Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.

Partial Retraction of: The EMBO Journal (2010) 29: 3607-3620. DOI: 10.1038/emboj.2010.237 | Published online 24 September 2010 Journal statement The journal contacted the authors in February 2022 about potential image insertions and duplications in Fig 4A and 4E. In the absence of source data, the authors are retracting Fig 4A, the lower panel of Fig 4E (LAMP1 immunoblot), and the following statements in the text that rely on these data: "Quantitative analysis showed that the percentage of Flotillin-1 associated with DRMs was increased in LSD endolysosomal membranes (Figure 4A), indicating an increased amount of cholesterol-enriched regions in these membrane samples." "LAMP1 also displayed a similar distribution profile in WT and LSD cells (Figure 4E)". Author statement The authors could not verify the aberrations in panel A of Fig 4 and the lower immunoblot (LAMP1) of 4E because the original source data are no longer available (12 years after publication, which is beyond the institute's 10-year data retention policy). The authors wish to clarify that the main conclusions of the paper are not affected by the retraction of Figure panels 4A and 4E for the following reasons: Figure panel 4A supports the observation that there are increased cholesterol-enhanced regions in LSD samples. This finding is also supported by data provided in figs 4B, 4C and 4D. Figure panel 4E: The LAMP1 blot in Fig 4E shows that the distribution of protein normally excluded from DRMs is not altered between Wt and LSD samples. This result is also supported by the upper blot in this panel (Transferrin receptor). The authors apologize for these errors and agree with this corrigendum; no response could be obtained from AL.

Signals from the lysosome: a control centre for cellular clearance and energy metabolism.

For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.

Brain Disorders Due to Lysosomal Dysfunction.

Recent studies of autophagic and lysosomal pathways have significantly changed our understanding of lysosomes; once thought to be simple degradative and recycling centers, lysosomes are now known to be organelles capable of influencing signal transduction, via the mammalian target of rapamycin complex 1 (mTORC1), and regulating gene expression, via transcription factor EB (TFEB) and other transcription factors. These pathways are particularly relevant to maintaining brain homeostasis, as dysfunction of the endolysosomal and autophagic pathways has been associated with common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, and lysosomal storage disorders, a group of inherited disorders characterized by the intralysosomal buildup of partially degraded metabolites. This review focuses on the cellular biology of lysosomes and discusses the possible mechanisms by which disruption of their function contributes to neurodegeneration. We also review and discuss how targeting TFEB and lysosomes may offer innovative therapeutic approaches for treating a wide range of neurological conditions.

The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease.

Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid ß progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs.

A Comprehensive Map of CNS Transduction by Eight Recombinant Adeno-associated Virus Serotypes Upon Cerebrospinal Fluid Administration in Pigs.

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.

Brain Targeting in MPS-IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a childhood metabolic neuropathology caused by the inherited deficiency of the lysosomal enzyme sulfamidase and is characterized by the accumulation of undegraded glycosaminoglycans in the lysosomes of cells and tissues of affected patients. MPS-IIIA represents one of the most common forms of lysosomal storage disorders (LSDs) and to date there is no cure. Since neurodegeneration is the most relevant pathological feature in MPS-IIIA patients, the treatment of the central nervous system (CNS) lesions represents the goal of any effective therapy for this devastating disorder. During the last years many advances have been made in developing and testing new therapies for brain involvement in MPS-IIIA. These studies have been possible because of the availability of mouse and dog models that recapitulate the MPS-IIIA neuropathological features. Some of these approaches are based on direct CNS administration routes through which the therapeutic molecules access the CNS via the parenchyma (intracerebral injections) or via the cerebrospinal fluid (intraventricular/intrathecal injections). These approaches are highly invasive and poorly suited for clinical use. Minimally invasive approaches are based on systemic injections into the blood stream of therapeutics capable of crossing the blood-brain barrier (BBB). This review will present the background of the clinic and pathology aspects of MPS-IIIA and will describe the current MPS-IIIA preclinical and clinical studies focusing on how a systemic therapeutic strategy based on crossing the BBB has been successfully used to treat CNS pathology and behavioral abnormalities in a mouse model of MPS-IIIA. Future clinical applications of this approach to MPS-IIIA patients will be also discussed together with the possibility of using similar strategies in other LSDs with neurological involvement.

Phosphorylation-regulated degradation of the tumor-suppressor form of PED by chaperone-mediated autophagy in lung cancer cells.

PED/PEA-15 is a death effector domain (DED) family member with a variety of effects on cell growth and metabolism. To get further insight into the role of PED in cancer, we aimed to find new PED interactors. Using tandem affinity purification, we identified HSC70 (Heat Shock Cognate Protein of 70 kDa)-which, among other processes, is involved in chaperone-mediated autophagy (CMA)-as a PED-interacting protein. We found that PED has two CMA-like motifs (i.e., KFERQ), one of which is located within a phosphorylation site, and demonstrate that PED is a bona fide CMA substrate and the first example in which phosphorylation modifies the ability of HSC70 to access KFERQ-like motifs and target the protein for lysosomal degradation. Phosphorylation of PED switches its function from tumor suppression to tumor promotion, and we show that HSC70 preferentially targets the unphosphorylated form of PED to CMA. Therefore, we propose that the up-regulated CMA activity characteristic of most types of cancer cell enhances oncogenesis by shifting the balance of PED function toward tumor promotion. This mechanism is consistent with the notion of a therapeutic potential for targeting CMA in cancer, as inhibition of this autophagic pathway may help restore a physiological ratio of PED forms.

Impaired parkin-mediated mitochondrial targeting to autophagosomes differentially contributes to tissue pathology in lysosomal storage diseases.

Dysfunctional mitochondria are a well-known disease hallmark. The accumulation of aberrant mitochondria can alter cell homeostasis, thus resulting in tissue degeneration. Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by the buildup of undegraded material inside the lysosomes that leads to autophagic-lysosomal dysfunction. In LSDs, autophagic stress has been associated to mitochondrial accumulation and dysfunction. However, the mechanisms underlying mitochondrial aberrations and how these are involved in tissue pathogenesis remain largely unexplored. In normal conditions, mitochondrial clearance occurs by mitophagy, a selective form of autophagy, which relies on a parkin-mediated mitochondrial priming and subsequent sequestration by autophagosomes. Here, we performed a detailed analysis of key steps of mitophagy in a mouse model of multiple sulfatase deficiency (MSD), a severe type of LSD characterized by both neurological and systemic involvement. We demonstrated that in MSD liver reduced parkin levels resulted in inefficient mitochondrial priming, thus contributing to the accumulation of giant mitochondria that are located outside autophagic vesicles ultimately leading to cytochrome c release and apoptotic cell death. Morphological and functional changes were also observed in mitochondria from MSD brain but these were not directly associated with neuronal cell loss, suggesting a secondary contribution of mitochondria to neurodegeneration. Together, these data shed new light on the mechanisms underlying mitochondrial dysfunction in LSDs and on their tissue-specific differential contribution to the pathogenesis of this group of metabolic disorders.

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.

Autophagy contributes to inflammation in patients with TNFR-associated periodic syndrome (TRAPS).

OBJECTIVES: Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is caused by TNFRSF1A mutations, known to induce intracellular retention of the TNFα receptor 1 (TNFR1) protein, defective TNFα-induced apoptosis, and production of reactive oxygen species. As downregulation of autophagy, the main cellular pathway involved in insoluble aggregate elimination, has been observed to increase the inflammatory response, we investigated whether it plays a role in TRAPS pathogenesis. METHODS: The possible link between TNFRSF1A mutations and inflammation in TRAPS was studied in HEK-293T cells, transfected with expression constructs for wild-type and mutant TNFR1 proteins, and in monocytes derived from patients with TRAPS, by investigating autophagy function, NF-κB activation and interleukin (IL)-1ß secretion. RESULTS: We found that autophagy is responsible for clearance of wild-type TNFR1, but when TNFR1 is mutated, the autophagy process is defective, probably accounting for mutant TNFR1 accumulation as well as TRAPS-associated induction of NF-κB activity and excessive IL-1ß secretion, leading to chronic inflammation. Autophagy inhibition due to TNFR1 mutant proteins can be reversed, as demonstrated by the effects of the antibiotic geldanamycin, which was found to rescue the membrane localisation of mutant TNFR1 proteins, reduce their accumulation and counteract the increased inflammation by decreasing IL-1ß secretion. CONCLUSIONS: Autophagy appears to be an important mechanism in the pathogenesis of TRAPS, an observation that provides a rationale for the most effective therapy in this autoinflammatory disorder. Our findings also suggest that autophagy could be proposed as a novel therapeutic target for TRAPS and possibly other similar diseases.

Efficacy of a combined intracerebral and systemic gene delivery approach for the treatment of a severe lysosomal storage disorder.

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.

Protein aggregation and autophagy dysfunction: new lessons from mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases with strong neurological involvement. MPSs are caused by defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs), which consequently accumulate into the lysosomes as primary storage. Macroautophagy/autophagy impairment is well known to drive neurodegeneration in MPSs, however, mechanisms underlying such dysfunction are still poorly understood. Recently, by studying a mouse model for MPS-III (Sanfilippo syndrome) we have shown that the progressive aggregation of amyloid proteins in neuronal cell bodies occurs downstream of the GAG storage and, in turn, impairs the autophagy pathway by affecting lysosomal-dependent autophagosome clearance.

In vivo Gene Therapy to the Liver and Nervous System: Promises and Challenges.

In vivo genetic engineering has recently shown remarkable potential as a novel effective treatment for an ever-growing number of diseases, as also witnessed by the recent marketing authorization of several in vivo gene therapy products. In vivo genetic engineering comprises both viral vector-mediated gene transfer and the more recently developed genome/epigenome editing strategies, as long as they are directly administered to patients. Here we first review the most advanced in vivo gene therapies that are commercially available or in clinical development. We then highlight the major challenges to be overcome to fully and broadly exploit in vivo gene therapies as novel medicines, discussing some of the approaches that are being taken to address them, with a focus on the nervous system and liver taken as paradigmatic examples.

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.

Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44.

Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple sulfatase deficiency, a rare recessive disorder in which all sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion.

Protein Aggregation and Dysfunction of Autophagy-Lysosomal Pathway: A Vicious Cycle in Lysosomal Storage Diseases.

Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In post-mitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often associated to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degradation function, thus generating a vicious cycle, which boost neurodegenerative cascades.

Retinal Degeneration in MPS-IIIA Mouse Model.

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development.

Enhancing the Therapeutic Potential of Sulfamidase for the Treatment of Mucopolysaccharidosis IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.