CLN8-Batten disease is a rare neurodegenerative disorder characterized phenotypically by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature death. Mutations in CLN8 result in characteristic Batten disease symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subtypes of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on CLN8 disease burden and progression, a Cln8mnd mouse model was utilized to measure the impact and progression of histopathological and motor-behavioral outcomes between sexes. Immunohistochemistry staining utilized markers for intracellular storage materials, astrocytes, and microglial cells; sections were obtained of the thalamus and the somatosensory cortex of Cln8mnd mice. Several notable sex differences were observed in the presentation of brain pathology, including Cln8mnd female mice consistently presenting with greater GFAP+ astrocytosis and CD68+ microgliosis in the somatosensory cortex and ventral posteromedial/ventral posterolateral nuclei of the thalamus when compared to Cln8mnd male mice. Female Cln8mnd mice experienced a diminished lifespan by 0.5 months compared to their male counterparts (p less than 0.05). Furthermore, sex differences in motor-behavioral assessments identified Cln8mnd female mice experience poorer motor performance in the Morris Water Maze assessment, reverse Morris Water Maze, and increased tremors. Female Cln8mnd mice perished earlier, performed worse on motor-behavioral assessments, and demonstrated marked microglial and astrocyte reactivity compared to their male counterparts. Taken together, the results provide further evidence of biological sex as a modifier of Batten disease progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeutic impact.

CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.

CLN8-Batten disease (CLN8 disease) is a rare neurodegenerative disorder characterized phenotypically by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature death. Mutations in CLN8 results in characteristic Batten disease symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subforms of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on CLN8 disease burden and progression, we utilized a Cln8mnd mouse model to measure the impact and progression of histopathological and behavioral outcomes between sexes. Several notable sex differences were observed in the presentation of brain pathology, including Cln8mnd female mice consistently presenting with greater GFAP+ astrocytosis and CD68+ microgliosis in the somatosensory cortex, ventral posteromedial/ventral posterolateral nuclei of the thalamus, striatum, and hippocampus when compared to Cln8mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8mnd female mice experience poorer motor performance and earlier death than their male counterparts. Cln8mnd mice treated with an AAV9-mediated gene therapy were also examined to assess sex differences on therapeutics outcomes, which revealed no appreciable differences between the sexes when responding to the therapy. Taken together, our results provide further evidence of biologic sex as a modifier of Batten disease progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeutic impact.

The CLN8 disease type refers to one of the neuronal ceroid lipofuscinoses (NCLs) which are the most common group of neurodegenerative diseases in childhood. The clinical phenotypes of this disease are progressive neurological deterioration that could lead to seizures, dementia, ataxia, visual failure, and various forms of abnormal movement. In the current study, we describe two patients who presented with atypical phenotypic manifestation and protracted clinical course of CLN8 carrying a novel compound heterozygous variant at the CLN8 gene. Our patients developed a mild phenotype of CLN8 disease: as they presented mild epilepsy, cognitive decline, mild learning disability, attention-deficit/hyperactivity disorder (ADHD), they developed a markedly protracted course of motor decline. Bioinformatic analyses of the compound heterozygous CLN8 gene variants were carried out. Most of the variants seem likely to act by compromising the structural integrity of regions within the protein. This in turn is expected to reduce the overall stability of the protein and render the protein less active to various degrees. The cases in our study confirmed and expanded the effect of compound heterozygous variants in CLN8 disease.

The endo-lysosomal system (ELS) comprises a set of membranous organelles responsible for transporting intracellular and extracellular components within cells. Defects in lysosomal proteins usually affect a large variety of processes and underlie many diseases, most of them with a strong neuronal impact. Mutations in the endoplasmic reticulum-resident CLN8 protein cause CLN8 disease. This condition is one of the 14 known neuronal ceroid lipofuscinoses (NCLs), a group of inherited diseases characterised by accumulation of lipofuscin-like pigments within lysosomes. Besides mediating the transport of soluble lysosomal proteins, recent research suggested a role for CLN8 in the transport of vesicles and lipids, and autophagy. However, the consequences of CLN8 deficiency on ELS structure and activity, as well as the potential impact on neuronal development, remain poorly characterised. Therefore, we performed CLN8 knockdown in neuronal and non-neuronal cell models to analyse structural, dynamic and functional changes in the ELS and to assess the impact of CLN8 deficiency on axodendritic development. CLN8 knockdown increased the size of the Golgi apparatus, the number of mobile vesicles and the speed of endo-lysosomes. Using the fluorescent fusion protein mApple-LAMP1-pHluorin, we detected significant lysosomal alkalisation in CLN8-deficient cells. In turn, experiments in primary rat hippocampal neurons showed that CLN8 deficiency decreased the complexity and size of the somatodendritic compartment. Our results suggest the participation of CLN8 in vesicular distribution, lysosomal pH and normal development of the dendritic tree. We speculate that the defects triggered by CLN8 deficiency on ELS structure and dynamics underlie morphological alterations in neurons, which ultimately lead to the characteristic neurodegeneration observed in this NCL. This is, to our knowledge, the first characterisation of the effects of CLN8 dysfunction on the structure and dynamics of the ELS. Moreover, our findings suggest a novel role for CLN8 in somatodendritic development, which may account at least in part for the neuropathological manifestations associated with CLN8 disease.