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Childhood Neurodegenerative Lysosomal Storage Disorders

• Anil B. Mukherjee, MD, PhD, Head, Section on Developmental Genetics
• Maria B. Bagh, PhD, Research Fellow
• Abhilash Appu, PhD, Visiting Fellow
• Avisek Mondal, PhD, Visiting Fellow
• Nisha Plavelil, PhD, Visiting Fellow
• Tamal Sadhukhan, PhD, Visiting Fellow
• Koyel Roy, MSc, Contract Biologist
• Sriparna B. Sadhukhan, MSc, Contract Biologist

We conduct both basic and translational research into a group of the most common childhood neurodegenerative lysosomal storage disorders (LSDs), called neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, diseases that mostly affect children, and there is no curative treatment for any of the NCLs. Mutations in at least 13 different genes (called CLNs) underlie various forms of NCLs. The infantile NCL (INCL), a fatal neuro-degenerative LSD, is caused by inactivating mutations in the CLN1 gene. Our investigations focus on understanding the molecular mechanism(s) of pathogenesis underlying INCL (CLN1 disease), juvenile NCL (JNCL: CLN3 disease), and congenital NCL (CNCL: CLN10 disease). Interestingly, all NCL types share some common clinical features such as epileptic seizures, progressive psychomotor decline, and visual impairment resulting from retinal degeneration. The pathologic features include intracellular accumulation of autofluorescent material, neuroinflammation, cortical atrophy, and shortened lifespan.

Several years ago, we initiated investigations on INCL. Numerous proteins, especially in the brain, require S-palmitoylation (also called S-acylation). While S-palmitoylation plays important roles in membrane anchorage of soluble proteins, protein-protein interactions, protein trafficking, and protein stability, such lipid-modified proteins must also be depalmitoylated for recycling or degradation and clearance by lysosomal hydrolases. Dynamic S-palmitoylation requires coordinated actions of two types of enzyme with opposing functions. The enzymes that catalyze S-palmitoylation are palmitoyl acyltransferases (PATs), which are zinc-finger proteins with a common DHHC (Asp-His-His-Cys) motif, and they are called ZDHHC PATs or simply ZDHHCs. The mammalian genome encodes a family of 23 ZDHHC PATS. Similarly, the palmitoyl thioesterases, which depalmitoylate S-acylated proteins, are localized either in the lysosomes like PPT1 or in the cytoplasm like acyl-protein thioesterase-1 (APT1). Recently, several protein depalmitoylases, called ABHD17, were identified that catalyze the turnover of N-Ras (a GTP-ase signal-transduction protein).

The aim of our translational research is to apply the knowledge gained from our basic laboratory investigations to develop novel therapeutic strategies for Batten disease. The results of our earlier investigations on INCL led to a bench-to-bedside clinical trial. Using Cln1–knockout (Cln1^–/–) mice, which recapitulate virtually all clinical and pathological features of INCL, we discovered that PPT1 deficiency causes endoplasmic-reticulum (ER) and oxidative stress, which at least in part causes neuronal death by apoptosis. During the past several years, we also delineated a mechanism by which PPT1 deficiency disrupts the recycling of synaptic-vesicle (SV) proteins, which are essential for generating fresh SVs to replenish the SV pool size at the nerve terminals so as to maintain uninterrupted neurotransmission. We also discovered that ER and oxidative stress contribute to neuronal apoptosis and neuro-inflammation in INCL. Further, we found that PPT1 deficiency causes mis-routing of the V0a1 subunit of v-ATPase (the proton pump on the lysosomal membrane), which dysregulates lysosomal acidification, causing elevated pH and thus adversely affecting lysosomal degradative function.

We also developed a non-invasive method, using MRI and MRS (magnetic resonance spectroscopy) to evaluate the progression of neurodegeneration in Cln1^–/– mice. The methods permit repeated evaluation of potential therapeutic agents in treated animals. Application of such methods in our clinical trial with INCL also allowed us to evaluate the progressive decline in brain volume and neurodegeneration. In collaboration with Wadih Zein, we are also conducting studies to determine whether electro-retinography can be used to assess the progressive retinal deterioration in Cln1^–/– as well as in Cln1–knockin (KI) mice, which carry the nonsense mutation in the CLN1 gene commonly found in the INCL patient population in the US. Moreover, we discovered that the blood-brain barrier is disrupted in Cln1^–/– mice and that this pathology is ameliorated by treatment with resveratrol, which has antioxidant properties. More recently, we discovered that a nucleophilic small molecule with antioxidant properties, N-(tert-butyl) hydroxylamine (NtBuHA), ameliorates the neurological abnormalities in Cln1^–/– mice and extends their lifespan. The compound is currently undergoing preclinical evaluation for the approval of an IND by the FDA. Intriguingly, we discovered that in Cln1^–/– mice the lysosomes contain insufficient amounts of PPT1 protein and PPT1–enzymatic activity, contributing to neuro-pathology in this disease. These and related studies provide insight into the complex mechanisms of heritable disorders of neurodegeneration such as CLN1 disease (INCL), as well as CLN3 disease (JNCL), and identify several potential therapeutic targets. Our results suggest that thioesterase-mimetic small molecules such as NtBuHA are potential therapeutics for INCL and may even be for JNCL. More recently, we discovered that cathepsin D (CD) deficiency in lysosomes is a common pathogenic link between CLN1 disease and CLN10 disease (CNCL). Our ongoing laboratory and translational investigations are attempting to advance our knowledge of INCL, JNCL, and congenital NCL (CNCL) diseases.

Mistargeted NPC1 protein to the plasma membrane promotes cholesterol-mediated mTORC1 activation, contributing to INCL pathogenesis.

As stated above, inactivating mutations in the CLN1 gene cause INCL. CLN1 encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal depalmitoylating enzyme. Numerous proteins, especially in the brain, undergo S-palmitoylation, which confers hydrophobicity, increases membrane-affinity, and promotes protein-protein interactions. Moreover, dynamic S-palmitoylation (palmitoylation-depalmitoylation) facilitates intracellular protein trafficking. Despite the discovery that inactivating mutations in the CLN1 gene encoding PPT1 cause INCL, a clear picture of the pathogenic mechanism of this devastating lysosomal storage disease (LSD) has not emerged for more than two decades. In addition to its degradative function, the lysosome plays a pivotal role in cholesterol homeostasis; it is the major cellular sorting station for dietary cholesterol. Cholesterol is transported to the late endosome/lysosome and is exported to diverse cellular compartments, including to the plasma membrane and the endoplasmic reticulum (ER). The Niemann Pick C1 (NPC1) protein, localized to the lysosomal limiting membrane, plays a critical role in sterol trafficking, and its inactivation causes the hereditary neurodegenerative lipid storage disorder Niemann-Pick type C (NPC). Recently, it was reported that cells from patients with NPC had increased cholesterol on the lysosomal limiting membrane, which mediates the activation of the mechanistic (mammalian) target of rapamycin complex 1 (mTORC1) protein kinase. The mTORC1 kinase integrates intracellular as well as environmental cues to regulate cell growth and metabolism. Aberrant activation of mTORC1 signaling negatively regulates autophagy, which is the principal pathway for lysosomal degradation and clearance of abnormal protein aggregates and damaged organelles. Remarkably, in all three types of autophagy, the lysosome plays pivotal roles in the degradation of cargo contained in the autophagosomes. Most notably, the dysregulation of autophagy has been implicated not only in the pathogenesis of common neurodegenerative diseases such as Alzheimer’s and Parkinson’s, but also in most of the LSDs, in which neurodegeneration is a frequent and devastating manifestation.

To understand the mechanism of INCL pathogenesis, we used the Cln1^–/– mouse, a reliable animal model of INCL. In the brain of these mice, total cholesterol levels have been reported to be significantly higher than those in their WT littermates. However, in that study the levels cholesterol in lysosomes were not evaluated. We thus first determined the cholesterol levels in total lysates of cortical tissues from 2-, 4- and 6-month-old wild-type (WT) and Cln1^–/– mice as well as in lysosomal fractions from those tissues. We found that cholesterol levels in total lysates as well as in lysosomal fractions from Cln1^–/– mice of all three age groups were significantly higher than those in their WT littermates. These results were further confirmed by confocal imaging in neurons from Cln1^–/– mouse brain, which a showed substantially higher level of colocalization of Filipin III–stained cholesterol with lysotracker red. Taken together, these results raised the possibility that in the brain of Cln1^–/– mice lysosomal cholesterol homeostasis is dysregulated. Cholesterol enters the cell in its esterified form packaged with lipoproteins. The low-density lipoprotein (LDL) receptor (LDLR)–bound cholesterol enters the cell via a receptor-mediated pathway. Once within the cell, cholesterol esters are hydrolyzed by lysosomal acid lipase, liberating cholesterol, which is then transported to various cellular structures, including the ER and the plasma membrane. The Niemann-Pick C1 (NPC1) and NPC2 proteins mediate lysosomal cholesterol egress and import, respectively. Moreover, a lysosomal integral membrane protein (LIMP) 2/SCARB2 has also been reported to bind cholesterol and, like NPC1, it transports cholesterol through a transglycocalyx tunnel, a membrane domain rich in glycoproteins and glycolipids. Under normal circumstances, the balance between the export and import maintains lysosomal cholesterol homeostasis. Thus, inactivating mutations in either the NPC1 or NPC2 gene dysregulates cellular cholesterol homeostasis, causing a profound neurological dysfunction, leading to a fatal neurodegenerative LSD, Niemann-Pick type C disease.

We found that Niemann-Pick C1 (NPC1) protein, which mediates lysosomal cholesterol egress, requires dynamic S-palmitoylation for trafficking to the lysosomal membrane. Intriguingly, in Cln1^–/– mice, NPC1 mistargeting to the plasma membrane caused increased oxysterol-binding protein (OSBP) on lysosomal membrane, activating cholesterol-mediated mTORC1 signaling. Activated mTORC1 signaling suppressed autophagy, contributing to neurodegeneration. Importantly, treatment of Cln1^–/– mice with a pharmacological inhibitor of OSBP suppressed mTORC1 activation, rescued autophagy, and ameliorated neuropathology. Our findings reveal a previously unrecognized pathway to INCL pathogenesis and suggest that suppression of cholesterol-mediated activation of mTORC1 signaling may have therapeutic implications.

Disruption of the lysosomal nutrient-sensing scaffold promotes aberrant activation of mTORC1 signaling via IGF1, contributing to neurodegeneration in a mouse model of INCL.

Emerging evidence has remarkably transformed our understanding of the lysosome as a terminal degradative organelle into a critical signaling hub for fundamental metabolic processes. Sensing of essential nutrients by lysosomes has emerged as an important function in coordinating cellular metabolism and growth. Signals from nutrients such as glucose, amino acids, fatty acids, and cholesterol are integrated by the lysosome, turning the cellular events from anabolic to catabolic processes such as autophagy. Whereas materials from extracellular sources are transported to the lysosome by endocytosis, those originating from intracellular sources are delivered by autophagy. Notably, activation of the mTORC1 pathway, situated at the crossroads of nutrient signaling, suppresses autophagy. The loss of autophagy in the central nervous system has been reported to cause neurodegeneration. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy, in all of which the lysosome plays a pivotal role in degrading the cargo contained in the autophagosomes. Indeed, dysregulation of autophagy has been implicated not only in the pathogenesis of common neurodegenerative diseases such as Alzheimer’s and Parkinson’s but also underlies pathogenesis in many members of a family of over 60 LSDs, for which neurodegeneration is a devastating manifestation. Neuronal ceroid lipofuscinoses (NCLs, Batten disease) constitute a group of the most common neurodegenerative LSDs, which mostly affect children. Mutations in at least 13 different genes (CLN1–CLN13) underlie various forms of NCL. Despite the discovery more than two decades ago that the LSD INCL is caused by inactivating mutations in the CLN1 gene, the mechanism of CLN1 disease pathogenesis has remained elusive. Children afflicted with CLN1 disease are phenotypically normal at birth, but by 6–18 months of age they manifest psychomotor retardation. Around two years of age, these children develop complete retinal degeneration, causing blindness. At around four years of age, an isoelectric electroencephalogram (EEG) attests to a vegetative state. They remain in this condition for several more years before eventual death. These grim facts underscore an urgent need for understanding the mechanism underlying pathogenesis of CLN1 disease, which may facilitate the development of an effective treatment.

The CLN1 gene encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal depalmitoylating enzyme. Many proteins in the brain undergo S-palmitoylation (also called S-acylation), a post-translational modification in which a 16-carbon, saturated fatty acid (generally palmitate) is attached to specific cysteine residues in polypeptides via thioester linkage. S-palmitoylation confers hydrophobicity to soluble proteins, increases membrane affinity, promotes protein-protein interactions, and facilitates protein trafficking. Dynamic S-palmitoylation (palmitoylation-depalmitoylation) facilitates endosomal trafficking and the localization of many proteins, especially in the brain. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHC-PATs or simply ZDHHCs), depalmitoylation is mediated by thioesterases. Inactivating mutations in the CLN1 gene causing PPT1 deficiency result in lysosomal accumulation of autofluorescent ceroid lipofuscin. When the ceroid lipofuscins are organized within lysosomes, they are called GRODS (granular osmiophilic deposits), a characteristic finding in neurons and other cell types from patients with CLN1 disease. The lysosomes are the dynamic regulators of the function of many proteins, especially in the brain, and their importance is underscored by the fact that impaired lysosomal function contributes to the pathogenesis of the LSDs. These inherited diseases are characterized by metabolic dysfunction, neurodegeneration, and shortened lifespan. Moreover, it has been suggested that the lysosomal pathway plays critical roles in many cellular functions, including signaling in response to environmental cues. We reasoned that impaired dynamic S-palmitoylation of important proteins that are likely substrates of Ppt1 may impair their ability to traffic to their destination. This abnormality may result in varying impairment of functions of these proteins, cumulatively contributing to neurodegeneration.

In the present study, we used cortical tissues from Cln1^–/– mice, a reliable animal model of CLN1 disease, because in a pilot study (www.clinicaltrials.gov; NCT00028262), magnetic resonance imaging (MRI) of the brain of children with CLN1 disease showed rapid degeneration of cortical tissues. We also used cultured cells from INCL patients to determine whether the loss of Cln1/Ppt1 causes aberrant activation mTORC1 signaling, suppressing autophagy and contributing to neurodegeneration. We found that in the brain of Cln1^–/– mice, which mimic CLN1 disease, Ppt1 deficiency caused aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1), which suppressed autophagy, contributing to neurodegeneration. Emerging evidence indicates that sensing essential nutrients is an important function of the lysosome. Intriguingly, Ppt1 deficiency disrupted the lysosomal nutrient-sensing scaffold (LNSS), upon which mTORC1 must attach to activate. Despite this defect, mTORC1 was activated by IGF1 via PI3K/Akt–mediated pathway. Importantly, treatment of Cln1^–/– mice with pharmacological inhibitors of PI3K/Akt suppressed mTORC1 activation, restored autophagy and improved motor function. Our findings reveal a previously unrecognized mechanism by which Cln1/Ppt1 deficiency contributes to pathogenesis of CLN1 disease.

Impaired lysosomal Ca²⁺ homeostasis contributes to pathogenesis in INCL mice.

The lysosome is an organelle long known for mediating degradation and clearance of cellular waste. In recent years, it has become evident that it is a highly dynamic structure that also plays important roles in cell metabolism in response to environmental cues. Impaired lysosomal degradative function leads to a family of about 60 inherited LSDs. Dysregulation of cellular Ca²⁺ homeostasis is reported to play important roles in the pathogenesis of several human diseases, including the LSDs. Defective lysosomal Ca²⁺ homeostasis has also been reported to impair autophagy. In most of the LSDs, defective autophagy leads to neurodegeneration.

The ER is the major Ca²⁺ repository in the cell, and Ca²⁺ plays a key regulatory role in autophagy. It is an intracellular degradative process that requires Ca²⁺–dependent lysosomal hydrolases for the degradation and clearance of the cargo contained in the autophagosomes. Lysosomal Ca²⁺ homeostasis is mediated by inositol 3-phosphate receptor 1(IP3R1)–mediated transport of Ca²⁺ from the ER to the lysosome. It has also been reported that selective interaction of IP3Rs with the ER–lysosome contact sites is required for the delivery of Ca²⁺ to the lysosome. Moreover, antagonists of IP3Rs rapidly and completely block lysosomal Ca²⁺ refilling. Interestingly, IP3R1 has been reported to undergo S-palmitoylation for regulating Ca²⁺ flux in immune cells. Furthermore, disruption of Ca²⁺ homeostasis may dysregulate neurotransmitter release, contributing to neurodegeneration. Autophagy is impaired by dysregulation of Ca²⁺homeostasis in many LSDs including in Cln1^–/– mice. We sought to test the hypothesis that CLN1 mutations dysregulate lysosomal Ca²⁺ homeostasis and suppress the catalytic activities of Ca²⁺–dependent lysosomal hydrolases, which impair the degradation of undigested cargo in autophagosomes, causing neuro-pathology in INCL.

We sought to determine the mechanism by which PPT1 deficiency impairs lysosomal degradative function and contributes to INCL pathogenesis. We found that in Cln1^–/– mice low levels of IP3R1 dysregulate lysosomal Ca²⁺ homeostasis. Intriguingly, the transcription factor NFATC4, which regulates IP3R1 expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Cln1^–/–mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1 expression, thereby dysregulating lysosomal Ca²⁺ homeostasis. Consequently, Ca²⁺–dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1 overexpression in Cln1^–/– mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Cln1/Ppt1 in regulating lysosomal Ca²⁺ homeostasis and suggest that the defect contributes to INCL pathogenesis.

Publications

1. Appu AP, Bagh MB, Sadhukhan T, Mondal A, Casey S, Mukherjee AB. Cln3-mutations underlying juvenile neuronal ceroid lipofuscinosis cause significantly reduced levels of palmitoyl-protein thioesterases-1 (Ppt1)-protein and Ppt1-enzyme activity in the lysosome. J Inherit Metab Dis 2019;42:944–954.
2. Mukherjee AB, Appu AP, Sadhukhan T, Casey S, Mondal A, Zhang Z, Bagh MB. Emerging new roles of the lysosome and neuronal ceroid lipofuscinoses. Mol Neurodegener 2019;14(1):4.
3. Sarkar C, Sadhukhan T, Bagh MB, Appu AP, Chandra G, Mondal A, Saha A, Mukherjee AB. Cln1-mutations suppress Rab7-RILP interaction and impair autophagy contributing to neuropathology in a mouse model of INCL. J Inherit Metab Dis 2020;43:1082–1101.
4. Sadhukhan T, Bagh MB, Sadhukhan S, Appu AP, Mondal A, Iben JR, Li T, Coon SL, Mukherjee AB. Ablation of microRNA-155 and neuroinflammation in a mouse model of CLN1-disease. Biochem Biophys Res Commun 2021;571:137–144.
5. Sadhukhan T, Bagh MB, Appu AP, Mondal A, Zhang W, Liu A, Mukherjee AB. In a mouse model of INCL reduced S-palmitoylation of cytosolic thioesterase APT1 contributes to microglia proliferation and neuroinflammation. J Inherit Metab Dis 2021;44:1051–1069.

Collaborators

• Steven L. Coon, PhD, Molecular Genetics Core, NICHD, Bethesda, MD
• Aiyi Liu, PhD, Biostatistics & Bioinformatics Branch, NICHD, Bethesda, MD
• Rafael M. Previde, PhD, Section on Cellular Signaling, NICHD, Bethesda, MD
• Stanko Stojilkovic, PhD, Section on Cellular Signaling, NICHD, Bethesda, MD
• Wadih M. Zein, MD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD

Contact

For more information, email mukherja@exchange.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/mukherjee.

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