to motor, sensory and cognitive deficits. MS symptoms are unpredictable and exceedingly variable. Pain is a frequent symptom of MS and manifests as nociceptive or neuropathic pain, even at early disease stages. Neuropathic pain is one of the most debilitating symptoms that reduces quality of life and interferes with daily activities, particularly because conventional pharmacotherapies do not adequately alleviate neuropathic pain. Despite advances, the mechanisms underlying neuropathic pain in MS remain elusive. The majority of the studies investigating the pathophysiology of MS-associated neuropathic pain have been performed in animal models that replicate some of the clinical and neuropathological features of MS. Experimental autoimmune encephalomyelitis (EAE) is one of the best-characterized and most commonly used animal models of MS. As in the case of individuals with MS, rodents affected by EAE manifest increased sensitivity to pain which can be assessed by well-established assays. Investigations on EAE provided valuable insights into the pathophysiology of neuropathic pain. Nevertheless, additional investigations are warranted to better understand the events that lead to the onset and maintenance of neuropathic pain in order to identify targets that can facilitate the development of more effective therapeutic interventions. The goal of the present review is to provide an overview of several mechanisms implicated in neuropathic pain in EAE by summarizing published reports. We discuss current knowledge gaps and future research directions, especially based on information obtained by use of other animal models of neuropathic pain such as nerve injury.

Contribution of astrocytes to neuropathology of neurodegenerative diseases.

Classically, the loss of vulnerable neuronal populations in neurodegenerative diseases was considered to be the consequence of cell autonomous degeneration of neurons. However, progress in the understanding of glial function, the availability of improved animal models recapitulating the features of the human diseases, and the development of new approaches to derive glia and neurons from induced pluripotent stem cells obtained from patients, provided novel information that altered this view. Current evidence strongly supports the notion that non-cell autonomous mechanisms contribute to the demise of neurons in neurodegenerative disorders, and glia causally participate in the pathogenesis and progression of these diseases. In addition to microglia, astrocytes have emerged as key players in neurodegenerative diseases and will be the focus of the present review. Under the influence of pathological stimuli present in the microenvironment of the diseased CNS, astrocytes undergo morphological, transcriptional, and functional changes and become reactive. Reactive astrocytes are heterogeneous and exhibit neurotoxic (A1) or neuroprotective (A2) phenotypes. In recent years, single-cell or single-nucleus transcriptome analyses unraveled new, disease-specific phenotypes beyond A1/A2. These investigations highlighted the complexity of the astrocytic responses to CNS pathology. The present review will discuss the contribution of astrocytes to neurodegenerative diseases with particular emphasis on Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia. Some of the commonalties and differences in astrocyte-mediated mechanisms that possibly drive the pathogenesis or progression of the diseases will be summarized. The emerging view is that astrocytes are potential new targets for therapeutic interventions. A comprehensive understanding of astrocyte heterogeneity and disease-specific phenotypic complexity could facilitate the design of novel strategies to treat neurodegenerative disorders.