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Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus 1 . 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. The lysosomal transmembrane protein CLN3 is required for the lysosomal clearance of glycerophosphodiesters in mice and in human cells, suggesting that the loss of CLN3 causes Batten disease in children due to defects in glycerophospholipid metabolism.

Neuronal Ceroid Lipofuscinosis (NCL), also known as Batten disease, is an incurable childhood brain disease. The thirteen forms of NCL are caused by mutations in thirteen CLN genes. Mutations in one CLN gene, CLN5, cause variant late-infantile NCL, with an age of onset between 4 and 7 years. The CLN5 protein is ubiquitously expressed in the majority of tissues studied and in the brain, CLN5 shows both neuronal and glial cell expression. Mutations in CLN5 are associated with the accumulation of autofluorescent storage material in lysosomes, the recycling units of the cell, in the brain and peripheral tissues. CLN5 resides in the lysosome and its function is still elusive. Initial studies suggested CLN5 was a transmembrane protein, which was later revealed to be processed into a soluble form. Multiple glycosylation sites have been reported, which may dictate its localisation and function. CLN5 interacts with several CLN proteins, and other lysosomal proteins, making it an important candidate to understand lysosomal biology. The existing knowledge on CLN5 biology stems from studies using several model organisms, including mice, sheep, cattle, dogs, social amoeba and cell cultures. Each model organism has its advantages and limitations, making it crucial to adopt a combinatorial approach, using both human cells and model organisms, to understand CLN5 pathologies and design drug therapies. In this comprehensive review, we have summarised and critiqued existing literature on CLN5 and have discussed the missing pieces of the puzzle that need to be addressed to develop an efficient therapy for CLN5 Batten disease.

CLN3 disease or juvenile neuronal ceroid lipofuscinosis (Batten disease), is a progressive, severe, neurodegenerative, lysosomal storage disorder. Previous studies have demonstrated that network-level excitability differences are present in mouse models prior to significant lysosomal storage accumulation. Here we sought to identify the earliest biochemical and functional markers of disease in the hippocampus, a brain region important in learning and memory and implicated in CLN3 disease. Using targeted hydrophilic interaction liquid chromatography high resolution mass spectrometry (LC-HRMS), we quantified levels of glycerophosphodiesters (GPDs), recently-described biomarkers of CLN3 disease, in early postnatal hippocampus. In addition, we assessed hippocampal excitability via in vitro voltage-sensitive dye imaging (VSDI) across the period of postnatal hippocampal maturation (p7, p14, p21). Finally, we completed longitudinal electroencephalogram (EEG) recordings to evaluate in vivo hippocampal circuit dynamics once the hippocampal circuit was matured. Intriguingly, glycercophosphoinositol (GPI or GroPIns), but not other GPDs, were significantly elevated in CLN3 disease hippocampus in early development at p11, further supporting the hypothesis that GPI plays a key role in disease pathogenesis. Functionally, the hippocampus was significantly hypoexcitable as early as p7 and showed a very atypical pattern of maturation across early development. This aberrant development resulted in abnormal in vivo circuit function, with pathologic slowing observed on EEG recordings at p30. Collectively these data underscore the potential link between pathologic metabolism of GPI and functional defects in CLN3 disease. In addition, this work highlights that CLN3 disease is an early neurodevelopmental, and not just neurodegenerative, disorder.

Lysosomes are implicated in a wide spectrum of human diseases, including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration, and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in patients remains challenging. Here, we report the development of the 'tagless LysoIP' method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell-derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients' lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms.

Haploinsufficiency of progranulin (PGRN) due to mutations in the granulin ( GRN ) gene causes frontotemporal lobar degeneration (FTLD), and complete loss of PGRN leads to a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests that PGRN is essential for proper lysosomal function, but the precise mechanisms involved are not known. Here, we show that PGRN facilitates neuronal uptake and lysosomal delivery of prosaposin (PSAP), the precursor of saposin peptides that are essential for lysosomal glycosphingolipid degradation. We found reduced levels of PSAP in neurons both in mice deficient in PGRN and in human samples from FTLD patients due to GRN mutations. Furthermore, mice with reduced PSAP expression demonstrated FTLD-like pathology and behavioural changes. Thus, our data demonstrate a role of PGRN in PSAP lysosomal trafficking and suggest that impaired lysosomal trafficking of PSAP is an underlying disease mechanism for NCL and FTLD due to GRN mutations. Mutations in the granulin gene are associated with frontotemporal lobe dementia (FTLD) and a lysosomal storage disease. The authors show that reduced progranulin levels leads to impaired neuronal uptake and lysosomal delivery of prosaposin, and that decreased prosaposin expression in mice leads to FTLD-like behaviour.

Neuronal ceroid lipofuscinoses (NCLs) are paediatric neurodegenerative disorders that primarily affect the central nervous system (CNS). The high prevalence of gastrointestinal (GI) symptoms has prompted researchers and clinicians to move beyond an exclusively “brain-centric” perspective. At the molecular level, mutations in CLN genes lead to lysosomal dysfunction and impaired autophagy, resulting in intracellular accumulation of storage material that disrupts both central and enteric neuronal homeostasis. To systematically examine current clinical and preclinical knowledge on gut involvement in NCLs, with a focus on recent findings related to the enteric nervous system and gut microbiota. We conducted a systematic review following the PRISMA guidelines using PubMed as the sole database. Both clinical (human) and preclinical (animal) studies were included. A total of 18 studies met the inclusion criteria, focusing on gastrointestinal dysfunction, nervous system involvement, and gut microbiota. We found that the nature of GI symptoms was multifactorial in NCLs, involving not only the CNS but also the autonomic and enteric nervous systems, which were affected early by lysosomal deposits and enteric neuron degeneration. Of note, preclinical studies showed that gene therapy could improve not only CNS manifestations but also GI ones, which may have beneficial implications for patient care. While the role of the ENS seems to be clearer, that of gut microbiota needs to be further clarified. Current evidence from preclinical models highlighted alterations in the composition of the microbiota and suggested a possible influence on the progression and modulation of neurological symptoms. However, these results need to be confirmed by further studies demonstrating the causality of this relationship. GI involvement is a key feature of NCLs, with early impact on the enteric nervous system and possible links to gut microbiota. Although preclinical findings—particularly on gene therapy—are encouraging due to their dual impact on both CNS and GI manifestations, the causal role of the gut microbiota remains to be fully elucidated. In this context, the development of sensitive and specific outcome measures to assess GI symptoms in clinical trials is crucial for evaluating the efficacy of future therapeutic interventions.

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment 1 – 3 . Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system 4 – 6 , but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease) 7 . ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8 . di Ronza et al. identify CLN8 as a cargo receptor for lysosomal enzymes required for their endoplasmic-reticulum-to-Golgi transport, linking Batten disease caused by CLN8 mutations to defects in organelle biogenesis.

The concept of lysosomal storage disorders (LSDs) has existed for over 50 years, but our understanding of the causes and pathobiology of these diseases have come to light only recently, following advances in genetic technology. In this Review, Rose-Mary Boustany summarizes current understanding of known LSDs, highlighting existing treatment approaches for patients with these often devastating disorders, and outlining the barriers to development of novel therapies. Since the discovery of the lysosome in 1955, advances have been made in understanding the key roles and functions of this organelle. The concept of lysosomal storage diseases (LSDs)—disorders characterized by aberrant, excessive storage of cellular material in lysosomes—developed following the discovery of α-glucosidase deficiency as the cause of Pompe disease in 1963. Great strides have since been made in understanding the pathobiology of LSDs and the neuronal ceroid lipofuscinoses (NCLs). The NCLs are neurodegenerative disorders that display symptoms of cognitive and motor decline, seizures, blindness, early death, and accumulation of lipofuscin in various cell types, and also show some similarities to 'classic' LSDs. Defective lysosomal storage can occur in many cell types, but the CNS and PNS are particularly vulnerable to LSDs and NCLs, being affected in two-thirds of these disorders. Most LSDs are inherited in an autosomal recessive manner, with the exception of X-linked Hunter disease, Fabry disease and Danon disease, and a variant type of adult NCL (Kuf disease). This Review provides a summary of known LSDs, and the pathways affected in these disorders. Existing therapies and barriers to development of novel and improved treatments for LSDs and NCLs are also discussed. Key Points The spectrum of lysosomal storage disease (LSD) includes defects in degradative and synthetic enzymes, lysosomal membrane defects, the neuronal ceroid lipofuscinoses (NCLs) and disorders of lysosome biogenesis and endosome–lysosome traffic LSDs result in excess cellular storage and cell death in the CNS, PNS, lungs, liver, bone, skeletal and cardiac muscle and the reticuloendothelial system; symptomatology includes neurocognitive decline, seizures, blindness and hepatosplenomegaly NCLs are typified by abnormal inclusions in brain, eye, liver, skin and the reticuloendothelial system, with clinical manifestations such as neurocognitive decline, blindness, seizures and early death Therapies involving enzyme replacement, substrate reduction and chaperone-mediated delivery, as well as haematopoetic and other stem-cell therapies, have had modest successes in the treatment of patients with LSDs LSDs and NCLs share pathological features: abnormal lipid trafficking, dysregulation of apoptosis and autophagy, prolonged inflammation, disturbed endoplasmic reticulum–cytosol calcium balance, cellular stress, and the unfolded protein response The biological underpinnings of LSDs and NCLs partly explain the limited success of 'direct' therapies for these disorders, but also provide novel targets for therapeutic approaches

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. CLN3 mutations cause Batten disease, a devastating neurodegenerative lysosomal storage disease. Here, the authors discovered that CLN3 plays a crucial role in both trafficking of lysosomal proteins and autophagic lysosomal reformation.

Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GCase), and is essential for proper GCase activity. GCase activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.