A Homozygous CNP Missense Mutation in Weimaraners With an Adult-Onset Neurodegenerative Disease, Myelin Abnormalities and Accumulation of Lipofuscin-Like Inclusions

1. Introduction

We recently reported a novel canine lysosomal storage disease (LSD) with similarities to the neuronal ceroid lipidoses in two Dalmatian littermates with a homozygous nullifying single-base deletion and reading-frame shift in CNP, the gene that encodes a protein with 2’,3’-cyclic-nucleotide 3’-phosphodiesterase enzyme activity (CNPase) and a structural role in myelin [1]. The affected littermates developed slowly progressing neurodegenerative signs that were first noticed when they were a year and a half old. The neurodegenerative signs included behavioral changes, cognitive decline, incoordination, and visual impairment. Results of magnetic resonance imaging of both littermates when 5-years old were consistent with diffuse cerebrocortical, cerebellar, and brainstem atrophy. After reaching eight years of age or older, some relatives of the littermates that were heterozygous for the CNP single-base deletion began showing neurologic signs including aggression, tremors, loss of appetite and weight, restlessness, behavioral changes, kyphosis, ataxia, sleep disturbance and loss of balance.

Due to the progression of neurodegenerative signs, the Dalmatian littermates with the homozygous deletion were euthanized when seven and eight years old. Fluorescence microscopic examination of unstained tissue from both littermates detected accumulations of cytoplasmic autofluorescent storage granules in the cerebellar cortex, cerebral cortex, optic nerve, and cardiac muscle. Immunohistochemical staining with antibodies raised to LAMP2 confirmed that the autofluorescence came from lysosomes. Electron microscopic examination of these same tissues found membrane-bounded cytoplasmic storage bodies with variable and complex ultrastructural appearances. In addition, the myelin sheaths surrounding the optic nerve axons exhibited abnormal ballooning between the layers of myelin membranes. Immunohistochemical staining with anti-CNPase antibodies produced pronounced staining in nerve fiber tracts of the cerebellum, cerebral cortex, optic nerve, and cardiac muscle from a control dog, but failed to detect CNPase antigen in the affected Dalmatian littermates [1].

We here describe a similar disorder that has occurred in a different dog breed, the Weimaraner. In addition, we report that the likely cause of the Weimaraner disease was a homozygous missense mutation in CNP.

2. Materials and Methods

A 5-year, 1-month old neutered male Weimaraner (proband) was presented for neurological evaluation by a veterinary neurologist (MS) for an approximately 3-month history of pelvic limb ataxia, episodes of fecal incontinence, and lethargy. On neurologic examination, the proband was ambulatory with moderate paraparesis and proprioceptive pelvic limb ataxia characterized by a long-strided pelvic limb gait. A myelopathy was suspected. Standard MR images (1.5 Tesla instrument) of the thoracolumbar and cervical spine was performed and no significant spinal cord abnormalities were identified. Additional MR imaging of the brain was performed (T2-weighted sagittal, dorsal, and transverse images; T1-weighted post-gadolinium contrast sagittal, dorsal, and transverse images; FLAIR and T2-weighted gradient echo transverse images), which revealed pronounced cerebral parenchymal atrophy (Figure 1). Cerebrospinal fluid analysis was unremarkable. Based on these findings a neurodegenerative disease process was suspected. Approximately 1.5 years after the initial onset of clinical signs, humane euthanasia was elected due to the progression of neurologic dysfunction that included increased falling, worsening fecal incontinence, cognitive decline, incoordination, decreased interest in food, changes in posture, and episodes of trance-like behavior.

Following euthanasia, the eyes, brain and heart ventricular wall were collected and preserved as described previously [2]. Chemically fixed slices of the retinas, optic nerves, cerebral cortex, cerebellar cortex, and cardiac muscle were cryo-embedded and sectioned with a cryostat. The unstained cryostat sections were examined for autofluorescence with a Zeiss Axiophot microscope equipped with an Olympus DP72 color digital camera as described previously [1]. Slices of fixed cerebral cortex, cerebellar cortex, and cardiac muscle were embedded in paraffin. Sections of the paraffin-embedded samples were immunostained using BioLegend anti-CNPase primary antibody (cat. no. 836403), Abcam anti-mitochondrial ATP synthase subunit c primary antibody (cat. no. ab180149), Abcam anti-ATPB primary antibody (cat. no. ab14730), Agilent Dako anti-GFAP primary antibody (cat. no. Z0334), and Fujifilm Wako anti-Iba1 primary antibody (cat. no. 019-19741). Immunostaining was performed as described previously [1,3]. Additional slices of these tissues and of the optic nerve were examined with electron microscopy as described previously [1].

Genomic DNA was prepared from EDTA-anticoagulated blood using a previously described procedure [4]. The proband’s DNA was submitted to the University of Missouri Genomics Technology Core Facility for library preparation and 2X150 bp paired-end sequencing on their Illumina NovaSeq 6000 sequencer. A previously described data-processing pipeline was used to align the sequence reads to a current canine reference genome assembly (Dog10K_Boxer_Tasha) and to analyze them with Ensembl annotation in conjunction with reads from 4,024 other whole genome sequences obtained from the NCBI Sequence Read Archive (SRA) [1]. The SRA BioSample identifiers for all 4,025 whole genome sequences used in this analysis are listed in Supplementary File 1. The amino acid positions for canine CNPase were numbered according to ENSCAFT00000102206.

We used an allelic discrimination assay to genotype individual dogs for a candidate variant at position 20,355,460 on chromosome 9. For this assay, the sequences of the PCR primers were5’-CAGAGCTGCAGTTTCCTTTCCT-3′ and 5′-AGCGTCTTGCACTCTTGCA-3′. The competing probes sequences were 5′-VIC-TGGCCACCGTCTCCT-NFQ-3′ (reference allele) and 5′-FAM-TGGCCACCATCTTCCT-NFQ-3′ (variant allele).

3. Results

3.1. Microscopic Findings

Clusters of autofluorescent granules were present in cells of the cerebral cortex and cerebellar cortex of the proband (Figure 2). In the cerebellum these inclusions were present primarily in the Purkinje cell and granule cell layers. In the cerebral cortex, the cells containing these inclusions were scattered throughout the gray matter. No inclusions with similar fluorescence properties were present in the neural retina, but autofluorescent granules were present in the optic nerve (Figure 3A). The granules in the optic nerve occurred either individually or as smaller clusters than seen in the brain tissues. Groups of autofluorescent inclusions were also present in the cardiac muscle, usually clustered in linear arrays within the muscle fibers (Figure 3B). The emission colors of the inclusions in these tissues ranged from golden yellow to orange as detected by a color digital camera that was matched to the spectral sensitivity of the human eye.

Electron microscopic examination of the cerebral cortex gray matter revealed the presence of membrane-bounded organelles containing mixtures of materials that included granular components of varying electron density, membranous components, and lipid-like inclusions (Figure 4 and Figure 5). Organelles with similar heterogeneous contents were present in the cerebellar cortex gray matter (Figure 6 and Figure 7), in the cerebral cortical and cerebellar white matter tracts (examples from the cerebral cortical white matter shown in Figure 8 and Figure 9), and in the optic nerve (Figure 10). The myelin sheaths of the optic nerve and of the brain white-matter axon tracts contained numerous areas of large abnormal gaps between the layers of myelin (Figure 11 and Figure 15). In the optic nerve, it appeared that some of abnormal myelin had budded off from the axonal sheaths and was taken up into the adjacent cells forming structures that contained whorls of membrane (Figure 12, Figure 13 and Figure 14). In the brain white matter tracts, some of the axons appeared degenerate, with the axoplasm replaced by myelin-derived membrane and membrane fragments that appeared to have collapsed inward into the axon (Figure 16).

Aggregates of abnormal electron-dense inclusion bodies were also present in the cardiac muscle fibers of the proband (Figure 17). These inclusions were localized to the mitochondria-rich regions of the cardiac muscle fibers. They were much more uniform in appearance than the inclusion bodies observed in the brain and optic nerve, consisting of parallel arrays of membrane-like constituents overlayed with patches of amorphous very electron-dense material (Figure 17 C and D).

A large fraction of the disease-related inclusions in the cerebral cortical gray matter were labeled with an antibody directed against the subunit c protein of mitochondrial ATP synthase (Figure 18). In contrast, only a very small subset of the disease-related inclusions in the cerebellar cortex were labeled with this antibody (Figure 19). In cardiac muscle, the disease-related inclusions exhibited strong immunolabeling with the subunit c antibody (Figure 20). None of the disease-related inclusions in either brain tissue or cardiac muscle exhibited labelling with an antibody that binds the beta subunit of mitochondrial synthase.

Immunolabeling of brain tissue samples of the proband indicated that the disease is characterized by neuroinflammation. Astrocyte activation, as indicated by immunolabeling for GFAP, was pronounced in both cerebral cortical gray matter and in the cerebellar cortex (Figure 21). Microglial activation, indicated by Iba1 immunolabeling, on the other hand, was pronounced in the cerebral cortex but not in the cerebellar cortex (Figure 22).

In Dalmatians with a disorder similar to that of the proband, a frame-shift variant CNP that encodes 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase) was accompanied by an absence of detectable CNPase protein in tissues of the affected dog [1]. Sections of cerebral cortex and cerebellar cortex from the proband, and from an unaffected dog were immunolabeled for CNPase localization. Abundant immunolabeling was observed in both tissues of the control dog but was not detectable in the tissues of the proband (Figure 23).

3.2. Molecular Genetic Findings

To identify the cause of the disease, we used DNA from the proband to generate a 32.2-fold average-coverage whole-genome sequence. This sequence contained 26,761 variants relative to the canine reference sequence that were predicted to alter the primary structure of the encoded gene products. Of these, 9,064 variants were in the homozygous state. When the proband’s homozygous variants were sorted according to the allele frequency among all 4,035 canine whole genome sequences included in the analysis, the lowest allele frequency (0.000376 or 3 of 7,986 called alleles) was a G-to-A transition at position 20,355,460 on chromosome 9, which was predicted to produce a Thr42Met missense mutation in CNPase. The validity of this variant call was confirmed by an Integrative-Genomics-Viewer-assisted inspection of aligned reads from the proband’s whole genome sequence to the Tasha reference sequence from position 20,355,440 to 20,355,479 on chromosome 9 (Figure 24) [5,6]. A previous report attributes a canine adult-onset, slowly progressive neurologic disease to a homozygous CNP single base deletion frame-shift variant [1]. A literature review failed to identify a similar disease phenotype in association with any of the other thirty-four genes that harbored variants with allele frequencies of 0.0003 or less. A blastp query [7] indicated that a threonine residue at an equivalent position was conserved in 100 mammalian CNP orthologs.

Two hundred-ninety-two of our archived DNA samples from Weimaraners were genotyped for the 9:20,355,460 G/A polymorphism in CNP. In addition to the proband, one other sample tested homozygous for the variant 9:20,355,460 A allele. The homozygous sample was from a spayed female that exhibited bilateral hindlimb weakness and fecal incontinence. The sample was submitted to us to evaluate a differential diagnosis of degenerative myelopathy [8], which was ruled out when the sample tested homozygous for the reference G allele at 31:26,532,306, the site of the G-to-A transition in SOD1 associated with degenerative myelopathy in many breeds [9,10]. When 8 years old, this Weimaraner presented for a 5-week history of pelvic limb weakness which started after stumbling down the stairs and a one-year history of progressive fecal incontinence (dropping stools 2-3 times a week). The initial examination identified an ambulatory paraparesis and generalized whole body tremors noted at rest and during movement (a pre-existing condition present since puppyhood). T2 magnetic resonance images of the neuraxis demonstrated significant generalized brain atrophy and areas of increased signal intensity in the cerebral cortex parenchyma similar to those observed in the proband (Figure 25). Cerebrospinal fluid analysis and cytology indicated a marginal increase in protein concentration (46.0 mg/dL, upper limit RR 45mg/dL) and normal nucleated cellularity and cell types. Genetic testing for variants previously associated with hypomyelination or spinal dysraphism demonstrated no mutations [11,12]. The patient was initially started on azathioprine (25 mg PO SID) as an empirical immunomodulatory therapy for suspected immune mediated inflammatory myopathy/ polyneuropathy. Azathioprine was then discontinued due to increased hepatocellular enzyme values and cyclosporine (100 mg PO SID for 30 days), a tapering dose of prednisone (15mg PO SID for 14 days to start), and Cerenia (30 mg PO SID PRN) therapy was initiated. Ten-months later the patient was again evaluated for increased symmetrical muscle atrophy, worsening pelvic limb paresis and generalized whole body tremors at rest and during movement or weight bearing. Because of progression in her clinical signs, the patient was euthanized, but no necropsy was performed.

Of the 292 archived Weimaraner DNA samples 9 were G/A heterozygotes at 9:20,355,460. One of these samples was from an asymptomatic 3-year-old dog. The other eight were from dogs that exhibited slowly progressive hindleg weakness and other signs of neurodegenerative disease. Their ages ranged from 8.8 years to 13.0 years (mean, 10.7 years). The samples from these 8 dogs were submitted to us for degenerative myelopathy SOD1 variant testing. All 8 of these samples were found to be homozygous for the SOD1 reference allele. The remaining 282 archived Weimaraner samples were homozygous for the CNP reference allele, 9:20,355,460 G. Thus, the frequency of the variant 9:20,355,460 A allele in the archived collection of Weimaraner samples was 0.019. All but 31 of the 279 Weimaraners homozygous for the CNP reference allele were submitted to us as degenerative myelopathy suspects because they exhibited adult-onset hindlimb weakness.

4. Discussion

For diagnostic purposes, we received clinical records, postmortem tissues, and blood as a source of DNA from a Weimaraner (the proband) with an adult-onset, slowly progressive neurodegenerative disease. Examination of the postmortem tissues by fluorescence-, light-, and electron-microscopy, and immunohistochemistry suggested that the dog had an LSD similar to neuronal ceroid lipofuscinosis (NCL) [13]; however, marked abnormalities in myelin structure distinguished the disease from most previously recognized LSDs. Examination of the proband’s whole genome sequence identified a candidate for causality: a rare homozygous missense mutation in CNP, the gene that encodes CNPase. This variant was recognized as a plausible causal candidate because a different homozygous CNP variant was known to cause a similar neurological disease of Dalmatian dogs that was also characterized by autofluorescent storage body accumulation and myelin abnormalities [1]. The absence of immunohistochemical staining of CNPase antigen in brain sections from the proband strongly supported the homozygous CNP missense mutation as the cause of the neurodegenerative disease.

Previous reports have described murine, human, and canine CNP deficiency diseases [1,14,15]. The CNP deficiency disease in transgenic mice was first described in 2003. Cnp nullizygous mice appeared normal through their first five months of life. After that, the mice developed progressive neurologic signs including ataxia, gait abnormalities, weight loss, hind-limb paralysis, convulsions, kyphosis and reduced life span. Among the post-mortem lesions were generalized brain atrophy with marked white-matter loss, axonal spheroids containing multivesicular storage bodies, myelin abnormalities including enlarged myelin inner tongues and disorganized paranodal structures, and both an astrogliosis and a microgliosis [15]. Subsequent examinations of the Cnp nullizygous mice found that structural myelin abnormalities preceded the overt clinical signs. Specifically, paranodal disorganization was detected at three months of age [16] and in the small-diameter fibers axonal degeneration, spheroid formation, redundant myelin loops, and swollen inner tongues (including some containing autophagic vacuoles) can be detected shortly after birth [17].

A 2020 report described an infantile-onset, progressive neurodegenerative disease in a male child with a homozygous CNP missense mutation identified by exome sequencing [14]. This patient appeared to have developed normally until 16-months of age when he became abnormally irritable. After that, he exhibited delayed or regressive development. He developed microcephaly, episodic body stiffness and dystonia, scoliosis, progressive hypotonia and died from aspiration pneumonia at five years of age. Brain MRI showed markedly reduced white matter volume. The patient’s apparently normal parents and a female sibling were heterozygous for the CNP missense mutation. The patient’s two earlier-born male siblings had similar clinical histories and died at seven and eight years of age. The genotypes of the latter siblings were not reported [14].

The putative causal mutation in the affected dogs predicts a substitution of methionine for threonine at position 42 in the CNPase isoform 2 (position 22 in isoform 1). Based on the immunohistochemical findings, this single amino acid substitution appears to have resulted in lack of detectable CNPase protein in brain tissues from the proband. The similar human neurological disorder that results from a Ser82Leu substitution resulted in an almost complete absence of CNP protein in cultured cells from an affected patient relative to a healthy control subject [14]. Together these findings suggest that both Thr42 and Ser82 are essential for maintaining the stability of the protein in both species (Supplementary File 2). In the human cells, inhibition of proteosome function did not rescue the lack of CNPase protein, indicating that the deficiency of the mutant protein was not due to accelerated degradation by proteosomes. Further research will be necessary to elucidate the mechanisms by which these amino acid substitutions destabilize the protein.

Mice that are heterozygous for a Cnp knockout allele appear to be phenotypically normal when younger, but by 19 months of age these heterozygotes accumulated axonal spheroids and developed a more intense microgliosis and astrogliosis in comparison to age-matched wildtype mice [18]. In addition, these aged heterozygotes exhibited abnormal behavior which may be reflective of depression and catatonia [18]. These findings suggest that individuals of other species that are heterozygous for deleterious CNP variants may be at risk for developing milder later-onset neurological signs. In the current study, our archived DNA samples from Weimaraners included eight from dogs that were at least 8 years old and heterozygous for the likely causal CNP missense mutation. All eight exhibited hindleg weakness that was not associated with a variant in SOD1 that underlies late-onset hindlimb weakness in many breeds [9]. It is possible that this later-onset sign is related to the CNP variant. It does not appear that the parents or other older relatives of the children with the CNP-related disorder were evaluated for subtle neurological abnormalities that might be expected based on the mouse data. Further research will be necessary to determine whether human subjects heterozygous for deleterious CNP variants develop late-onset neurological signs.

CNP encodes at least two forms of CNPase protein that have apparently unrelated functions: enzymatic catalysis of nucleoside 2′,3′-cyclic monophosphates hydrolysis to nucleoside 2′-monophosphates [19,20] and a structural role in myelin where CNPase accounts for approximately 5% of the myelin-associated protein in the central nervous system and 0.3% of the protein in the peripheral nervous system [19,20,21]. Alternative first exons and alternative initiation start sites express transcripts that encode two major CNPase isoforms, sometimes referred to as CNPase1 and CNPase2, with identical amino acid sequences except that CNPase2 has an additional 20-amino-acid extension at the N-terminal end [22,23]. When unphosphorylated, the N-terminal extension functions as a mitochondrial targeting signal [24]. Although it has been localized to the mitochondrial inner and outer membranes, the precise roles of CNPase in the mitochondria remain to be elucidated. However, studies indicate that is involved in regulating the functioning of the mitochondrial transition pore [20]. Abnormal regulation of the mitochondrial transition pore can result in cell damage or death [25,26]. Thus, deficiency in CNP protein would be predicted to alter both myelin structure and mitochondrial function. Clearly the structure of the myelin sheaths surrounding axons is profoundly abnormal in both Dalmatians and Weimaraners that lack CNP as a result of different mutations, indicating that CNP protein is necessary for maintaining the normal organization of myelin sheaths. The mechanism by which it does so remains to be elucidated.

Based on ultrastructural analyses, it appears that some of the abnormal myelin is taken up into neurons and accumulates in storage bodies. Mitochondrial function was not assessed in either of the canine disorders or in the human subjects with a CNP deficiency. However, the disease-related accumulation of storage bodies of both the affected Dalmatians and Weimaraner occurs within the mitochondria-rich regions of the cardiac muscles suggesting that these inclusions may be derived from damaged mitochondria. Storage bodies in both cardiac muscle and nervous tissue of CNP-deficient dogs of both breeds contain subunit c protein from the mitochondrial inner membrane, consistent with a mitochondrial origin of at least some of the contents of the disease-related storage bodies. Abnormal or damaged mitochondria are likely incorporated into disease-related storage bodies via autophagocytosis.

The accumulation of subunit c-containing autofluorescent storage bodies as a result of CNPase deficiency resembles that which occurs in many of the NCL disorders [13,27,28]. However, in the NCLs the storage body accumulation is usually linked to a mutation that results in a direct impairment of lysosomal function. The CNP protein has not been shown to directly influence lysosomal function, so the storage body accumulation that occurs as a result of CNP deficiency is likely to be secondary to formation of myelin- and mitochondrial-derived substrates that get incorporated into phagolysosomes and cannot be degraded efficiently. The accumulation of these storage bodies, in addition to myelin abnormalities and mitochondrial dysfunction, may contribute to the development the neurological signs associated with CNP deficiency. The slow progression of disease signs may be explained by a gradual accumulation of storage material in neurons over time and the effects of this accumulation on cell function.