The CLN2 form of neuronal ceroid lipofuscinosis is a neurodegenerative disease that results from mutations in the TPP1 gene. Affected children exhibit progressive declines in most neurological functions including vision. Functional declines are accompanied by progressive brain and retinal atrophy. TPP1 encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Dachshunds with a TPP1 null mutation exhibit a disorder very similar to human CLN2 disease. Periodic infusion of recombinant TPP1 protein or a single injection of a TPP1 gene therapy vector into the cerebrospinal fluid of affected dogs significantly delays the onset and progression of neurological signs but does not slow vision loss or retinal degeneration. Studies were conducted to determine whether intravitreal implantation of autologous bone marrow derived stem cells transduced with a TPP1 expression construct would inhibit retinal degeneration in the canine model. A single injection of the transduced cells at an early stage in the disease progression substantially inhibited the development of disease-related retinal function deficits and structural changes. No adverse effects of the treatment were detected. These findings indicate that ex vivo gene therapy using autologous stem cells is an effective means of achieving sustained delivery of therapeutic compounds to tissues such as the retina for which systemic administration would be ineffective.

A recessive, adult-onset neuronal ceroid-lipofuscinosis (NCL) occurs in Tibetan terriers. A genome-wide association study restricted this NCL locus to a 1.3Mb region of canine chromosome 2 which contains canine ATP13A2. NCL-affected dogs were homozygous for a single-base deletion in ATP13A2, predicted to produce a frameshift and premature termination codon. Homozygous truncating mutations in human ATP13A2 have been shown by others to cause Kufor-Rakeb syndrome (KRS), a rare neurodegenerative disease. These findings suggest that KRS is also an NCL, although analysis of KRS brain tissue will be needed to confirm this prediction. Generalized brain atrophy, behavioral changes, and cognitive decline occur in both people and dogs with ATP13A2 mutations; however, other clinical features differ between the species. For example, Tibetan terriers with NCL develop cerebellar ataxia not reported in KRS patients and KRS patients exhibit parkinsonism and pyramidal dysfunction not observed in affected Tibetan terriers. To see if ATP13A2 mutations could be responsible for some cases of human adult-onset NCL (Kufs disease), we resequenced ATP13A2 from 28 Kufs disease patients. None of these patients had ATP13A2 sequence variants likely to be causal for their disease, suggesting that mutations in this gene are not common causes of Kufs disease.

The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases characterized by progressive neurodegeneration and accumulation of autofluorescent storage granules. A 9-month-old Miniature Dachshund presented with NCL-like signs that included disorientation, ataxia, weakness, visual impairment, and behavioral changes. Neurons throughout the CNS contained autofluorescent lysosomal inclusions with granular osmiophilic deposit (GROD) ultrastructure characteristic of classical infantile NCL (INCL). Human INCL is an autosomal recessive disorder that results from mutations in PPT1, a gene that encodes the enzyme palmitoyl protein thioesterase 1 (PPT1; EC 3.1.22). Resequencing of PPT1 from the affected dog revealed that the dog was homozygous for a single nucleotide insertion in exon 8 (PPT1 c.736_737insC), upstream from the His289 active site. Brain tissue from this dog lacked PPT1 activity. The sire and dam of the propositus were heterozygous for the c.736_737insC mutation; whereas, 127 unrelated Dachshunds were homozygous for the wild-type allele. This is the first reported instance of canine NCL caused by a mutation in PPT1.

CLN2 neuronal ceroid lipofuscinosis is a rare recessive hereditary retinal and neurodegenerative disease resulting from deleterious sequence variants in TPP1 that encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Children with this disorder develop normally, but starting at 2-4 years of age begin to exhibit neurological signs and visual deficits. Vision loss that progresses to blindness is associated with progressive retinal degeneration and impairment of retinal function. Similar progressive loss of retinal function and retinal degeneration occur in a dog CLN2 disease model with a TPP1 null sequence variant. Studies using the dog model were conducted to determine whether intravitreal injection of recombinant human TPP1 (rhTPP1) administered starting after onset of retinal functional impairment could slow or halt the progression of retinal functional decline and degeneration. TPP1-null dogs received intravitreal injections of rhTPP1 in one eye and vehicle in the other eye beginning at 23.5-25 weeks of age followed by second injections at 34-40 weeks in 3 out of 4 dogs. Ophthalmic exams, in vivo ocular imaging, and electroretinography (ERG) were repeated regularly to monitor retinal structure and function. Retinal histology was evaluated in eyes collected from these dogs when they were euthanized at end-stage neurological disease (40-45 weeks of age). Intravitreal rhTPP1 injections were effective in preserving retinal function (as measured with the electroretinogram) and retinal morphology for as long as 4 months after a single treatment. These findings indicate that intravitreal injection of rhTPP1 administered after partial loss of retinal function is an effective treatment for preserving retinal structure and function in canine CLN2 disease.

The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage disorders characterized by progressive neurodegeneration and declines in neurological functions. Pathogenic sequence variants in at least 13 genes underlie different forms of NCL, almost all of which are recessively inherited. To date 13 sequence variants in 8 canine orthologs of human NCL genes have been found to occur in 11 dog breeds in which they result in progressive neurological disorders similar to human NCLs. Canine NCLs can serve as models for preclinical evaluation of therapeutic interventions for these disorders. In most NCLs, the onset of neurological signs occurs in childhood, but some forms have adult onsets. Among these is CLN12 disease, also known as Kufor-Rakeb syndrome, PARK9, and spastic paraplegia78. These disorders result from variants in ATP13A2 which encodes a putative transmembrane ion transporter important for lysosomal function. Three Australian Cattle Dogs (a female and two of her offspring) were identified with a progressive neurological disorder with an onset of clinical signs at approximately 6 years of age. The affected dogs exhibited clinical courses and histopathology characteristic of the NCLs. Whole genome sequence analysis of one of these dogs revealed a homozygous c.1118C > T variant in ATP13A2 that predicts a nonconservative p.(Thr373Ile) amino acid substitution. All 3 affected dogs were homozygous for this variant, which was heterozygous in 42 of 394 unaffected Australian Cattle Dogs, the remainder of which were homozygous for the c.1118C allele. The high frequency of the mutant allele in this breed suggests that further screening for this variant should identify additional homozygous dogs and indicates that it would be advisable to perform such screening prior to breeding Australian Cattle Dogs.

A neutered male domestic medium-haired cat presented at a veterinary neurology clinic at 20 months of age due to progressive neurological signs that included visual impairment, focal myoclonus, and frequent severe generalized seizures that were refractory to treatment with phenobarbital. Magnetic resonance imaging revealed diffuse global brain atrophy. Due to the severity and frequency of its seizures, the cat was euthanized at 22 months of age. Microscopic examination of the cerebellum, cerebral cortex and brainstem revealed pronounced intracellular accumulations of autofluorescent storage material and inflammation in all 3 brain regions. Ultrastructural examination of the storage material indicated that it consisted almost completely of tightly-packed membrane-like material. The clinical signs and neuropathology strongly suggested that the cat suffered from a form of neuronal ceroid lipofuscinosis (NCL). Whole exome sequence analysis was performed on genomic DNA from the affected cat. Comparison of the sequence data to whole exome sequence data from 39 unaffected cats and whole genome sequence data from an additional 195 unaffected cats revealed a homozygous variant in CLN6 that was unique to the affected cat. This variant was predicted to cause a stop gain in the transcript due to a guanine to adenine transition (ENSFCAT00000025909:c.668G > A; XM_003987007.5:c.668G > A) and was the sole loss of function variant detected. CLN6 variants in other species, including humans, dogs, and sheep, are associated with the CLN6 form of NCL. Based on the affected cat's clinical signs, neuropathology and molecular genetic analysis, we conclude that the cat's disorder resulted from the loss of function of CLN6. This study is only the second to identify the molecular genetic basis of a feline NCL. Other cats exhibiting similar signs can now be screened for the CLN6 variant. This could lead to establishment of a feline model of CLN6 disease that could be used in therapeutic intervention studies.

CLN2 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disorder characterized by progressive vision loss, neurological decline, and seizures. CLN2 disease results from mutations in TPP1 that encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Children with CLN2 neuronal ceroid lipofuscinosis experience ocular disease, characterized by progressive retinal degeneration associated with impaired retinal function and gradual vision loss culminating in total blindness. A similar progressive loss of retinal function is also observed in a dog CLN2 model with a TPP1 null mutation. A study was conducted to evaluate the efficacy of periodic intravitreal injections of recombinant human (rh) TPP1 in inhibiting retinal degeneration and preserving retinal function in the canine model. TPP1 null dogs received periodic intravitreal injections of rhTPP1 in one eye and vehicle in the other eye beginning at approximately 12 weeks of age. Ophthalmic exams, in vivo ocular imaging, and electroretinography (ERG) were repeated regularly to monitor retinal structure and function. Retinal histology was evaluated in eyes collected from these dogs when they were euthanized at end-stage neurological disease (43-46 weeks of age). Intravitreal rhTPP1 dosing prevented disease-related declines in ERG amplitudes in the TPP1-treated eyes. At end-stage neurologic disease, TPP1-treated eyes retained normal morphology while the contralateral vehicle-treated eyes exhibited loss of inner retinal neurons and photoreceptor disorganization typical of CLN2 disease. The treatment also prevented the development of disease-related focal retinal detachments observed in the control eyes. Uveitis occurred secondary to the administration of the rhTPP1 but did not hinder the therapeutic benefits. These findings demonstrate that periodic intravitreal injection of rhTPP1 preserves retinal structure and function in canine CLN2 disease.

CLN2 disease is one of a group of lysosomal storage disorders called the neuronal ceroid lipofuscinoses (NCLs). The disease results from mutations in the TPP1 gene that cause an insufficiency or complete lack of the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). TPP1 is involved in lysosomal protein degradation, and lack of this enzyme results in the accumulation of protein-rich autofluorescent lysosomal storage bodies in numerous cell types including neurons throughout the central nervous system and the retina. CLN2 disease is characterized primarily by progressive loss of neurological functions and vision as well as generalized neurodegeneration and retinal degeneration. In children the progressive loss of neurological functions typically results in death by the early teenage years. A Dachshund model of CLN2 disease with a null mutation in TPP1 closely recapitulates the human disorder with a progression from disease onset at approximately 4 months of age to end-stage at 10-11 months. Delivery of functional TPP1 to the cerebrospinal fluid (CSF), either by periodic infusion of the recombinant protein or by a single administration of a TPP1 gene therapy vector to the CSF, significantly delays the onset and progression of neurological signs and prolongs life span but does not prevent the loss of vision or modest retinal degeneration that occurs by 11 months of age. In this study we found that in dogs that received the CSF gene therapy treatment, the degeneration of the retina and loss of retinal function continued to progress during the prolonged life spans of the treated dogs. Eventually the normal cell layers of the retina almost completely disappeared. An exception was the ganglion cell layer. In affected dogs that received TPP1 gene therapy to the CSF and survived an average of 80 weeks, ganglion cell axons were present in numbers comparable to those of normal Dachshunds of similar age. The selective preservation of the retinal ganglion cells suggests that while TPP1 protein delivered via the CSF may protect these cells, preservation of the remainder of the retina will require delivery of normal TPP1 more directly to the retina, probably via the vitreous body.

Late-infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a hereditary neurological disorder characterized by progressive retinal degeneration and vision loss, cognitive and motor decline, seizures, and pronounced brain atrophy. This fatal pediatric disease is caused by mutations in the CLN2 gene which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Utilizing a TPP1-/- Dachshund model of CLN2 disease, studies were conducted to assess the effects of TPP1 enzyme replacement administered directly to the CNS on disease progression. Recombinant human TPP1 (rhTPP1) or artificial cerebrospinal fluid vehicle was administered to CLN2-affected dogs via infusion into the CSF. Untreated and vehicle treated affected dogs exhibited progressive declines in pupillary light reflexes (PLRs) and electroretinographic (ERG) responses to light stimuli. Studies were undertaken to determine whether CSF administration of rhTPP1 alters progression of the PLR and ERG deficits in the canine model. rhTPP1 administration did not inhibit the decline in ERG responses, as rhTPP1 treated, vehicle treated, and untreated dogs all exhibited similar progressive and profound declines in ERG amplitudes. However, in some of the dogs treated with rhTPP1 there were substantial delays in the appearance and progression of PLR deficits compared with untreated or vehicle treated affected dogs. These findings indicate that CSF administration of TPP1 can attenuate functional impairment of neural pathways involved in mediating the PLR but does not prevent loss of retinal responses detectable with ERG.

Mutations in the human protein palmitoyl thioesterase-1 (PPT-1) gene result in an autosomal recessive neurodegenerative disorder designated neuronal ceroid lipofuscinosis (NCL), type CLN1, or infantile NCL. Among the symptoms of the CLN1 disease are accumulation of autofluorescent lysosomal storage bodies in neurons and other cell types, seizures, motor and cognitive decline, blindness, and premature death. Development of an effective therapy for this disorder will be greatly assisted by the availability of suitable animal models. A mouse PPT-1 gene knockout model has recently been generated. Studies were performed to determine whether the mouse model exhibits ocular features of the human CLN1 disorder. A progressive accumulation of autofluorescent storage material in all layers of the retina was observed in the PPT-1 knockout mice. Accompanying the storage body accumulation was a modest loss of cells with nuclei in the outer and inner nuclear layers. As indicated by electroretinogram (ERG) responses, retinal function was only mildly impaired at 4 months of age but was severely impaired by 8 months, despite only modest changes in retinal morphology. The pupillary light reflex (PLR), on the other hand, was exaggerated in the knockout mice. The apparent anomaly between the ERG and the PLR findings suggests that disease-related PLR changes may be due to changes in extraocular signal processing. The pronounced ocular phenotype in the PPT-1 knockout mice makes these animals a good model for testing therapeutic interventions for treatment of the human CLN1 disorder.

CLN5 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disease characterized by progressive neurological decline, vision loss and seizures. Visual impairment in children with CLN5 disease is attributed to a progressive decline in retinal function accompanied by retinal degeneration as well as impaired central nervous system function associated with global brain atrophy. We studied visual system pathology in five Golden Retriever littermates homozygous for the CLN5 disease allele previously identified in the breed. The dogs exhibited signs of pronounced visual impairment by 21-22 months of age. Electroretinogram recordings showed a progressive decline in retinal function primarily affecting cone neural pathways. Altered visual evoked potential recordings indicated that disease progression affected visual signal processing in the brain. Aside from several small retinal detachment lesions, no gross retinal abnormalities were observed with in vivo ocular imaging and histologically the retinas did not exhibit apparent abnormalities by 23 months of age. However, there was extensive accumulation of autofluorescent membrane-bound lysosomal storage bodies in almost all retinal layers, as well as in the occipital cortex, by 20 months of age. In the retina, storage was particularly pronounced in retinal ganglion cells, the retinal pigment epithelium and in photoreceptor cells just interior to the outer limiting membrane. The visual system pathology of CLN5-affected Golden Retrievers is similar to that seen early in the human disease. It was not possible to follow the dogs to an advanced stage of disease progression due to the severity of behavioral and motor disease signs by 23 months of age. The findings reported here indicate that canine CLN5 disease will be a useful model of visual system disease in CLN5 neuronal ceroid lipofuscinosis. The baseline data obtained in this investigation will be useful in future therapeutic intervention studies. The findings indicate that there is a fairly broad time frame after disease onset within which treatments could be effective in preserving vision.

The CLN2 form of neuronal ceroid lipofuscinosis is an autosomal recessively inherited lysosomal storage disease that is characterized by progressive vision loss culminating in blindness, cognitive and motor decline, neurodegeneration, and premature death. CLN2 disease results from mutations in the gene that encodes the soluble lysosomal enzyme tripeptidyl peptidase-1. A null mutation in the TPP1 gene encoding this enzyme causes a CLN2-like disease in Dachshunds. Dachshunds that are homozygous for this mutation serve as a model for human CLN2 disease, exhibiting clinical signs and neuropathology similar to those of children with this disorder. Affected dogs reach end-stage terminal disease status at 10-11 months of age. In addition to retinal changes typical of CLN2 disease, a retinopathy consisting of multifocal, bullous retinal detachment lesions was identified in 65% of (TPP1-/-) dogs in an established research colony. These lesions did not occur in littermates that were heterozygous or homozygous for the normal TPP1 allele. Retinal changes and the functional effects of this multifocal retinopathy were examined objectively over time using ophthalmic examinations, fundus photography, electroretinography (ERG), quantitative pupillary light response (PLR) recording, fluorescein angiography, optical coherence tomography (OCT) and histopathology. The retinopathy consisted of progressive multifocal serous retinal detachments. The severity of the disease-related retinal thinning was no more serious in most detached areas than in adjacent areas of the retina that remained in close apposition to the retinal pigment epithelium. The retinopathy observed in these dogs was somewhat similar to canine multifocal retinopathy (CMR), a disease caused by a mutation of the bestrophin gene BEST1. ERG a-wave amplitudes were relatively preserved in the Dachshunds with CLN2 disease, whether or not they developed the multifocal retinopathy. The retinopathy also had minimal effects on the PLR. Histological evaluation indicated that the CLN2 disease-related retinal degeneration was not exacerbated in areas where the retina was detached except where the detached areas were very large. DNA sequence analysis ruled out a mutation in the BEST1 exons or splice junctions as a cause for the retinopathy. Perfect concordance between the TPP1 mutation and the retinopathy in the large number of dogs examined indicates that the retinopathy most likely occurs as a direct result of the TPP1 mutation. Therefore, inhibition of the development and progression of these lesions can be used as an indicator of the efficacy of therapeutic interventions currently under investigation for the treatment of CLN2 disease in the Dachshund model. In addition, these findings suggest that TPP1 mutations may underlie multifocal retinopathies of unknown cause in animals and humans.

An autosomal recessive disease of Black Russian Terriers was previously described as a juvenile-onset, laryngeal paralysis and polyneuropathy similar to Charcot Marie Tooth disease in humans. We found that in addition to an axonal neuropathy, affected dogs exhibit microphthalmia, cataracts, and miotic pupils. On histopathology, affected dogs exhibit a spongiform encephalopathy characterized by accumulations of abnormal, membrane-bound vacuoles of various sizes in neuronal cell bodies, axons and adrenal cells. DNA from an individual dog with this polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV) was used to generate a whole genome sequence which contained a homozygous RAB3GAP1:c.743delC mutation that was absent from 73 control canine whole genome sequences. An additional 12 Black Russian Terriers with POANV were RAB3GAP1:c.743delC homozygotes. DNA samples from 249 Black Russian Terriers with no known signs of POANV were either heterozygotes or homozygous for the reference allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM), a severe developmental disorder characterized by abnormalities of the eye, genitals and nervous system including a predominantly axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, axonal transport, autophagy and synaptic transmission. The neuronal vacuolation and membranous inclusions and vacuoles in axons seen in this canine disorder likely reflect alterations of these processes. Thus, this canine disease could serve as a model for WARBM and provide insight into its pathogenesis and treatment.

We obtained DNA, brains, and eyes from American Bulldogs with neurodegeneration due to neuronal ceroid lipofuscinosis (NCL). The diagnosis of NCL was confirmed by detection of autofluorescent cytoplasmic inclusions within neurons throughout the brains, in retinal ganglion cells, and along outer limiting membranes of the retinas. Electron microscopy revealed that the inclusions had coarsely granular matrices surrounding well-delineated spherical structures and that the inclusions near the retinal outer limiting membranes were within photoreceptor cells, mostly cones. Affected American Bulldogs were homozygous for the A allele of a G to A transition in the cathepsin D gene (CTSD), which predicts the conversion of methionine-199 to an isoleucine. Only the G allele was detected in DNA samples from 131 randomly selected dogs representing 108 breeds other than American Bulldog; however, the A allele had a frequency of 0.28 among 123 genotyped American Bulldogs. Transmission analysis in a 99 dog pedigree of American Bulldogs indicated a probability of less than 10(-7) that alleles from any mutation unlinked to CTSD would be concordant with the pedigree and phenotypes of the dogs. Brain samples from affected dogs had 36% of the cathepsin D-specific enzymatic activity found in control dog brains; whereas, specific enzymatic activities of 15 other lysosomal enzymes were unchanged or increased. Compared to previously described NCLs in mice and sheep that completely lack cathepsin D activity, the clinical course of NCL in the American Bulldogs was less severe and more closely resembled that of many human NCLs.

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of β-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a β subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two β subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.

Development of effective treatments for amyotrophic lateral sclerosis (ALS) would be facilitated by identification of early events in the pathological cascade of disease progression. Degenerative myelopathy (DM), a naturally occurring disease in dogs, is quite similar to forms of ALS associated with SOD1 mutations and is likely to be a good model for these forms of the human disease. The sequence of histopathological changes that occur in DM was characterized by analyzing tissue samples obtained from affected dogs euthanized at various stages of disease progression. Cervical spinal cord and the associated spinal nerve roots, ulnar nerve, and the extensor carpi radialis (ECR) muscle were obtained from Pembroke Welsh Corgi dogs (PWCs) with early and late stage DM and from age-matched unaffected PWCs. In early stage disease there was a substantial change in the ratio of the two main muscle fiber types and an increase in mean muscle fiber area in the ECR. DM, even in late stage disease, was not accompanied by changes in the number of motor neuron cell bodies, in the number of axons in the motor or sensory nerve roots, or in the ulnar nerve. In addition, no disease-related denervation of the acetylcholine receptors of the ECR was observed at any stage of the disease. On the other hand, axon densities in both motor and sensory nerve tracts in the cervical cord were reduced in affected dogs. SOD1-immunoreactive aggregates were observed in spinal cord motor neuron cell bodies only in late stage disease. These findings suggest that some of the earliest pathological changes in DM occur in the muscle fibers and upper motor and sensory neuron tracts in the spinal cord. Targeting therapeutic interventions to these early events in the disease are most likely to be effective in slowing disease progression for DM and may translate to therapy of SOD1-related forms of ALS.

Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is a rare, progressive, fatal neurodegenerative pediatric disorder resulting from deficiencies of the lysosomal enzyme tripeptidyl peptidase 1 that are caused by mutations in TPP1. Identifying biomarkers of CLN2 disease progression will be important in assessing the efficacy of therapeutic interventions for this disorder. Neurofilament light is an intrinsic component of healthy neurons; elevated circulating extracellular neurofilament light is a biomarker of neuropathology in several adult-onset neurological diseases. Our objective was to assess whether circulating neurofilament light is a biomarker that is responsive to enzyme replacement therapy (ERT) in CLN2 disease.

Rod and cone photoreceptors are specialized retinal neurons that have a fundamental role in visual perception, capturing light and transducing it into a neuronal signal. Aberrant functioning of rod and/or cone photoreceptors can ultimately lead to progressive degeneration and eventually blindness. In man, many rod and rod-cone degenerative diseases are classified as forms of retinitis pigmentosa (RP). Dogs also have a comparable disease grouping termed progressive retinal atrophy (PRA). These diseases are generally due to single gene defects and follow Mendelian inheritance.We collected 51 DNA samples from Miniature Schnauzers affected by PRA (average age of diagnosis ∼3.9 ±1 years), as well as from 56 clinically normal controls of the same breed (average age ∼6.6 ±2.8 years). Pedigree analysis suggested monogenic autosomal recessive inheritance of PRA. GWAS and homozygosity mapping defined a critical interval in the first 4,796,806 bp of CFA15. Whole genome sequencing of two affected cases, a carrier and a control identified two candidate variants within the critical interval. One was an intronic SNV in HIVEP3, and the other was a complex structural variant consisting of the duplication of exon 5 of the PPT1 gene along with a conversion and insertion (named PPT1dci ). PPT1dci was confirmed homozygous in a cohort of 22 cases, and 12 more cases were homozygous for the CFA15 haplotype. Additionally, the variant was found homozygous in 6 non-affected dogs of age higher than the average age of onset. The HIVEP3 variant was found heterozygous (n = 4) and homozygous wild-type (n = 1) in cases either homozygous for PPT1dci or for the mapped CFA15 haplotype. We detected the wildtype and three aberrant PPT1 transcripts in isolated white blood cell mRNA extracted from a PRA case homozygous for PPT1dci , and the aberrant transcripts involved inclusion of the duplicated exon 5 and novel exons following the activation of cryptic splice sites. No neurological signs were detected among the dogs homozygous for the PPT1dci variant. Therefore, we propose PPT1dci as causative for a non-syndromic form of PRA (PRA PPT1 ) that shows incomplete penetrance in Miniature Schnauzers, potentially related to the presence of the wild-type transcript. To our knowledge, this is the first case of isolated retinal degeneration associated with a PPT1 variant.

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.

Late-infantile neuronal ceroid lipofuscinosis (CLN2) is a hereditary neurological disorder characterized by progressive retinal degeneration and vision loss, cognitive and motor decline, seizures, and pronounced brain atrophy. The progressive loss of neurological functions eventually leads to death, usually by the early teenage years. Utilizing a canine model of CLN2, therapeutic studies to inhibit the brain and retinal degenerations are currently under way. Using this dog model, studies were undertaken to compare quantitative assessments of the pupillary light reflex (PLR) and electroretinography (ERG) as tools for evaluating the effects of the disease on retinal function. The PLR and ERG were recorded in normal and CLN2-affected Dachshunds at 2 month intervals between the ages of 4 and 10 months. Using custom instrumentation for quantitative PLR assessments, a series of white light stimuli of varying intensity was used to elicit pupil constriction, and pupil images were recorded using continuous infrared illumination and an infrared-sensitive camera. Electroretinography was used to evaluate retinal function in the same dogs. As the disease progressed, affected dogs exhibited progressive and profound declines in ERG amplitudes under both scotopic and photopic conditions. With low intensity light stimuli, CLN2 was also accompanied by progressive deficits in the PLR. Changes in the PLR to dim light stimuli included significant deficits in latency, constriction velocity, constriction amplitude, and redilation velocity. However, despite the almost complete loss of detectable ERG responses by disease end stage, the PLR to bright stimuli was well preserved throughout the disease progression. These findings demonstrate that the PLR is much more sensitive than the ERG in detecting residual retinal function in animal models of retinal degenerative disease. The preservation of the PLR in dogs with profoundly depressed ERGs correlates with a preservation of visually-mediated behavior even late in the disease progression. Quantitative analysis of the PLR has potential as a biomarker in animal models of retinal degenerative diseases and in evaluating the efficacy of therapeutic interventions in preserving retinal function.