Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in Batten disease.

Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.

Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease.

The ceroid lipofuscinosis neuronal 1 (CLN1) disease, formerly called infantile neuronal ceroid lipofuscinosis, is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1 disease has remained elusive. Here, we report that in the brain of Cln1-/- mice, which mimic CLN1 disease, the mechanistic target of rapamycin complex-1 (mTORC1) kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1-/- mice, two constituent proteins of the Ragulator complex (vacuolar (H+)-ATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1 deficiency in Cln1-/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1 activation, restored autophagy, and ameliorated neurodegeneration in Cln1-/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1 activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease.

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

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.

Mechanistic Insights into S-Depalmitolyse Activity of Cln5 Protein Linked to Neurodegeneration and Batten Disease: A QM/MM Study.

Ceroid lipofuscinosis neuronal protein 5 (Cln5) is encoded by the CLN5 gene. The genetic variants of this gene are associated with the CLN5 form of Batten disease. Recently, the first crystal structure of Cln5 was reported. Cln5 shows cysteine palmitoyl thioesterase S-depalmitoylation activity, which was explored via fluorescent emission spectroscopy utilizing the fluorescent probe DDP-5. In this work, the mechanism of the reaction between Cln5 and DDP-5 was studied computationally by applying a QM/MM methodology at the ωB97X-D/6-31G(d,p):AMBER level. The results of our study clearly demonstrate the critical role of the catalytic triad Cys280-His166-Glu183 in S-depalmitoylation activity. This is evidenced through a comparison of the pathways catalyzed by the Cys280-His166-Glu183 triad and those with only Cys280 involved. The computed reaction barriers are in agreement with the catalytic efficiency. The calculated Gibb's free-energy profile suggests that S-depalmitoylation is a rate-limiting step compared to the preceding S-palmitoylation, with barriers of 26.1 and 25.3 kcal/mol, respectively. The energetics were complemented by monitoring the fluctuations in the electron density distribution through NBO charges and bond strength alterations via local mode stretching force constants during the catalytic pathways. This comprehensive protocol led to a more holistic picture of the reaction mechanism at the atomic level. It forms the foundation for future studies on the effects of gene mutations on both the S-palmitoylation and S-depalmitoylation steps, providing valuable data for the further development of enzyme replacement therapy, which is currently the only FDA-approved therapy for childhood neurodegenerative diseases, including Batten disease.

Dem-Aging: autophagy-related pathologies and the "two faces of dementia".

Neuronal ceroid lipofuscinosis (NCL) is an umbrella term referring to the most frequent childhood-onset neurodegenerative diseases, which are also the main cause of childhood dementia. Although the molecular mechanisms underlying the NCLs remain elusive, evidence is increasingly pointing to shared disease pathways and common clinical features across the disease forms. The characterization of pathological mechanisms, disease modifiers, and biomarkers might facilitate the development of treatment strategies.The DEM-AGING project aims to define molecular signatures in NCL and expedite biomarker discovery with a view to identifying novel targets for monitoring disease status and progression and accelerating clinical trial readiness in this field. In this study, we fused multiomic assessments in established NCL models with similar data on the more common late-onset neurodegenerative conditions in order to test the hypothesis of shared molecular fingerprints critical to the underlying pathological mechanisms. Our aim, ultimately, is to combine data analysis, cell models, and omic strategies in an effort to trace new routes to therapies that might readily be applied in the most common forms of dementia.

Integrative human and murine multi-omics: Highlighting shared biomarkers in the neuronal ceroid lipofuscinoses.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative disorders whose molecular mechanisms remain largely unknown. Omics approaches are among the methods that generate new information on modifying factors and molecular signatures. Moreover, omics data integration can address the need to progressively expand knowledge around the disease and pinpoint specific proteins to promote as candidate biomarkers. In this work, we integrated a total of 62 proteomic and transcriptomic datasets originating from humans and mice, employing a new approach able to define dysregulated processes across species, stages and NCL forms. Moreover, we selected a pool of differentially expressed proteins and genes as species- and form-related biomarkers of disease status/progression and evaluated local and spatial differences in most affected brain regions. Our results offer promising targets for potential new therapeutic strategies and reinforce the hypothesis of a connection between NCLs and other forms of dementia, particularly Alzheimer's disease.

Neuronal progenitor cells-based metabolomics study reveals dysregulated lipid metabolism and identifies putative biomarkers for CLN6 disease.

Neuronal ceroid lipofuscinosis 6 (CLN6) is a rare and fatal autosomal recessive disease primarily affecting the nervous system in children. It is caused by a pathogenic mutation in the CLN6 gene for which no therapy is available. Employing an untargeted metabolomics approach, we analyzed the metabolic changes in CLN6 subjects to see if this system could potentially yield biomarkers for diagnosis and monitoring disease progression. Neuronal-like cells were derived from human fibroblast lines from CLN6-affected subjects (n = 3) and controls (wild type, n = 3). These were used to assess the potential of a neuronal-like cell-based metabolomics approach to identify CLN6 distinctive and specific biomarkers. The most impacted metabolic profile is associated with sphingolipids, glycerophospholipids metabolism, and calcium signaling. Over 2700 spectral features were screened, and fifteen metabolites were identified that differed significantly between both groups, including the sphingolipids C16 GlcCer, C24 GlcCer, C24:1 GlcCer and glycerophospholipids PG 40:6 and PG 40:7. Of note, these fifteen metabolites were downregulated in the CLN6 disease group. This study is the first to analyze the metabolome of neuronal-like cells with a pathogenic mutation in the CLN6 gene and to provide insights into their metabolomic alterations. This could allow for the development of novel biomarkers for monitoring CLN6 disease.

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.

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.

Extracellular Vesicles Released by Genetically Modified Macrophages Activate Autophagy and Produce Potent Neuroprotection in Mouse Model of Lysosomal Storage Disorder, Batten Disease.

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.

Enhanced expression of the autophagosomal marker LC3-II in detergent-resistant protein lysates from a CLN3 patient's post-mortem brain.

• Neuronal Ceroido Lipofuscinoses (NCL) are inherited, neurodegenerative disorders associated with lysosomal storage. • Impaired autophagy plays a pathogenetic role in several NCL forms, including CLN3 disease, but study on human brains are lacking. • In post-mortem brain samples of a CLN3 patient the LC3-I to LC3-II shift was consistent with activated autophagy. However, the autophagic process seemed to be ineffective due to the presence of lysosomal storage markers. • After fractionation with buffers of increasing detergent-denaturing strength, a peculiar solubility pattern of LC3-II was observed in CLN3 patient's samples, suggesting a different lipid composition of the membranes where LC3-II is stacked.

Recent insights into the networking of CLN genes and proteins in mammalian cells.

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.

Abnormal triaging of misfolded proteins by adult neuronal ceroid lipofuscinosis-associated DNAJC5/CSPα mutants causes lipofuscin accumulation.

Mutations in DNAJC5/CSPα are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage materials (AFSMs) termed lipofuscin. Functionally, DNAJC5 has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how DNAJC5 dysfunction causes lipofuscinosis and neurodegeneration is unclear. Here we report two functionally distinct but coupled chaperoning activities of DNAJC5, which jointly regulate lysosomal homeostasis: While endolysosome-associated DNAJC5 promotes ESCRT-dependent microautophagy, a fraction of perinuclear and non-lysosomal DNAJC5 mediates MAPS. Functional proteomics identifies a previously unknown DNAJC5 interactor SLC3A2/CD98hc that is essential for the perinuclear DNAJC5 localization and MAPS but dispensable for microautophagy. Importantly, uncoupling these two processes, as seen in cells lacking SLC3A2 or expressing ANCL-associated DNAJC5 mutants, generates DNAJC5-containing AFSMs resembling NCL patient-derived lipofuscin and induces neurodegeneration in a Drosophila ANCL model. These findings suggest that MAPS safeguards microautophagy to avoid DNAJC5-associated lipofuscinosis and neurodegeneration.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AFSM: autofluorescent storage materials; ANCL: adult neuronal ceroid lipofuscinosis; Baf. A1: bafilomycin A1; CLN: ceroid lipofuscinosis neuronal; CLU: clusterin; CS: cysteine string domain of DNAJC5/CSPα; CUPS: compartment for unconventional protein secretion; DN: dominant negative; DNAJC5/CSPα: DnaJ heat shock protein family (Hsp40) member C5; eMI: endosomal microautophagy; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INCL: infant neuronal ceroid lipofuscinosis; JNCL: juvenile neuronal ceroid lipofuscinosis; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAPTM4B: lysosomal protein transmembrane 4 beta; LN: linker domain of DNAJC5/CSPα; MAPS: misfolding-associated protein secretion; mCh/Ch: mCherry; mCi/Ci: mCitrine; MTOR: mechanistic target of rapamycin kinase; NCL: neuronal ceroid lipofuscinosis; PPT1: palmitoyl-protein thioesterase 1; PQC: protein quality control; SBP: streptavidin binding protein; SGT: small glutamine-rich tetratricopeptide repeat; shRNA: short hairpin RNA; SLC3A2/CD98hc: solute carrier family 3 member 2; SNCA/α-synuclein: synuclein alpha; TMED10: transmembrane p24 trafficking protein 10; UV: ultraviolet; VPS4: vacuolar protein sorting 4 homolog; WT: wild type.

A Caenorhabditis elegans model of autosomal dominant adult-onset neuronal ceroid lipofuscinosis identifies ethosuximide as a potential therapeutic.

Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rare neurodegenerative disorder characterized by progressive dementia and premature death. Four ANCL-causing mutations have been identified, all mapping to the DNAJC5 gene that encodes cysteine string protein α (CSPα). Here, using Caenorhabditis elegans, we describe an animal model of ANCL in which disease-causing mutations are introduced into their endogenous chromosomal locus, thereby mirroring the human genetic disorder. This was achieved through CRISPR/Cas9-mediated gene editing of dnj-14, the C. elegans ortholog of DNAJC5. The resultant homozygous ANCL mutant worms exhibited reduced lifespans and severely impaired chemotaxis, similar to isogenic dnj-14 null mutants. Importantly, these phenotypes were also seen in balanced heterozygotes carrying one wild-type and one ANCL mutant dnj-14 allele, mimicking the heterozygosity of ANCL patients. We observed a more severe chemotaxis phenotype in heterozygous ANCL mutant worms compared with haploinsufficient worms lacking one copy of CSP, consistent with a dominant-negative mechanism of action. Additionally, we provide evidence of CSP haploinsufficiency in longevity, as heterozygous null mutants exhibited significantly shorter lifespan than wild-type controls. The chemotaxis phenotype of dnj-14 null mutants was fully rescued by transgenic human CSPα, confirming the translational relevance of the worm model. Finally, a focused compound screen revealed that the anti-epileptic drug ethosuximide could restore chemotaxis in dnj-14 ANCL mutants to wild-type levels. This suggests that ethosuximide may have therapeutic potential for ANCL and demonstrates the utility of this C. elegans model for future larger-scale drug screening.

Lysosomal dysfunction, autophagic defects, and CLN5 accumulation underlie the pathogenesis of KCTD7-mutated neuronal ceroid lipofuscinoses.

Lysosomes are essential catabolic organelles responsible for the degradation of biomacromolecules into low-molecular-weight materials for subsequent reuse. Neuronal ceroid lipofuscinoses (NCLs) are a group of fatal neurodegenerative lysosomal storage disorders characterized by the intracellular accumulation of lipoprotein aggregates (called ceroid lipofuscin) in neurons and other tissues. Mutations in KCTD7, which encodes a substrate-binding adaptor for the CUL3-RING E3 (CRL3) ubiquitin ligase complex, are categorized as a unique NCL subtype. However, the molecular mechanisms underlying the KCTD7-mutated NCLs remain unclear. In our recent study, we showed that KCTD7 deficiency leads to the accumulation of lysosomal storage deposits owing to lysosomal dysfunction and macroautophagic/autophagic defects. We identified CLN5 as an authentic substrate of CRL3-KCTD7 E3s. Wild-type KCTD7 targets CLN5 for ubiquitination and proteasomal degradation, whereas NCL patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive CLN5 accumulation in the endoplasmic reticulum. Accumulated CLN5 disrupts the interaction between CLN6-CLN8 and lysosomal enzymes, leading to impaired ER-to-Golgi trafficking of lysosomal enzymes. Thus, our findings indicate that KCTD7 is a key player in maintaining lysosomal and autophagic homeostasis and demonstrate that KCTD7 and CLN5, two NCL causative genes, are biochemically linked and function in a common neurodegenerative pathway.

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.

KCTD7 mutations impair the trafficking of lysosomal enzymes through CLN5 accumulation to cause neuronal ceroid lipofuscinoses.

Lysosomes are central organelles for cellular degradation and energy metabolism. Neuronal ceroid lipofuscinoses (NCLs) are a group of the most common neurodegenerative lysosomal storage disorders characterized by intracellular accumulation of ceroid in neurons. Mutations in KCTD7, a gene encoding an adaptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, are categorized as a unique NCL subtype. However, the underlying mechanisms remain elusive. Here, we report various lysosomal and autophagic defects in KCTD7-deficient cells. Mechanistically, the CRL3-KCTD7 complex degrades CLN5, whereas patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive accumulation of CLN5. The accumulated CLN5 disrupts the interaction between CLN6/8 and lysosomal enzymes at the endoplasmic reticulum (ER), subsequently impairing ER-to-Golgi trafficking of lysosomal enzymes. Our findings reveal previously unrecognized roles of KCTD7-mediated CLN5 proteolysis in lysosomal homeostasis and demonstrate that KCTD7 and CLN5 are biochemically linked and function in a common neurodegenerative pathway.

TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1, and Batten disease.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.

CLN8 Gene Compound Heterozygous Variants: A New Case and Protein Bioinformatics Analyses.

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

Lysosomal Proteomics Links Disturbances in Lipid Homeostasis and Sphingolipid Metabolism to CLN5 Disease.

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5-/- mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments.