The NAMPT inhibitor FK866 reverts the damage in spinal cord injury
© Esposito et al; licensee BioMed Central Ltd. 2012
Received: 9 January 2012
Accepted: 10 April 2012
Published: 10 April 2012
Emerging data implicate nicotinamide phosphoribosyl transferase (NAMPT) in the pathogenesis of cancer and inflammation. NAMPT inhibitors have proven beneficial in inflammatory animal models of arthritis and endotoxic shock as well as in autoimmune encephalitis. Given the role of inflammatory responses in spinal cord injury (SCI), the effect of NAMPT inhibitors was examined in this setting.
We investigated the effects of the NAMPT inhibitor FK866 in an experimental compression model of SCI.
Twenty-four hr following induction of SCI, a significant functional deficit accompanied widespread edema, demyelination, neuron loss and a substantial increase in TNF-α, IL-1β, PAR, NAMPT, Bax, MPO activity, NF-κB activation, astrogliosis and microglial activation was observed. Meanwhile, the expression of neurotrophins BDNF, GDNF, NT3 and anti-apoptotic Bcl-2 decreased significantly. Treatment with FK866 (10 mg/kg), the best known and characterized NAMPT inhibitor, at 1 h and 6 h after SCI rescued motor function, preserved perilesional gray and white matter, restored anti-apoptotic and neurotrophic factors, prevented the activation of neutrophils, microglia and astrocytes and inhibited the elevation of NAMPT, PAR, TNF-α, IL-1β, Bax expression and NF-κB activity.
We show for the first time that FK866, a specific inhibitor of NAMPT, administered after SCI, is capable of reducing the secondary inflammatory injury and partly reduce permanent damage. We also show that NAMPT protein levels are increased upon SCI in the perilesional area which can be corrected by administration of FK866.
Our findings suggest that the inflammatory component associated to SCI is the primary target of these inhibitors.
Nicotinamide phosphoribosyl transferase (NAMPT) is an enzyme that catalyzes the synthesis of nicotinamide mononucleotide (NMN) from nicotinamide (NM) and 5'-phosphoribosyl-1'-pyrophosphate (PRPP), thus playing an important role in the cyclic biosynthetic pathway of nicotinamide adenine dinucleotide (NAD). As such, this enzyme is central to cellular bioenergetics, and may control indirectly a number of signalling pathways that depend on NAD levels, such as Poly (ADP-ribose) polymerase (PARP) and sirtuin activation . A secreted form has been described, which is also known as visfatin or Pre-B cell colony-enhancing factor (PBEF) . Indeed, it has also been postulated that NAMPT behaves as an adipokine secreted from visceral fat tissues  or may have an important role in immune system. For example, NAMPT has been originally identified as an extracellular proinflammatory cytokine, able to induce cellular expression of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 and to promote pre-B cell colony formation [4, 5]. Lymphocytes, dendritic cells, monocytes and macrophages express NAMPT when presented inflammatory stimuli [6–8]. This suggests that NAMPT, either via the "NAD salvage pathway" or by other unknown mechanisms relating to its secreted form, may modulate innate or acquired immune functions.
Emerging data implicate PBEF/NAMPT/Visfatin in the pathogenesis of a number of different human diseases, in particular in the field of cancer and inflammation . The link between NAMPT and inflammation is rapidly strengthening. Indeed, visfatin levels have been found elevated in the systemic circulation of patients suffering from diseases with inflammatory components, such as type 2 diabetes , acute pancreatitis , osteoarthritis , sepsis , atherosclerosis  and psoriasis .
The search for novel antitumoral drugs has led to the identification of two inhibitors of NAMPT, FK866 (also known as APO866) and CHS828, which have now entered Phase II clinical trials. Given the role of NAMPT in inflammatory processes, an important question is whether these agents may protect from inflammatory damage. While in vitro evidences would support the use of these drugs in inflammatory processes [12, 15], in vivo evidence is limited. Indeed, it has been shown that FK866 is able to reduce disease burden in inflammatory animal models of arthritis and endotoxic shock [15, 16] as well as in experimental autoimmune encephalitis . These studies have highlighted that NAMPT inhibitors exert their effects in a pleiotropic manner, by reducing cytokine release, as well as offsetting PARP and sirtuin activation.
Spinal cord injury (SCI) is a highly debilitating pathology . Although innovative medical care has improved patient outcome, advances in pharmacotherapy for minimizing neuronal injury and promoting regeneration are limited. The complex pathophysiology of SCI may explain the difficulty in finding a suitable therapy. The primary traumatic mechanical injury to the SC causes the death of a number of neurons that cannot be regenerated: neurons continue to die for hours following traumatic SCI . The events that characterize this successive phase to mechanical injury are called "secondary damage" characterized by cellular, molecular, and biochemical cascades. The presence of a local inflammatory response maintains and amplifies the secondary damage . When SCI occurs, microglia in parenchyma is activated and macrophages in circulation get across blood-brain barrier (BBB) to act as intrinsic spinal phagocytes. These cells release different pro-inflammatory mediators such as proinflammatory cytokines  and reactive oxygen species (ROS) and nitrogen species . Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) modulate the secondary inflammatory response following traumatic SCI . ROS and peroxynitrite also cause DNA damage, which results in the activation of the nuclear enzyme PARP, depletion of NAD+ and ATP and ultimately cell death . Therefore, recently it has been demonstrated that SCI induced PARP activation .
In the light of these evidences and given the high therapeutic unmet need of SCI, we investigated the possible contribution of NAMPT in this condition. We now show that pharmacological inhibition of NAMPT given after the injury leads to a significant reduction of inflammatory cytokines, a histological improvement of the perilesional area and a significant recovery of locomotor activity.
Male Adult CD1 mice (25-30 g, Harlan, Milan, Italy) were housed in a controlled environment. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (DM 116192) as well as with the EEC regulations (DL 116/92, application of the European Communities Council Directive 86/609/EEC). All efforts were made to minimize animal suffering and the number of animals used.
We used the clip compression model described by Rivlin and Tator . Mice were anesthetized using chloral hydrate (400 mg/kg body weight). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles. These muscles were dissected away exposing T5-T8 vertebrae. The spinal cord was exposed via a four-level T5-T8 laminectomy and SCI was produced by extradural compression at T6-T7 level using an aneurysm clip with a closing force of 24 g. In all injured groups, the spinal cord was compressed for 1 min. Sham animals were only subjected to laminectomy. Following surgery, 1.0 cc of saline was administered subcutaneously in order to replace the blood volume lost during the surgery. During recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. The mice were individually housed in a temperature-controlled room at 27°C for a survival period of 20 days. Food and water were provided to the mice ad libitum. During this time period, the animals' bladders were manually voided twice a day until the mice were able to regain normal bladder function.
Experimental groups and treatments
Mice were randomly allocated into the following groups: (i) sham + vehicle group. Mice were subjected to laminectomy but the aneurysm clip was not applied, and treated intraperitoneally with vehicle (N = 30). (ii) Sham + FK866 group. Identical to sham + vehicle group except for intraperitoneal administration of FK866 (10 mg/kg) 1 h and 6 h after laminectomy (N = 30). (iii) SCI + vehicle group. Mice were subjected to SCI and were administered vehicle at 1 h and 6 h after SCI (N = 30). (iv) SCI + FK866 group. Mice were subjected to SCI and administered FK866 (10 mg/kg, intraperitoneally) at 1 h and 6 h after SCI (N = 30).
Timing of administration of FK866 was similar to that proposed for methylprednisolone at high concentrations (the current therapy available for traumatic SCI). FK866 were synthesized as reported . The dose of FK866 used here was based on previous in vivo studies [15–17]. Alongside, all experiments were also performed with an analogue of FK866, named GPP78, and data were super-imposable to those obtained with FK866 (data not shown).
In a separate set of experiments to investigate the motor score, additional animals (10 animals/each group) were observed until 20 days after SCI. FK866 (10 mg/kg) was administered 1 h and 6 h after SCI and daily until day 19.
At the end of the experimental period (24 hrs), animals were deeply anesthetized with sodium pentobarbital and then perfused transcardially with cold phosphate-buffered saline (PBS, 0.1 M) followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. SC tissues were removed under magnified vision. Tissue segments containing the lesion (1 cm on each side of the lesion) were paraffin embedded, cut into longitudinal sections for posterior area of SC, and processed for different immunohistochemical procedures.
Different tissues were also collected for Golgi-Cox staining, which highlights neurons and processes
For immunoblotting analysis, mice (N = 10) were anaesthetized and exsanguinated via cardiac puncture. SCs were dissected, and cleaned from meninges and nerve roots. One centimeter of the cord centered at the injury site was homogenized for preparing cytosolic and nuclear extracts.
Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined in SC tissues as previously described . MPO activity was expressed in units/mg protein.
Measurement of TNF-α and IL-1β
SC tissues, collected at 24 hrs after SCI, were homogenized in PBS containing 2 mmol/L of phenyl-methyl sulfonyl fluoride and tissue TNF-α and IL-1β levels were evaluated by using a colorimetric commercial kit DuoSet ELISA Development System (R&D Systems, Milan, Italy).
Immunohistochemical localization of Bax, Bcl-2, GFAP, CD11-β, BDNF, GDNF, NT-3, PAR, and NAMPT
Sections (8 μm) were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% hydrogen peroxide in 60% methanol for 30 minutes. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 minutes. Sections were incubated overnight with anti-Bax (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-Bcl-2 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GFAP (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD11-β (1:500; AbD Serotec, Raleigh, NC, USA), anti-BDNF (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GDNF (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NT3 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PAR (10 μg/ml; Merck Millipore, Billerica, MA, USA), and NAMPT (10 μg/ml; Novus Biologicals, Littleton, CO, USA). Sections were incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (Vector Lab. Inc., Burlingame, CA, USA). Immunocytochemistry photographs (n = 5 photos from each samples collected from all mice in each experimental group) were assessed by densitometry as previously described [29, 30] by using Optilab (Graftek, France) software on a Macintosh personal computer (CPU G3-266; Apple, Inc., Cupertino, CA, USA).
The tissues were fixed in 10% buffered formalin, and placed on a layer of glass wool in a large volume of Golgi-Cox solution and incubated at 37°C. After two months of incubation, the tissue samples were washed, dehydrated, cleared, wax impregnated and ultimately embedded in molten paraffin wax. 50 μ-thick sections were cut with the help of rotary microtome. The sections were collected in warm water and then mounted on albumenised slides. The slides were kept in oven at 50°C for half an hour. The paraffin wax was removed by immersing the slides in xylene. The slides were passed through descending grades of alcohol and ultimately dipped in distilled water. Blackening was done by placing the sections in 5% ammonia solution for one hour and subsequently washed thoroughly in distilled water. The tissue sections were dehydrated, cleared and mounted.
Section 5 μm-thick sections were deparaffinized with xylene, stained with Haematoxylin/Eosin (H&E), with Luxol Fast Blue with cresyl violet (Klüver and Barrera stain) counterstaining (used to assess demyelination) and studied using light microscopy (Dialux 22 Leitz, Milan Italy). Damaged neurons were counted and the histopathologic changes of the gray matter were scored on a 6-point scale : 0, no lesion observed, 1, gray matter contained 1 to 5 eosinophilic neurons; 2, gray matter contained 5 to 10 eosinophilic neurons; 3, gray matter contained more than 10 eosinophilic neurons; 4, small infarction (less than one third of the gray matter area); 5, moderate infarction; (one third to one half of the gray matter area); 6, large infarction (more than half of the gray matter area). The scores from all the sections from each spinal cord were averaged to give a final score for an individual mice.
Terminal Deoxynucleotidyltransferase-Mediated UTP End Labeling (TUNEL) Assay
TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer's instruction (Apotag, HRP kit DBA, Milan, Italy), as previously reported . The signals were visualized with diaminobenzidine.
Preparation of spinal cord extracts and Western blot analysis for phospho-NF-κB p65 (serine 536), NF-κB p65, Bax and Bcl-2
Cytosolic and nuclear extracts were prepared as previously described  with slight modifications. The filters were blocked with 5% non-fat dried milk (AppliChem GmbH, Germany) in PBS (PM) for 40 minutes at room temperature (RT) and subsequently probed with specific Abs phospho-NFκB p65 (serine 536) (Cell Signaling, 1:1,000), or anti-Bax (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), or anti-Bcl-2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), or anti-NFκB p65 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) in PM with 0.1% Tween-20 (Sigma-Aldrich, Milan, Italy) (PMT) at 4°C, overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2,000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at RT. To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the Abs against β-actin and lamin A/C (1:10,000 Sigma-Aldrich, Milan, Italy). The relative expression of the protein bands of phospho NFκB (75 kDa), NFκB p65 (65 kDa), Bax (approximately 23 kDa), Bcl-2 (approximately 29 kDa) was quantified by densitometric scanning of the X-ray films with Imaging Densitometer (GS-700, Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst, IBM, Milan, Italy).
Grading of motor disturbance
The motor function of mice subjected to compression trauma was assessed once a day for 20 days after injury. Recovery from motor disturbance was graded using the Basso Mouse Scale (BMS) open-field score , since the BMS has been shown to be a valid locomotor rating scale for mice. The evaluations were made by two blind observers for all analyzed groups. Briefly, the BMS is a nine-point scale that provides a gross indication of locomotor ability and determines the phases of locomotor recovery and features of locomotion. The BMS scale ranges from 0 (indicating complete paralysis) to 9 (indicating normal hindlimb function), rating locomotion on aspects of hindlimb function such as weight support, stepping ability, coordination, and toe clearance. The BMS score was determined for ten mice in each group.
All compounds, except for FK866 and GPP78, were obtained from Sigma-Aldrich (Milan, Italy). FK866 was synthesized in house as described previously and was over 99% pure . All chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Italy, UK).
All values in the figures and text are expressed as mean ± standard error of the mean (SEM) of N observations. For the in vivo studies N represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least 3 experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. BMS scale data were analyzed by the Mann-Whitney test and considered significant when p-value was < 0.05
Results and discussion
FK866 and GPP78 treatments reduce the severity of spinal cord trauma
FK866 modulates cytokines expression and neutrophil infiltration after SCI
Effect of FK866 on phosphorylation of p65 on Ser536 and nuclear NF-κB p65
Instead, IκB-α expression did not seem to be affected by FK866 treatment at 24 h after the injury (data not shown).
Effect of FK866 on NAMPT and PAR formation
FK866 decreased the degree of positive staining for NAMPT in the spinal cord from mice subjected to SCI (Figure 5c). Moreover, immunohistochemistry for PAR, as an indicator of in vivo PARP activation, revealed the occurrence of positive staining for PAR localized in nuclei of Schwann cells in the white and gray matter of the spinal cord tissues from mice subjected to SCI (Figure 5e). FK866 treatment reduced the degree of positive staining for PAR (Figure 5f) in the spinal cord.
Effect of FK866 on apoptosis in spinal cord after injury
To test whether the tissue damage was associated with the induction of apoptosis, we evaluated at 24 h after injury, the possible changes in apoptotic proteins (Bax and Bcl-2) and the DNA fragmentation (TUNEL-like staining).
Effect of FK866 on neurotrophic factors
FK866 treatment reduces astrocyte and microglial activation after SCI
SCI can result in severe disability, and it is at present a disease with a high therapeutic unmet need. The pathophysiology of SCI can be divided in two separate distinct phases: a primary injury elicited by the compression that determines a patient's prognosis and a secondary phase which can exacerbate damage and limit restorative processes, and therefore, contribute to overall morbidity and mortality. Unambiguously, inflammatory response following SCI contributes substantially to these secondary devastating effects. SCI-induced inflammation can lead to further reduction in functional recovery because of the development of scar tissue, as well as necrosis or apoptosis of neurons and myelin forming oligodendrocytes .
In the present manuscript, we have investigated the effect of the inhibition of an enzyme involved in NAD metabolism, NAMPT, in SCI recovery. NAMPT exists in two major forms, an intracellular one which has been implicated directly in NAD homeostasis, and an extracellular one, which is also named visfatin or PBEF . Whether visfatin exerts its functions via modulating NAD homeostasis is at present unclear, and a number of other mechanisms have been proposed ; . Furthermore, it cannot be ruled out that extracellular NMN, the product of NAMPT, may have biological functions. What is beyond doubt is that the extracellular form of visfatin displays plasma increases in a number of human inflammatory diseases.
We now show that NAMPT protein levels are increased upon SCI in the peri-lesional area. This could be due to the invasion of activated immune cells with upregulated NAMPT expression  into the injured spinal tissue. More importantly, we show for the first time that NAMPT inhibition corrects elevated NAMPT levels and ablates various inflammatory aspects of SCI-associated secondary damage by targeting neutrophils, macrophages, microglia and astrocytes, thereby leading to a significant recovery of the motor performance of injured animals.
Neutrophils are the first inflammatory cells to arrive at the site of injury and are implicated in the modulation of the secondary injury by release of reactive mediators that damage endothelial cells and increase vascular permeability thereby exaggerating immune cell invasion to the spinal cord leading to further damage . Neutrophils infiltration, as measured by MPO activity was significantly increased 24 h post trauma in spinal cord. In FK866-treated animals, SCI-evoked elevated MPO level was profoundly decreased and was accompanied by an overall attenuation of inflammatory mediators and motor function in mice.
Microglia and macrophages dominate the innate immune response elicited by SCI and sustain the secondary damage in animal models of SCI  as macrophage activation and their cytokine release, notably TNF-α and IL-1β, contribute to axonal demyelination  and cell death . Twenty-four h after traumatic SCI, spinal content of the proinflammatory TNF-α and IL-1β as well as CD11b positive cells displayed a substantial rise, while NAMPT inhibition by administration of FK866 prevented the elevation of inflammatory markers. These findings paralleled the reversal by FK866 of SCI-induced myelin degradation in spinal cord tissues as verified by less myelin phagocytosis by macrophages in Oil Red staining and less degenerating myelin in Luxol fast blue staining. Moreover, in spinal cord sections from injured-mice a dramatic astrogliosis denoted by increased immunoreactivity of GFAP was observed. As reactive astrocytes gives rise to an astroglial scar that acts as a physical and/or chemical barrier to axonal regeneration , its inhibition achieved by administration of FK866 is beneficial as was associated with more intact white matter in histologic evaluation.
Apoptosis is another component of secondary injury that can be triggered by a variety of insults including cytokines, inflammatory injury and free radical damage. Apoptosis in oligodendrocytes contributes to post-SCI demyelination while apoptosis in neurons contributes to cell loss [43, 44]. In our study, apoptosis was evident in both white and gray matter of injured spinal cord as evidenced by TUNEL positive cells and augmented tissue Bax/Bcl-2 ratio. Interestingly in FK866-treated mice, no apoptosis was detected, pro-apoptotic Bax significantly decreased and anti-apoptotic Bcl-2 restored. These observations were accompanied by the complete replenishment of the neurotrophic factors BDNF, GDNF and NT-3 as well as amelioration of neuron morphology in perilesional spinal cord sections from mice receiving FK866.
The beneficial properties of FK866 observed herein would be in accord with recent reports indicating that NAMPT inhibitors can ameliorate animal model symptomatology of inflammatory diseases such as arthritis, endotoxic shock and autoimmune encephalitis [15–17]. While the mechanism by which this class of drugs act as anti-tumoral agents is thought to involve energy deprivation , how FK866 should reduce inflammation is less straightforward. It has been recently shown that a reduction of PARP and/or SIRT activity might in part explain this phenomenon . Indeed, this would also be conceivable in SCI. PARP is a ubiquitous, chromatin-bound enzyme abundantly present in the nuclei of numerous cell types (Szabó and Dawson, 1998). Continuous or excessive activation of PARP produces extended chains of ADP-ribose on nuclear proteins and results in a substantial depletion of intracellular NAD+ and subsequently, adenosine triphosphate (ATP), leading to cellular dysfunction and ultimately, cell death . Consistent with our observation of a drastic rise in PAR level, an indicator of PARP activation, 24 h following SCI it has been demonstrated that SCI induces PARP activation  and that pharmacologic or genetic ablation of PARP activity can reduce the degree of tissue injury associated with spinal cord trauma . Of note, treatment with FK866 blunted the SCI-evoked PARP activation indicating PARP as a potential signaling pathway targeted by NAMPT inhibitors to elicit their neuroprotection. SIRTs constitute another group of NAD-dependent enzymes with divergent roles in neuronal survival. SIRT1 elicits anti-apoptotic effects while SIRT2, 3 and 6 have been shown to promote cell death . Specifically, SIRT2 is reportedly increased upon damage to the spinal cord in rat following a proteomic approach, while its pharmacological or genetic inhibition protects against neurotoxicity in models of Parkinson's disease . Therefore, it is plausible that NAD depletion following administration of NAMPT inhibitors decreases SIRT2 deacetylase activity and confers protection against deleterious stimuli in SCI. Of importance, recent evidence also suggests that cell survival promoting properties of SIRT1 might be mediated by a non-catalytic mechanism and, hence, can be preserved in presence of NAMPT inhibitors.
While the processes downstream of NAD depletion have not been evaluated in the present manuscript, we have observed that (i) inflammatory cytokines (eg. TNF-α and IL-1β) are increased upon SCI and are significantly reduced by NAMPT inhibition; (ii) NF-κB expression was also significantly increased upon SCI and normalized by NAMPT inhibition and (iii) that neutrophil infiltration and reactive gliosis, two hallmarks of the inflammatory process which takes place in the injured spinal cord, were significantly reduced by FK866 treatment. These data, taken together, would suggest that the inflammatory component of the injury is the primary target of these inhibitors. FK866 could (also) alleviate SCI by inhibiting TNF-α secretion by macrophages and microglia, thereby reducing inflammation and thus preventing the damage.
This study suggests that FK866, a specific inhibitor of NAMPT, administered after SCI, is capable of reducing the secondary injury and partly reduce permanent damage. The specificity of the effect is supported by the fact that another inhibitor of NAMPT, termed GPP78, elicits the same effects. Both drugs have been administered after the trauma was elicited, in a setting that mimics the clinical situation, and therefore our results may have clinical implications.
The authors would like to thank Carmelo La Spada for his excellent technical assistance during this study, Mrs Caterina Cutrona for secretarial assistance and Miss Valentina Malvagni for editorial assistance with the manuscript.
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