Pathology of General Proprioception in a Canine Disease Model of Amyotrophic Lateral Sclerosis

1. Introduction

Amyotrophic lateral sclerosis (ALS) is a group of disorders characterized by progressive loss of motor function, usually culminating in paralysis and death [1,2] . These disorders generally have a late-adult onset and usually progress to respiratory failure within 3 to 5 years of the first appearance of clinical signs. Variants in numerous genes have been associated with different forms of ALS [3–6]. Among these is the superoxide dismutase 1 gene (SOD1), variants of which have been associated with some familial forms of ALS. There is considerable heterogeneity in disease phenotype among patients with SOD1-associated ALS, with variations in age of onset, disease duration, penetrance, and clinical manifestations, even among members of the same family [7,8]. Depending on the specific underlying SOD1 variant, the disease can have an upper or lower motor neuron onset and can vary in rate of progression from onset to end-stage disease [7].

Canine degenerative myelopathy (DM) is a naturally occurring disease that has similarities to upper motor neuron onset ALS. Like some familial ALS cases, DM has been associated with missense variants in SOD1. SOD1-associated DM therefore represents a potential model for SOD1-associated ALS. As a result of phenotypic and genetic similarities, SOD1-associated DM has been employed to study disease pathogenesis with the goal of identifying therapeutic intervention targets for ALS. The utility of this model is enhanced by the fact that, unlike in human ALS, the onset and progression of DM is relatively homogeneous among affected dogs, likely reflecting a uniformity in the underlying pathophysiology.

Although much of the focus of ALS research has been on motor neurons, sensory neuron pathologies occur in parallel with motor neuron pathology. However, the sites where disease pathology initiates have not been identified. The earliest clinical signs of ALS could, at least in part, be the result of primary pathology in sensory neuronal pathways that then spread to motor neurons and higher order neurons of the brain. Indeed, in some ALS patients, sensory deficits and pathology occur early in the disease progression [ 23,28,61]. Presymptomatic peripheral and central degeneration of sensory pathways also have been reported in a mouse model for SOD1-related ALS [26,27,30], further suggesting that the sensory defects occur early in the disease process.

In ALS and DM, the initial signs are usually localized and then spread until there is multisystemic involvement at late-stage disease. It has been hypothesized that in ALS and DM this spread is mediated by variant forms of proteins, such as SOD1, that are released into the extracellular space and internalized by other cells where they seed protein aggregation and cellular pathology. It is possible that therapeutic intervention at an early stage could prevent this spread by inhibiting the production and release of the aberrant proteins or peptides from the initial sites of formation. Therefore, it is important to identify where the pathology occurs early in the disease progression.

Since DM has a relatively homogeneous phenotype and affected dogs are euthanized at various stages of disease progression, the disorder presents an opportunity to examine pathologic progression from early- to late-stage disease. The canine disease model has the advantage over transgenic mouse models in that the canine nervous system is closer to that of humans in size and complexity and the ages of onset and durations of disease progression are more similar between ALS and DM.

Clinical signs of DM begin with general proprioceptive ataxia and spastic paresis in the pelvic limbs and eventually spread to affect sensory and upper and lower motor neuron functions in other parts of the body if the dogs are kept alive until terminal stage disease. Sensory transmission in general proprioceptive neuronal pathways that detect position and movement in muscles and joints include (1) unconscious proprioceptive information to the cerebellum and (2) conscious proprioception to the somatosensory cortex of the brain. Components of the pelvic limb general proprioceptive pathway are illustrated in Figure 1. The cell bodies for each general proprioceptive afferent are in the dorsal root ganglia (DRG), axons of which traverse the dorsal roots of the spinal cord. Cerebellar projections from the muscle spindle receptors involve second order neurons located in the intermediate gray matter of the spinal cord at the base of the medial dorsal horn constituting the nucleus thoracicus, which extends from T1 to L3–4. Ipsilateral sensory axons course in the dorsal spinocerebellar tract located in the dorsal portion of the lateral funiculus in the spinal cord. Axons from the ipsilateral nucleus thoracicus reach the cerebellum through the caudal cerebellar peduncle. Axons also decussate in the white commissure from the nucleus thoracicus and ascend in the ventral spinocerebellar tract to enter the cerebellum through the rostral cerebellar peduncle. Somatosensory projections from touch receptors involve a long collateral from the DRG, which ascends in the dorsal funiculus, in the fasciculus gracilis caudal to T5 or fasciculus cuneatus cranial to T6. At the lumbar level the fasciculus gracilis occupies the entire dorsal funiculus. Since most axons leave the fasciculus to synapse in the nucleus thoracicus, the dorsal funiculus decreases in size at the thoracic level. Axons from the pelvic limbs eventually ascend into the caudal medulla to synapse in the nucleus gracilis as the second order neuron, then axons from the second order neurons decussate and ascend in the medial lemniscus to the ventral caudolateral nucleus of the thalamus to synapse on the third order neuron synapse with final projection to the somatosensory cortex. Pathology in any of the components of this pathway could contribute to the pelvic limb proprioceptive signs that characterize DM.

To understand the mechanisms that initiate disease progression, it will be important to develop a better understanding of the pathology underlying pelvic limb proprioceptive dysfunction, particularly early in the disease process. Therefore, this study was undertaken to characterize the somatosensory and cerebellar projection pathways involving pelvic limb general proprioception in dogs with DM.

2. Materials and methods

3. Results

4. Discussion

ALS is often referred to as a motor neuron disorder [1,2]. Although for most forms of ALS, motor neuron loss or pathology is present at end-stage disease, it is not known whether this pathology is primary or secondary to pathology in other components of the neuromuscular pathways. In familial forms of the disease associated with SOD1 sequence variants, the initial clinical signs can be consistent with pathology in motor neurons, sensory neurons, or in a combination of these [7,8,22–25]. For example, in patients with the D90A SOD1 variant, the presenting signs are usually sensory, including lower back pain, hip and knee joint pain, muscle pain, focal paresthesia, and abnormal heat sensations [7,23]. Growing evidence from studies on ALS patients and the mouse SOD1^G93A model indicate that primary pathology in sensory neurons, including those in proprioceptive pathways, may play key roles in ALS early disease progression [22,23,26–33]. More research is needed to clarify the initial sites of pathology in the various forms of ALS. Because companion dogs with DM are euthanized at various stages of disease progression and clinical signs are relatively homogeneous, the canine disease model of SOD1-associated ALS presents an opportunity to characterize disease pathology in all the tissues that could play key roles in early development and progression of the disease. Studies using the naturally occurring canine disease model may provide insights into ALS pathogenesis that cannot be obtained from the study of transgenic mouse models, particularly since the pathogenic processes may be influenced by body size, which affects axon length, as well as the complexity of the nervous system, lifespan, age at disease onset and rate of disease progression.

ALS is characterized by the progression from regional to wide-spread impairment in motor and sensory pathways [34]. Typically, clinical signs begin in one body region and spread contiguously [35–37]. Evidence suggests that this spread occurs via a prion-like mechanism that in the case of DM and SOD1-associated ALS may be mediated by misfolding of the variant SOD1 protein [38–46]. According to this model, the SOD1 mutations result in misfolding of the protein and the misfolded protein in turn seeds the intracellular aggregation of SOD1 and other proteins. This initial step in pathogenesis is proposed to occur first in specific cell types, which may include neurons in pathways associated with proprioception. Over time, it is proposed that the cells that are initially affected begin to release the aberrant proteins, or peptides derived from them, in aggregated or soluble forms. Internalization of these substances by other cells is proposed to seed aggregate formation and release of the resulting products, thus propagating the pathology until many vulnerable cells are affected [39]. Based on this mechanism, identification of the early sites of aggregate accumulation and pathology will be important in developing early therapeutic intervention strategies.

Our findings suggest that one of the early sites of SOD1 aggregate formation occurs in the sensory neurons of the dorsal root ganglia (DRG). SOD1 aggregate formation was observed in the lumbar DRG of early-stage DM affected dogs and by late-stage, almost all the DRG neurons contained aggregates. SOD1 aggregation was also present in the axons of the DRG neurons (sensory roots) even in dogs not exhibiting clinical signs, but appeared to become more abundant and condensed in clinically affected dogs as the disease progressed, at which point, SOD1 positive immunolabeling appeared in the myelin sheaths of some sensory root axons, in addition to its presence within the axons. The increase in the proportion of DRG neurons that were affected over the course of disease and the appearance of strong SOD1 immunolabeling in the myelin sheaths in late-stage disease are consistent with a prion-like spreading mechanism. While this aggregate accumulation did not appear to result in overt sensory neuron loss or morphological evidence of neuronal death, the aggregate formation may play a role in the clinical presentation of general proprioceptive ataxia by altering important cell functions. Previous studies have shown that accumulation of SOD1 aggregates is toxic to cells [47,48], so the presence of these aggregates in both the cell bodies and the myelinated axons of the sensory neurons could result in functional impairment.

Like the lumbar sensory root axons, the motor root axons exhibited SOD1 immunolabeling in clinically unaffected dogs. However, the changes that occurred in the lumbar DRG over the course of disease progression were not observed in the motor neurons, suggesting that SOD1 accumulation is less pronounced in the motor neurons than in the sensory neurons in the lumbar cord. The lumbar motor neuron cell bodies did not display any morphological signs of degeneration, nor did they contain SOD1 aggregates at any disease stage. Yet, pronounced SOD1 aggregate accumulation occurs in thoracic motor neurons in DM-affected PWCs [14] . Likewise in lumbar sensory roots, DM was not accompanied by a decrease in axon numbers. However, in a previous study significant sensory root axon loss in the thoracic cord was found in late-stage PWCs [14] . This was accompanied by the appearance of pyknotic neurons in the T8 thoracic DRGs. The basis for this regional difference in spinal cord pathology is not known.

Taken together, these regionally differing patterns of pathology suggest that selective vulnerability in DM is not dictated solely by neuronal subtype, but also by rostro-caudal location within the neuroaxis. The preferential involvement of thoracic motor neurons, sensory roots, and DRG, coupled with relative sparing of their lumbar counterparts, raises the possibility that features like local microenvironmental factors and metabolic demand may influence susceptibility to SOD1-associated injury. This interpretation is supported by earlier work demonstrating that axonal degeneration, demyelination, and marked astrogliosis are most severe in the mid- to lower- thoracic spinal cord in DM affected PWC [18,19].

The observation of SOD1 accumulation within myelin sheaths suggests potential involvement of oligodendrocytes and Schwann cells in DM. The apparently stronger immunolabeling observed in early-stage samples may reflect an initial phase of protein accumulation/redistribution. Given the qualitative nature of this assessment, this interpretation should be considered preliminary and warrants further investigation. Evidence indicates that Schwann cell pathology occurs in SOD1-related ALS. Studies manipulating mutant SOD1 expression specifically in Schwann cells have demonstrated that although the presence of the abnormal protein in these cells alone is not sufficient to drive disease, it can modulate disease onset and progression, highlighting the role of these cells as contributors to neurodegeneration [51–53]. Additional work has shown dysfunction of both perisynaptic and myelinating Schwann cells in SOD1 disease models, including impaired repair capacity at the neuromuscular junction, altered calcium signaling, and loss of dedifferentiation of myelinating Schwann cell populations [54–57]. These findings underscore the importance of glial contributions to ALS pathogenesis and support the concept that disease progression arises from complex interactions between neurons and their supporting cells. To date, Schwann cell involvement has not been systematically evaluated in DM; however, the presence of SOD1 immunoreactivity within myelin in our study suggests a potential parallel mechanism. This represents an additional overlap between DM and SOD1-associated ALS and highlights the need for further investigation into the role of Schwann cells in DM pathogenesis.

Although DM was not accompanied by loss of L4 motor or sensory neurons, the findings do not preclude the possibility of distal axonal pathology in or functional impairment of the motor or sensory axons that innervate the pelvic limbs. Indeed, it has been reported that axonal dystrophy occurs in the fibular nerves of PWCs with DM [58]. Because the fibular nerve is a mixed nerve that includes both motor, sensory and autonomic axons, it was not possible to deduce whether the pathology observed was primarily in axons of motor neurons, sensory neurons, or in both. The sural nerve is a cutaneous sensory nerve relaying information from the lower limbs [59,60]. Morphometric analysis of sural nerve biopsies from patients with ALS revealed evidence of nerve fiber pathology (e.g. axonal degeneration, fiber loss, and increased remyelination), that correlated with disease duration [61–63]. Although interpretation was limited in the current study by suboptimal fixation in some cases, degenerating axons and presumptive regenerative clusters were observed in the sural nerve of a late-stage DM-affected dog. Regenerative clusters typically arise when an axon undergoes degeneration and adjacent intact axons sprout to reinnervate the denervated territory, reflecting an attempted compensatory response. Similar clusters have been reported in the fibular nerve of late-stage PWCs, further supporting the presence of chronic, ongoing peripheral nerve involvement in this disease [58]. Further research will be necessary to confirm whether pure sensory nerve axonal pathology occurs in DM. However, in the mouse SOD1^G93A model of ALS, it has been shown that functional impairment of proprioceptive neurons occurs early in the disease process. In addition, it was reported that the sensory nerve endings of muscle spindles degenerate prior to symptom onset in the same mouse model [30]. Reduced innervation of muscle spindles has also been reported in ALS patients, although it was not known whether the disease in any of the patients studied was related to pathogenic SOD1 variants [64]. The findings from dog and mouse studies are consistent with the hypothesis that proprioceptive neurons may be among the earliest to be affected in the SOD1-associated disorders.

In the current study, pathology in the FG and the dorsal and ventral spinocerebellar tracts of lumbar spinal cord were observed in dogs with early-stage DM, consistent with the findings of other studies [18,19]. In addition to the decrease in axon numbers seen in the spinal tracts, changes in myelin (splitting, ballooning, thinning and condensation) and a visual drop out of large caliber axons were occasionally observed. These qualitative observations warrant further investigation to provide a more comprehensive understanding of axonal pathology in DM. These alterations in the somatosensory pathways likely play important roles in early-stage DM. Therefore, to gain a better understanding of early disease pathogenesis, it will be important to elucidate the mechanisms responsible for these pathological changes.

Degeneration of neurons of the nucleus thoracicus has been reported in ALS patients, but the tissues examined were from subjects with end-stage disease [65]. We documented that SOD1 aggregate accumulation in neurons of the nucleus thoracicus begins in early DM. If similar accumulation also occurs in early progression of human SOD1-related ALS, inhibiting this pathology would be an important target for therapeutic intervention.

ALS is characterized by neuroinflammation as reflected by activation of microglia and astrocytes in the central nervous system [66–70]. Both astrocytic and microglial activation were observed in the nucleus thoracicus of PWCs with both early- and late-stage DM. Activated microglia and astrocytes have also been documented in the ventral horn of the spinal cords in DM affected dogs [19,71,72]. Microglial activation has been correlated with disease progression in both transgenic rodent ALS models and ALS patients [73–78]. Thus, it appears that canine DM can complement studies of the role of neuroinflammation in the pathogenesis of ALS and for testing the hypothesis that suppression of neuroinflammation would be of therapeutic benefit in treating ALS [79–82].

All but one of the PWCs in this study that did not develop clinical signs of DM were homozygous for the SOD1 risk allele, and the remaining unaffected dog was heterozygous (Supplementary File 2). Although most control dogs were homozygous for the risk allele, in addition to not exhibiting disease signs, these dogs did not exhibit the characteristic thoracic spinal cord histopathologic hallmarks used in differential DM diagnosis (i.e., SOD1 positive aggregates in motor neurons, and axonal degeneration and gliosis), supporting their appropriateness as controls in this study. The high prevalence of the SOD1 risk allele within the PWC population limited the availability of homozygous normal dogs of the same breed [11]. The phenotypically unaffected dogs ranged in age from 10.8 to 15.8 years of age at the time of euthanasia. Most of these dogs were older than the dogs with late-stage DM at the time of euthanasia. Thus, it appears that the onset of disease signs in this group of dogs was dramatically delayed compared to the phenotypically affected dogs with the same genotype. As reported previously, among PWCs homozygous for the risk variant, additional individuals were identified that reached 15 years of age without exhibiting any signs of DM [11,83]. Dogs of other breeds that are homozygous for the SOD1 risk allele also do not develop DM by 10 or more years of age [11]. In some breeds, the frequency of homozygosity for the SOD1 risk allele is very high, but DM has not been reported as a health concern. For example, of 79 Wire Fox Terriers genotyped for the SOD1 disease variant, almost 90% were homozygous for the risk allele [11], but the American Kennel Club does not list DM as among the health concerns that have been reported for this breed. These observations led to the hypothesis that there are genetic variants that modify the risk of developing DM associated with the SOD1 risk variant. A variant in SP110 that encodes a nuclear body protein was identified as such a modifier in PWCs [83]. There are likely to be additional genetic variants that modify the risk of developing DM among dogs that are homozygous for the SOD1 risk variant. Identification of such modifier genes would likely be helpful in developing therapeutic interventions for SOD1-associated DM and ALS if we could mimic the effects of these modifiers in delaying disease onset and progression.

The phenotypically unaffected dogs that were homozygous for the SOD1 risk allele exhibited some morphological features that were observed in the affected dogs, although to a lesser degree. For example, the axons nerves in the asymptomatic dogs exhibited SOD1 immunolabeling, but this appeared to be less pronounced than in the dogs that were phenotypically affected. This suggests that the factors that delayed the onset of clinical disease signs also delayed the accumulation of SOD1 aggregates but did not completely prevent it. Research is currently underway to identify additional potential genetic factors that may slow disease progression in dogs homozygous for the SOD1 risk allele.

5. Conclusion

The data from this and previous studies on DM indicate that pronounced pathology occurs in the somatosensory and cerebellar projection pathways for general proprioception early in the disease progression. These findings indicate that increased emphasis should be placed on testing the hypothesis that primary pathology in proprioceptive and other sensory neurons play roles in SOD1-related ALS pathogenesis that may be as important as the recognized role of motor neuron pathology in these diseases. Our data indicate that pathology in the general proprioceptive pathway is a potential target for early therapeutic interventions that could be tested in the DM model. Interventions that could inhibit the spread of pathology from the first order neurons to the rest of the neuromuscular system could slow the progression of ALS signs. Identification of factors that delayed disease progression in some dogs homozygous for the SOD1 risk allele may provide important insights into potential therapeutic intervention strategies.

Supplementary Material