Childhood Neurodegenerative Lysosomal Storage Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

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, CLN-3 disease and CLN-10 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 neuronal deat