Cellular prion protein protects from inflammatory and neuropathic pain
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Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours.
We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 months old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mechanical and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behaviour observed in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a critical role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC.
KeywordsPrion protein pain knockout mice NMDA receptor spinal cord
The dorsal horn of spinal cord is an important site for pain transmission and modulation of incoming nociceptive information arriving from peripheral nociceptors [1, 2]. Glutamate is the key neurotransmitter released by the primary afferent fibers [3, 4] and plays an important role in nociceptor sensitization and in the modulation of allodynia . Glutamate receptors (GluRs) such as N-methyl-D-aspartate (NMDA) receptors contribute in various ways to pain induction, transmission and control [5, 6, 7]. Consequently, NMDA receptor inhibitors exhibit antinociceptive and analgesic effects in rodents [8, 9] as well in humans , however, their clinical use for the treatment of pain has been hampered by their CNS side-effects [11, 12]. For this reason, strategies such as src interfering peptides have been proposed as ways to interfere with NMDAR hyperactivity in the pain pathway without affecting basal NMDA receptor function .
NMDA receptors are regulated by a plethora of cellular signaling pathways that could potentially be targeted for therapeutic intervetion . Along these lines, our laboratory has recently shown  that NMDA receptor activity in mouse hippocampal neurons is regulated by cellular prion protein (PrPc). Specifically, NMDA receptors expressed in mice lacking PrPC show slowed current decay kinetics, and spontaneous synaptic NMDA currents in pyramidal neurons displayed increased current amplitude . We subsequently showed that PrPC protects from depressive like behavior by tonically inhibiting NMDA receptor activity , thus suggesting that the altered NMDA currents in PrPC null mice are associated with a clear behavioral phenotype. Given the important role of NMDA receptors in the afferent pain pathway, we hypothesized that absence of PrPC may give rise to pain hypersensitivity. Here, we show that PrPC null mice exhibit a decreased nociceptive threshold, both under basal conditions, as well as in models of inflammatory and neuropathic pain. These effects were reversed by treatment of the animals with the NMDA receptor antagonist MK-801, thus implicating NMDA receptor dysregulation in the observed pain phenotype.
Mechanical and thermal withdrawal threshold of PrP+/+ and PrP-/- mice
Nociceptive response of PrP+/+ and PrP-/- mice under acute stimulation
We also directly injected glutamate (3 μmol or 10 μmol) into the paws of wild type and null mice, and determined the time that the animals spent licking and biting over a 15 minute time course. As shown in Figure 2E, the higher dose of glutamate resulted in a significantly greater increase in response time compared to wild type animals.
Nociceptive response of PrP+/+ and PrP-/- mice in response to NMDA treatment
Nociceptive response of PrP+/+ and PrP-/- mice under neuropathic pain
It is well established that glutamate is involved in nociceptive processing in the spinal cord in both normal conditions and under certain pathological nociceptive processes [4, 11, 17] with NMDA receptors being the a key player in the binding of glutamate and activation of postsynaptic responses . Under conditions of neuropathic pain, NMDA receptors are upregulated to mediate sensitization in second order neurons give rise to a phenomenon termed "wind up" [19, 20, 21, 22] leading to allodynia and hyperalgesia . Consequently, NMDA receptor antagonists have been known to decrease neuronal hyperexitability and reduce pain, and the efficacy of several NMDA receptor antagonists has been investigated in preclinical [8, 9, 23] and clinical pain studies . Conversely, processes that augment NMDA receptor function would be expected to be pronociceptive.
We have recently shown that PrPC physically interacts with NMDA receptors to inhibit NMDA receptor activity in the brain . The absence of PrPC resulted in increased amplitudes and durations of synaptic NMDA currents  whereas AMPA receptors were unaffected . The data presented here are consistent with a similar NMDA receptor hyperfunction in the afferent pain pathway. Mice lacking PrPc displayed increased nociceptive responses in thermal and mechanical tests, and showed an increased susceptibility to the development of inflammatory pain. Of note, an intrathecal dose of NMDA (30 pmol/i.t.), which does not induce in wild-type animals any pain behaviour per se, produced nociceptive behaviour in PrPC null mice that was comparable to that exhibited by wild-type animals injected with a 10 fold higher dose of NMDA (300 pmol/i.t.). These data fit with NMDA receptor hyperactivity, and suggest that PrPC null mice behave as if they are in a basal neuropathic pain state.
It is well established that nociceptive behaviour observed during first phase of formalin pain is a result of the direct chemical activation of peripheral nociceptors and by the release of local glutamate by the primary afferent, whereas the second phase results from central sensitization of dorsal horn neurons induced by primary afferent activity and peripheral inflammatory response . However, the nociception caused by intraplnatar (i.pl.) injection of glutamate involves peripheral, spinal and supraspinal sites of action and is greatly mediated by both NMDA and non-NMDA receptors as well as by the release of nitric oxide . Furthermore, it is also mediated by capsaicin-sensitive fibres and by release of neurokinins from sensory neurons that activate NK2 and B1 receptors . NMDA receptor antagonists are effective in attenuating both phases of the nociception induced by formalin in rodents when delivered systemically [28, 29] or spinally [30, 31]. Moreover, NMDA receptor inhibitors reduced windup in dorsal horn neurons in animals with peripheral nerve injury [32, 33, 34], and NMDA receptor inhibition may impair wind-up in spinal neurons that relay C fiber input to the primary somatosensory cortex . Our data showing sensitivity of the all of the observed PrPC null mouse pain phenotypes to NMDA receptor blockers thus fit with these previous studies. Also in agreement with our data are results from Meotti and co-workers  showing that PrPC null mice exhibited higher number of abdominal constrictions following intraperitoneal acetic acid injection when compared to wild-type mice. On the other hand, the same study showed that PrPC null mice appeared to exhibit less pain in the tail-flick test, and had no difference in latency response in the hot-plate test, in contrast with our findings.
Although much attention has been focused on the infectious (misfolded) scrapie form of PrPC (termed PrPSc) it is becoming increasingly evident that normal PrPC plays an important role in the normal physiology of the nervous system . This includes neuroprotection [15, 38] protection from epileptic seizures [39, 40] and from development of depressive like behaviour  all of which have been linked to NMDA receptors. Our findings showing increased pain in PrPC null mice fit with the concept of PrPC playing a beneficial physiological role. It is widely recognized that misfolding of PrPC into a β-sheet rich infectious prion mediates severe neurological phenotypes, such as Creutzfeldt-Jakob disease (CJD) and variant CJD [41, 42, 43]. In these disorders, normal PrPC is progressively converted into infectious prions, which then form plaques and cause severe neurodegeneration, ultimately leading to death. It is possible that the conversion of normal PrPC results in altered NMDA receptor currents in these patients, either by a reduction in the levels of normal PrPC, or by binding of misfolded PrPSc to the receptor complex. Such a mechanism may indeed fit with data showing that neuronal cultures infected with PrPSc are protected from cell death by NMDA receptor blockers . In this context, it is interesting to note that a substantial fraction of new variant CJD patients show increased pain sensitivity  including limb pain . Furthermore, there is a case report of a woman who developed vulvodynia despite normal vulvo-vaginal examination, and this patient was subsequently diagnosed with CJD . While it is unknown as to whether this is due to augmentation of spinal NMDA receptor function in these patients due to compromised PrPC regulation of the receptors, such a mechanism would be consistent with our observations in mice.
In summary, our data indentify PrPC as an important negative regulator of pain signalling. Considering that PrPC physically interacts with the receptor complex to depress current amplitude, it may perhaps be possible to mimic this inhibition with small organic molecules interacting at the PrPC interaction site on the receptor.
Materials and methods
All experiments were conducted following the protocol approved by the Institutional Animal Care and Use Committee (protocol #M09100) and all efforts were made to minimize animal suffering. Unless stated otherwise, 10 week old male mice (C57BL/6J wild type and PrP null weighing 25-30 g) were used. Animals were housed at a maximum of five per cage (30 × 20 × 15 cm) with food and water ad libitum. They were kept in 12 h light/dark cycles (lights on at 7:00 a.m.) at a temperature of 23 ± 1°C. All manipulations were carried out between 11.00 am and 3:00 pm. Different cohorts of mice were used for each test and each mouse was used only once. The observer was blind to the experimental conditions in the experiment examining the age dependence of the pain phenotype. Mice with a targeted disruption of the prion gene (PrP) of the Zürich 1 strain  were obtained from the European Mouse Mutant Archive (EM:0158; European Mouse Mutant Archive, Rome) and out-bred to generate PrP-/- (PrP-null) littermates used in the experiments. Genotyping was performed by gel electrophoresis of PCR products obtained from genomic DNA that was isolated from tail samples. Primers and PCR parameters were similar to those used previously .
Drugs and treatment
The following drugs were used in the study: L-glutamic acid hydrochloride, MK-801, N-methyl-D-aspartatic acid, Formaldehyde (Sigma Chemical Company, St. Louis, MO, USA). All drugs were dissolved in PBS. When drugs were delivered by the intraperitoneal (i.p.) route, a constant volume of 10 ml/kg body weight was injected. When drugs were administered by the intrathecal (i.t.) route, volumes of 5 μl were injected. Appropriate vehicle-treated groups were also assessed simultaneously. The choice of the doses of each drug was based on preliminary experiments in our laboratory.
The formalin test is a widely used model that allows us to evaluate two different types of pain: neurogenic pain is caused by direct activation of nociceptive nerve terminals, while the inflammatory pain phase is mediated by a combination of peripheral input and spinal cord sensitization [49, 50, 51]. Animals received 20 μl of different concentrations of formalin solution (1.25% or 2.5%) made up in PBS injected intraplantarly (i.pl.) in the ventral surface of the right hindpaw. We observed animals individually from 0-5 min (neurogenic phase) and 15-30 min (inflammatory phase). Following i.pl. injection of formalin, the animals were immediately placed individually in observation chambers and the time spent licking or biting the injected paw was recorded with a chronometer and considered as nociceptive response. In experiemnts involving MK-801, mice were treated by intrathecal delivery 10 minutes prior to formalin injection (1.25%) at a dose of 3 nmol.
Intraplantar glutamate injection
The procedure used was similar to that described previously . Briefly, a volume of 20 μl of glutamate (3 nmol/paw or 10 nmol/paw prepared in PBS) was injected i.pl. into the ventral surface of the right hindpaw. Animals were observed individually for 15 min following glutamate injection. The amount of time spent licking the injected paw was recorded with a chronometer and was considered as nociceptive response.
Intrathecal NMDA injection
To directly investigate the role of spinal glutamate receptors in the nociceptive behaviour observed for PrPc knockout mice, we compared the nociceptive behavior of wild-type and PrPc knockout mice after a single intrathecal injection of NMDA. Animals received an i.t. injection of 5 μl of NMDA solution. Injections were given to non-anaesthetized animals using the method described by Hylden and Wilcox . Briefly, animals were restrained manually and a 30-gauge needle attached to a 25-μl microsyringe was inserted through the skin and between the vertebrae into the subdural space of the L5-L6 spinal segments. NMDA injections (30 pmol or 300 pmol) were given over a period of 5 seconds. The amount of time that animals spent biting or licking their hind paws, tail or abdomen was determined with a chronometer and considered as nociceptive response. In experiments involving MK-801, mice were treated by intraperitoneal delivery 30 minutes prior to NMDA injection at a dose of 30 pmol/site.
Chronic constriction injury (CCI)-induced neuropathy
For neuropathic pain, we used a sciatic nerve injury model according to the method described by Bennett and Xie  with minor modifications. Briefly, mice were anaesthetized (isoflurane 5% indution, 2.5% maintenence) and the right sciatic nerve was exposed at the level of the thigh by blunt dissection through the biceps femoris. Proximal to the sciatic nerve trifurcation, about 12 mm of nerve was freed of adhering tissue and 3 loose ligatures (silk suture 6-0) were loosely tied around it with about 1 mm spacing so that the epineural circulation was preserved. In sham-operated rats, the nerve was exposed but not injuried. Mechanical and thermal withdrawal thresholds were determined 3 days after surgery. In another series of experiments, PrPC null mice received MK-801 (3 pmol/i.t.) and mechanical withdrawal threshold was evaluated 10 min after drug delivery.
Mechanical withdrawal threshold
To assess changes in sensation or in the development of mechanical allodynia, sensitivity to tactile stimulation was measured using the DPA (Ugo Basile, Varese, Italy). Animals were placed individually in a small enclosed testing arena (20 cm × 18.5 cm × 13 cm, length × width × height) with a wire mesh floor for 60 min. The DPA device was positioned beneath the animal, so that the filament was directly under the plantar surface of the foot to be tested. Each paw was tested three times per session. For experiment 1, the same cohort of both wild-type and PrPC null mice were tested at age 1, 2, 3, 4 and 5 months. For neuropathic pain, testing was performed on the ispsilateral (ligated) paw before ligation (day 0) and then on the 3rd day after ligation.
Thermal withdrawal threshold
Thermal hyperalgesia was examined by measuring the latency to withdrawal of the hind paws from a focused beam of radiant heat applied to the plantar surface using a Plantar Test apparatus (Ugo Basile). Three trials each for the right hind paws were performed and for each reading, the apparatus was set at a cut-off time of 20 s. As with mechanical pain testing, the same cohorts of either wild-type or PrPC null mice were used at 1, 2, 3, 4 and 5 months. Thermal withdrawal threshold was tested 1 day after they were used for mechanical testing. For neuropathic pain, testing was performed on the ispsilateral (ligated) paw before ligation (day 0) and then on the 4th day after nerve injury.
Data were presented as means ± SEM and evaluated by t-tests, two-way or three-way analysis of variance (ANOVA) followed by Tukey test when appropriate. A value of P < 0.05 was considered to be significant.
This work was supported by an operating grant to GWZ from the PrioNet Canada. GWZ is a Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR) and a Canada Research Chair in Molecular Neurobiology. VMG is supported by an AHFMR Fellowship and by a Fellowship from the Hotchkiss Brain Institute (HBI). We thank Dr. Clint Doering for genotyping and breeding paradigms, Dr. Frank R. Jirik for providing the outbred PrP null mouse line, and Stephan Bonfield for the blind analysis for experiment 1.
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