Congenital tremor and myopathy secondary to novel MYBPC1 variant.

Congenital myopathy with tremor (MYOTREM) is a recently described disorder characterized by mild myopathy and a postural and intention tremor present since early infancy. MYOTREM is associated with pathogenic variants in MYBPC1 which encodes slow myosin-binding protein C, a sarcomere protein with regulatory and structural roles. Here, we describe a family with three generations of variably affected members exhibiting a novel variant in MYBPC1 (c.656 T > C, p.Leu219Pro). Among the unique features of affected family members is the persistence of tremor in sleep. We also present the first muscle magnetic resonance images for this disorder, and report muscle atrophy and fatty infiltration.

Further characterization of CEP85L-associated lissencephaly type 10: Report of a three-generation family and review of the literature.

Lissencephaly type 10 is a recently reported condition characterized by posterior predominant abnormalities in gyration with associated seizures, developmental delays or intellectual disability. We report a boy who presented at 5 years of age with epilepsy and developmental delays. His family history was notable for epilepsy in two prior generations associated with variable developmental and cognitive impact. Exome sequencing identified a novel missense variant in CEP85L [NM_001042475.2; c.196A>G, p.(Thr66Ala)] which segregated in four affected family members across three generations. Brain imaging of the proband demonstrated a posterior lissencephaly pattern with pachygyria, while other affected family members demonstrated a similar subcortical band heterotopia. This report expands the phenotypic spectrum of this rare disorder by describing a novel variant in CEP85L in a family with variable clinical and neuroimaging findings.

The Benefit of Multigene Panel Testing for the Diagnosis and Management of the Genetic Epilepsies.

With the increasing use of genetic testing in pediatric epilepsy, it is important to describe the diagnostic outcomes as they relate to clinical care. The goal of this study was to assess the diagnostic yield and impact on patient care of genetic epilepsy panel testing. We conducted a retrospective chart review of patients at the Children's Hospital of Eastern Ontario (CHEO) who had genetic testing between the years of 2013-2020. We identified 227 patients that met criteria for inclusion. The majority of patients had their testing performed as "out-of-province" tests since province-based testing during this period was limited. The diagnostic yield for multi-gene epilepsy panel testing was 17% (39/227) and consistent with the literature. Variants of unknown significance (VUS) were reported in a significant number of undiagnosed individuals (77%; 128/163). A higher diagnostic rate was observed in patients with a younger age of onset of seizures (before one year of age; 32%; 29/90). A genetic diagnosis informed prognosis, recurrence risk counselling and expedited access to resources in all those with a diagnosis. A direct change in clinical management as a result of the molecular diagnosis was evident for 9% (20/227) of patients. The information gathered in this study provides evidence of the clinical benefits of genetic testing in epilepsy and serves as a benchmark for comparison with the current provincial Ontario Epilepsy Genetic Testing Program (OEGTP) that began in 2020.

Spinal A3 adenosine receptor activation acutely restores morphine antinociception in opioid tolerant male rats.

Opioids are potent analgesics, but their pain-relieving effects diminish with repeated use. The reduction in analgesic potency is a hallmark of opioid analgesic tolerance, which hampers opioid pain therapy. In the central nervous system, opioid analgesia is critically modulated by adenosine, a purine nucleoside implicated in the beneficial and detrimental actions of opioid medications. Here, we focus on the A3 adenosine receptor (A3 AR) in opioid analgesic tolerance. Intrathecal administration of the A3 AR agonist MRS5698 with daily systemic morphine in male rats attenuated the reduction in morphine antinociception over 7 days. In rats with established morphine tolerance, intrathecal MRS5698 partially restored the antinociceptive effects of morphine. However, when MRS5698 was discontinued, these animals displayed a reduced antinociceptive response to morphine. Our results suggest that MRS5698 acutely and transiently potentiates morphine antinociception in tolerant rats. By contrast, in morphine-naïve rats MRS5698 treatment did not impact thermal nociceptive threshold or affect antinociceptive response to a single injection of morphine. Furthermore, we found that morphine-induced adenosine release in cerebrospinal fluid was blunted in tolerant animals, but total spinal A3 AR expression was not affected. Collectively, our findings indicate that spinal A3 AR activation acutely potentiates morphine antinociception in the opioid tolerant state.

Evaluating and Enhancing the Preparation of Patients and Families before Pediatric Surgery.

Surgery can be a difficult and unfamiliar experience for children and their families. We examined the ability of existing information to help families feel better prepared for surgery at the Alberta Children's Hospital (ACH) and evaluated the best way to enhance its content and accessibility. We developed an online survey for families who have had surgery at ACH. Participants were recruited through pre-existing patient networks and from the ACH Short Stay Unit (SSU) between October 2018 and October 2019. The survey asked participants to evaluate the information available to prepare them for surgery and requested suggestions for improvement. Our survey results show that those who completed the in-person Surgery 101 program felt significantly more prepared for surgery. Of those who did not attend; 40% would have been interested in participating but were unaware that the program existed; and 17% planned to attend but were unable to; due to work or travel distance. Participants felt additional resources via online content or paper handouts would be most valuable. We used this information to prepare an online accessible summary of the Surgery 101 program and tour in the form of a video to reach more Albertan families preparing for surgery for their children.

An overview of the adverse effects of cannabis use for Canadian physicians.

PURPOSE: Cannabis is the most widely used illicit substance and one of the most commonly used psychoactive substances in the world, preceded only by alcohol, tobacco and caffeine. Recent changes in legislation regarding cannabis use in Canada and potential upcoming changes worldwide may have a further impact on the prevalence of cannabis use. Thus, it is critical to understand the risks and potential adverse health effects of acute and long-term cannabis use. Current literature is lacking in many areas surrounding cannabis use, and for the most part is unable to provide clear associations once confounding variables are considered. Here we provide a general overview of the history of cannabis, the physical and mental health consequences, and the risks to specific groups. SOURCE: A scoping search of published articles in PubMed from the start date (1946) until 2018. PRINCIPAL FINDING: Current evidence supports an association between cannabis use and mild respiratory and cardiac effects, but no clear increased risk of cancer. Psychiatric disorders, including schizophrenia and anxiety, show associations with cannabis use; however, a causal effect of cannabis use is unclear. While no evidence for increased risk in pregnancy has been found, risk is still undetermined. Youth may be at a greater risk as earlier initiation of use increases the risk of adverse health effects. CONCLUSION: Overall, evidence for direct and long-term adverse effects of cannabis use is minimal and additional longitudinal studies will be required to better delineate unidentified effects.

Scientific overview on CSCI-CITAC Annual General Meeting and 2017 Young Investigators' Forum.

The 2017 Annual General Meeting of the Canadian Society of Clinician Investigators (CSCI) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was a national Annual General Meeting (AGM) held in Toronto, Ontario November 20-22, 2017, in conjunction with the University of Toronto Clinician Investigator Program Research Day. The theme for this year's meeting was "Roll up your sleeves-How to manage your physician scientist career", emphasizing lectures and workshops that were designed to provide tools for being proactive and successful in career planning. The keynote speakers were Dr. Rod McInnes (McGill University and Canadian Institutes of Health Research Acting President), who was the Distinguished Scientist Award recipient, Dr. David Goltzman (McGill University), who was the 2017 Henry Friesen Award recipient, Dr. Gillian Hawker (University of Toronto), Dr. Mike Sapieha (Université de Montréal), who was the 2017 Joe Doupe Award recipient, and Dr. Alex MacKenzie (Children's Hospital of Eastern Ontario Research Institute, University of Ottawa). The workshops, focusing on career development for clinician scientists, were hosted by Dr. Lisa Robinson, Dr. Nicola Jones, Kevin Vuong, Fran Brunelle, Dr. Jason Berman and Dr. Alan Underhill. Further to this, the Young Investigators' Forum encompasses presentations from scientist-clinician trainees from across the country. All scientific abstracts are summarized in this review. There were over 100 abstracts showcased at this year's meeting during the highlighted poster sessions, with six outstanding abstracts selected for oral presentations during the President's Forum.

Intrathecal delivery of a palmitoylated peptide targeting Y382-384 within the P2X7 receptor alleviates neuropathic pain.

Pain hypersensitivity resulting from peripheral nerve injury depends on pathological microglial activation in the dorsal horn of the spinal cord. This microglial activity is critically modulated by P2X7 receptors (P2X7R) and ATP stimulation of these receptors produces mechanical allodynia, a defining feature of neuropathic pain. Peripheral nerve injury increases P2X7R expression and potentiates its cation channel function in spinal microglia. Here, we report a means to preferentially block the potentiation of P2X7R function by delivering a membrane permeant small interfering peptide that targets Y382-384, a putative tyrosine phosphorylation site within the P2X7R intracellular C-terminal domain. Intrathecal administration of this palmitoylated peptide (P2X7R379-389) transiently reversed mechanical allodynia caused by peripheral nerve injury in both male and female rats. Furthermore, targeting Y382-384 suppressed P2X7R-mediated release of cytokine tumor necrosis factor alpha and blocked the adoptive transfer of mechanical allodynia caused by intrathecal injection of P2X7R-stimulated microglia. Thus, Y382-384 site-specific modulation of P2X7R is an important microglial mechanism in neuropathic pain.

Microglial pannexin-1 channel activation is a spinal determinant of joint pain.

Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5'-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1ß (IL-1ß) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide 10panx suppressed the aberrant discharge of spinal laminae I-II neurons evoked by innocuous mechanical hindpaw stimulation in arthritic rats. Furthermore, mice with a microglia-specific genetic deletion of Panx1 were protected from developing mechanical allodynia. Treatment with probenecid, a clinically used broad-spectrum Panx1 blocker, resulted in a striking attenuation of MIA-induced mechanical allodynia and normalized responses in the dynamic weight-bearing test, without affecting acute nociception. Probenecid reversal of mechanical allodynia was also observed in rats 13 weeks after anterior cruciate ligament transection, a model of posttraumatic osteoarthritis. Thus, Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.

Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance.

Tolerance to the analgesic effects of opioids is a major problem in chronic pain management. Microglia are implicated in opioid tolerance, but the core mechanisms regulating their response to opioids remain obscure. By selectively ablating microglia in the spinal cord using a saporin-conjugated antibody to Mac1, we demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats. Increased P2X7 receptor (P2X7R) activity is a cardinal feature of microglial activation, and in this study we found that morphine potentiates P2X7R-mediated Ca2+ responses in resident spinal microglia acutely isolated from morphine tolerant rats. The increased P2X7R function was blocked in cultured microglia by PP2, a Src family protein tyrosine kinase inhibitor. We identified Src family kinase activation mediated by µ-receptors as a key mechanistic step required for morphine potentiation of P2X7R function. Furthermore, we show by site-directed mutagenesis that tyrosine (Y382-384) within the P2X7R C-terminus is differentially modulated by repeated morphine treatment and has no bearing on normal P2X7R function. Intrathecal administration of a palmitoylated peptide corresponding to the Y382-384 site suppressed morphine-induced microglial reactivity and preserved the antinociceptive effects of morphine in male rats. Thus, site-specific regulation of P2X7R function mediated by Y382-384 is a novel cellular determinant of the microglial response to morphine that critically underlies the development of morphine analgesic tolerance.SIGNIFICANCE STATEMENT Controlling pain is one of the most difficult challenges in medicine and its management is a requirement of a large diversity of illnesses. Although morphine and other opioids offer dramatic and impressive relief of pain, their impact is truncated by loss of efficacy (analgesic tolerance). Understanding why this occurs and how to prevent it are of critical importance in improving pain therapies. We uncovered a novel site (Y382-384) within the P2X7 receptor that can be targeted to blunt the development of morphine analgesic tolerance, without affecting normal P2X7 receptor function. Our findings provide a critical missing mechanistic piece, site-specific modulation by Y382-384, that unifies P2X7R function to the activation of spinal microglia and the development of morphine tolerance.

Genetic deletion of microglial Panx1 attenuates morphine withdrawal, but not analgesic tolerance or hyperalgesia in mice.

Opioids are among the most powerful analgesics for managing pain, yet their repeated use can lead to the development of severe adverse effects. In a recent study, we identified the microglial pannexin-1 channel (Panx1) as a critical substrate for opioid withdrawal. Here, we investigated whether microglial Panx1 contributes to opioid-induced hyperalgesia (OIH) and opioid analgesic tolerance using mice with a tamoxifen-inducible deletion of microglial Panx1. We determined that escalating doses of morphine resulted in thermal pain hypersensitivity in both Panx1-expressing and microglial Panx1-deficient mice. In microglial Panx1-deficient mice, we also found that acute morphine antinociception remained intact, and repeated morphine treatment at a constant dose resulted in a progressive decline in morphine antinociception and a reduction in morphine potency. This reduction in morphine antinociceptive potency was indistinguishable from that observed in Panx1-expressing mice. Notably, morphine tolerant animals displayed increased spinal microglial reactivity, but no change of microglial Panx1 expression. Collectively, our findings indicate microglial Panx1 differentially contributes to opioid withdrawal, but not the development of opioid-induced hyperalgesia or tolerance.

An evolutionary switch in ND2 enables Src kinase regulation of NMDA receptors.

The non-receptor tyrosine kinase Src is a key signalling hub for upregulating the function of N-methyl D-aspartate receptors (NMDARs). Src is anchored within the NMDAR complex via NADH dehydrogenase subunit 2 (ND2), a mitochondrially encoded adaptor protein. The interacting regions between Src and ND2 have been broadly identified, but the interaction between ND2 and the NMDAR has remained elusive. Here we generate a homology model of ND2 and dock it onto the NMDAR via the transmembrane domain of GluN1. This interaction is enabled by the evolutionary loss of three helices in bilaterian ND2 proteins compared to their ancestral homologues. We experimentally validate our model and demonstrate that blocking this interaction with an ND2 fragment identified in our experimental studies prevents Src-mediated upregulation of NMDAR currents in neurons. Our findings establish the mode of interaction between an NMDAR accessory protein with one of the core subunits of the receptor.

Blocking microglial pannexin-1 channels alleviates morphine withdrawal in rodents.

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (10panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia.

Animal models of chronic pain: Advances and challenges for clinical translation.

Chronic pain is a global problem that has reached epidemic proportions. An estimated 20% of adults suffer from pain, and another 10% are diagnosed with chronic pain each year (Goldberg and McGee, ). Despite the high prevalence of chronic pain (an estimated 1.5 billion people are afflicted worldwide), much remains to be understood about the underlying causes of this condition, and there is an urgent requirement for better pain therapies. The discovery of novel targets and the development of better analgesics rely on an assortment of preclinical animal models; however, there are major challenges to translating discoveries made in animal models to realized pain therapies in humans. This review discusses common animal models used to recapitulate clinical chronic pain conditions (such as neuropathic, inflammatory, and visceral pain) and the methods for assessing the sensory and affective components of pain in animals. We also discuss the advantages and limitations of modeling chronic pain in animals as well as highlighting strategies for improving the predictive validity of preclinical pain studies. © 2016 Wiley Periodicals, Inc.

Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physical dependence.

The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4). Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal) were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (-) naloxone, an opioid receptor antagonist, and (+) naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.