Childhood Neurodegenerative Lysosomal Storage Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Summary Neuronal ceroid lipofuscinoses (NCLs), as a group are commonly called Batten disease. These are the most common neurodegenerative lysosomal storage disorders (LSDs) that mostly affect children. As a group, these diseases have no curative treatment. Mutations in at least 13 different genes (called the CLNs) underlie various forms of NCLs. The infantile NCL (or INCL), also called infantile Batten disease,is one of the most devastating neurodegenerative LSDs caused by inactivating mutations in the CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme that catalyzes depalmitoylation of S-palmitoylated (S-acylated) proteins (constituents of ceroid) for their recycling or degradation by lysosomal hydrolases. The deficiency of PPT1 prevents the degradation of S-acylated proteins causing the accumulation of ceroid in lysosomes, which leads to INCL. There are several pathological features (e.g. elevated lysosomal pH, accumulation of intracellular autofluorescent material (called GRODs), seizures and shortened life span. These features are common to virtually all NCLs. These findings prompted us to investigate whether there are common pathogenic mechanisms that are shared by all NCLs. We have previously reported that cathepsin D (CD)-deficiency is a common pathogenic link between congenital NCL (CLN10-disease), caused by mutations in the CLN10 gene encoding cathepsin D (CD), and INCL caused by mutations in the CLN1 gene encoding PPT1. Thus, in both INCL (CLN1-disease) and CLN10-disease lysosomal accumulation of ceroid contributes to pathogenesis. During the past year, we uncovered that in the lysosomes of Cln3-/- mice, which mimic juvenile NCL (JNCL), there is lysosomal insufficiency of Ppt1-protein and Ppt1-enzyme activity suggesting that there might be a pathogenic link between INCL (CLN1-disease) and juvenile NCL (CLN3-disease). Defective lysosomal acidification contributes to pathogenesis of virtually all lysosomal storage disorders (LSDs). It is also a contributory factor in the pathogenesis of common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite the critical importance of lysosomal acidification, the mechanism(s) underlying the dysregulation of lysosomal acidification in these diseases until now remained poorly understood. The cellular proton pump, vacuolar-ATPase (v-ATPase), is known to regulate lysosomal pH. A multi-subunit protein complex, v-ATPase is composed of a cytosolic V1-sector and a lysosomal membrane-anchored V0-sector. The V1 subunit breaks down ATP generating energy required for the V0 sector to transport protons from the cytoplasm to the lysosomal lumen to maintain acidic pH. We found that in the brain tissues of Cln1-/- mice, reduced v-ATPase activity correlated with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires S-palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Unexpectedly, we discovered that in Ppt1-deficient Cln1-/- mice, V0a1 is misrouted to the plasma membrane instead of its normal location on lysosomal membrane. Notably, treatment of the Cln1-/- mice with a thioesterase (Ppt1)-mimetic, non-toxic small molecule, N-tert (Butyl) hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal an unanticipated role of Cln1/Ppt1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase activity may dysregulate lysosomal acidification in other LSDs including various forms of the NCLs and common neurodegenerative diseases. It is increasingly evident that without understanding the precise molecular mechanism(s) of the NCLs, the development of mechanism-based effective therapies is difficult. Despite the discovery that CLN1 mutations causing lysosomal PPT1-deficiency underlies INCL, the precise molecular mechanism(s) of pathogenesis has remained elusive for more than two decades. Thus, our research efforts have been focused on understanding the mechanism(s) of pathogenesis underlying CLN1-disease, CLN3 disease and CLN10 disease. We found that autophagy is dysregulated in Cln1-/- mice, which mimic INCL, and in postmortem brain tissues as well as cultured fibroblasts from INCL patients. Moreover, Rab7, a small GTPase, critical for autophagosome-lysosome fusion, requires S-palmitoylation for trafficking to the late endosomal/lysosomal membrane where it interacts with Rab-interacting lysosomal protein (RILP), essential for autophagosome-lysosome fusion. Intriguingly, PPT1-deficiency in Cln1 -/- mice, dysregulated Rab7-RILP interaction and prevents autophagosome-lysosome fusion and impaired degradative functions of the autolysosome leading to INCL pathogenesis. Importantly, treatment of Cln1 -/- mice with a brain-penetrant, PPT1-mimetic, small molecule, N-tert (butyl)hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal a previously unrecognized role of CLN1/PPT1 in autophagy and suggest that small molecules functionally mimicking PPT1 may have therapeutic implications. In virtually all neurodegenerative disorders, neuronal death is followed by proliferation and activation of astrocytes and microglia (hereafter called astroglia). These activated astroglia secrete cytokines that are neurotoxic causing death of viable neurons, which leads to progressive neurodegeneration. Using two different mouse models of INCL, we found that astroglia activation occurs in an age-dependent manner. It has recently been reported that cytokines secreted by the activated microglia stimulates the differentiation and activation of a special type of astrocytes called Astrocyte A1. These astrocytes secrete as yet uncharacterized, extremely potent neurotoxins, which leads to further neuronal death and progressive neurodegeneration. We are attempting to isolate homogeneous cultures of A1 Astrocytea and characterize the neurotoxins. The Cln1-/- mice, which mimic human INCL, develop progressive neuroinflammation contributing to neurodegeneration. However, the underlying mechanism of neuroinflammation in INCL and in Cln1-/- mice has remained elusive. Previously, it has been reported that microRNA-155 (miR-155) regulates inflammation and miR-profiling in Cln1-/- mouse brain showed that the level of miR-155 was upregulated. Thus, we sought to determine whether ablation of miR-155 in Cln1-/- mice may suppress neuroinflammation in these mice. Towards this goal, we generated Cln1-/-/miR-155-/- double-knockout mice and evaluated the inflammatory signatures in the brain. We found that the brains of double-KO mice manifest progressive neuroinflammatory changes virtually identical to those found in Cln1-/- mice. We conclude that ablation of miR-155 in Cln1-/- mice does not alter the neuroinflammatory trajectory in INCL mouse model. To explore the mechanism underlying INCL pathogenesis we studied lysosomal calcium (Ca++) homeostasis to determine whether dysregulation of lysosomal Ca++ homeostasis contributes to neuropathology in this disease. We found that in Cln1-/- mice, which mimic INCL, low level of IP3R1, which mediates Ca++-transport from the endoplasmic reticulum (ER) to the lysosome, dysregulates lysosomal Ca++ homeostasis. Intriguingly, the transcription factor NFATC4, which promotes IP3R1-expression, requires S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8 as the enzymes that catalyze S-palmitoylation of NFATC4. Remarkably, in Cln1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly suppressed S-palmitoylated NFATC4 level in the nucleus, which suppressed IP3R1-expression, thereby, dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++-dependent lysosomal enzyme activities were suppressed impaired autophagy,causing pathogenesis. We previously reported that in Cln1-/- mice, which mimic human CLN1-disease, endoplasmic reticulum (ER)-stress contributes to neurodegeneration. However, the mechanism underlying ER-stress has remained elusive. Newly synthesized proteins in the ER are transported to the Golgi via COPII (coat protein complex II) vesicles, whereas the retrograde transport (Golgi to ER) is mediated by COPI vesicles. We hypothesized that defective anterograde (ER to the Golgi) trafficking of proteins leads to ER-stress in Cln1-/- mice. We found that the levels of five COPII vesicle-associated proteins (i.e. Sar1, Sec23, Sec24, Sec13 and Sec31) are significantly higher in the ER-fractions of cortical tissues from Cln1-/- mice compared with those from their WT littermates. Remarkably, all COPII proteins, except Sec13, undergo S-palmitoylation. Moreover, CLN8, a Batten disease-protein, also requires dynamic S-palmitoylation (palmitoylation-depalmitoylation) for ER-Golgi trafficking. Intriguingly, Ppt1-deficiency in Cln1-/- mice impairs ER-Golgi trafficking of Cln8-protein along with several COPII-associated proteins. We propose that defective anterograde trafficking causes excessive accumulation of proteins in the ER at least in part contributes to ER-stress in CLN1 disease. 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. Numerous proteins in the brain require dynamic S-palmitoylation (palmitoylation-depalmitoylation) for trafficking to their destination. Although PPT1 depalmitoylates S-palmitoylated proteins and its deficiency causes CLN1 disease, the underlying pathogenic mechanism has remained elusive. We report that Niemann-Pick C1 (NPC1), a polytopic membrane protein mediating lysosomal cholesterol egress, requires dynamic S-palmitoylation for trafficking to the lysosome. In Cln1-/- mice, Ppt1 deficiency misroutes NPC1-dysregulating lysosomal cholesterol homeostasis. Along with this defect, increased oxysterol-binding protein (OSBP) promotes cholesterol-mediated activation of mechanistic target of rapamycin C1 (mTORC1), which inhibits autophagy contributing to neurodegeneration. Pharmacological inhibition of OSBP suppresses mTORC1 activation, rescues autophagy, and ameliorates neuropathology in Cln1-/- mice. Our findings reveal a previously unrecognized role of CLN1/PPT1 in lysosomal cholesterol homeostasis and suggest that suppression of mTORC1 activation may be beneficial for CLN1 disease. Neurodegeneration is a devastating manifestation in most of the lysosomal storage disorders (LSDs). Neuronal ceroid lipofuscinoses (NCLs), commonly called Batten disease, constitute a group of fatal neurodegenerative LSDs that mostly affect children and young adults. Although loss-of-function mutations in 13 different genes (CLN1-CLN8 and CLN10-CLN14) underlie various types of NCLs. These diseases share several clinical and pathological features including progressive psychomotor decline, visual impairment, epileptic seizures and characteristic intracellular accumulation of autofluorescent ceroid lipofuscin. The CLN1 disease is caused by inactivating mutations in the CLN1 gene encoding palmitoyl-protein thioesterase-1, which catalyzes depalmitoylation of S-palmitoylated (or S-acylated) proteins, which are constituents of autofluorescent ceroid lipofuscin. Previously, we reported that in Cln1-/- mice, which mimic human CLN1 disease, and in cultured fibroblasts from CLN1 disease patients increased lysosomal cholesterol mediated mTORC1-hyperactivation and suppressed autophagy. Interestingly, the inhibition of autophagy in mouse central nervous system has been reported to cause neurodegeneration. Here we report that lysosomal cholesterol levels in cultured fibroblasts from patients with CLN1-, CLN2-, CLN3-, CLN6- and CLN8-disease are elevated. Moreover, the levels of phosphorylated-S6K1 (pS6K1) and p4E-BP1, the canonical markers of mTORC1-activation, are also significantly elevated in all five NCL patient fibroblasts compared with those in unaffected control cells. Furthermore, autophagy was markedly suppressed in all NCL patient fibroblasts. Importantly, pharmacological inhibition of mTORC1 in CLN disease fibroblasts and in Cln1-/- mice by Rapalink-1 rescued autophagy and ameliorated neuropathology. We propose that elevated lysosomal cholesterol mediates mTORC1-activation, a targetable pathway that suppresses autophagy, contributes to neurodegeneration in all CLN diseases. Commercial Application Translational Research: Small molecules with nucleophilic properties disrupt thioester linkage in S-acylated proteins (constituents of ceroid). Thus, these small molecules functionally mimic the enzyme (called PPT1), which is missing or defective in INCL. Another advantage of these molecules is that they cross the blood-brain-barrier. Therefore, nucleophilic small molecules may have therapeutic potential for INCL. Accordingly, we screened a panel of nucleophilic small molecules and identified one compound, N-tert (Butyl)hydroxylamine (NtBuHA)that has anti-oxidant and anti-inflammatory properties and functionally mimic the Ppt1 enzyme. Testing of this molecule in PPT1-deficient mice has shown that it is non-toxic and has efficasy in removing ceroid deposits, ameliorating neuropathology and extending lifespan. A US patent application for NtBuHA has been approved and a patent has been issued. This compound is currently undergoing preclinical evaluation. Ongoing research is attempting to characterize this and other similar molecules in the hope that PPT1-mimetic small molecules may be clinically useful for the treatment of thioesterase-deficiency diseases including INCL (CLN1 disease).