Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

Characterization of seizures and EEG findings in creatine transporter deficiency due to SLC6A8 mutation.

Seizures occur in up to 59% of boys with creatine transporter deficiency (CTD). While seizure phenotypes have been previously described, electroencephalogram (EEG) findings have only been reported in several case reports. In this prospective observational study, we report seizure characteristics and EEG findings in combination with neurobehavioral and SLC6A8 pathogenic variants in twenty males with CTD. Eighteen study participants (SP) underwent video-EEG, and seven had follow-up EEG recordings. Seizures typically occurred by age of 2 years. Thirteen (65%) had non-febrile seizures, requiring anti-seizure medications in nine. Four had febrile seizures. Seizures were bilateral tonic-clonic in 7 SP and focal impaired awareness in 5 SP; often responding to 1 to 2 antiseizure medications. EEG showed slowing in 5 SP, beta activity in 6 SP, and focal/multifocal, and/or generalized epileptiform activity in 9 SP. Follow-up EEGs in 7 SP showed emergence of epileptiform activity in 1 SP, and increased activity in 2 SP. In conclusion, seizures were frequent in our cohort but tended to respond to antiseizure medications. Longitudinal follow up provided further insight into emergence of seizures and EEG abnormalities soliciting future studies with long term follow up. Biomarkers of epileptogenicity in CTD are needed to predict seizures in this population.

Corrigendum to "Elevated amyloid beta peptides and total tau in cerebrospinal fluid in individuals with Creatine transporter deficiency".

[This corrects the article DOI: 10.1016/j.ymgmr.2023.101001.].

Elevated amyloid beta peptides and total tau in cerebrospinal fluid in individuals with Creatine transporter deficiency.

Background: Creatine transporter deficiency (CTD) is a rare X-linked disorder of creatine transport caused by pathogenic variants in SLC6A8 (Xq28). The disorder is marked by developmental delay, especially speech delay. The biomarkers Aß40, Aß42 and total tau are abnormal in Alzheimer disease (AD), a common neurodegenerative disorder pathologically characterized by Aß peptide containing amyloid plaques and tau neurofibrillary tangles. Although CTD results in neuronal energy deficiency, the pathological processes underlying the CTD phenotype are not fully characterized. Methods: Cerebral spinal fluid (CSF) was collected as an optional part of a natural history study of CTD. Aß40, Aß42 and total tau levels were quantified in CSF from individuals with CTD and from age-appropriate comparison samples. Neuro3-Plex enzyme-linked immunoassay was performed on a Quanterix SR-X instrument. The Vineland Adaptive Behavior Scale, 3rd Edition was used to determine an overall Adaptive Behavior Composite (ABC) standard score. Results: CSF from 12 individuals with CTD and 23 age appropriate non-CTD comparison samples were analyzed. We found that levels of total tau [t(32) = 4.05, p = 0.0003], Aß40 [t(31) = 6.11, p < 0.0001], and Aß42 [t(32) = 3.20, p = 0.003] were elevated in the participants with CTD relative to the comparison group. Additionally, except for one individual that we considered an outlier, all three biomarkers correlated inversely with the adaptive behavior score (total tau: ρ = -0.60 [-0.88, 0.005]; Aß40: ρ = -0.67 [-0.91, -0.12]; Aß42: ρ = -0.62 [-0.89, -0.02]). Conclusion: We describe here the novel finding of elevated protein biomarkers in the CSF of individuals with CTD. Aß40, Aß42 and total tau are markedly elevated in individuals with CTD compared to comparison samples, and increased levels of these biomarkers inversely correlated with ABC scores. We hypothesize that elevated CSF levels of Aß40 and Aß42 are due to cellular energy deficiency. Elevated CSF total tau levels may indicate ongoing neuronal damage. The observed inverse correlation of Vineland ABC scores with increased biomarker levels needs to be confirmed in a larger CTD cohort; however, our observation of increased Aß40, Aß42 and total tau levels in CSF from individuals with CTD may provide insight into pathological mechanisms contributing to the CTD phenotype and may prove useful as supportive data in future therapeutic trials.

Rare disease variant curation from literature: assessing gaps with creatine transport deficiency in focus.

BACKGROUND: Approximately 4-8% of the world suffers from a rare disease. Rare diseases are often difficult to diagnose, and many do not have approved therapies. Genetic sequencing has the potential to shorten the current diagnostic process, increase mechanistic understanding, and facilitate research on therapeutic approaches but is limited by the difficulty of novel variant pathogenicity interpretation and the communication of known causative variants. It is unknown how many published rare disease variants are currently accessible in the public domain. RESULTS: This study investigated the translation of knowledge of variants reported in published manuscripts to publicly accessible variant databases. Variants, symptoms, biochemical assay results, and protein function from literature on the SLC6A8 gene associated with X-linked Creatine Transporter Deficiency (CTD) were curated and reported as a highly annotated dataset of variants with clinical context and functional details. Variants were harmonized, their availability in existing variant databases was analyzed and pathogenicity assignments were compared with impact algorithm predictions. 24% of the pathogenic variants found in PubMed articles were not captured in any database used in this analysis while only 65% of the published variants received an accurate pathogenicity prediction from at least one impact prediction algorithm. CONCLUSIONS: Despite being published in the literature, pathogenicity data on patient variants may remain inaccessible for genetic diagnosis, therapeutic target identification, mechanistic understanding, or hypothesis generation. Clinical and functional details presented in the literature are important to make pathogenicity assessments. Impact predictions remain imperfect but are improving, especially for single nucleotide exonic variants, however such predictions are less accurate or unavailable for intronic and multi-nucleotide variants. Developing text mining workflows that use natural language processing for identifying diseases, genes and variants, along with impact prediction algorithms and integrating with details on clinical phenotypes and functional assessments might be a promising approach to scale literature mining of variants and assigning correct pathogenicity. The curated variants list created by this effort includes context details to improve any such efforts on variant curation for rare diseases.

Elevated cerebrospinal fluid ubiquitin C-terminal hydrolase-L1 levels correlate with phenotypic severity and therapeutic response in Niemann-Pick disease, type C1.

BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is an ultrarare, recessive disorder due to pathological variants of NPC1. The NPC1 phenotype is characterized by progressive cerebellar ataxia and cognitive impairment. Although classically a childhood/adolescent disease, NPC1 is heterogeneous with respect to the age of onset of neurological signs and symptoms. While miglustat has shown to be clinically effective, there are currently no FDA approved drugs to treat NPC1. Identification and characterization of biomarkers may provide tools to facilitate therapeutic trials. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is a protein which is highly expressed by neurons and is a biomarker of neuronal damage. We thus measured cerebrospinal fluid (CSF) levels of UCHL1 in individuals with NPC1. METHODS: CSF levels of UCHL1 were measured using a Quanterix Neuroplex 4 assay in 94 individuals with NPC1 and 35 age-appropriate comparison samples. Cross-sectional and longitudinal CSF UCHL1 levels were then evaluated for correlation with phenotypic measures and treatment status. RESULTS: CSF UCHL1 levels were markedly elevated (3.3-fold) in individuals with NPC1 relative to comparison samples. The CSF UCHL1 levels showed statistically significant (adj p < 0.0001), moderate, positive correlations with both the 17- and 5-domain NPC Neurological Severity Scores and the Annual Severity Increment Scores. Miglustat treatment significantly decreased (adj p < 0.0001) CSF UCHL1 levels by 30% (95% CI 17-40%). CONCLUSIONS: CSF UCHL1 levels are elevated in NPC1, increase with increasing clinical severity and decrease in response to therapy with miglustat. Based on these data, UCHL1 may be a useful biomarker to monitor disease progression and therapeutic response in individuals with NPC1.

Cerebrospinal Fluid Protein Biomarker Discovery in CLN3.

Syndromic CLN3-Batten is a fatal, pediatric, neurodegenerative disease caused by variants in CLN3, which encodes the endolysosomal transmembrane CLN3 protein. No approved treatment for CLN3 is currently available. The protracted and asynchronous disease presentation complicates the evaluation of potential therapies using clinical disease progression parameters. Biomarkers as surrogates to measure the progression and effect of potential therapeutics are needed. We performed proteomic discovery studies using cerebrospinal fluid (CSF) samples from 28 CLN3-affected and 32 age-similar non-CLN3 individuals. Proximal extension assay (PEA) of 1467 proteins and untargeted data-dependent mass spectrometry [MS; MassIVE FTP server (ftp://MSV000090147@massive.ucsd.edu)] were used to generate orthogonal lists of protein marker candidates. At an adjusted p-value of <0.1 and threshold CLN3/non-CLN3 fold-change ratio of 1.5, PEA identified 54 and MS identified 233 candidate biomarkers. Some of these (NEFL, CHIT1) have been previously linked with other neurologic conditions. Others (CLPS, FAM217B, QRICH2, KRT16, ZNF333) appear to be novel. Both methods identified 25 candidate biomarkers, including CHIT1, NELL1, and ISLR2 which had absolute fold-change ratios >2. NELL1 and ISLR2 regulate axonal development in neurons and are intriguing new candidates for further investigation in CLN3. In addition to identifying candidate proteins for CLN3 research, this study provides a comparison of two large-scale proteomic discovery methods in CSF.

Brain proton MR spectroscopy measurements in CLN3 disease.

BACKGROUND: CLN3 is an autosomal recessive lysosomal disorder with intracellular accumulation of ceroid-lipofuscins. CLN3 classically has onset around 4-6 years of age involving vision loss, followed by developmental regression and seizures. Symptoms are progressive and result in premature death. Because treatments are under development, here we explore magnetic resonance spectroscopy (MRS) measurements of metabolite levels in the brain as a potential objective outcome measures. METHODS: Individuals with genetically confirmed CLN3 were enrolled from October 2017-November 2021 in a prospective natural history study (NCT033007304). Baseline concentrations of brain metabolites measured by MRS were compared to concurrently collected dimensional assessment measures: Vineland-3 Adaptive Behavior Composite (ABC) score, verbal intelligence quotient (VIQ), and the Physical, Capability with actual vision, and Clinical global impression of change sub-domains of the Unified Batten Disease Rating Scale (UBDRS). RESULTS: 27 participants with typical CLN3 presentation (15F:12M; ages 6.0-20.7 years) completed MRS, ABC, and UBDRS; 20 (12F:8M; ages 6.5-20.7 years) also completed the VIQ assessment. N-acetyl aspartate [B(95% CI) = -0.61(-0.78;-0.45)] and glutamine/glutamate/GABA [B(95% CI) = -0.82(-1.04;-0.6)] in the parietal gray matter (PGM) decreased across the ages. The strongest correlations between MRS metabolite measurements and the clinical severity assessments were found with N-acetyl aspartate [VIQ (ρ = 0.58), Vineland-3 ABC (ρ = 0.59), UBDRS |ρ| range = (0.57;0.7)] and glutamine/glutamate/GABA [VIQ (ρ = 0.57), Vineland-3 ABC (ρ = 0.60), UBDRS |ρ| range = (0.59;0.77)] measured in the midline PGM. These correlations were accounted for when age was considered. CONCLUSIONS: Based on their correlations to established assessments, NAA and glutamine/glutamate/GABA measured in the midline parietal gray matter may be useful indicators of CLN3 disease state. In a clinical trial, divergence of the MRS measurements and clinical severity markers from age may be useful as surrogate measures for treatment responses.

Identification of cerebral spinal fluid protein biomarkers in Niemann-Pick disease, type C1.

BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is an ultrarare, recessive, lethal, lysosomal disease characterized by progressive cerebellar ataxia and cognitive impairment. Although the NPC1 phenotype is heterogeneous with variable age of onset, classical NPC1 is a pediatric disorder. Currently there are no therapies approved by the FDA and therapeutics trials for NPC1 are complicated by disease rarity, heterogeneity, and the relatively slow rate of neurological decline. Thus, identification of disease relevant biomarkers is necessary to provide tools that can support drug development efforts for this devastating neurological disease. METHODS: Proximal extension assays (O-link® Explore 1536) were used to compare cerebrospinal fluid (CSF) samples from individuals with NPC1 enrolled in a natural history study and non-NPC1 comparison samples. Relative expression levels of 1467 proteins were determined, and candidate protein biomarkers were identified by evaluating fold-change and adjusted Kruskal-Wallis test p-values. Selected proteins were orthogonally confirmed using ELISA. To gain insight into disease progression and severity we evaluated the altered protein expression with respect to clinically relevant phenotypic aspects: NPC Neurological Severity Score (NPC1 NSS), Annual Severity Increment Score (ASIS) and age of neurological onset. RESULTS: This study identified multiple proteins with altered levels in CSF from individuals with NPC1 compared to non-NPC1 samples. These included proteins previously shown to be elevated in NPC1 (NEFL, MAPT, CHIT1, CALB1) and additional proteins confirmed by orthogonal assays (PARK7, CALB2/calretinin, CHI3L1/YKL-40, MIF, CCL18 and ENO2). Correlations with clinically relevant phenotypic parameters demonstrated moderate negative (p = 0.0210, r = -0.41) and possible moderate positive (p = 0.0631, r = 0.33) correlation of CSF CALB2 levels with age of neurological onset and ASIS, respectively. CSF CHI3L1 levels showed a moderate positive (p = 0.0183, r = 0.40) correlation with the concurrent NPC1 NSS. A strong negative correlation (p = 0.0016, r = -0.648) was observed between CSF CCL18 and age of neurological onset for childhood/adolescent cases. CSF CCL18 levels also showed a strong positive correlation (p = 0.0017, r = 0.61) with ASIS. CONCLUSION: Our study identified and validated multiple proteins in CSF from individuals with NPC1 that are candidates for further investigation in a larger cohort. These analytes may prove to be useful as supportive data in therapeutic trials. TRIAL REGISTRATIONS: NCT00344331, NCT00001721, NCT02931682.

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease, Type C1.

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.

Elevated oxysterol and N-palmitoyl-O-phosphocholineserine levels in congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) and Niemann-Pick type C (NPC) disease are inborn errors of metabolism that can both present with infantile-onset severe liver disease and other multisystemic manifestations. Plasma bile acid and N-palmitoyl-O-phosphocholineserine (PPCS) are screening biomarkers with proposed improved sensitivity and specificity for NPC. We report an infant with ATP6AP1-CDG who presented with cholestatic liver failure and elevated plasma oxysterols and bile acid, mimicking NPC clinically and biochemically. On further investigation, PPCS, but not the bile acid derivative N-(3ß,5α,6ß-trihydroxy-cholan-24-oyl) glycine (TCG), were elevated in plasma samples from individuals with ATP6AP1-, ALG1-, ALG8-, and PMM2-CDG. These findings highlight the importance of keeping CDG within the diagnostic differential when evaluating children with early onset severe liver disease and elevated bile acid or PPCS to prevent delayed diagnosis and treatment.

Anesthesia outcomes in lysosomal disorders: CLN3 and GM1 gangliosidosis.

Natural history studies of pediatric rare neurometabolic diseases are important to understand disease pathophysiology and to inform clinical trial outcome measures. Some data collections require sedation given participants' age and neurocognitive impairment. To evaluate the safety of sedation for research procedures, we reviewed medical records between April 2017 and October 2019 from a natural history study for CLN3 (NCT03307304) and one for GM1 gangliosidosis (NCT00029965). Twenty-two CLN3 individuals underwent 28 anesthetic events (age median 11.0, IQR 8.4-15.3 years). Fifteen GM1 individuals had 19 anesthetic events (9.8, 7.1-14.7). All participants had the American Society of Anesthesiology classification of II (8/47) or III (39/47). Mean sedation durations were 186 (SD = 54; CLN3) and 291 (SD = 33; GM1) min. Individuals with GM1 (6/19, 31%) were more frequently prospectively intubated for sedation (CLN3 3/28, 11%). Minor adverse events associated with sedation occurred in 8/28 (28%, CLN3) and 6/19 (32%, GM1) individuals, frequencies within previously reported ranges. No major adverse clinical outcomes occurred in 47 anesthetic events in pediatric participants with either CLN3 or GM1 gangliosidosis undergoing research procedures. Sedation of pediatric individuals with rare neurometabolic diseases for research procedures is safe and allows for the collection of data integral to furthering their understanding and treatment.

Neurofilament light chain in cerebrospinal fluid as a novel biomarker in evaluating both clinical severity and therapeutic response in Niemann-Pick disease type C1.

PURPOSE: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal disorder caused by pathogenic variants in NPC1. Disease progression is monitored using the NPC Neurological Severity Scale, but there are currently no established validated or qualified biomarkers. Neurofilament light chain (NfL) is being investigated as a biomarker in multiple neurodegenerative diseases. METHODS: Cross-sectional and longitudinal cerebrospinal fluid (CSF) samples were obtained from 116 individuals with NPC1. NfL levels were measured using a solid-phase sandwich enzyme-linked immunosorbent assay and compared with age-appropriate non-NPC1 comparison samples. RESULTS: Median levels of NfL were elevated at baseline (1152 [680-1840] pg/mL) in NPC1 compared with controls (167 [82-372] pg/mL; P < .001). Elevated NfL levels were associated with more severe disease as assessed by both the 17-domain and 5-domain NPC Neurological Severity Score. Associations were also observed with ambulation, fine motor, speech, and swallowing scores. Although treatment with the investigational drug 2-hydroxypropyl-ß-cyclodextrin (adrabetadex) did not decrease CSF NfL levels, miglustat therapy over time was associated with a decrease (odds ratio = 0.77, 95% CI = 0.62-0.96). CONCLUSION: CSF NfL levels are increased in individuals with NPC1, associated with clinical disease severity, and decreased with miglustat therapy. These data suggest that NfL is a biomarker that may have utility in future therapeutic trials.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Glycerophosphoinositol is Elevated in Blood Samples From CLN3 Δex7-8 pigs, Cln3 Δex7-8 Mice, and CLN3-Affected Individuals.

Introduction: CLN3 Batten disease is a rare pediatric neurodegenerative lysosomal disorder caused by biallelic disease-associated variants in CLN3. Despite decades of intense research, specific biofluid biomarkers of disease status have not been reported, hindering clinical development of therapies. Thus, we sought to determine whether individuals with CLN3 Batten disease have elevated levels of specific metabolites in blood. Methods: We performed an exhaustive metabolomic screen using serum samples from a novel minipig model of CLN3 Batten disease and validated findings in CLN3 pig serum and CSF and Cln3 mouse serum. We further validate biomarker candidates with a retrospective analysis of plasma and CSF samples collected from participants in a natural history study. Plasma samples were evaluated from 22 phenotyped individuals with CLN3 disease, 15 heterozygous carriers, and 6 non-affected non-carriers (NANC). Results: CLN3 pig serum samples from 4 ages exhibited large elevations in 4 glycerophosphodiester species: glycerophosphoinositol (GPI), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), and glycerophosphoserine (GPS). GPI and GPE exhibited the largest elevations, with similar elevations found in CLN3 pig CSF and Cln3 mouse serum. In plasma samples from individuals with CLN3 disease, glycerophosphoethanolamine and glycerophosphoinositol were significantly elevated with glycerophosphoinositol exhibiting the clearest separation (mean 0.1338 vs 0.04401 nmol/mL for non-affected non-carriers). Glycerophosphoinositol demonstrated excellent sensitivity and specificity as a biomarker, with a receiver operating characteristic area under the curve of 0.9848 (P = .0003). Conclusions: GPE and GPI could have utility as biomarkers of CLN3 disease status. GPI, in particular, shows consistent elevations across a diverse cohort of individuals with CLN3. This raises the potential to use these biomarkers as a blood-based diagnostic test or as an efficacy measure for disease-modifying therapies.

Phenotypic expression of swallowing function in Niemann-Pick disease type C1.

BACKGROUND: Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive disease characterized by endolysosomal accumulation of unesterified cholesterol with progressive deterioration in swallowing, often leading to premature death. Although documented, the natural history of NPC1 swallowing dysfunction has yet to be delineated systematically. This manuscript aims to provide a comprehensive characterization of the phenotypic spectrum and progression of swallowing dysfunction in NPC1. METHODOLOGY: The National Institutes of Health (NIH) NPC1 natural history study (NCT00344331) enrolled 120 patients, who underwent comprehensive interpretative swallow assessments for swallowing safety, dietary modifications, and aspiration risk. Longitudinal statistical modeling accounted for all outcomes with NPC1 disease covariates (first symptom onset, age at neurological symptom onset, seizure history, duration of neurological symptoms) as well as miglustat use (a glucosylceramide synthase inhibitor) and NIH study duration (NIHSD; the length of time an individual participated in the NIH study). Probabilities for disease progression and time to swallowing decline were conducted for the entire cohort. RESULTS: Time to swallowing decline with American Speech-Language-Hearing Association National Outcome Measure (ASHA-NOMS) and the NIH-adapted Penetration Aspiration Scale (NIH-PAS) were identified: [Formula: see text] person-years and [Formula: see text] person-years, respectively. NIHSD and seizure history consistently and significantly were associated with decline (ORNIHSD = 1.34-2.10, 95% CI 1.04-3.4, p = 0.001-0.026; ORSeizure = 3.26-18.22, 1.03-167.79; p = 0.001-0.046), while miglustat use revealed protection (ORMiglustat = 0.01-0.43, 0.007-0.98; p = 0.001-0.044). The probability of decline with NPC1 neurological severity scale and annual severity increment scale were established with the aforementioned covariates, varying amongst subgroups. CONCLUSION: This study represents the most extensive collection of prospective, instrumental swallowing assessments in NPC1 to date with an interpretive analysis providing an improved understanding of NPC1 disease progression with swallowing function-serving as a foundation for clinical management and future NPC1 therapeutics.

CLN3 is required for the clearance of glycerophosphodiesters from lysosomes.

Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.

Modeling SARS-CoV-2 and influenza infections and antiviral treatments in human lung epithelial tissue equivalents.

There is a critical need for physiologically relevant, robust, and ready-to-use in vitro cellular assay platforms to rapidly model the infectivity of emerging viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens.

Complex N-Linked Glycosylation: A Potential Modifier of Niemann-Pick Disease, Type C1 Pathology.

Complex asparagine-linked glycosylation plays key roles in cellular functions, including cellular signaling, protein stability, and immune response. Previously, we characterized the appearance of a complex asparagine-linked glycosylated form of lysosome-associated membrane protein 1 (LAMP1) in the cerebellum of Npc1-/- mice. This LAMP1 form was found on activated microglia, and its appearance correlated both spatially and temporally with cerebellar Purkinje neuron loss. To test the importance of complex asparagine-linked glycosylation in NPC1 pathology, we generated NPC1 knock-out mice deficient in MGAT5, a key Golgi-resident glycosyl transferase involved in complex asparagine-linked glycosylation. Our results show that Mgat5-/-:Npc1-/- mice were smaller than Mgat5+/+:Npc1-/- mice, and exhibited earlier NPC1 disease onset and reduced lifespan. Western blot and lectin binding analyses of cerebellar extracts confirmed the reduction in complex asparagine-linked glycosylation, and the absence of the hyper-glycosylated LAMP1 previously observed. Western blot analysis of cerebellar extracts demonstrated reduced calbindin staining in Mgat5-/-:Npc1-/- mice compared to Mgat5+/+:Npc1-/- mutant mice, and immunofluorescent staining of cerebellar sections indicated decreased levels of Purkinje neurons and increased astrogliosis in Mgat5-/-:Npc1-/- mice. Our results suggest that reduced asparagine-linked glycosylation increases NPC1 disease severity in mice, and leads to the hypothesis that mutations in genes involved in asparagine-linked glycosylation may contribute to disease severity progression in individuals with NPC1. To examine this with respect to MGAT5, we analyzed 111 NPC1 patients for two MGAT5 SNPs associated with multiple sclerosis; however, we did not identify an association with NPC1 phenotypic severity.