Neuronal ceroid lipofuscinosis type 6 (CLN6) is a fatal, autosomal recessive neurodegenerative disorder characterized by cognitive/motor impairment, vision loss, as well as neuronal loss and gliosis in the brain, and premature death. Onset typically occurs in childhood. No approved pharmacological treatments exist that halt or reverse disease progression. A novel flupirtine benzyl carbamate was orally administered to male and female Cln6nclf mice from 4 to 28 weeks of age to evaluate its neuroprotective and antispastic effects. Drug treatment produced significant, sex-dependent phenotypic improvements. Treated mice of both sexes exhibited reduced hindlimb spasticity, but only treated males demonstrated diminution in locomotor hyperactivity and recovery of visuospatial performance. In the brains of male and female Cln6nclf mice, flupirtine benzyl carbamate significantly decreased astrocytosis, microgliosis and mitochondrial ATP synthase subunit C (SCMAS) accumulation, increased neuronal marker expression and reduced the number of TUNEL-positive cells. The treatment failed to rescue photoreceptor loss or clear retinal SCMAS storage. These outcomes result in distinct sex-specific differences in neuronal vulnerability and drug responsiveness. Overall, these findings demonstrate that flupirtine benzyl carbamate diminishes key motor, visual and pathological deficits in CLN6 disease, highlighting its promise as a potential disease-modifying therapy for CLN6 in humans despite sex-specific differences.

Neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurogenetic disorders caused by mutations in 14 different genes. CLN6 disease manifests as variant late-infantile NCL (vLINCL) or as an adult variant. In childhood, symptoms include speech delay, vision loss, cognitive and motor decline, seizures, and early death. An in-depth characterization of a naturally occurring Cln6 mutant mouse (Cln6nclf) is presented, with implications for translational research. The expanded phenotype provides data showing early death, vision loss, and motor deficits in male and female Cln6nclf mice. Diminished visual acuity in Cln6nclf mice was noted at 28 weeks of age, but the pathological loss of retinal layers began as early as 2 weeks or postnatal day 14 (P14). Apoptosis was confirmed by TUNEL staining in the Cln6nclf mouse brain at P8 and in the retina at P12. A peak in glial fibrillary acidic protein (GFAP) expression was established as a normal developmental phenomenon in the wild-type and Cln6nclf mouse brain cerebellum and the CA2-CA3 regions of the hippocampus at P8. In Cln6nclf mice, GFAP levels were elevated at P12 in the cerebellum and hippocampus. In the retina, a developmental peak in gliosis was absent, with increased astrogliosis noted at P6 and P8 in female and male Cln6nclf mice, respectively. This highlights the lack of a sex-dependent response in wild-type mice. These novel data position the Cln6nclf mouse model as a useful tool for screening potential therapeutics for human CLN6 disease.

Neuronal ceroid lipofuscinoses (NCLs) are genetically heterogeneous neurodegenerative disorders, characterized by progressive cognitive and motor decline, epilepsy, visual impairment, and shortened life-expectancy. CLN6-related NCLs include both late-infantile and adult myoclonic form. We report a 21-year-old patient, with mild developmental delay, who developed occipital seizures at 14 years, and subsequently cognitive decline, cortical myoclonus, and photosensitivity at low and higher frequencies. Overall, the picture suited progressive myoclonus epilepsy. Electroretinogram was normal. A skin biopsy revealed a mixed storage of curvilinear and fingerprint profiles. A brain MRI showed severe cortical atrophy. Performing genetic analyses, two biallelic variants were identified in the CLN6 gene, each inherited from one of the healthy parents, one c.722T>C, p.(Met241Thr) already described in the late-infantile form and the other one c.486+28T>C, intronic and novel, causing aberrant splicing. In the patient, the expression of the allele containing c.722T>C variant was increased, in comparison with the carrier parent. The peculiar genetic pattern observed in the patient could explain a milder clinical picture when compared with late-infantile form, since CLN6 expression was partially preserved. However, the presence of a delay, and the early cognitive decline suggested a continuum phenotype connecting late-infantile and adult CLN6-related forms. PLAIN LANGUAGE SUMMARY: We report a patient with CLN6 disease who developed symptoms at an intermediate age: 9 years for mild intellectual disability and 14 years for occipital seizures and progressive myoclonus epilepsy, without visual impairment. The patient is compound heterozygous for a CLN6 missense variant c.722T>C, p.(Met241Thr) already described in the late-infantile form and for a novel intronic variant c.486+28T>C, causing aberrant splicing. In the patient, the expression of the allele containing c.722T>C variant was increased, compared with the carrier parent. The splice site variant had a milder effect. The peculiar genetic pattern may explain the continuum phenotype between late-infantile and adult forms.

Mutations in the CLN6 gene cause late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease of childhood onset. Clinically, individuals present with progressive motor and cognitive regression, ataxia, and early death. The aim of this study is to establish natural history data of individuals with classic, late-infantile-onset (age less than five years) CLN6 disease. We analyzed the natural history of 25 patients with late-infantile-onset CLN6, utilizing the Hamburg motor-language scale to measure disease progression. The key outcomes were CLN6 disease progression, assessed by rate of decline in motor and language clinical domain summary scores (0 to 6 total points); onset and type of first symptom; onset of first seizure; and time from first symptom to complete loss of function. Median age of total motor and language onset of decline was 42 months (interquartile range 36 to 48). The estimated rate of decline in total score was at a slope of -1.20 (S.D. 0.30) per year, after the start of decline. Complete loss of both motor and language function was found to be, on average, 88.1 months (S.D. 13.5). To our knowledge, this is the largest international study that monitors the longitudinal natural history and progression of CLN6 disease. These data may serve as a template for future interventional trials targeted to slow the progression of this devastating disease. The neuronal ceroid lipofuscinoses (NCL), also known as Batten disease, are a group of inherited lysosomal storage disorders that share similar pathological and clinical features. They are characterized by accumulation of autofluorescent storage material within the lysosome and the death of neurons. Clinical presentation includes medically refractory epilepsy, visual failure, and motor and cognitive decline, usually beginning in childhood and ending in premature death. Each NCL is caused by mutations in a single gene and is associated with a typical age of onset and disease progression. However, all NCL also have a broader age of onset and disease course. Considerable expertise has been developed in the care and management of patients. There is one treatment in the clinic and others are in development.

A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband's DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of CLN6. Other mutations in CLN6 cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog's genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the CLN6 disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.