Intra-articular sprouting of nociceptors accompanies progressive osteoarthritis: comparative evidence in four murine models.

Objective: Knee joints are densely innervated by nociceptors. In human knees and rodent models, sprouting of nociceptors has been reported in late-stage osteoarthritis (OA). Here, we sought to describe progressive nociceptor remodeling in early and late-stage OA, using four distinct experimental mouse models. Methods: Sham surgery, destabilization of the medial meniscus (DMM), partial meniscectomy (PMX), or non-invasive anterior cruciate ligament rupture (ACLR) was performed in the right knee of 10-12-week old male C57BL/6 NaV1.8-tdTomato mice. Mice were euthanized (1) 4, 8 or 16 weeks after DMM or sham surgery; (2) 4 or 12 weeks after PMX or sham; (3) 1 or 4 weeks after ACLR injury or sham. Additionally, a cohort of naïve male wildtype mice was evaluated at age 6 and 24 months. Mid-joint cryosections were assessed qualitatively and quantitatively for NaV1.8+ or PGP9.5+ innervation. Cartilage damage, synovitis, and osteophytes were assessed. Results: Progressive OA developed in the medial compartment after DMM, PMX, and ACLR. Synovitis and associated neo-innervation of the synovium by nociceptors peaked in early-stage OA. In the subchondral bone, channels containing sprouting nociceptors appeared early, and progressed with worsening joint damage. Two-year old mice developed primary OA in the medial and the lateral compartment, accompanied by nociceptor sprouting in the synovium and the subchondral bone. All four models showed increased nerve signal in osteophytes. Conclusion: These findings suggest that anatomical neuroplasticity of nociceptors is intrinsic to OA pathology. The detailed description of innervation of the OA joint and its relationship to joint damage might help in understanding OA pain.

The role of intra-articular neuronal CCR2 receptors in knee joint pain associated with experimental osteoarthritis in mice.

BACKGROUND: C-C chemokine receptor 2 (CCR2) signaling plays a key role in pain associated with experimental murine osteoarthritis (OA) after destabilization of the medial meniscus (DMM). Here, we aimed to assess if CCR2 expressed by intra-articular sensory neurons contributes to knee hyperalgesia in the early stages of the model. METHODS: DMM surgery was performed in the right knee of 10-week-old male wild-type (WT), Ccr2 null, or Ccr2RFP C57BL/6 mice. Knee hyperalgesia was measured using a Pressure Application Measurement device. CCR2 receptor antagonist (CCR2RA) was injected systemically (i.p.) or intra-articularly (i.a.) at different times after DMM to test its ability to reverse knee hyperalgesia. In vivo Ca2+ imaging of the dorsal root ganglion (DRG) was performed to assess sensory neuron responses to CCL2 injected into the knee joint cavity. CCL2 protein in the knee was measured by ELISA. Ccr2RFP mice and immunohistochemical staining for the pan-neuronal marker, protein gene product 9.5 (PGP9.5), or the sensory neuron marker, calcitonin gene-related peptide (CGRP), were used to visualize the location of CCR2 on intra-articular afferents. RESULTS: WT, but not Ccr2 null, mice displayed knee hyperalgesia 2-16 weeks after DMM. CCR2RA administered i.p. alleviated established hyperalgesia in WT mice 4 and 8 weeks after surgery. Intra-articular injection of CCL2 excited sensory neurons in the L4-DRG, as determined by in vivo calcium imaging; responses to CCL2 increased in mice 20 weeks after DMM. CCL2, but not vehicle, injected i.a. rapidly caused transient knee hyperalgesia in naïve WT, but not Ccr2 null, mice. Intra-articular CCR2RA injection also alleviated established hyperalgesia in WT mice 4 and 7 weeks after surgery. CCL2 protein was elevated in the knees of both WT and Ccr2 null mice 4 weeks after surgery. Co-expression of CCR2 and PGP9.5 as well as CCR2 and CGRP was observed in the lateral synovium of naïve mice; co-expression was also observed in the medial compartment of knees 8 weeks after DMM. CONCLUSIONS: The findings suggest that CCL2-CCR2 signaling locally in the joint contributes to knee hyperalgesia in experimental OA, and it is in part mediated through direct stimulation of CCR2 expressed by intra-articular sensory afferents.

Cemented versus uncemented fixation of second-generation Trabecular Metal glenoid components: minimum 5-year outcomes.

BACKGROUND: Total shoulder arthroplasty (TSA) with second-generation Trabecular Metal™ implants (Zimmer, Warsaw, IN, USA) has shown good short-term outcomes. Differences in outcomes between cemented and uncemented fixation are unknown. This study compared the clinical, radiographic, and patient-rated outcomes of TSA with cemented vs. uncemented TM glenoids at minimum 5-year follow-up. METHODS: Patients who underwent anatomic TSA with second-generation TM glenoid components for primary osteoarthritis were identified for minimum 5-year follow-up. The patients were divided into 2 groups: cemented and uncemented glenoid fixation. Outcome measures included implant survival, patient-rated outcome scores (Patient-Reported Outcomes Measurement Information System [PROMIS] and American Shoulder and Elbow Surgeons scores), shoulder range of motion, and radiographic analysis. Findings were compared between groups. RESULTS: The study included 55 shoulders: 27 in the cemented group (21 with full radiographic follow-up) and 28 in the uncemented group (22 with full radiographic follow-up). Both groups had similar follow-up times (6.6 years in cemented group vs. 6.7 years in uncemented group, P = .60). Moreover, the groups did not differ significantly in sex composition, age at the time of surgery, or preoperative Walch glenoid grade distribution. No patients required revision surgery. The 2 groups had similar preoperative range of motion, but patients in the uncemented group had greater follow-up forward flexion (P = .03), external rotation (P < .01), and lateral elevation (P = .03) than did patients in the cemented group. PROMIS scores were not significantly different between groups. American Shoulder and Elbow Surgeons scores were similar (89.8 in cemented group vs. 94.1 in uncemented group, P = .21). Mid-term radiographs showed a metal debris rate of 24% in the cemented group and 27% in the uncemented group. Although these values were not significantly different (P = .90), the frequency of mild metal debris (grade 1-2), when present, was greater in the uncemented group (grade 2 in 6 shoulders) than in the cemented group (grade 1 in 4 and grade 2 in 1, P = .02). There was a greater presence of mild (grade 1) radiolucent lines in the uncemented group (64%) than in the cemented group (29%, P < .01). No glenoid had evidence of loosening (defined by a change in position or radiolucent lines > 2 mm). The presence of metal debris and radiolucent lines did not have a significant effect on clinical outcomes. CONCLUSION: At minimum 5-year follow-up, TSA patients with TM glenoids demonstrated excellent clinical and patient-reported outcomes with a 100% implant survival rate, regardless of cemented vs. uncemented fixation. However, the uncemented group showed a significantly higher rate of radiolucent lines and a higher frequency of mild metal debris. These radiographic findings did not affect the clinical outcomes, and their implications for long-term outcomes and prosthesis survival is unknown.

Preoperative PROMIS Scores Predict Postoperative PROMIS Score Improvement for Patients Undergoing Hand Surgery.

Background: Patient-Reported Outcomes Measurement Information System (PROMIS) can be used alongside preoperative patient characteristics to set postsurgery expectations. This study aimed to analyze whether preoperative scores can predict significant postoperative PROMIS score improvement. Methods: Patients undergoing hand and wrist surgery with initial and greater than 6-month follow-up PROMIS scores were assigned to derivation or validation cohorts, separating trauma and nontrauma conditions. Receiver operating characteristic curves were calculated for the derivation cohort to determine whether preoperative PROMIS scores could predict postoperative PROMIS score improvement utilizing minimal clinically important difference principles. Results: In the nontrauma sample, patients with baseline Physical Function (PF) scores below 31.0 and Pain Interference (PI) and Depression scores above 68.2 and 62.2, respectively, improved their postoperative PROMIS scores with 95%, 96%, and 94% specificity. Patients with baseline PF scores above 52.1 and PI and Depression scores below 49.5 and 39.5, respectively, did not substantially improve their postoperative PROMIS scores with 94%, 93%, and 96% sensitivity. In the trauma sample, patients with baseline PF scores below 34.8 and PI and Depression scores above 69.2 and 62.2, respectively, each improved their postoperative PROMIS scores with 95% specificity. Patients with baseline PF scores above 52.1 and PI and Depression scores below 46.6 and 44.0, respectively, did not substantially improve their postoperative scores with 95%, 94%, and 95% sensitivity. Conclusions: Preoperative PROMIS PF, PI, and Depression scores can predict postoperative PROMIS score improvement for a select group of patients, which may help in setting expectations. Future work can help determine the level of true clinical improvement these findings represent.

Preoperative Patient-Reported Outcomes Measurement Information System (PROMIS) scores predict postoperative outcome in total shoulder arthroplasty patients.

BACKGROUND: The Patient-Reported Outcomes Measurement Information System (PROMIS) has recently been validated in orthopedic patients with upper-extremity disease. The purpose of this study was to describe preoperative and postoperative PROMIS scores in total shoulder arthroplasty (TSA) patients, compare PROMIS physical function (PF) scores with clinical functional measurements, and determine whether preoperative PROMIS scores could predict achievement of the minimal clinically important difference (MCID) postoperatively. METHODS: Preoperative and postoperative (>3 months) PROMIS scores in patients who underwent primary anatomic TSA were reviewed. Preoperative and postoperative shoulder forward flexion and external rotation were also collected. PROMIS PF, pain interference (PI), and depression scores were compared. Accuracy analyses determined whether preoperative PROMIS scores from each domain could predict postoperative achievement of the MCID in the same domain. RESULTS: The study included 62 patients. Significant improvements in PROMIS PF, PI, and depression scores, as well as forward flexion and external rotation, were found postoperatively (P < .001). The multivariate model demonstrated that preoperative PROMIS PF, PI, and depression scores were predictive of postoperative achievement of the MCID (area under the receiver operating characteristic curve, 0.70-0.87). Ninety percent cutoff scores showed that patients with a preoperative PF score lower than 31.7, PI score greater than 66.9, and depression score greater than 55.5 were more likely to achieve the MCID. CONCLUSIONS: In TSA patients, preoperative PROMIS PF, depression, and PI scores demonstrated strong to excellent abilities to predict postoperative achievement of the MCID. PROMIS PF scores were responsive to the functional improvements observed clinically. The reported cutoff scores allow surgeons to identify patients with increased or decreased probabilities of achieving a clinically meaningful improvement after TSA.

An aggrecan fragment drives osteoarthritis pain through Toll-like receptor 2.

Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E373-374A). Further cleavage by MMPs (N341-342F) releases a 32-amino-acid aggrecan fragment (32-mer). We investigated the role of this 32-mer in driving joint pain. We found that the 32-mer excites dorsal root ganglion nociceptive neurons, both in culture and in intact explants. Treatment of cultured sensory neurons with the 32-mer induced expression of the proalgesic chemokine CCL2. These effects were mediated through TLR2, which we demonstrated was expressed by nociceptive neurons. In addition, intra-articular injection of the 32-mer fragment provoked knee hyperalgesia in WT but not Tlr2-null mice. Blocking the production or action of the 32-mer in transgenic mice prevented the development of knee hyperalgesia in a murine model of osteoarthritis. These findings suggest that the aggrecan 32-mer fragment directly activates TLR2 on joint nociceptors and is an important mediator of the development of osteoarthritis-associated joint pain.

Visualization of Peripheral Neuron Sensitization in a Surgical Mouse Model of Osteoarthritis by In Vivo Calcium Imaging.

OBJECTIVE: To develop a method for analyzing sensory neuron responses to mechanical stimuli in vivo, and to evaluate whether these neuronal responses change after destabilization of the medial meniscus (DMM). METHODS: DMM or sham surgery was performed in 10-week-old male C57BL/6 wild-type or Pirt-GCaMP3+/- mice. All experiments were performed 8 weeks after surgery. Knee and hind paw hyperalgesia were assessed in wild-type mice. The retrograde label DiI was injected into the ipsilateral knee to quantify the number of knee-innervating neurons in the L4 dorsal root ganglion (DRG) in wild-type mice. In vivo calcium imaging was performed on the ipsilateral L4 DRG of Pirt-GCaMP3+/- mice as mechanical stimuli (paw pinch, knee pinch, or knee twist) were applied to the ipsilateral hind limb. RESULTS: Eight weeks after surgery, mice subjected to DMM had more hyperalgesia in the knee and hind paw compared to mice subjected to sham surgery. Intraarticular injection of DiI labeled similar numbers of neurons in the L4 DRG of mice subjected to sham surgery and mice subjected to DMM. Increased numbers of sensory neurons responded to all 3 mechanical stimuli in mice subjected to DMM, as assessed by in vivo calcium imaging. The majority of responses in mice subjected to sham surgery and mice subjected to DMM were in small to medium-sized neurons, consistent with the size of nociceptors. The magnitude of responses was similar between mice subjected to sham surgery and mice subjected to DMM. CONCLUSION: Our findings indicate that increased numbers of small to medium-sized DRG neurons respond to mechanical stimuli 8 weeks after DMM surgery, suggesting that nociceptors have become sensitized by lowering the response threshold.

Is cannabis an effective treatment for joint pain?

Cannabis has been used to treat pain for thousands of years. However, since the early part of the 20th century, laws restricting cannabis use have limited its evaluation using modern scientific criteria. Over the last decade, the situation has started to change because of the increased availability of cannabis in the United States for either medical or recreational purposes, making it important to provide the public with accurate information as to the effectiveness of the drug for joint pain among other indications. The major psychotropic component of cannabis is Δ9-tetrahydrocannabinol (THC), one of some 120 naturally occurring phytocannabinoids. Cannabidiol (CBD) is another molecule found in herbal cannabis in large amounts. Although CBD does not produce psychotropic effects, it has been shown to produce a variety of pharmacological effects. Hence, the overall effects of herbal cannabis represent the collective activity of THC, CBD and a number of minor components. The action of THC is mediated by two major G-protein coupled receptors, cannabinoid receptor type 1 (CB1) and CB2, and recent work has suggested that other targets may also exist. Arachidonic acid derived endocannabinoids are the normal physiological activators of the two cannabinoid receptors. Natural phytocannabinoids and synthetic derivatives have produced clear activity in a variety of models of joint pain in animals. These effects are the result of both inhibition of pain pathway signalling (mostly CB1) and anti-inflammatory effects (mostly CB2). There are also numerous anecdotal reports of the effectiveness of smoking cannabis for joint pain. Indeed, it is the largest medical request for the use of the drug. However, these reports generally do not extend to regulated clinical trials for rheumatic diseases. Nevertheless, the preclinical and human data that do exist indicate that the use of cannabis should be taken seriously as a potential treatment of joint pain.

Chemogenetic Inhibition of Pain Neurons in a Mouse Model of Osteoarthritis.

OBJECTIVE: To determine the ability of drugs that activate inhibitory G protein-coupled receptors (GPCRs) expressed in peripheral voltage-gated sodium channel 1.8 (NaV 1.8)-positive sensory neurons to control osteoarthritis (OA)-associated pain. METHODS: We used designer receptors exclusively activated by a designer drug (DREADD) technology, which employs engineered GPCRs to activate or inhibit neurons upon binding the synthetic ligand clozapine N-oxide (CNO). NaV 1.8-Pdi C57BL/6 mice were generated to express the inhibitory DREADD receptor Pdi in NaV 1.8-expressing sensory neurons. Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male mice. Four, 8, 12, or 16 weeks after surgery, knee hyperalgesia or hind paw mechanical allodynia was tested. Subsequently, CNO or vehicle was administered, and the effect on pain-related behaviors was measured by a blinded observer. Morphine was used as a control. RESULTS: Immunohistochemistry and electrophysiology confirmed functional expression of the inhibitory DREADD receptor Pdi by NaV 1.8-positive sensory neurons. Acute inhibition of NaV 1.8-expressing neurons in mice treated with CNO reduced knee hyperalgesia 4 weeks after DMM surgery and reduced mechanical allodynia 8 weeks after DMM surgery. Inhibition had no effect on pain-related behaviors 12 and 16 weeks after DMM surgery. Morphine, a drug that activates GPCRs in the peripheral and central nervous systems, was still effective in the later stage of experimental OA. CONCLUSION: Chemogenetic inhibition of NaV 1.8-expressing neurons blocks knee hyperalgesia and mechanical allodynia in early experimental OA, but is no longer efficacious in the later stages. These data indicate that activation of inhibitory GPCRs located solely outside the central nervous system may be ineffective in treating chronic OA pain.

Discovery and characterization of novel small-molecule CXCR4 receptor agonists and antagonists.

The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV-1 infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. While previous efforts have identified a number of CXCR4 antagonists, there have been no small molecule agonists reported. Herein, we describe the identification of a novel series of CXCR4 modulators, including the first small molecules to display agonist behavior against this receptor, using a combination of structure- and ligand-based virtual screening. These agonists produce robust calcium mobilization in human melanoma cell lines which can be blocked by the CXCR4-selective antagonist AMD3100. We also demonstrate the ability of these new agonists to induce receptor internalization, ERK activation, and chemotaxis, all hallmarks of CXCR4 activation. Our results describe a new series of biologically relevant small molecules that will enable further study of the CXCR4 receptor and may contribute to the development of new therapeutics.

Role of adult hippocampal neurogenesis in persistent pain.

The full role of adult hippocampal neurogenesis (AHN) remains to be determined, yet it is implicated in learning and emotional functions, and is disrupted in negative mood disorders. Recent evidence indicates that AHN is decreased in persistent pain consistent with the idea that chronic pain is a major stressor, associated with negative moods and abnormal memories. Yet, the role of AHN in development of persistent pain has remained unexplored. In this study, we test the influence of AHN in postinjury inflammatory and neuropathic persistent pain-like behaviors by manipulating neurogenesis: pharmacologically through intracerebroventricular infusion of the antimitotic AraC; ablation of AHN by x-irradiation; and using transgenic mice with increased or decreased AHN. Downregulating neurogenesis reversibly diminished or blocked persistent pain; oppositely, upregulating neurogenesis led to prolonged persistent pain. Moreover, we could dissociate negative mood from persistent pain. These results suggest that AHN-mediated hippocampal learning mechanisms are involved in the emergence of persistent pain.

Pattern of CXCR7 Gene Expression in Mouse Brain Under Normal and Inflammatory Conditions.

The chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 acting via its G-protein coupled receptor (GPCR) CXCR4 has been implicated in neurogenesis, neuromodulation, brain inflammation, HIV-1 encephalopathy and tumor growth. CXCR7 was identified as an alternate receptor for SDF-1/CXCL12. Characterization of CXCR7-deficient mice demonstrated a role for CXCR7 in fetal endothelial biology, cardiac development, and B-cell localization. Despite its ligand binding properties, CXCR7 does not seem to signal like a conventional GPCR. It has been suggested that CXCR7 may not function alone but in combination with CXCR4. Here, we investigated the regional localization of CXCR7 receptors in adult mouse brain using CXCR7-EGFP transgenic mice. We found that the receptors were expressed in various brain regions including olfactory bulb, cerebral cortex, hippocampus, subventricular zone (SVZ), hypothalamus and cerebellum. Extensive CXCR7 expression was associated with cerebral blood vessels. Using cell type specific markers, CXCR7 expression was found in neurons, astrocytes and oligodendrocyte progenitors. GAD-expressing neurons exhibited CXCR7 expression in the hippocampus. Expression of CXCR7 in the dentate gyrus included cells that expressed nestin, GFAP and cells that appeared to be immature granule cells. In mice with Experimental Autoimmune Encephalomyelitis (EAE), CXCR7 was expressed by migrating oligodendrocyte progenitors in the SVZ. We then compared the distribution of SDF-1/CXCL12 and CXCR7 using bitransgenic mice expressing both CXCR7-EGFP and SDF-1-mRFP. Enhanced expression of SDF-1/CXCL12 and CXCR7 was observed in the corpus callosum, SVZ and cerebellum. Overall, the expression of CXCR7 in normal and pathological nervous system suggests CXCR4-independent functions of SDF-1/CXCL12 mediated through its interaction with CXCR7.

Damage-associated molecular patterns generated in osteoarthritis directly excite murine nociceptive neurons through Toll-like receptor 4.

OBJECTIVE: To determine whether selected damage-associated molecular patterns (DAMPs) present in the osteoarthritic (OA) joints of mice excite nociceptors through Toll-like receptor 4 (TLR-4). METHODS: The ability of S100A8 and α2 -macroglobulin to excite nociceptors was determined by measuring the release of monocyte chemoattractant protein 1 (MCP-1) by cultured dorsal root ganglion (DRG) cells as well as by measuring the intracellular calcium concentration ([Ca(2+) ]i ) in cultured DRG neurons from naive mice or from mice that had undergone surgical destabilization of the medial meniscus (DMM) 8 weeks previously. The role of TLR-4 was assessed using TLR-4(-/-) cells or a TLR-4 inhibitor. The [Ca(2+) ]i in neurons within ex vivo intact DRGs was measured in samples from Pirt-GCaMP3 mice. Neuronal expression of the Tlr4 gene was determined by in situ hybridization. DMM surgery was performed in wild-type and TLR-4(-/-) mice; mechanical allodynia was monitored, and joint damage was assessed histologically after 16 weeks. RESULTS: DRG neurons from both naive and DMM mice expressed Tlr4. Both S100A8 and α2 -macroglobulin stimulated release of the proalgesic chemokine MCP-1 in DRG cultures, and the neurons rapidly responded to S100A8 and α2 -macroglobulin with increased [Ca(2+) ]i . Blocking TLR-4 inhibited these effects. Neurons within intact DRGs responded to the TLR-4 agonist lipopolysaccharide. In both of the calcium-imaging assays, it was primarily the nociceptor population of neurons that responded to TLR-4 ligands. TLR-4(-/-) mice were not protected from mechanical allodynia or from joint damage associated with DMM. CONCLUSION: Our experiments suggest a role of TLR-4 signaling in the excitation of nociceptors by selected DAMPs. Further research is needed to delineate the importance of this pathway in relation to OA pain.

Localized CCR2 Activation in the Bone Marrow Niche Mobilizes Monocytes by Desensitizing CXCR4.

Inflammatory (classical) monocytes residing in the bone marrow must enter the bloodstream in order to combat microbe infection. These monocytes express high levels of CCR2, a chemokine receptor whose activation is required for them to exit the bone marrow. How CCR2 is locally activated in the bone marrow and how their activation promotes monocyte egress is not understood. Here, we have used double transgenic lines that can visualize CCR2 activation in vivo and show that its chemokine ligand CCL2 is acutely released by stromal cells in the bone marrow, which make direct contact with CCR2-expressing monocytes. These monocytes also express CXCR4, whose activation immobilizes cells in the bone marrow, and are in contact with stromal cells expressing CXCL12, the CXCR4 ligand. During the inflammatory response, CCL2 is released and activates the CCR2 on neighboring monocytes. We demonstrate that acutely isolated bone marrow cells co-express CCR2 and CXCR4, and CCR2 activation desensitizes CXCR4. Inhibiting CXCR4 by a specific receptor antagonist in mice causes CCR2-expressing cells to exit the bone marrow in absence of inflammatory insults. Taken together, these results suggest a novel mechanism whereby the local activation of CCR2 on monocytes in the bone marrow attenuates an anchoring signalling provided by CXCR4 expressed by the same cell and mobilizes the bone marrow monocyte to the blood stream. Our results also provide a generalizable model that cross-desensitization of chemokine receptors fine-tunes cell mobility by integrating multiple chemokine signals.

A critical role of Src family kinase in SDF-1/CXCR4-mediated bone-marrow progenitor cell recruitment to the ischemic heart.

The G protein-coupled receptor CXCR4 and its ligand stromal-cell derived factor 1 (SDF-1) play a crucial role in directing progenitor cell (PC) homing to ischemic tissue. The Src family protein kinases (SFK) can be activated by, and serve as effectors of, G proteins. In this study we sought to determine whether SFK play a role in SDF-1/CXCR4-mediated PC homing. First, we investigated whether SDF-1/CXCR4 signaling activates SFK. Bone-marrow mononuclear cells (BM MNCs) were isolated from WT and BM-specific CXCR4-KO mice and treated with SDF-1 and/or CXCR4 antagonist AMD3100. SDF-1 treatment rapidly induced phosphorylation (activation) of hematopoietic Src (i.e., Lyn, Fgr, and Hck) in WT cells but not in AMD3100-treated cells or CXCR4-KO cells. Then, we investigated whether SFK are involved in SDF-1/CXCR4-mediated PC chemotaxis. In a combined chemotaxis and endothelial-progenitor-cell (EPC) colony assay, Src inhibitor SU6656 dose-dependently inhibited the SDF-1-induced migration of colony-forming EPCs. Next, we investigated whether SFK play a role in SDF-1/CXCR4-mediated BM PC homing to the ischemic heart. BM MNCs from CXCR4BAC:eGFP reporter mice were i.v. injected into WT and SDF-1BAC:SDF1-RFP transgenic mice following surgically-induced myocardial infarction (MI). eGFP(+) MNCs and eGFP(+)c-kit(+) PCs that were recruited in the infarct border zone in SDF-1BAC:SDF1-RFP recipients were significantly more than that in WT recipients. Treatments of mice with SU6656 significantly reduced eGFP(+) and eGFP(+)c-kit(+) cell recruitment in both WT and SDF-1BAC:RFP recipients and abrogated the difference between the two groups. Remarkably, PCs isolated from BM-specific C-terminal Src kinase (CSK)-KO (Src activated) mice were recruited more efficiently than PCs from WT PCs in the WT recipients. In conclusion, SFK are activated by SDF-1/CXCR4 signaling and play an essential role in SDF-1/CXCR4-mediated BM PC chemotactic response and ischemic cardiac recruitment.

Ubiquitin-specific protease 14 regulates c-Jun N-terminal kinase signaling at the neuromuscular junction.

BACKGROUND: Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14's catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14's catalytic activity is required for neuromuscular junction (NMJ) structure and function. RESULTS: Mice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice. CONCLUSIONS: USP14's catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.

Integrin/Chemokine receptor interactions in the pathogenesis of experimental autoimmune encephalomyelitis.

Excessive infiltration of leukocytes and the elaboration of inflammatory cytokines are believed to be responsible for the observed damage to neurons and oligodendrocytes during multiple sclerosis (MS). Blocking adhesion molecules or preventing the effects of chemotactic mediators such as chemokines can be exploited to prevent immune cell recruitment to inflamed tissues. An anti-α4 integrin antibody (anti-VLA-4mAb/natalizumab (Tysabri®)) has been used as a treatment for MS and reduces leukocyte influx into the brain. In patients, anti-VLA-4 reduces relapses and disability progression. However, its mechanism of action in the brain is not completely understood. The anti-VLA-4mAb was demonstrated to mobilize hematopoietic progenitor cells. Interestingly, the chemokine SDF-1/CXCL12 and its receptor CXCR4 are also key factors regulating the migration of hematopoietic stem cells. Moreover, studies have revealed a crosstalk between SDF-1/CXCR4 and VLA-4 signaling in regulating cell migration. In this study, we address the effects of anti-VLA-4 on chemokine signaling in the brain during MS. We assessed the ability of anti-VLA-4 to regulate Experimental Autoimmune Encephalomyelitis (EAE) and chemokine/receptor signaling. Preclinical administration of anti-VLA-4 delayed clinical signs of EAE. We found that anti-VLA-4 treatment reduced chemokine expression. In order to further explore the interaction of anti-VLA-4 with chemokine/receptor signaling we used dual color transgenic mice. After EAE induction, the expression of both SDF-1/CXCL12 and CXCR4 receptor was upregulated, treatment with anti-VLA-4 inhibited this effect. The effects of anti-VLA-4 on chemokine signaling in the CNS may be of importance when considering its mechanism of action and understanding the pathogenesis of EAE.

Stromal derived growth factor-1 (CXCL12) modulates synaptic transmission to immature neurons during post-ischemic cerebral repair.

In response to ischemic injury, the brain mounts a repair process involving the development of new neurons, oligodendrocytes, and astrocytes. However, the manner in which new neurons integrate into existing brain circuitry is not well understood. Here we observed that during the four weeks after transient middle cerebral artery occlusion (MCAO), doublecortin (DCX)-expressing neural progenitors originating in the subventricular zone (SVZ) were present in the ischemic lesion borderzone, where they received γ-aminobutyric acid (GABA) inputs, a feature that is common to newly developing neurons. The chemokine stromal derived factor-1 (SDF-1 or CXCL12) was enriched in lesional endothelial and microglial cells for up to four weeks after transient MCAO, and application of SDF-1 to acute brain slices enhanced GABAergic inputs to the new neurons. These observations suggest that SDF-1 is in a position to coordinate neovascularization and neurogenesis during the repair process after cerebral ischemia-reperfusion.

Calcium entry and α-synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons.

The core motor symptoms of Parkinson's disease (PD) are attributable to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed a major factor in PD pathogenesis. Previous work has shown that activity-dependent calcium entry through L-type channels elevates perinuclear mitochondrial oxidant stress in SNc dopaminergic neurons, providing a potential basis for their selective vulnerability. What is less clear is whether this physiological stress is present in dendrites and if Lewy bodies, the major neuropathological lesion found in PD brains, exacerbate it. To pursue these questions, mesencephalic dopaminergic neurons derived from C57BL/6 transgenic mice were studied in primary cultures, allowing for visualization of soma and dendrites simultaneously. Many of the key features of in vivo adult dopaminergic neurons were recapitulated in vitro. Activity-dependent calcium entry through L-type channels increased mitochondrial oxidant stress in dendrites. This stress progressively increased with distance from the soma. Examination of SNc dopaminergic neurons ex vivo in brain slices verified this pattern. Moreover, the formation of intracellular α-synuclein Lewy-body-like aggregates increased mitochondrial oxidant stress in perinuclear and dendritic compartments. This stress appeared to be extramitochondrial in origin, because scavengers of cytosolic reactive oxygen species or inhibition of NADPH oxidase attenuated it. These results show that physiological and proteostatic stress can be additive in the soma and dendrites of vulnerable dopaminergic neurons, providing new insight into the factors underlying PD pathogenesis.