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Targeting of the CB2 receptor results in neuroprotection in the SOD1G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). The neuroprotective effects of CB2 receptors are facilitated by their upregulation in the spinal cord of the mutant mice. Here, we investigated whether similar CB2 receptor upregulation, as well as parallel changes in other endocannabinoid elements, is evident in the spinal cord of dogs with degenerative myelopathy (DM), caused by mutations in the superoxide dismutase 1 gene (SOD1). We used well-characterized post-mortem spinal cords from unaffected and DM-affected dogs. Tissues were used first to confirm the loss of motor neurons using Nissl staining, which was accompanied by glial reactivity (elevated GFAP and Iba-1 immunoreactivity). Next, we investigated possible differences in the expression of endocannabinoid genes measured by qPCR between DM-affected and control dogs. We found no changes in expression of the CB1 receptor (confirmed with CB1 receptor immunostaining) or NAPE-PLD, DAGL, FAAH and MAGL enzymes. In contrast, CB2 receptor levels were significantly elevated in DM-affected dogs determined by qPCR and western blotting, which was confirmed in the grey matter using CB2 receptor immunostaining. Using double-labelling immunofluorescence, CB2 receptor immunolabelling colocalized with GFAP but not Iba-1, indicating upregulation of CB2 receptors on astrocytes in DM-affected dogs. Our results demonstrate a marked upregulation of CB2 receptors in the spinal cord in canine DM, which is concentrated in activated astrocytes. Such receptors could be used as a potential target to enhance the neuroprotective effects exerted by these glial cells., Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Ministerio Economia y competitividad, GW Pharmaceuticals, Depto. de Bioquímica y Biología Molecular, Fac. de Medicina, TRUE, pub

Altered microglia function contributes to loss of CNS homeostasis during aging in the brain. Few studies have evaluated age-related alterations in spinal cord microglia. We previously demonstrated that lumbar spinal cord microglial expression of inducible nitric oxide synthase (iNOS) was equivalent between aging, neurologically normal dogs and dogs with canine degenerative myelopathy (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157). This unexpected finding suggested that microglia in aging spinal cord have a pro-inflammatory polarization. In this study, we reexamined our microglial results (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157) within the context of aging rather than disease by comparing microglia in aging versus young adult dogs. For both aging and young adult dogs, the density of microglia was significantly higher closest to the motor neuron cell body. However, there was no difference in densities between aging versus young adult dogs at all distances except for the furthest distance analyzed. The number of motor neurons with polarized microglia was higher in aging dogs; yet, the density per motor neuron of arginase-1-expressing microglia was reduced in aging dogs compared with young adult dogs. Finally, aging dogs had increased steady-state mRNA levels for genes consistent with activated microglia compared with young adult dogs. However, altered mRNA levels were limited to the lumbar spinal cord. These data suggested that aging dog spinal cord microglia exhibit regional immunophenotypic differences, which may render lumbar motor neurons more susceptible to age-related pathological insults.

Altered microglia function contributes to loss of CNS homeostasis during aging in the brain. Few studies have evaluated age-related alterations in spinal cord microglia. We previously demonstrated that lumbar spinal cord microglial expression of inducible nitric oxide synthase (iNOS) was equivalent between aging, neurologically normal dogs and dogs with canine degenerative myelopathy (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157). This unexpected finding suggested that microglia in aging spinal cord have a pro-inflammatory polarization. In this study, we reexamined our microglial results (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157) within the context of aging rather than disease by comparing microglia in aging versus young adult dogs. For both aging and young adult dogs, the density of microglia was significantly higher closest to the motor neuron cell body. However, there was no difference in densities between aging versus young adult dogs at all distances except for the furthest distance analyzed. The number of motor neurons with polarized microglia was higher in aging dogs; yet, the density per motor neuron of arginase-1-expressing microglia was reduced in aging dogs compared with young adult dogs. Finally, aging dogs had increased steady-state mRNA levels for genes consistent with activated microglia compared with young adult dogs. However, altered mRNA levels were limited to the lumbar spinal cord. These data suggested that aging dog spinal cord microglia exhibit regional immunophenotypic differences, which may render lumbar motor neurons more susceptible to age-related pathological insults.

Altered microglia function contributes to loss of CNS homeostasis during aging in the brain. Few studies have evaluated age-related alterations in spinal cord microglia. We previously demonstrated that lumbar spinal cord microglial expression of inducible nitric oxide synthase (iNOS) was equivalent between aging, neurologically normal dogs and dogs with canine degenerative myelopathy (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157). This unexpected finding suggested that microglia in aging spinal cord have a pro-inflammatory polarization. In this study, we reexamined our microglial results (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157) within the context of aging rather than disease by comparing microglia in aging versus young adult dogs. For both aging and young adult dogs, the density of microglia was significantly higher closest to the motor neuron cell body. However, there was no difference in densities between aging versus young adult dogs at all distances except for the furthest distance analyzed. The number of motor neurons with polarized microglia was higher in aging dogs; yet, the density per motor neuron of arginase-1-expressing microglia was reduced in aging dogs compared with young adult dogs. Finally, aging dogs had increased steady-state mRNA levels for genes consistent with activated microglia compared with young adult dogs. However, altered mRNA levels were limited to the lumbar spinal cord. These data suggested that aging dog spinal cord microglia exhibit regional immunophenotypic differences, which may render lumbar motor neurons more susceptible to age-related pathological insults.

Toxicity within superoxide dismutase-1 (SOD1)-associated familial amyotrophic lateral sclerosis (ALS) is non-cell autonomous with direct contribution from microglia. Microglia exhibit variable expression of neuroprotective and neurotoxic molecules throughout disease progression. The mechanisms regulating microglial phenotype within ALS are not well understood. This work presents a first study to examine the specific microglial phenotypic response in close association to motor neurons in a naturally occurring disease model of ALS, canine degenerative myelopathy (DM). Microglia closely associated with motor neurons were increased in all stages of DM progression, although only DM Late reached statistical significance. Furthermore, the number of arginase-1 expressing microglia per motor neuron were significantly increased in early stages of DM, whereas the number of inducible nitric oxide synthase (iNOS)-expressing microglia per motor neuron was indistinguishable from aged controls at all stages of disease. Fractalkine, a chemotactic molecule for microglia, was expressed in motor neurons, and the fractalkine receptor was specifically localized to microglia. However, we found no correlation between microglial response and lumbar spinal cord fractalkine levels. Taken together, these data suggest that arginase-1-expressing microglia are recruited to the motor neuron early in DM disease through a fractalkine-independent mechanism.

Toxicity within superoxide dismutase-1 (SOD1)-associated familial amyotrophic lateral sclerosis (ALS) is non-cell autonomous with direct contribution from microglia. Microglia exhibit variable expression of neuroprotective and neurotoxic molecules throughout disease progression. The mechanisms regulating microglial phenotype within ALS are not well understood. This work presents a first study to examine the specific microglial phenotypic response in close association to motor neurons in a naturally occurring disease model of ALS, canine degenerative myelopathy (DM). Microglia closely associated with motor neurons were increased in all stages of DM progression, although only DM Late reached statistical significance. Furthermore, the number of arginase-1 expressing microglia per motor neuron were significantly increased in early stages of DM, whereas the number of inducible nitric oxide synthase (iNOS)-expressing microglia per motor neuron was indistinguishable from aged controls at all stages of disease. Fractalkine, a chemotactic molecule for microglia, was expressed in motor neurons, and the fractalkine receptor was specifically localized to microglia. However, we found no correlation between microglial response and lumbar spinal cord fractalkine levels. Taken together, these data suggest that arginase-1-expressing microglia are recruited to the motor neuron early in DM disease through a fractalkine-independent mechanism.

Toxicity within superoxide dismutase-1 (SOD1)-associated familial amyotrophic lateral sclerosis (ALS) is non-cell autonomous with direct contribution from microglia. Microglia exhibit variable expression of neuroprotective and neurotoxic molecules throughout disease progression. The mechanisms regulating microglial phenotype within ALS are not well understood. This work presents a first study to examine the specific microglial phenotypic response in close association to motor neurons in a naturally occurring disease model of ALS, canine degenerative myelopathy (DM). Microglia closely associated with motor neurons were increased in all stages of DM progression, although only DM Late reached statistical significance. Furthermore, the number of arginase-1 expressing microglia per motor neuron were significantly increased in early stages of DM, whereas the number of inducible nitric oxide synthase (iNOS)-expressing microglia per motor neuron was indistinguishable from aged controls at all stages of disease. Fractalkine, a chemotactic molecule for microglia, was expressed in motor neurons, and the fractalkine receptor was specifically localized to microglia. However, we found no correlation between microglial response and lumbar spinal cord fractalkine levels. Taken together, these data suggest that arginase-1-expressing microglia are recruited to the motor neuron early in DM disease through a fractalkine-independent mechanism.

Canine degenerative myelopathy (DM) is a naturally occurring neurodegenerative disease with similarities to some forms of amyotrophic lateral sclerosis (ALS). Most dogs that develop DM are homozygous for a common superoxide dismutase 1 gene (SOD1) mutation. However, not all dogs homozygous for this mutation develop disease. We performed a genome-wide association analysis in the Pembroke Welsh Corgi (PWC) breed comparing DM-affected and -unaffected dogs homozygous for the SOD1 mutation. The analysis revealed a modifier locus on canine chromosome 25. A haplotype within the SP110 nuclear body protein (SP110) was present in 40% of affected compared with 4% of unaffected dogs (P = 1.5 x 10(-5)), and was associated with increased probability of developing DM (P = 4.8 x 10(-6)) and earlier onset of disease (P = 1.7 x 10(-5)). SP110 is a nuclear body protein involved in the regulation of gene transcription. Our findings suggest that variations in SP110-mediated gene transcription may underlie, at least in part, the variability in risk for developing DM among PWCs that are homozygous for the disease-related SOD1 mutation. Further studies are warranted to clarify the effect of this modifier across dog breeds.

Table of contentsA1 One health advances and successes in comparative medicine and translational researchCheryl StroudA2 Dendritic cell-targeted gorilla adenoviral vector for cancer vaccination for canine melanomaIgor Dmitriev, Elena Kashentseva, Jeffrey N. Bryan, David T. CurielA3 Viroimmunotherapy for malignant melanoma in the companion dog modelJeffrey N. Bryan, David Curiel, Igor Dmitriev, Elena Kashentseva, Hans Rindt, Carol Reinero, Carolyn J. HenryA4 Of mice and men (and dogs!): development of a commercially licensed xenogeneic DNA vaccine for companion animals with malignant melanomaPhilip J. BergmanA5 Successful immunotherapy with a recombinant HER2-expressing Listeria monocytogenes in dogs with spontaneous osteosarcoma paves the way for advances in pediatric osteosarcomaNicola J. Mason, Josephine S. Gnanandarajah, Julie B. Engiles, Falon Gray, Danielle Laughlin, Anita Gaurnier-Hausser, Anu Wallecha, Margie Huebner, Yvonne PatersonA6 Human clinical development of ADXS-HER2Daniel O'ConnorA7 Leveraging use of data for both human and veterinary benefitLaura S. TremlA8 Biologic replacement of the knee: innovations and early clinical resultsJames P. StannardA9 Mizzou BioJoint Center: a translational success storyJames L. CookA10 University and industry translational partnership: from the lab to commercializationMarc JacobsA11 Beyond docking: an evolutionarily guided OneHealth approach to drug discoveryGerald J. Wyckoff, Lee Likins, Ubadah Sabbagh, Andrew SkaffA12 Challenges and opportunities for data applications in animal health: from precision medicine to precision husbandryAmado S. GuloyA13 A cloud-based programmable platform for healthHarlen D. HaysA14 Comparative oncology: One Health in actionAmy K. LeBlancA15 Companion animal diseases bridge the translational gap for human neurodegenerative diseaseJoan R. Coates, Martin L. Katz, Leslie A. Lyons, Gayle C. Johnson, Gary S. Johnson, Dennis P. O'BrienA16 Duchenne muscular dystrophy gene therapyDongsheng DuanA17

Table of contentsA1 One health advances and successes in comparative medicine and translational researchCheryl StroudA2 Dendritic cell-targeted gorilla adenoviral vector for cancer vaccination for canine melanomaIgor Dmitriev, Elena Kashentseva, Jeffrey N. Bryan, David T. CurielA3 Viroimmunotherapy for malignant melanoma in the companion dog modelJeffrey N. Bryan, David Curiel, Igor Dmitriev, Elena Kashentseva, Hans Rindt, Carol Reinero, Carolyn J. HenryA4 Of mice and men (and dogs!): development of a commercially licensed xenogeneic DNA vaccine for companion animals with malignant melanomaPhilip J. BergmanA5 Successful immunotherapy with a recombinant HER2-expressing Listeria monocytogenes in dogs with spontaneous osteosarcoma paves the way for advances in pediatric osteosarcomaNicola J. Mason, Josephine S. Gnanandarajah, Julie B. Engiles, Falon Gray, Danielle Laughlin, Anita Gaurnier-Hausser, Anu Wallecha, Margie Huebner, Yvonne PatersonA6 Human clinical development of ADXS-HER2Daniel O'ConnorA7 Leveraging use of data for both human and veterinary benefitLaura S. TremlA8 Biologic replacement of the knee: innovations and early clinical resultsJames P. StannardA9 Mizzou BioJoint Center: a translational success storyJames L. CookA10 University and industry translational partnership: from the lab to commercializationMarc JacobsA11 Beyond docking: an evolutionarily guided OneHealth approach to drug discoveryGerald J. Wyckoff, Lee Likins, Ubadah Sabbagh, Andrew SkaffA12 Challenges and opportunities for data applications in animal health: from precision medicine to precision husbandryAmado S. GuloyA13 A cloud-based programmable platform for healthHarlen D. HaysA14 Comparative oncology: One Health in actionAmy K. LeBlancA15 Companion animal diseases bridge the translational gap for human neurodegenerative diseaseJoan R. Coates, Martin L. Katz, Leslie A. Lyons, Gayle C. Johnson, Gary S. Johnson, Dennis P. O'BrienA16 Duchenne muscular dystrophy gene therapyDongsheng DuanA17

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Typically, the initial progressive upper motor neuron spastic and general proprioceptive ataxia in the pelvic limbs occurs at 8 years of age or older. If euthanasia is delayed, the clinical signs will ascend, causing flaccid tetraparesis and other lower motor neuron signs. DNA samples from 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls were used for genome-wide association mapping, which produced the strongest associations with markers on CFA31 in a region containing the canine SOD1 gene. SOD1 was considered a regional candidate gene because mutations in human SOD1 can cause amyotrophic lateral sclerosis (ALS), an adult-onset fatal paralytic neurodegenerative disease with both upper and lower motor neuron involvement. The resequencing of SOD1 in normal and affected dogs revealed a G to A transition, resulting in an E40K missense mutation. Homozygosity for the A allele was associated with DM in 5 dog breeds: Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, German Shepherd dog, and Chesapeake Bay retriever. Microscopic examination of spinal cords from affected dogs revealed myelin and axon loss affecting the lateral white matter and neuronal cytoplasmic inclusions that bind anti-superoxide dismutase 1 antibodies. These inclusions are similar to those seen in spinal cord sections from ALS patients with SOD1 mutations. Our findings identify canine DM to be the first recognized spontaneously occurring animal model for ALS.