Introduction

Disorders of myelin in dogs and cats are infrequently diagnosed yet are important diseases to be able to recognize and diagnose.¹ They present either early in life as familial or inherited disorders or as acquired disorders in mature animals although exceptions to this rule can be seen. The critical issue is to be able to identify which of these disorders will have a poor prognosis versus those where the animal may recover spontaneously or return to normal if the cause is discovered and is reversible. This chapter will summarize what myelin is and why it is important for normal neurologic function, and the known inherited and acquired myelin disorders of the dog and cat.

Myelin

Myelin is the multilamellar, compact membrane that surrounds axons of a certain caliber in the central and peripheral nervous systems (CNS and PNS). It is synthesized by the oligodendrocyte in the CNS and the Schwann cell in the PNS. At birth, the CNS of the dog and cat is incompletely myelinated hence their inability to ambulate for up to 2 weeks. During the early neonatal period, oligodendrocytes ensheath growing axons who signal the ensheathing cell (neuregulins control this process in the PNS) to surround them with their multiple processes in the CNS, where one oligodendrocyte most often myelinates multiple segments (internodes) along different axons. In the PNS, the Schwann cells develop the so-called 1:1 relationship with an axon, myelinating single internodes. In the dog, we have shown that development of myelin in the corticospinal tract is time dependent.² Very little myelin is present at birth, but by 28 days at least 50% of axons are myelinated though complete myelination will require months. Oligodendrocytes develop from oligodendrocyte progenitors (OPCs) which in turn are derived from neural stem cells. The timing of this developmental process is critical and is under tight genetic control. As oligodendrocytes mature they synthesize vast amounts of lipids and to a lesser extent, proteins. The myelin proteins however are critical toward normal myelin development and there is little room for error in their expression. In the CNS, the major myelin proteins are proteolipid protein (PLP) and myelin basic protein, while in the PNS, protein zero predominates.

The highly lipid-rich myelin membrane acts primarily as an insulator for axons, allowing impulses to be transmitted in a fast and energy efficient manner. The failure to develop myelin (hypomyelination or dysmyelination) or its loss (demyelination) results in profound neurologic dysfunction. If localized to certain areas of the CNS, symptoms will relate to that anatomic site. Most often in animals however, the myelin disturbance is generalized and a wide range of neurologic symptoms are seen.

While myelin is best known for its role in saltatory impulse conduction, it is becoming increasingly clear that it is important also in the maintenance of the axon. Thus important glial-axon interactions occur both during development and in maintaining the myelin-axon unit in the mature nervous system.

Inherited Myelin Disease

The majority of myelin disorders in small animals are of genetic origin. Indeed there are many genetic disorders of myelination in rodents and humans, some of which have similarities to certain canine or feline diseases. The best characterized of inherited myelin disorders are those described in mice and rats where the generic term, the myelin mutant is used.^3,4 In animals and humans, these disorders, are inherited either as X-linked or autosomal recessive disorders. Overall, inherited disorders of CNS myelin in humans and animals are most frequently inherited as X-linked traits. In animals with genetic myelin disorders, the most common symptomatology is a delay or absence in ambulation along with the presence of tremor which can vary in severity. At its worst, the tremor can affect the head, trunk and limbs with the inability to stand and ambulate and at its least, a mild tremor in animals that are able to stand and walk. There is usually no evidence of an intention tremor and in mild cases this tremor may lessen or disappear with time. In severely affected cases, the tremor can persist and most importantly, seizures may develop. Two well described inherited disorders occur in dogs:

1. The Shaking Pup (shp)

This was first described in 1981 by Griffiths, et al in Glasgow.^5-9 The disorder is found in Welsh Springer Spaniels and is inherited as an X-linked recessive disease. We have studied this disorder for over 25 years and have determined through many generations that the disease is inherited in true Mendelian fashion with close to 50% of males being hemizygous i.e., affected. Male pups with the disorder can be identified with the presence of tremor as early as 5-6 days of age and unequivocally by 14 days. They have a severe truncal tremor and are unable to stand. The tremor is not present at rest or when the pups are asleep. Between 4-6 months, affected dogs develop generalized seizures which can however be well controlled with phenobarbitone therapy. The tremor changes in character with age becoming a coarse, whole body tremor. From 9-12 months shaking pups will develop extensor rigidity and spasticity in all four limbs. Pups are able to suckle early in life and with hand feeding and careful husbandry can live for over two years but remain unable to stand and severely neurologically impaired.

Examination of the CNS macro- and microscopically shows a severe absence of myelin through the entire white matter but most noticeably in the brain. With time the brain shows increasing discrepancy in size compared to controls due to lack of myelin and perhaps axon loss, although the latter as not been documented. Severe ventricular dilatation is found in older dogs. Microscopic evaluation of the CNS shows generalized lack but not absence of myelin, at least in the spinal cord. With time, more myelin is found but nowhere near control levels and this varies from dog to dog. The genetic disorder in this mutant was identified in 1990, when we showed that there is a point mutation in the second exon of the PLP gene with a proline for histidine substitution likely leading to a misfolded protein that is poorly transported in the cell.¹⁰

Diagnosis of this disorder is relatively straightforward. Firstly it is inherited as an X-linked trait, i.e., only males in a litter should be affected and around half of male pups. The tremor will be severe and pups cannot ambulate and there will likely be little or no improvement with time. MRI will confirm a myelin disturbance in the brain. In addition, brain stem auditory evoked potentials in hemizygous dogs will show a normal wave I (PNS origin and not affected by this PLP mutation) but delayed and poor amplitude CNS conduction of waves II-V.¹¹ As the mutation is known, we identify affected males and carrier females by PCR on DNA from blood early in life. Future identification of such a disorder in Spaniels would suggest a PLP mutation, but other breeds may also present with such a disorder and sequencing of the PLP gene may be diagnostic. Indeed a previous report in the Samoyed was likely a X-linked disease with a mutation in the PLP gene (immunolabeling of the spinal cord showed an absence of PLP--Duncan, unpublished), although DNA was not available to confirm this.²

An interesting feature of this disorder is that in our colony the heterozygous female carriers of the trait may display mild/moderate signs of the disorder. Indeed practically all female carriers develop a tremor of variable severity, at 12-14 days of age. However they are able to ambulate and the tremor disappears over a 4-12 week period. The cause of the tremor is skewed X-linked inactivation (Duncan and Dlouhy, in preparation) which leads to over 50% of oligodendrocytes arising from the maternal X chromosome (Lyon's hypothesis). This results in a mosaic myelin abnormality as we have demonstrated in the brain stem of heterozygous females.¹¹ The importance here is to recognize that these females will recover but it signifies that they are carriers and should be neutered.

2. Weimaraner/Chow Chow Hypomyelination

This disorder is more common in the Weimaraner breed but clinically and pathologically it appears identical in both breeds. It is relatively common in Weimaraners being reported in most parts of the United States and also in certain European countries. Signs are first noted at 10-12 days when both male and female pups develop a marked tremor.¹³ They are able to ambulate but walk with a 'bouncy' gait. When resting and asleep the tremor is lost. There is no evidence of an intention tremor on eating. Occasionally the pups may need supplemental feeding but this is uncommon. Over a period of 4 weeks to 3 months, the tremor resolves although those dogs with the most severe tremor may have a mild tremor for life. The disease is inherited as an autosomal recessive trait, therefore around 25% of a litter will be affected, including both males and females. Single litters may not have such a clear-cut percentage of affected dogs, but studies over extended generations define this a true Mendelian defect.

The microscopic abnormality in these dogs is unique. In the brain there appears to be a generalized paucity of myelin compared to controls but not nearly as severe as seen in the shaking pup. In the spinal cord however, there is a marked area of poor myelin formation around the peripheral parts of the lateral and ventral column of the spinal cord. In the dorsal column, only a small area of reduced myelination is seen in the sub-pial part of the fasciculus gracilis tract. Deep spinal cord white matter is normally myelinated. With time, the areas of hypomyelination become myelinated, although myelin sheaths remain thinner than normal.

In the Chow Chow, the disorder has an identical clinical and pathological bases but the disease is much less frequently reported.^14-15 We have also seen a similar disorder in two feline litter-mates with a similar defect in myelination in the spinal cord though much more severe than in the two dog breeds noted above. The genetic basis of these disorders is not known but it is clear that this disorder may be seen in many breeds as well as in the cat.

In the Weimaraner, our goal is to map and identify the causative gene. We are currently using a genome wide association study strategy to accomplish this with the goal of developing a bench-test that will identify carriers and allow the breed to selectively breed non-carrier dogs hence removing the disease from the Weimaraner breed.

3. Canine Spongiform Leukoencephalomyelopathy

This uncommon disorder was described by us in two breeds of dogs, Australian cattle dogs and Shetland sheepdogs.¹⁶ The initial symptoms in the Australian cattle dogs were seen at 3-4 weeks of age with the development of a moderate to severe whole body tremor with the presence of dysmetric gait. Occasional pups developed symptoms at a later time point however. Unlike the previous described conditions above, these dogs worsened with time, developing spasticity, paresis and becoming unable to ambulate. Additionally, severe and widespread cranial nerve deficits are seen although vision and audition are intact. Electrophysiological testing showed abnormalities in spinal cord and brainstem auditory evoked potentials suggestive of a demyelinating disease. CSF examination showed significant increases in lactate, pyruvate and 3-OH butyric acid which were thought to be associated with mitochondrial dysfunction. The neurologic abnormalities in the Shetland sheepdogs and disease progression were similar to the Australian cattle dogs, but in addition, a CT scan suggested diffuse brain hypomyelination.

In the Australian cattle dog, one female was responsible for giving birth to three litters with affected dogs which were of both sexes. She mated with two different dogs. This breeding history, the variable time of onset of disease and variable disease progression strongly suggested a disease of maternal origin and possibly a mitochondrial disorder. The disease severity was so severe that all dogs were euthanized. The hallmark of the pathology was a severe but variable vacuolation of the white matter in the brain and spinal cord with scattered but modest demyelination. Sequencing of the mitochondrial DNA of these dogs showed a mutation in mitochondrial encoded cytochrome b gene.¹⁶ To our knowledge, this was the first demonstration of a mitochondrial DNA (mtDNA) mutation in animals. Such disorders are found in humans affecting many mtDNA genes.

From a veterinary neurology standpoint, these disorders while initially resembling the shaking pup and Weimaraner diseases are readily distinguished due to their progression and mode of inheritance. In life, MRI would be diagnostically helpful and a CSF tap to examine for mitochondrial defect could be performed. Many human mtDNA disorders have characteristic changes in skeletal muscle with accumulation of mitochondria beneath the sarcolemma ('ragged red fibers'). Thus if mitochondrial disease is suspected, a muscle biopsy could be useful.

4. Polyneuropathy in Siamese Cats

In 1989, we reported an interesting disorder in three cats, two of whom were related and all were Siamese or Siamese origin.¹⁷ The disorder presented in all cats at 4-7 months of age. All had neurologic evidence of polyneuropathy with tetraparesis, ataxia, reduced reflexes and hypotonia and in one case, tremor. Nerve conduction studies showed marked slowing in both motor and sensory nerves with only mild EMG abnormalities. All three cats showed progression of clinical signs with a mild head tremor developing in one cat. Cranial nerve function was normal. As a result of the severe progression, all three cats were euthanized and mild splenomegaly and moderate hepatomegaly noted at necropsy. In the CNS, mild demyelination and axon degeneration were seen but in the PNS there was severe and generalized disturbance in myelination with extensive demyelination and remyelination. Schwann cells were noted to contain membrane-bound lamellar inclusions suggesting a lysosomal storage disease. Biochemical analysis of brain and viscera showed a severe reduction in sphingomyelinase activity to 5% of control. This and other biochemical data identified these as cases of Niemann-Pick Type A disease. Since this report, this disorder has not been further documented yet the possibility of its recurrence, especially in Siamese cats is present. The progression of this inherited disorder sets it apart from the previously described diseases and is typical of a lysosomal storage disorder. Future cases of this disease could be identified at time of onset by biopsy of a visceral organ and the pathological process identified by nerve biopsy.

Acquired Myelin Disorder

In humans, the most important acquired myelin disorder is multiple sclerosis (MS). It is a disorder primarily of young adults, most frequently women. A similar spontaneous disease in animals has yet to be reported. Indeed only one acquired myelin disease has been reported to-date, and this is in cats that are fed a diet of irradiated food. Cats develop a severe and progressive neurologic disease after 4-5 months of ingesting irradiated food. This disorder has now been reported in Ireland,^18-19 USA²⁰ and Australia.²¹ Affected cats present with ataxia and paresis of the hind limbs which may progress to paraplegia. In addition, cats may develop visual disturbances and even blindness. We showed that when cats are returned to a non-irradiated diet, they can recover completely. This was also later reported in the outbreak of this disease in Australia.

The pathologic basis of this disorder is extensive vacuolation of white matter in the brain, optic nerves, and spinal cord leading to extensive demyelination. There is practically no axon loss though in cats fed an irradiated diet for long periods this may occur as some animals fail to recover. Recovery is due solely to remyelination which is extensive along the spinal cord and optic nerves.

This disorder is important for two major reasons. Firstly, it proves that remyelination alone will restore function, a basic finding that had not been proven in experimental and human demyelinating disorders.²⁰ Secondly in relation to this feline disorder, it proved that returning cats to a non-irradiated diet, with careful management of severe neurologically sick cats, results in their gradual recovery to normality after 3-4 months. While their initial severe neurologic dysfunction may suggest a poor prognosis, the diagnosis of a demyelinating disease such as may change the outlook provided the cause of the demyelination is terminated.

Summary

It is clear from this brief review, that disorders of myelin in the dog and cat are not common, and they may present a diagnostic dilemma however as they remain an arcane part of veterinary neurology. In the case of potential inherited disease, genetic counseling and further test breeding may be needed to prove an inherited basis. To diagnose a myelin deficiency as the cause of the problem however, the presence of tremor and MRI evoked potential studies should be confirmatory. Necropsy is required to absolutely confirm the diagnosis and characterize the disease and frozen tissue (brain and spinal cord for RNA and blood or other tissue for DNA, if those are only available) should be collected and frozen. If such careful approaches are used in the future it is highly likely that new disorders of myelin will be discovered and characterized in the dog and cat.

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