Photosensitisation and green egg yolks in laying hens caused by the feeding of ensiled alfalfa leaves.

1. The present study was carried out to determine the effects of feeding ensiled alfalfa leaves (ALS) as an alternative protein source to laying hens under the terms of an organic diet. Due to the occurrence of unexpected negative health effects and undesirable egg yolk pigmentation in the test groups the trial was prematurely stopped and further analysis was conducted to evaluate the responsible substances.2. Body weights of the test groups decreased significantly already in week 2 of the trial. Performance variables dropped. Olive green pigmented egg yolks were found in groups fed diets containing ALS. Severe comb necrosis occurred in the experimental group receiving the highest level of ALS (20%) combined with the option of free-range access and therefore natural light exposure.3. The noxious agent found in ALS, blood serum and egg yolk was the photosensitising chlorophyll derivate pheophorbid a (PPBa), deriving from a strong depletion of chlorophyll contained in the alfalfa leaves. PPBa caused the olive-green pigmentation found in yolks and led to photosensitivity in groups with the highest level of ALS in the diet in combination with light exposure.4. By aiming for high protein and amino acid levels, harvesting and processing have, unintentionally and initially unnoticed, led to a strong accumulation of phototoxic PPBa. From these results it is strongly advised not to include ensiled alfalfa leaves as a protein source in organic laying hen diets.

Expression of Autophagy-Related Proteins in the Spinal Cord of Pembroke Welsh Corgi Dogs With Canine Degenerative Myelopathy.

Canine degenerative myelopathy (DM) is a progressive neurodegenerative disease frequently found in Pembroke Welsh Corgi (PWC) dogs, and it has clinical and pathologic similarities to human amyotrophic lateral sclerosis. Autophagy is a major intracellular protein degradation system. Abnormalities of autophagy--resulting in cell death through mechanisms called type II programmed cell death--have recently been reported to occur in various neurodegenerative diseases, including amyotrophic lateral sclerosis. Thus, the distribution and expression levels of proteins involved in autophagy were examined in the spinal cords of 8 PWC dogs suffering from DM with superoxide dismutase mutation, 5 non-DM PWC dogs, and 6 Beagle dogs without neurologic signs. There was no significant difference in the ratio of neurons with microtubule-associated protein light chain 3 (LC3)-positive somata relative to those that were LC3 negative among the 3 groups, whereas the number of LC3-positive neurites was significantly increased in DM dogs. Punctate LC3 immunoreactivity did not colocalize with a lysosome marker, LAMP2 (lysosome-associated membrane protein 2). NBR1 (neighbor of BRCA gene 1) was localized mostly in reactive astrocytes, whereas there were p62 (p62/A170/SQSTM1)-positive foci in the neuropil of the spinal cord of DM dogs. Western blotting revealed in DM dogs the decreased expression of Beclin1 and Atg16 L, which are molecules involved in formation of the isolation membrane. These findings suggest that altered autophagosome degradation may result in LC3 and p62 accumulation in the DM spinal cord, whereas the early stage of membrane formation is likely to be downregulated.

Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy, a potential disease model of amyotrophic lateral sclerosis.

Canine degenerative myelopathy (DM) is a progressive, adult-onset, multisystem degenerative disease with many features in common with amyotrophic lateral sclerosis (ALS). As with some forms of ALS, DM is associated with mutations in superoxide dismutase 1 (SOD1). Clinical signs include general proprioceptive ataxia and spastic upper motor neuron paresis in pelvic limbs, which progress to flaccid tetraplegia and dysphagia. The purpose of this study was to characterize DM as a potential disease model for ALS. We previously reported that intercostal muscle atrophy develops in dogs with advanced-stage DM. To determine whether other components of the thoracic motor unit (MU) also demonstrated morphological changes consistent with dysfunction, histopathologic and morphometric analyses were conducted on thoracic spinal motor neurons (MNs) and dorsal root ganglia (DRG) and in motor and sensory nerve root axons from DM-affected boxers and Pembroke Welsh corgis (PWCs). No alterations in MNs or motor root axons were observed in either breed. However, advanced-stage PWCs exhibited significant losses of sensory root axons, and numerous DRG sensory neurons displayed evidence of degeneration. These results indicate that intercostal muscle atrophy in DM is not preceded by physical loss of the motor neurons innervating these muscles, nor of their axons. Axonal loss in thoracic sensory roots and sensory neuron death suggest that sensory involvement may play an important role in DM disease progression. Further analysis of the mechanisms responsible for these morphological findings would aid in the development of therapeutic intervention for DM and some forms of ALS.

C9orf72 poly-GA proteins impair neuromuscular transmission.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron disease, in which lower motoneurons lose control of skeletal muscles. Degeneration of neuromuscular junctions (NMJs) occurs at the initial stage of ALS. Dipeptide repeat proteins (DPRs) from G4C2 repeat-associated non-ATG (RAN) translation are known to cause C9orf72-associated ALS (C9-ALS). However, DPR inclusion burdens are weakly correlated with neurodegenerative areas in C9-ALS patients, indicating that DPRs may exert cell non-autonomous effects, in addition to the known intracellular pathological mechanisms. Here, we report that poly-GA, the most abundant form of DPR in C9-ALS, is released from cells. Local administration of poly-GA proteins in peripheral synaptic regions causes muscle weakness and impaired neuromuscular transmission in vivo. The NMJ structure cannot be maintained, as evidenced by the fragmentation of postsynaptic acetylcholine receptor (AChR) clusters and distortion of presynaptic nerve terminals. Mechanistic study demonstrated that extracellular poly-GA sequesters soluble Agrin ligands and inhibits Agrin-MuSK signaling. Our findings provide a novel cell non-autonomous mechanism by which poly-GA impairs NMJs in C9-ALS. Thus, targeting NMJs could be an early therapeutic intervention for C9-ALS.

Diagnosis and management of dogs with degenerative myelopathy: A survey of neurologists and rehabilitation professionals.

BACKGROUND: Antemortem diagnosis of degenerative myelopathy (DM) in dogs is presumptive and there are no accepted guidelines for the management of this condition. HYPOTHESIS/OBJECTIVES: Describe current practices of neurology clinicians and physical rehabilitation professionals in the diagnosis and management of DM. ANIMALS: None. METHODS: Online surveys examining diagnosis and management of DM were constructed and distributed via neurology and rehabilitation listservs. RESULTS: One hundred ninety neurology and 79 rehabilitation professionals from 20 countries participated. Most neurology (142/189) and rehabilitation (23/39) respondents required genetic testing for the superoxide dismutase 1 (SOD1) mutation and 82/189 neurologists also required spinal magnetic resonance imaging (MRI) for presumptive DM diagnosis. Most neurology respondents recommended exercise (187/190) and physical rehabilitation (184/190). Over 50% (102/190) of neurology respondents perform rechecks on dogs diagnosed with DM. Rehabilitation respondents reported preservation or improvement of strength (78/79) and coordination (77/79) as therapeutic goals. At-home exercises (75/79), underwater treadmill (64/79), gait training (55/79), and strength building exercises (65/79) were used to maintain strength (58/79), coordination (56/79), muscle mass (56/79), and improve overall wellbeing (54/79). Neurology respondents reported that owners elect euthanasia when dogs become nonambulatory paraparetic whereas rehabilitation respondents report euthanasia when paraplegia and incontinence develop. CONCLUSION AND CLINICAL IMPORTANCE: The majority of dogs diagnosed with DM have not undergone advanced imaging, the combination of history, neurological findings, and genetic testing is heavily relied upon. Whereas the diagnosis of DM is frequently made by veterinary neurologists, continued care is often performed by rehabilitation professionals or primary veterinarians.

Effect of platelet lysate on Schwann-like cell differentiation of equine bone marrow-derived mesenchymal stem cells.

Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 106/µl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time PCR analysis of the expression levels of neuronal markers showed that the ePL group tended to express higher levels of Nestin, Musashi1, and Pax3 than the FBS group. p75 was expressed at low levels in both groups. Immunostaining results showed localization of Nestin in both groups of differentiated cells, but the positive cell rate was significantly higher in the ePL group than in the FBS group. Overall, the ePL gro showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease. This knowledge could be applied translationaly in the treatment of amyotrophic lateral sclerosis in humans.Overall, the ePL group showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease and in the treatment of amyotrophic lateral sclerosis in humans.

The inhibitory effects of MIF on accumulation of canine degenerative myelopathy-associated mutant SOD1 aggregation.

Canine degenerative myelopathy (DM) is a progressive neurodegenerative disorder, which is commonly associated with c.118G > A (p. E40K) missense mutation in the superoxide dismutase 1 (SOD1) gene. Mutant SOD1 protein (SOD1E40K) is likely to be misfolded, acquire insolubility, aggregate in the cytoplasm of neural cells, and lead to degeneration of the nervous tissues. Along with a chaperone activity, macrophage migration inhibitory factor (MIF) is a multifunctional protein that has been shown to directly inhibit human mutant SOD1 misfolding and enhance survival of mutant SOD1-expressing motor neurons. The purpose of this study was to determine whether MIF also inhibits DM-related SOD1E40K misfolding and accumulation of SOD1 aggregates. Human embryonic kidney 293A cells were transfected SOD1cWT or SOD1E40K with or without MIF. The percentages of cells containing transfected SOD1 aggregates were measured by immunocytochemistry, and the amount of SOD1E40K in the insoluble fraction was evaluated by immunoblotting. The percentage of cells with SOD1E40K aggregates and the amount of insoluble SOD1E40K protein decreased in the presence of MIF. Because the chaperone activity of MIF assists in SOD1E40K folding and enhances the refolding and degradation of misfolded SOD1E40K, the results of this study suggests that MIF regulates the accumulation of SOD1 aggregates by its chaperone activity. We propose that enhancing intracellular MIF chaperone activity could be an effective therapeutic strategy for DM.

Brain diffusion tensor imaging in dogs with degenerative myelopathy.

BACKGROUND: Degenerative myelopathy (DM) in dogs shares similarities with superoxide dismutase 1-associated human amyotrophic lateral sclerosis (ALS). Brain microstructural lesions are quantified using diffusion tensor imaging (DTI) in ALS patients. OBJECTIVE: Characterize brain neurodegenerative changes in DM-affected dogs using DTI. ANIMALS: Sixteen DM-affected and 8 control dogs. METHODS: Prospective observational study. Brain DTI was performed at baseline and every 3 months on DM-affected dogs and compared to controls. Fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity were calculated on specified regions of interest. Gait scores (0, normal to 14, tetraplegia) were assigned at each scan. Diffusion tensor imaging values in DM-affected dogs were compared to controls, gait scores, and evaluated over time. RESULTS: Mean age was 5.7 years (SD 3.2) in controls and 9.7 years (SD 1.4) in DM-affected dogs. In DM-affected dogs, mean baseline gait score was 4 (SD 1), and mean score change from baseline to last scan was 4.82 (SD 2.67). Nine dogs had ≤3 scans; 7 had >3 scans. Accounting for age, no differences in DTI indices were identified for any brain or proximal spinal cord regions between DM-affected dogs and controls (P > .05). Diffusion tensor imaging values poorly correlated with gait scores (R2 < .2). No significant changes were identified in diffusion indices over time (P > .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Diffusion tensor imaging indices did not differentiate DM-affected from control dogs, detect longitudinal changes, or differentiate disease severity. Findings do not yet support brain DTI as an imaging biomarker.

Treatment with lithium carbonate does not improve disease progression in two different strains of SOD1 mutant mice.

It has been shown that chronic treatment with lithium carbonate (Li(2)CO(3)) in presymptomatic SOD1G93A transgenic male mice, a model of ALS, was able to remarkably increase their lifespan through the activation of autophagy and the promotion of mitochondriogenesis and neurogenesis. This prompted us to test the lithium effect also in female SOD1G93A mice with two phenotypes of different disease severity. Female SOD1G93A mice of C57BL/6J or 129S2/Sv genetic background were treated daily with Li(2)CO(3) 37 mg/kg (1 mEq/kg) i.p. starting from age 75 days until death. Grip strength, latency to fall on rotarod and body weight were monitored twice weekly. At the time of death the spinal cord was removed to assess the number of motor neurons and to measure the expression of a marker of autophagy (LCII) and the activity of mitochondrial complex IV. We observed a significant anticipation of the onset and reduced survival in 129Sv/G93A and no effect in C57/G93A mice treated with lithium compared to vehicle treated mice. Moreover, lithium neither exerted neuroprotective effects nor increased the expression of LCII and the activity of mitochondrial complex IV in the spinal cord. The present study does not identify any therapeutic or neuroprotective effect of lithium in SOD1G93A female mice.

Localization of a mutant SOD1 protein in E40K-heterozygous dogs: Implications for non-cell-autonomous pathogenesis of degenerative myelopathy.

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disorder. Dogs with typical clinical signs carry homozygous mutations in the superoxide dismutase 1 (SOD1) gene; therefore, DM is regarded as a naturally-occurring model of amyotrophic lateral sclerosis (ALS). Despite the presence of a toxic mutant protein, E40K-SOD1 heterozygotes rarely develop clinical signs. Therefore, E40K-heterozygotes may provide insights into the subclinical and early phase of mutant SOD1-related pathology. In order to identify the distribution of mutant SOD1 in the spinal cords of E40K-heterozygotes, we developed a monoclonal antibody 16G9 that reacts to the mutant E40K-SOD1 protein. We found that the spinal cords of E40K-heterozygotes display white matter degeneration, the severity of which was markedly less than that in E40K-homozygotes. In E40K-heterozygotes, 16G9-reactive SOD1 accumulated predominantly in reactive astrocytes, while spinal neurons remained almost completely free of this form of SOD1 proteins. In contrast, all symptomatic E40K-homozygotes contained 16G9-reactive SOD1 in their spinal neurons and reactive astrocytes. These results suggest that mutant SOD1 proteins accumulate in reactive astrocytes during the early phase of DM pathology, which may contribute to subclinical neurodegeneration. The early involvement of reactive astrocytes in the pathogenesis of DM is strongly suspected and warrants further investigations in the context of non-cell autonomous neuronal death, as proposed for ALS.

Achados clínicos, histopatológicos e moleculares da mielopatia degenerativa canina: relato de caso / Clinical, histopathological and molecular findings of canine degenerative myelopathy: case report

Objetivou-se descrever os achados clínicos, histopatológicos e moleculares associados à MDC em um cão da raça Pastor-Suiço. O cão possuía uma paraparesia progressiva em membros pélvicos e foi submetido a avaliações clínicas, pelas quais se obteve, entre outros diferenciais, o diagnóstico presuntivo de MDC. Com a evolução dos sinais, o tutor optou pela eutanásia. Os achados histopatológicos da medula espinhal foram compatíveis com uma degeneração segmentar axonal e mielínica. O diagnóstico molecular foi realizado por meio da extração do DNA obtido por swab oral. Uma PCR foi otimizada utilizando-se primers descritos em literatura para amplificar a região do gene SOD1. A amostra foi, então, submetida a sequenciamento unidirecional, que revelou que o animal em questão era homozigoto para o alelo A para a mutação c.118G>A no éxon 2 do gene SOD1. O diagnóstico clínico presuntivo da MDC no presente caso foi esclarecido por meio dos achados histopatológicos, associados aos achados clínicos, e da sua caracterização molecular. Ressalta-se a contribuição deste relato, que traz aspectos clínicos, histopatológicos e moleculares associados à MDC na raça Pastor-Suíço, para a qual, até o presente momento, na literatura consultada, não há relato dessa enfermidade.(AU)

The objective of this study was to describe the clinical, histopathological and molecular findings associated with MDC in a Swiss Shepherd dog. The dog had a progressive paraparesis in pelvic limbs and was submitted to clinical evaluations where, among other differentials, the presumptive diagnosis of MDC was obtained. With the progression of the nervous deficits tutor opted for euthanasia. The histopathological findings of the spinal cord were compatible with axonal and myelinic segmental degeneration. Molecular diagnosis was performed by extracting the DNA obtained by oral swab. PCR was optimized using primers described in the literature to amplify the SOD1 gene region. The sample was then subjected to one-way sequencing which revealed that the animal in question was homozygous for the A allele for the c.118G>A mutation in exon 2 of the SOD1 gene. The presumptive diagnosis of MDC in the present case was clarified by histopathological findings, as well as by its molecular characterization. The contribution of this report brings clinical, histopathological and molecular aspects associated with canine degenerative myelopathy in the Swiss Shepherd breed, that until this moment, in the literature consulted, there is no report of this disease in the breed mentioned.(AU)

Canine degenerative myelopathy: biochemical characterization of superoxide dismutase 1 in the first naturally occurring non-human amyotrophic lateral sclerosis model.

Mutations in canine superoxide dismutase 1 (SOD1) have recently been shown to cause canine degenerative myelopathy, a disabling neurodegenerative disorder affecting specific breeds of dogs characterized by progressive motor neuron loss and paralysis until death, or more common, euthanasia. This discovery makes canine degenerative myelopathy the first and only naturally occurring non-human model of amyotrophic lateral sclerosis (ALS), closely paralleling the clinical, pathological, and genetic presentation of its human counterpart, SOD1-mediated familial ALS. To further understand the biochemical role that canine SOD1 plays in this disease and how it may be similar to human SOD1, we characterized the only two SOD1 mutations described in affected dogs to date, E40K and T18S. We show that a detergent-insoluble species of mutant SOD1 is present in spinal cords of affected dogs that increases with disease progression. Our in vitro results indicate that both canine SOD1 mutants form enzymatically active dimers, arguing against a loss of function in affected homozygous animals. Further studies show that these mutants, like most human SOD1 mutants, have an increased propensity to form aggregates in cell culture, with 10-20% of cells possessing visible aggregates. Creation of the E40K mutation in human SOD1 recapitulates the normal enzymatic activity but not the aggregation propensity seen with the canine mutant. Our findings lend strong biochemical support to the toxic role of SOD1 in canine degenerative myelopathy and establish close parallels for the role mutant SOD1 plays in both canine and human disorders.

Canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is an adult-onset fatal neurodegenerative disease that occurs in many breeds. The initial upper motor neuron spastic paraparesis and general proprioceptive ataxia in the pelvic limbs progress to a flaccid lower motor neuron tetraparesis. Recently, a missense mutation in the superoxide dismutase 1 (SOD1) gene was found to be a risk factor for DM, suggesting that DM is similar to some forms of human amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease). This article reviews the current knowledge of canine DM with regard to its signalment, clinical spectrum, diagnostic approach, and treatment. The implications of the SOD1 mutation on both diseases are discussed, comparing pathogenic mechanisms while conveying perspectives to translational medicine.