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Background: Batten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common pathogenic variant shared by most patients is the "1-kb" deletion which removes two internal coding exons (7 and 8) in CLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the 1-kb deletion: the 'major' and 'minor' transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigate CLN3 transcription in "healthy" samples without juvenile CLN3 disease. Methods: We leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range of CLN3 transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources. Results: We found that a readthrough gene affects the quantification and annotation of CLN3. After taking this into account, we detected over 100 novel CLN3 transcripts, with no dominantly expressed CLN3 transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated 'major' transcripts are detected. Together, they have median usage of 1.5% across 22 samples. Furthermore, we identified 48 CLN3 ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third of CLN3 transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data. Conclusion: Overall, these findings provide valuable insights into the complexity of CLN3 transcription, highlighting the importance of studying both canonical and non-canonical CLN3 protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) of CLN3. This knowledge is essential for investigating the impact of the 1-kb deletion and rare pathogenic variants on CLN3 transcription and disease pathogenesis. [ABSTRACT FROM AUTHOR]

Childhood dementias are a group of rare, fatal neurodegenerative disorders, characterised by global cognitive decline, loss of previously acquired developmental skills and behaviours and psychological symptoms of dementia (BPSD). Batten disease, or neuronal ceroid lipofuscinosis, and Sanfilippo syndrome, or mucopolysaccharidosis type III, are two of the more common forms of childhood dementia disorders worldwide. While psychosocial interventions are the best available therapeutic approach for BPSD management in adult-onset dementia, there is very limited literature or clinical experience in the context of childhood dementia. To address this gap, we conducted a descriptive case analysis of BPSD profiles, associated contributing factors and targeted psychosocial interventions in two cases with childhood dementia disorders (Sanfilippo syndrome and CLN3 (juvenile onset) Batten disease) who were referred to Dementia Support Australia, a national dementia behaviour support service in Australia. Primary BPSD identified in these disorders included physical and verbal aggression and irritability/lability. In these cases, contributing factors to the development of BPSD were not monolithic, encompassing pain, caregiver's approach and over or under-stimulation. Improvement in BPSD were observed using the Neuropsychiatric Inventory-Quesionnaire and globally noted as per the qualitative feedback reported by family and caregivers. Person-centred, multimodal psychosocial interventions were recognised as effective therapies in resolving BPSD in these cases. In conclusion, the case studies described the nature and presentation of BPSD in two common forms of childhood dementia and demonstrated the potential benefits of person-centred psychosocial interventions (delivered through national dementia-specific support programs) in alleviating BPSD such as irritability and aggression in these disorders. [ABSTRACT FROM AUTHOR]

Abstract Background Batten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common pathogenic variant shared by most patients is the “1-kb” deletion which removes two internal coding exons (7 and 8) in CLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the 1-kb deletion: the ‘major’ and ‘minor’ transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigate CLN3 transcription in “healthy” samples without juvenile CLN3 disease. Methods We leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range of CLN3 transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources. Results We found that a readthrough gene affects the quantification and annotation of CLN3. After taking this into account, we detected over 100 novel CLN3 transcripts, with no dominantly expressed CLN3 transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated ‘major’ transcripts are detected. Together, they have median usage of 1.5% across 22 samples. Furthermore, we identified 48 CLN3 ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third of CLN3 transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data. Conclusion Overall, these findings provide valuable insights into the complexity of CLN3 transcription, highlighting the importance of studying both canonical and non-canonical CLN3 protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) of CLN3. This knowledge is essential for investigating the impact of the 1-kb deletion and rare pathogenic variants on CLN3 transcription and disease pathogenesis.

Neuronal ceroid lipofuscinoses (NCL) are among the most prevalent neurodegenerative disorders of early life in humans. Disease‐causing variants have been described for 13 different NCL genes. In this study, a refined pathological characterization of a female rabbit with progressive neurological signs reminiscent of NCL was performed. Cytoplasmic pigment present in neurons was weakly positive with Sudan black B and autofluorescent. Immunohistology revealed astrogliosis, microgliosis and axonal degeneration. During the subsequent genetic investigation, the genome of the affected rabbit was sequenced and examined for private variants in NCL candidate genes. The analysis revealed a homozygous ~10.7 kb genomic duplication on chromosome 15 comprising parts of the MFSD8 gene, NC_013683.1:g.103,727,963_103,738,667dup. The duplication harbors two internal protein coding exons and is predicted to introduce a premature stop codon into the transcript, truncating ~50% of the wild‐type MFSD8 open reading frame encoding the major facilitator superfamily domain containing protein 8, XP_002717309.2:p.(Glu235Leufs*23). Biallelic loss‐of‐function variants in MFSD8 have been described to cause NCL7 in human patients, dogs and a single cat. The available clinical and pathological data, together with current knowledge about MFSD8 variants and their functional impact in other species, point to the MFSD8 duplication as a likely causative defect for the observed phenotype in the affected rabbit. [ABSTRACT FROM AUTHOR]

Abstract Background CLN3 disease (also known as CLN3 Batten disease or Juvenile Neuronal Ceroid Lipofuscinosis) is a rare pediatric neurodegenerative disorder caused by biallelic mutations in CLN3. While extensive efforts have been undertaken to understand CLN3 disease etiology, pathology, and clinical progression, little is known about the impact of CLN3 disease on parents and caregivers. Here, we investigated CLN3 disease progression, clinical care, and family experiences using semi-structured interviews with 39 parents of individuals with CLN3 disease. Analysis included response categorization by independent observers and quantitative methods. Results Parents reported patterns of disease progression that aligned with previous reports. Insomnia and thought- and mood-related concerns were reported frequently. “Decline in visual acuity” was the first sign/symptom noticed by n = 28 parents (70%). A minority of parents reported “behavioral issues” (n = 5, 12.5%), “communication issues” (n = 3, 7.5%), “cognitive decline” (n = 1, 2.5%), or “seizures” (n = 1, 2.5%) as the first sign/symptom. The mean time from the first signs or symptoms to a diagnosis of CLN3 disease was 2.8 years (SD = 4.1). Misdiagnosis was common, being reported by n = 24 participants (55.8%). Diagnostic tests and treatments were closely aligned with observed symptoms. Desires for improved or stabilized vision (top therapeutic treatment concern for n = 14, 32.6%), cognition (n = 8, 18.6%), and mobility (n = 3, 7%) dominated parental concerns and wishes for therapeutic correction. Family impacts were common, with n = 34 (81%) of respondents reporting a financial impact on the family and n = 20 (46.5%) reporting marital strain related to the disease. Conclusions Collectively, responses demonstrated clear patterns of disease progression, a strong desire for therapies to treat symptoms related to vision and cognition, and a powerful family impact driven by the unrelenting nature of disease progression.

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. [ABSTRACT FROM AUTHOR]

Background CLN3 disease (also known as CLN3 Batten disease or Juvenile Neuronal Ceroid Lipofuscinosis) is a rare pediatric neurodegenerative disorder caused by biallelic mutations in CLN3. While extensive efforts have been undertaken to understand CLN3 disease etiology, pathology, and clinical progression, little is known about the impact of CLN3 disease on parents and caregivers. Here, we investigated CLN3 disease progression, clinical care, and family experiences using semi-structured interviews with 39 parents of individuals with CLN3 disease. Analysis included response categorization by independent observers and quantitative methods. Results Parents reported patterns of disease progression that aligned with previous reports. Insomnia and thought-and mood-related concerns were reported frequently. “Decline in visual acuity” was the first sign/symptom noticed by n=28 parents (70%). A minority of parents reported “behavioral issues” (n=5, 12.5%), “communication issues” (n=3, 7.5%), “cognitive decline” (n=1, 2.5%), or “seizures” (n=1, 2.5%) as the first sign/symptom. The mean time from the first signs or symptoms to a diagnosis of CLN3 disease was 2.8 years (SD=4.1). Misdiagnosis was common, being reported by n=24 participants (55.8%). Diagnostic tests and treatments were closely aligned with observed symptoms. Desires for improved or stabilized vision (top therapeutic treatment concern for n=14, 32.6%), cognition (n=8, 18.6%), and mobility (n=3, 7%) dominated parental concerns and wishes for therapeutic correction. Family impacts were common, with n=34 (81%) of respondents reporting a financial impact on the family and n=20 (46.5%) reporting marital strain related to the disease. Conclusions Collectively, responses demonstrated clear patterns of disease progression, a strong desire for therapies to treat symptoms related to vision and cognition, and a powerful family impact driven by the unrelenting nature of disease progression. [ABSTRACT FROM AUTHOR]

Batten disease is a rare, progressive neurogenetic disorder composed of 13 genotypes that often presents in childhood. Children present with seizures, vision loss, and developmental regression. Neurorehabilitation services (i.e., physical therapy, occupational therapy, and speech-language therapy) can help improve the quality of life for children and their families. Owing to the rarity of Batten disease, there are no standardized clinical recommendations or outcome assessments. To describe developmental profiles, current dose of neurorehabilitation, and outcome assessments used clinically for children diagnosed with Batten disease. Electronic medical records of 70 children with Batten disease (subtypes n = 5 CLN1; n = 25 CLN2; n = 23 CLN3; n = 17 CLN6) were reviewed (7.0 ± 3.4 years). Descriptive statistics were used to describe clinical features, developmental skills, dose of neurorehabilitation, and outcome assessment use. Across CLN subtypes, most children experienced vision impairments (61%) and seizures (68%). Most children demonstrated delays in fine motor (65%), gross motor (80%), cognitive (63%), and language skills (83%). The most common frequency of neurorehabilitation was weekly (42% to 43%). Two standardized outcome assessments were used to track developmental outcomes: Peabody Developmental Motor Scales, second edition (30% of children completed this assessment) and Preschool Language Scales, fifth edition (27.4% of children completed this assessment). Neurorehabilitation professionals should understand the clinical features and prognosis for children with Batten disease. The child's clinical features and family preferences should guide the rehabilitation plan of care. Future work needs to be completed to define dosing parameters and validate outcome assessments for neurorehabilitation services. [ABSTRACT FROM AUTHOR]

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal‐lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP‐3 complex mu subunit, the lysosomal‐trafficking regulator LYST, mucopilin‐1, and the Wiskott–Aldrich syndrome‐associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation‐independent mannose‐6‐phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans. [ABSTRACT FROM AUTHOR]

Introduction: Late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), is a neurodegenerative autosomal recessive disease caused by TPP1 gene variants, with a spectrum of classic and atypical phenotypes. The aim of treatment is to slow functional decline as early as possible in an attempt to improve quality of life and survival. This study describes the clinical characteristics as well as the response to treatment with cerliponase alfa. Materials and methods: A retrospective study was conducted in five Latin-American countries, using clinical records from patients with CLN2. Clinical follow-up and treatment variables are described. A descriptive and bivariate statistical analysis was performed. Results: A total of 36 patients were observed (range of follow-up of 61–110 weeks post-treatment). At presentation, patients with the classic phenotype (n = 16) exhibited regression in language (90%), while seizures were the predominant symptom (87%) in patients with the atypical phenotype (n = 20). Median age of symptom onset and time to first specialized consultation was 3 (classical) and 7 (atypical) years, while the median time interval between onset of symptoms and treatment initiation was 4 years (classical) and 7.5 (atypical). The most frequent variant was c.827 A > T in 17/72 alleles, followed by c.622C > T in 6/72 alleles. All patients were treated with cerliponase alfa, and either remained functionally stable or had a loss of 1 point on the CLN2 scale, or up to 2 points on the Wells Cornel and Hamburg scales, when compared to pretreatment values. Discussion and conclusion: This study reports the largest number of patients with CLN2 currently on treatment with cerliponase alfa in the world. Data show a higher frequency of patients with atypical phenotypes and a high allelic proportion of intron variants in our region. There was evidence of long intervals until first specialized consultation, diagnosis, and enzyme replacement therapy. Follow-up after the initiation of cerliponase alfa showed slower progression or stabilization of the disease, associated with adequate clinical outcomes and stable functional scores. These improvements were consistent in both clinical phenotypes.

Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In humans, homozygous mutations in MFSD8 cause a late-infantile form of neuronal ceroid lipofuscinosis (NCL) called CLN7 disease. In the social amoeba Dictyostelium discoideum, Mfsd8 localizes to cytoplasmic puncta and vesicles, and regulates conserved processes during the organism’s life cycle. Here, we used D. discoideum to examine the effect of mfsd8-deficiency on the secretome during the early stages of multicellular development. Mass spectrometry revealed 61 proteins that were differentially released by cells after 4 and 8 h of starvation. Most proteins were present in increased amounts in mfsd8- conditioned buffer compared to WT indicating that loss of mfsd8 deregulates protein secretion and/or causes the release of proteins not normally secreted by WT cells. GO term enrichment analyses showed that many of the proteins aberrantly released by mfsd8- cells localize to compartments and regions of the cell associated with the endo-lysosomal and secretory pathways. Mass spectrometry also revealed proteins previously known to be impacted by the loss of mfsd8 (e.g., cathepsin D), as well as proteins that may underlie mfsd8-deficiency phenotypes during aggregation. Finally, we show that mfsd8-deficiency reduces intracellular proteasome 20S activity due to the abnormal release of at least one proteasomal subunit. Together, this study reveals the impact of mfsd8 loss on the secretome during D. discoideum aggregation and lays the foundation for follow up work that investigates the role of altered protein release in CLN7 disease.

Ceroid lipofuscinosis neuronal (CLN) genes encode 13 proteins that localize throughout the endomembrane system to regulate a variety of cellular processes. In humans, mutations in CLN genes cause a devastating form of neurodegeneration called neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease. Each CLN gene is associated with a specific subtype of the disease that differ from each other in severity and age of onset. The NCLs affect all ages and ethnicities worldwide but primarily affect children. The pathology underlying the NCLs is poorly understood, which has prevented the development of a cure or effective therapy for most subtypes of the disease. A growing body of literature supports the networking of CLN genes and proteins within cells, which aligns with the broadly similar cellular and clinical manifestations among the different subtypes of NCL. Here, all relevant literature is reviewed to provide a comprehensive overview of our current understanding of how CLN genes and proteins are networked in mammalian cells with an aim toward revealing new molecular targets for therapy development. Intriguingly, CLN gene and protein networking extends beyond the NCLs as recent work has linked several CLN genes and proteins to other forms of neurodegeneration such as Alzheimer's disease and Parkinson's disease. Thus, a deeper understanding of the pathways and cellular processes impacted by mutations in CLN genes will not only strengthen our knowledge of the pathological mechanisms underlying the NCLs but may also provide new insight into related forms of neurodegeneration. [ABSTRACT FROM AUTHOR]

Over the recent decades, the use of extracellular vesicles (EVs) has attracted considerable attention. Herein, we report the development of a novel EV-based drug delivery system for the transport of the lysosomal enzyme tripeptidyl peptidase-1 (TPP1) to treat Batten disease (BD). Endogenous loading of macrophage-derived EVs was achieved through transfection of parent cells with TPP1-encoding pDNA. More than 20% ID/g was detected in the brain following a single intrathecal injection of EVs in a mouse model of BD, ceroid lipofuscinosis neuronal type 2 (CLN2) mice. Furthermore, the cumulative effect of EVs repetitive administrations in the brain was demonstrated. TPP1-loaded EVs (EV-TPP1) produced potent therapeutic effects, resulting in efficient elimination of lipofuscin aggregates in lysosomes, decreased inflammation, and improved neuronal survival in CLN2 mice. In terms of mechanism, EV-TPP1 treatments caused significant activation of the autophagy pathway, including altered expression of the autophagy-related proteins LC3 and P62, in the CLN2 mouse brain. We hypothesized that along with TPP1 delivery to the brain, EV-based formulations can enhance host cellular homeostasis, causing degradation of lipofuscin aggregates through the autophagy–lysosomal pathway. Overall, continued research into new and effective therapies for BD is crucial for improving the lives of those affected by this condition. [ABSTRACT FROM AUTHOR]

Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.

Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life. Here, we summarize emerging links between the most common late life neurodegenerative disease, Alzheimer’s disease, and the most common early life neurodegenerative diseases, neuronal ceroid lipofuscinoses. Genetic studies reported an overlap of clinically diagnosed Alzheimer’s disease and mutations in genes known to cause neuronal ceroid lipofuscinoses. Accumulating data strongly suggest dysfunction of intracellular trafficking mechanisms and the autophagy-endolysosome system in both types of neurodegenerative disorders. This suggests shared cytopathological processes underlying these different types of neurodegenerative diseases. A better understanding of the common mechanisms underlying the different diseases is important as this might lead to the identification of novel targets for therapeutic concepts, the transfer of therapeutic strategies from one disease to the other and therapeutic approaches tailored to patients with specific mutations. Here, we review dysfunctions of the endolysosomal autophagy pathway in Alzheimer’s disease and neuronal ceroid lipofuscinoses and summarize emerging etiologic and genetic overlaps.

Abstract Background 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 Cln8 mnd mouse model to measure the impact and progression of histopathological and behavioral outcomes between sexes. Results Several notable sex differences were observed in the presentation of brain pathology, including Cln8 mnd 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 Cln8 mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8 mnd female mice experience poorer motor performance and earlier death than their male counterparts. Cln8 mnd 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. Conclusions 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 neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of fatal neurodegenerative disorders that primarily affect children. The etiology of Batten disease is linked to mutations in 13 genes that encode distinct CLN proteins, whose functions have yet to be fully elucidated. The social amoeba Dictyostelium discoideum has been adopted as an efficient and powerful model system for studying the diverse cellular roles of CLN proteins. The genome of D. discoideum encodes several homologs of human CLN proteins, and a growing body of literature supports the conserved roles and networking of CLN proteins in D. discoideum and humans. In humans, CLN proteins have diverse cellular roles related to autophagy, signal transduction, lipid homeostasis, lysosomal ion homeostasis, and intracellular trafficking. Recent work also indicates that CLN proteins play an important role in protein secretion. Remarkably, many of these findings have found parallels in studies with D. discoideum. Accordingly, this review will highlight the translatable value of novel work with D. discoideum in the field of NCL research and propose further avenues of research using this biomedical model organism for studying the NCLs.

Pathogenic variation in MAPT, GRN, and C9ORF72 accounts for at most only half of frontotemporal lobar degeneration (FTLD) cases with a family history of neurological disease. This suggests additional variants and genes that remain to be identified as risk factors for FTLD. We conducted a case-control genetic association study comparing pathologically diagnosed FTLD patients (n = 94) to cognitively normal older adults (n = 3541), and found suggestive evidence that gene-wide aggregate rare variant burden in MFSD8 is associated with FTLD risk. Because homozygous mutations in MFSD8 cause neuronal ceroid lipofuscinosis (NCL), similar to homozygous mutations in GRN, we assessed rare variants in MFSD8 for relevance to FTLD through experimental follow-up studies. Using post-mortem tissue from middle frontal gyrus of patients with FTLD and controls, we identified increased MFSD8 protein levels in MFSD8 rare variant carriers relative to non-variant carrier patients with sporadic FTLD and healthy controls. We also observed an increase in lysosomal and autophagy-related proteins in MFSD8 rare variant carrier and sporadic FTLD patients relative to controls. Immunohistochemical analysis revealed that MFSD8 was expressed in neurons and astrocytes across subjects, without clear evidence of abnormal localization in patients. Finally, in vitro studies identified marked disruption of lysosomal function in cells from MFSD8 rare variant carriers, and identified one rare variant that significantly increased the cell surface levels of MFSD8. Considering the growing evidence for altered autophagy in the pathogenesis of neurodegenerative disorders, our findings support a role of NCL genes in FTLD risk and suggest that MFSD8-associated lysosomal dysfunction may contribute to FTLD pathology.

Ceroid lipofuscinosis type 3 (CLN3) is an autosomal recessive, neurodegenerative metabolic disease. Typical clinical symptoms include progressive visual loss, epilepsy of unknown etiology and dementia. Presence of lipofuscin deposits with typical pattern of 'fingerprints' and vacuolized lymphocytes suggest the diagnosis of CLN3. Cause of CLN3 are mutations in the CLN3 gene, among which the most frequently found is the large deletion 1.02 kb spreading on exons 7 and 8. We present 4 patients from 2 families, in whom the deterioration of visual quality and acuity was observed as first clinical sign, when they were a few years old and it was successively accompanied by symptoms of neurologic deterioration (like generalized convulsions with consciousness impairment). In all patients the 1.02 kb deletion in the CLN3 gene was detected in homo- or heterozygosity with other CLN3 pathogenic variant. Ultrastructural studies revealed abnormal structures corresponding to 'fingerprint' profiles (FPPs) in conjunctival endothelial cells. It should be emphasized that in patients with blindness of unknown cause the diagnosis of ceroid lipofuscinosis should be considered and in older children—especially CLN3. The facility of the analysis for the presence of 1.02 kb deletion and economic costs are a solid argument for intensive use of this test in the diagnostic procedure of CLN3. [ABSTRACT FROM AUTHOR]

Neuronal ceroid lipofuscinosis type 2 (CLN2) disease is a very rare neurodegenerative lysosomal storage disorder. Progression is rapid and irreversible, making early diagnosis crucial for timely treatment. A group of pediatric neurologists and neuroradiologists with expertise in CLN2 convened to discuss early electroencephalogram (EEG) and magnetic resonance imaging (MRI) findings in CLN2 diagnosis. Of 18 CLN2 cases, 16 (88.9%) had background slowing and 16 (88.9%) had epileptiform discharges on initial EEG. Seven of 17 (41.2%) patients who received intermittent low-frequency photic stimulation had a photoparoxysmal response. Initial MRIs showed subtle cerebellar (n = 14, 77.8%) or cerebral (n = 9, 50.0%) atrophy, white matter abnormalities (n = 11, 61.1%), and basal ganglia T2 hypointensity (n = 6, 33.3%), which became more apparent on follow-up MRI. The recognition of even subtle cerebellar atrophy and white matter signal changes in children aged 2-5 years who present with language delay, new-onset seizures, and an EEG with epileptiform discharges and background slowing should prompt investigation for CLN2. Because these early signs are not unique to CLN2, genetic testing is essential early in the diagnostic journey. [ABSTRACT FROM AUTHOR]

Abstract Background Ceroid lipofuscinoses neuronal 6 (CLN6) disease belongs to the neuronal ceroid lipofuscinoses (NCLs), complex and genetically heterogeneous disorders with wide geographical and phenotypic variation. The first clinical signs usually appear between 18 months and 8 years, but examples of later-onset have also been reported. Common manifestations include ataxia, seizures, vision impairment, and developmental regression. Because these are shared by other neurological diseases, identification of CLN6 genetic variants is imperative for early diagnosis. Results We present one of the largest cohorts to date of genetically diagnosed CLN6 patients screened at a single center. In total 97 subjects, originating from 20 countries were screened between 2010 and 2020. They comprised 86 late-infantile, eight juvenile, and three adult-onset cases (two patients with Kufs disease type A, and one with teenage progressive myoclonic epilepsy). The male to female ratio was 1.06: 1.00. The age at referral was between six months and 33 years. The time from disease onset to referral ranged from less than 1 month to 8.3 years. The clinical phenotype consisted of a combination of symptoms, as reported before. We characterized a total of 45 distinct variants defining 45 distinct genotypes. Twenty-four were novel variants, some with distinct geographic associations. Remarkably, c.257A > G (p.H86R) was present in five out of 23 unrelated Egyptian individuals but in no patients from other countries. The most common genotype was homozygosity for the c.794_796del in-frame deletion. It was present in about one-third of CLN6 patients (28 unrelated cases, and 2 familial cases), all with late-infantile onset. Variants with a high likelihood of causing loss of CLN6 function were found in 21% of cases and made up 33% of all distinct variants. Forty-four percent of variants were classified as pathogenic or likely pathogenic. Conclusions Our study significantly expands the number of published clinical cases and the mutational spectrum of disease-associated CLN6 variants, especially for the Middle Eastern and North African regions. We confirm previous observations regarding the most prevalent symptoms and recommend including CLN6 in the genetic diagnosis of patients presenting with early-onset abnormalities of the nervous system, musculoskeletal system, and eye.

Infantile neuronal ceroid lipofuscinosis (CLN1 Batten Disease) is a devastating pediatric lysosomal storage disease caused by pathogenic variants in the CLN1 gene, which encodes the depalmitoylation enzyme, palmitoyl-protein thioesterase 1 (PPT1). CLN1 patients present with visual deterioration, psychomotor dysfunction, and recurrent seizures until neurodegeneration results in death, typically before fifteen years of age. Histopathological features of CLN1 include aggregation of lysosomal autofluorescent storage material (AFSM), as well as profound gliosis. The current management of CLN1 is relegated to palliative care. Here, we examine the therapeutic potential of a small molecule PPT1 mimetic, N-tert-butyl hydroxylamine (NtBuHA), in a Cln1 −/− mouse model. Treatment with NtBuHA reduced AFSM accumulation both in vitro and in vivo. Importantly, NtBuHA treatment in Cln1 −/− mice reduced neuroinflammation, mitigated epileptic episodes, and normalized motor function. Live cell imaging of Cln1 −/− primary cortical neurons treated with NtBuHA partially rescued aberrant synaptic calcium dynamics, suggesting a potential mechanism contributing to the therapeutic effects of NtBuHA in vivo. Taken together, our findings provide supporting evidence for NtBuHA as a potential treatment for CLN1 Batten Disease. [ABSTRACT FROM AUTHOR]

Abstract Background Commonly known as Batten disease, the neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of rare pediatric lysosomal storage disorders characterized by the intracellular accumulation of autofluorescent material (known as lipofuscin), progressive neurodegeneration, and neurological symptoms. In 2002, a disease-causing NCL mutation in the CLN6 gene was identified (c.214G > T) in the Costa Rican population, but the frequency of this mutation among local Batten disease patients remains incompletely characterized, as do clinical and demographic attributes for this rare patient population. Objective To describe the main sociodemographic and clinical characteristics of patients with a clinical diagnosis for Batten Disease treated at the National Children's Hospital in Costa Rica and to characterize via molecular testing their causative mutations. Methods DNA extracted from buccal swabs was used for CLN6 gene sequencing. Participants’ sociodemographic and clinical characteristics were also obtained from their medical records. Results Nine patients with a clinical diagnosis of Batten disease were identified. Genetic sequencing determined the presence of the previously described Costa Rican homozygous mutation in 8 of 9 cases. One patient did not have mutations in the CLN6 gene. In all cases where the Costa Rican CLN6 mutation was present, it was accompanied by a substitution in intron 2. Patients were born in 4 of the 7 Costa Rican provinces, with an average onset of symptoms close to 4 years of age. No parental consanguinity was present in pedigrees. Initial clinical manifestations varied between patients but generally included: gait disturbances, language problems, visual impairment, seizures and psychomotor regression. Cortical and cerebellar atrophy was a constant finding when neuroimaging was performed. Seizure medication was a common element of treatment regimens. Conclusions This investigation supports that the previously characterized c.214G > T mutation is the most common causative NCL mutation in the Costa Rican population. This mutation is geographically widespread among Costa Rican NCL patients and yields a clinical presentation similar to that observed for CLN6 NCL patients in other geographies.

Abstract Introduction: Neuronal ceroid lipofuscinosis (NCLs) is an autosomal recessive neurodegenerative disorders group. We report the first case in Colombia involving a new genetically confirmed variant of a homozygous CLN6 mutation. Case report: 12-year-old male, history of blood parents and average growth until 5 years of age. At this age began focal crises, progressive regression of neurodevelopment, severe cognitive deficit, and swallowing disorder that led to gastrostomy. Clinical exome + CNVs + mitochondrial DNA genetic study identified variant NM_017882.3 (CLN6): c. 22C>T, p. (Gln8*) in homozygous, deleterious. Late-onset infantile neuronal ceroid lipofuscinosis was diagnosed. Discussion: Mutations in the CLN6 gene are associated with late-onset infantile lipofuscinosis of autosomal recessive inheritance. This variant has not been previously described in the medical literature nor is it listed in the population databases, which indicates that it is extremely rare. The treatment focuses on the control of seizures, sleep disturbances, extrapyramidal symptoms, behavioral disorders, anxiety, and psychosis. Conclusion: To date, this variant of the CLN6 gene has not been reported in the world. There are currently no etiological or disease-specific therapeutic approaches. The use of exome/whole genome sequencing can be very useful for etiological diagnosis and differential diagnosis. An early diagnosis opens the door to future care and treatment.

ObjectiveSeveral small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function.MethodsNovel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples.ResultsPatients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology.InterpretationBiallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.

Background: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of neurodegenerative lysosomal storage diseases that affect all ages and ethnicities globally. Previous research showed that CLN5 participates in a variety of cellular processes. However, the precise function of CLN5 in the cell and the pathway(s) regulating its function are not well understood. In the model organism Dictyostelium discoideum, loss of the CLN5 homolog, cln5, impacts various cellular and developmental processes including cell proliferation, cytokinesis, aggregation, cell adhesion, and terminal differentiation. In this study, we used comparative transcriptomics to identify differentially expressed genes underlying cln5-deficiency phenotypes during growth and the early stages of multicellular development. During growth, genes associated with protein ubiquitination/deubiquitination, cell cycle progression, and proteasomal degradation were affected, while genes linked to protein and carbohydrate catabolism were affected during early development. We followed up this analysis by showing that loss of cln5 alters the intracellular and extracellular amounts of proliferation repressors during growth and increases the extracellular amount of conditioned medium factor, which regulates cAMP signalling during the early stages of development. Additionally, cln5- cells displayed increased intracellular and extracellular amounts of discoidin, which is involved in cell-substrate adhesion and migration. Previous work in mammalian models reported altered lysosomal enzyme activity due to mutation or loss of CLN5. Here, we detected altered intracellular activities of various carbohydrate enzymes and cathepsins during cln5- growth and starvation. Notably, cln5- cells displayed reduced ß-hexosaminidase activity, which aligns with previous work showing that D. discoideum Cln5 and human CLN5 can cleave the substrate acted upon by ß-hexosaminidase. Finally, consistent with the differential expression of genes associated with proteasomal degradation in cln5- cells, we also observed elevated amounts of a proteasome subunit and reduced proteasome 20S activity during cln5- growth and starvation. Overall, this study reveals the impact of cln5-deficiency on gene expression in D. discoideum, provides insight on the genes and proteins that play a role in regulating Cln5-dependent processes, and sheds light on the molecular mechanisms underlying CLN5 disease. [ABSTRACT FROM AUTHOR]

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions.

Background: CLN3 disease is a neurodegenerative condition presenting in the first decade of life typically leading to death in the third decade. The earliest symptom is rapidly progressive visual impairment followed by intellectual and motor impairments, epilepsy and behavioural disturbances. There are limited data on how the condition affects the family system or the role of family resilience in paediatric neurodegenerative diseases. Methods: Semi‐structured interviews were conducted with eight parents (five mothers and three fathers) of five children with CLN3. Interview questions focused on the experience of having a child with CLN3, its impact on the family system as well as the concept of family resilience. Data were analysed via thematic analysis. Results: The thematic analysis resulted in four main themes. The theme 'recurring losses' included the feeling of losing a healthy child, the child's loss of abilities and loss of relationships. The theme 'disruption to the family system' included that siblings could be 'side‐lined', the potential negative impact on romantic relationships and difficulties finding time to oneself. The theme 'Society is not developed for a progressive disease' highlighted the difficulties parents faced with respect to contacts with the health and/or social insurance system. The paediatric health care system was seen as supportive, but the adult health care system was not seen as fit for the purpose. Regarding family resilience, parents felt that the disease forced them to reconsider what was important in life. Several parents described that they learned to value small moments of joy and create deep connections through involvement in family routines and rituals. Conclusions: CLN3 places a very significant burden on the family system including parental feelings of loss, impact on family relationships and lack of understanding within the health/social insurance systems. The concept of family resilience may be useful in understanding the experiences of families affected by paediatric neurodegenerative conditions. [ABSTRACT FROM AUTHOR]

Ceroid lipofuscinosis neuronal (CLN) encompasses rare inherited neurodegenerative disorders that present in childhood with clinical features including epilepsy, psychomotor delay, progressive vision loss, and premature death. Published experience utilizing umbilical cord blood transplant (UCBT) for these disorders is limited. This retrospective analysis includes patients with CLN (2, 3, and 5) who underwent UCBT from 2012 to 2020. All subjects (n = 8) received standard-of-care myeloablative conditioning. Four also enrolled in clinical trial NCT02254863 and received intrathecal DUOC-01 cells posttransplant. Median age at UCBT was 5.9 years. All subjects achieved neutrophil engraftment with >95% donor chimerism at a median of 28.5 days. Sinusoidal obstructive syndrome was not observed. Severe acute graft-versus-host disease occurred in 12.5%. Other complications included autoimmune hemolytic anemia (25%) and viral reactivation/infection (62.5%). No transplant-related mortality was observed. Two CLN2 patients died, 1 from progressive disease and 1 from unknown cause at days +362 and +937, respectively. With median follow-up of 8 years, overall survival at 100 days and 24 months was 100% and 88%, respectively. Three of 4 CLN3 subjects stabilized Hamburg motor and language scores. While UCBT appears safe and feasible in these patients, given the variable expression and natural history, extended follow-up and further studies are needed to elucidate the potential impact of UCBT on clinical outcomes., (© The Author(s) 2024. Published by Oxford University Press.)

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of neurodegenerative lysosomal storage diseases that affect all ages and ethnicities globally. Previous research showed that CLN5 participates in a variety of cellular processes. However, the precise function of CLN5 in the cell and the pathway(s) regulating its function are not well understood. In the model organism Dictyostelium discoideum, loss of the CLN5 homolog, cln5, impacts various cellular and developmental processes including cell proliferation, cytokinesis, aggregation, cell adhesion, and terminal differentiation. In this study, we used comparative transcriptomics to identify differentially expressed genes underlying cln5-deficiency phenotypes during growth and the early stages of multicellular development. During growth, genes associated with protein ubiquitination/deubiquitination, cell cycle progression, and proteasomal degradation were affected, while genes linked to protein and carbohydrate catabolism were affected during early development. We followed up this analysis by showing that loss of cln5 alters the intracellular and extracellular amounts of proliferation repressors during growth and increases the extracellular amount of conditioned medium factor, which regulates cAMP signalling during the early stages of development. Additionally, cln5- cells displayed increased intracellular and extracellular amounts of discoidin, which is involved in cell-substrate adhesion and migration. Previous work in mammalian models reported altered lysosomal enzyme activity due to mutation or loss of CLN5. Here, we detected altered intracellular activities of various carbohydrate enzymes and cathepsins during cln5- growth and starvation. Notably, cln5- cells displayed reduced β-hexosaminidase activity, which aligns with previous work showing that D. discoideum Cln5 and human CLN5 can cleave the substrate acted upon by β-hexosaminidase. Finally, consistent with the differential expression of genes associated with proteasomal degradation in cln5- cells, we also observed elevated amounts of a proteasome subunit and reduced proteasome 20S activity during cln5- growth and starvation. Overall, this study reveals the impact of cln5-deficiency on gene expression in D. discoideum, provides insight on the genes and proteins that play a role in regulating Cln5-dependent processes, and sheds light on the molecular mechanisms underlying CLN5 disease.

Purpose: To report a case of bilateral chorioretinal scarring due to CLN3 heterozygous deletion in an asymptomatic patient. Observations: A 63 year-old patient with a history of well-controlled diabetes presented as a referral for diabetic retinopathy. He was asymptomatic with 20/20 visual acuity in both eyes. Exam revealed bilateral multifocal chorioretinal scarring left worse than right, sparing the fovea. He was unable to provide a family history due to adoption, and his remaining medical history and review of systems were noncontributory. Inflammatory and infectious workup was negative; however, genetic testing revealed heterozygous deletion of CLN3 exons 8 and 9. His disease has been nonprogressive at all follow-up appointments. Conclusions and importance: Mutations of CLN3 can present with retina-specific findings including bull's-eye maculopathy and electroretinogram (ERG) deficits; to our knowledge this patient's presentation is unique among those with CLN3 mutations.

Background: Ceroid lipofuscinoses neuronal 6 (CLN6) disease belongs to the neuronal ceroid lipofuscinoses (NCLs), complex and genetically heterogeneous disorders with wide geographical and phenotypic variation. The first clinical signs usually appear between 18 months and 8 years, but examples of later-onset have also been reported. Common manifestations include ataxia, seizures, vision impairment, and developmental regression. Because these are shared by other neurological diseases, identification of CLN6 genetic variants is imperative for early diagnosis. Results: We present one of the largest cohorts to date of genetically diagnosed CLN6 patients screened at a single center. In total 97 subjects, originating from 20 countries were screened between 2010 and 2020. They comprised 86 late-infantile, eight juvenile, and three adult-onset cases (two patients with Kufs disease type A, and one with teenage progressive myoclonic epilepsy). The male to female ratio was 1.06: 1.00. The age at referral was between six months and 33 years. The time from disease onset to referral ranged from less than 1 month to 8.3 years. The clinical phenotype consisted of a combination of symptoms, as reported before. We characterized a total of 45 distinct variants defining 45 distinct genotypes. Twenty-four were novel variants, some with distinct geographic associations. Remarkably, c.257A > G (p.H86R) was present in five out of 23 unrelated Egyptian individuals but in no patients from other countries. The most common genotype was homozygosity for the c.794_796del in-frame deletion. It was present in about one-third of CLN6 patients (28 unrelated cases, and 2 familial cases), all with late-infantile onset. Variants with a high likelihood of causing loss of CLN6 function were found in 21% of cases and made up 33% of all distinct variants. Forty-four percent of variants were classified as pathogenic or likely pathogenic. Conclusions: Our study significantly expands the number of published clinical cases and the mutational spectrum of disease-associated CLN6 variants, especially for the Middle Eastern and North African regions. We confirm previous observations regarding the most prevalent symptoms and recommend including CLN6 in the genetic diagnosis of patients presenting with early-onset abnormalities of the nervous system, musculoskeletal system, and eye. [ABSTRACT FROM AUTHOR]

Cardiac magnetic resonance imaging (MRI) is an essential tool for the study of hypertrophic cardiomyopathies (HCM) and for differentiating HCM from conditions with increased ventricular wall thickness, such as cardiac storage diseases. Although cardiac MRI is already used for the diagnosis and characterization of some forms of storage diseases involving the myocardium, it has not yet been used to study myocardial involvement in neuronal ceroid lipofuscinosis (NCL). Here, we describe comprehensive cardiac MRI findings in a patient with the CLN3 form of NCL showing basal inferior interventricular septal hypertrophy with maintained indexed LV mass within reference values and low T1-native values. MRI findings support a finding of abnormal storage material within themyocardiumin CLN3 disease. We recommend the possible routine use of cardiac MRI for early diagnosis of cardiac involvement in CLN3 disease (also termed juvenile NCL) and to monitor the effects of emerging CLN3 therapies on the myocardium as well. [ABSTRACT FROM AUTHOR]

This study assessed the effectiveness of genetic testing in shortening the time to diagnosis of late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease. Individuals who received epilepsy gene panel testing through Behind the Seizure®, a sponsored genetic testing program (Cohort A), were compared to children outside of the sponsored testing program during the same period (Cohort B). Two cohorts were analyzed: children aged ≥24 to ≤60 months with unprovoked seizure onset at ≥24 months between December 2016 and January 2020 (Cohort 1) and children aged 0 to ≤60 months at time of testing with unprovoked seizure onset at any age between February 2019 and January 2020 (Cohort 2). The diagnostic yield in Cohort 1A (n = 1814) was 8.4% (n = 153). The TPP1 diagnostic yield within Cohort 1A was 2.9‐fold higher compared to Cohort 1B (1.0%, n = 18/1814 vs..35%, n = 8/2303; p =.0157). The average time from first symptom to CLN2 disease diagnosis was significantly shorter than previously reported (9.8 vs. 22.7 months, p <.001). These findings indicate that facilitated access to early epilepsy gene panel testing helps to increase diagnostic yield for CLN2 disease and shortens the time to diagnosis, enabling earlier intervention. [ABSTRACT FROM AUTHOR]

Synapses are an early pathological target in many neurodegenerative diseases ranging from well-known adult onset conditions such as Alzheimer and Parkinson disease to neurodegenerative conditions of childhood such as spinal muscular atrophy (SMA) and neuronal ceroid lipofuscinosis (NCLs). However, the reasons why synapses are particularly vulnerable to such a broad range of neurodegeneration inducing stimuli remains unknown. To identify molecular modulators of synaptic stability and degeneration, we have used the Cln3 -/- mouse model of a juvenile form of NCL. We profiled and compared the molecular composition of anatomically-distinct, differentially-affected pre-synaptic populations from the Cln3 -/- mouse brain using proteomics followed by bioinformatic analyses. Identified protein candidates were then tested using a Drosophila CLN3 model to study their ability to modify the CLN3-neurodegenerative phenotype in vivo. We identified differential perturbations in a range of molecular cascades correlating with synaptic vulnerability, including valine catabolism and rho signalling pathways. Genetic and pharmacological targeting of key 'hub' proteins in such pathways was sufficient to modulate phenotypic presentation in a Drosophila CLN3 model. We propose that such a workflow provides a target rich method for the identification of novel disease regulators which could be applicable to the study of other conditions where appropriate models exist.

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.

ObjectiveJuvenile neuronal ceroid lipofuscinosis (JNCL), or juvenile Batten disease, is a pediatric lysosomal storage disease caused by autosomal recessive mutations in CLN3, typified by blindness, seizures, progressive cognitive and motor decline, and premature death. Currently, there is no treatment for JNCL that slows disease progression, which highlights the need to explore novel strategies to extend the survival and quality of life of afflicted children. Cyclic adenosine monophosphate (cAMP) is a second messenger with pleiotropic effects, including regulating neuroinflammation and neuronal survival. Here we investigated whether 3 phosphodiesterase-4 (PDE4) inhibitors (rolipram, roflumilast, and PF-06266047) could mitigate behavioral deficits and cell-specific pathology in the Cln3Δex7/8 mouse model of JNCL.MethodsIn a randomized, blinded study, wild-type (WT) and Cln3Δex7/8 mice received PDE4 inhibitors daily beginning at 1 or 3 months of age and continuing for 6 to 9 months, with motor deficits assessed by accelerating rotarod testing. The effect of PDE4 inhibitors on cAMP levels, astrocyte and microglial activation (glial fibrillary acidic protein and CD68, respectively), lysosomal pathology (lysosomal-associated membrane protein 1), and astrocyte glutamate transporter expression (glutamate/aspartate transporter) were also examined in WT and Cln3Δex7/8 animals.ResultscAMP levels were significantly reduced in the Cln3Δex7/8 brain, and were restored by PF-06266047. PDE4 inhibitors significantly improved motor function in Cln3Δex7/8 mice, attenuated glial activation and lysosomal pathology, and restored glutamate transporter expression to levels observed in WT animals, with no evidence of toxicity as revealed by blood chemistry analysis.InterpretationThese studies reveal neuroprotective effects for PDE4 inhibitors in Cln3Δex7/8 mice and support their therapeutic potential in JNCL patients. Ann Neurol 2016;80:909-923.

Cardiac magnetic resonance imaging (MRI) is an essential tool for the study of hypertrophic cardiomyopathies (HCM) and for differentiating HCM from conditions with increased ventricular wall thickness, such as cardiac storage diseases. Although cardiac MRI is already used for the diagnosis and characterization of some forms of storage diseases involving the myocardium, it has not yet been used to study myocardial involvement in neuronal ceroid lipofuscinosis (NCL). Here, we describe comprehensive cardiac MRI findings in a patient with the CLN3 form of NCL showing basal inferior interventricular septal hypertrophy with maintained indexed LV mass within reference values and low T1-native values. MRI findings support a finding of abnormal storage material within the myocardium in CLN3 disease. We recommend the possible routine use of cardiac MRI for early diagnosis of cardiac involvement in CLN3 disease (also termed juvenile NCL) and to monitor the effects of emerging CLN3 therapies on the myocardium as well.

MFSD8 is a transmembrane protein that has been reported to transport chloride ions across the lysosomal membrane. Mutations in MFSD8 are associated with a subtype of Batten disease called CLN7 disease. Batten disease encompasses a family of 13 inherited neurodegenerative lysosomal storage diseases collectively referred to as the neuronal ceroid lipofuscinoses (NCLs). Previous work identified an ortholog of human MFSD8 in the social amoeba D. discoideum (gene: mfsd8, protein: Mfsd8), reported its localization to endocytic compartments, and demonstrated its involvement in protein secretion. In this study, we further characterized the effects of mfsd8 loss during D. discoideum growth and early stages of multicellular development. During growth, mfsd8− cells displayed increased rates of proliferation, pinocytosis, and expansion on bacterial lawns. Loss of mfsd8 also increased cell size, inhibited cytokinesis, affected the intracellular and extracellular levels of the quorum-sensing protein autocrine proliferation repressor A, and altered lysosomal enzyme activity. During the early stages of development, loss of mfsd8 delayed aggregation, which we determined was at least partly due to impaired cell-substrate adhesion, defects in protein secretion, and alterations in lysosomal enzyme activity. Overall, these results show that Mfsd8 plays an important role in modulating a variety of processes during the growth and early development of D. discoideum.

Batten disease is a family of rare, fatal, neuropediatric diseases presenting with memory/learning decline, blindness, and loss of motor function. Recently, we reported the use of an AAV9-mediated gene therapy that prevents disease progression in a mouse model of CLN6-Batten disease (Cln6nclf), restoring lifespans in treated animals. Despite the success of our viral-mediated gene therapy, the dosing strategy was optimized for delivery to the brain parenchyma and may limit the therapeutic potential to other disease-relevant tissues, such as the eye. Here, we examine whether cerebrospinal fluid (CSF) delivery of scAAV9.CB.CLN6 is sufficient to ameliorate visual deficits in Cln6nclf mice. We show that intracerebroventricular (i.c.v.) delivery of scAAV9.CB.CLN6 completely prevents hallmark Batten disease pathology in the visual processing centers of the brain, preserving neurons of the superior colliculus, thalamus, and cerebral cortex. Importantly, i.c.v.-delivered scAAV9.CB.CLN6 also expresses in many cells throughout the central retina, preserving many photoreceptors typically lost in Cln6nclf mice. Lastly, scAAV9.CB.CLN6 treatment partially preserved visual acuity in Cln6nclf mice as measured by optokinetic response. Taken together, we report the first instance of CSF-delivered viral gene reaching and rescuing pathology in both the brain parenchyma and retinal neurons, thereby partially slowing visual deterioration.

Over the recent decades, the use of extracellular vesicles (EVs) has attracted considerable attention. Herein, we report the development of a novel EV-based drug delivery system for the transport of the lysosomal enzyme tripeptidyl peptidase-1 (TPP1) to treat Batten disease (BD). Endogenous loading of macrophage-derived EVs was achieved through transfection of parent cells with TPP1-encoding pDNA. More than 20% ID/g was detected in the brain following a single intrathecal injection of EVs in a mouse model of BD, ceroid lipofuscinosis neuronal type 2 (CLN2) mice. Furthermore, the cumulative effect of EVs repetitive administrations in the brain was demonstrated. TPP1-loaded EVs (EV-TPP1) produced potent therapeutic effects, resulting in efficient elimination of lipofuscin aggregates in lysosomes, decreased inflammation, and improved neuronal survival in CLN2 mice. In terms of mechanism, EV-TPP1 treatments caused significant activation of the autophagy pathway, including altered expression of the autophagy-related proteins LC3 and P62, in the CLN2 mouse brain. We hypothesized that along with TPP1 delivery to the brain, EV-based formulations can enhance host cellular homeostasis, causing degradation of lipofuscin aggregates through the autophagy–lysosomal pathway. Overall, continued research into new and effective therapies for BD is crucial for improving the lives of those affected by this condition.

Batten disease is unique among lysosomal storage disorders for the early and profound manifestation in the central nervous system, but little is known regarding potential neuron-specific roles for the disease-associated proteins. We demonstrate substantial overlap in the protein interactomes of three transmembrane Batten proteins (CLN3, CLN6, and CLN8), and that their absence leads to synaptic depletion of key partners (i.e., SNAREs and tethers) and altered synaptic SNARE complexing in vivo , demonstrating a novel shared etiology. [ABSTRACT FROM AUTHOR]