Studies on Childhood Neurodegenerative Lysosomal Storage Disorders

The Section on Developmental Genetics conducts both basic laboratory studies as well as clinical investigations. Our research interests are primarily focused on a group of the most common (1 in 12,500 births) neurodegenerative lysosomal storage disorders (LSDs) called Neuronal Ceroid Lipofuscinoses (NCLs), also known as Batten disease. Mutations in ~13 different genes (called CLNs) underlie various types of NCLs. For the past several years, our research has been primarily focused on infantile NCL (or INCL), one of the most devastating childhood neurodegenerative LSDs caused by mutations in the CLN1 gene, which encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal depalmitoylating enzyme. Because some of the pathologic features are common to all NCLs, it is generally believed that there may be a common link among all NCLs. However, despite intense investigations a common link among the NCLs, until now, remained poorly understood. We recently uncovered that lysosomal cathepsin D-deficiency is a common pathogenic link between CLN1-disease (INCL) and CLN10-disease (congenital NCL). Our long-term goals are to facilitate the development of mechanism-based therapeutic strategies for these NCLs.

There are more than 50 different LSDs, which are characterized by lysosomal accumulation of undigested cargo from both intracellular and extracellular sources are delivered to the lysosome for degradation. Phenotypes of individual LDSs vary widely, depending on the specific cell types affected. Enzyme-replacement therapy (ERT) has been moderately effective for some LSDs, in which the disease manifestation occurs primarily in the visceral organs. However, ERT has not been very successful for neurodegenerative LSDs because the blood-brain barrier prevents the free flow of macromolecules from the blood to the brain. Similar difficulties have been encountered with gene therapy in animal models of neurodegenerative LSDs. For several years, we focused on understanding the function of the molecular mechanism(s) of INCL using the Cln1^-/- mice, which mimic INCL. Palmitoylation (also called S-acylation) is a reversible posttranslational modification in which long-chain fatty acids (predominantly palmitate) are attached to cysteine residues via thioester linkage. Palmitoylation has emerged as an important regulatory mechanism for protein function. The thioester linkage is labile and can be cleaved by nucleophilic attack. Thus, our strategy has been to identify thioesterase (Ppt1)-mimetic small molecules as potential therapeutic agents for INCL. Previously, we reported that nucleophilic compounds such as cysteamine and N-acetylcysteine cleave thioester linkage in palmitoylated proteins (major components of ceroid) and mediate ceroid depletion in cultured cells from INCL patients and in brain tissues of Cln1^-/- mice. A bench-to-bedside clinical trial to determine whether a combination of these two drugs is beneficial for INCL patients has recently been completed and the major findings are published in Lancet Neurology.

Recently, we uncovered an unanticipated role of Cln1/Ppt1 in regulating lysosomal acidification. This is an important discovery because dysregulation of lysosomal acidification is a fundamental defect that contributes to pathogenesis in virtually all LSDs and in common neurodegenerative diseases like Alzheimer’s and Parkinson’s. The vacuolar H+-transporting ATPase (v-ATPase) regulates lysosomal acidification. However, despite intense studies the mechanism of lysosomal acidification defect in these diseases remains largely unclear. The v-ATPase is a multi-subunit protein complex with a cytosolic V1 sector and lysosomal membrane-anchored V0 sector. We found that subunit a1 of the V0 sector (V0a1) undergoes palmitoylation for anchoring to the lysosomal membrane. More interestingly, in Cln1^-/- mice, which mimics INCL, disruption of endosomal sorting and trafficking of V0a1 misrouts it to the plasma membrane instead of its normal localization on the lysosome. This defect caused v-ATPase-deficiency on lysosomal membrane and dysregulated lysosomal acidification in Cln1^-/- mice. Another important discovery is the identification and characterization of a potent, non-toxic, anti-oxidant, thioesterase-mimetic small molecule, N-tert (Butyl) hydroxylamine (NtBuHA), which readily crossed the blood-brain barrier, cleaved thioester linkage in S-acylated proteins (constituents of ceroid) and mediated ceroid depletion in cultured cells from INCL patients and in the brain tissues of Cln1^-/- mice. Importantly, NtBuHA-treatment improved motor coordination, preserved exploratory behavior and modestly extended lifespan of Cln1^-/- mice. This compound also improved the lysosomal pH to near normal. These findings have been published in Nature Neuroscience and Nature Communications, respectively. Currently, preclinical studies are being planned. Our efforts are directed to developing effective therapies for rare neurodegenerative LSDs like INCL and JNCL. Investigations on raregeneticdiseases may provide a windowintomore common neurodegenerative disorders facilitating the development of novel treatment strategies.