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Heritable Neurodegenerative Lysosomal Storage Disorders

Anil Mukherjee, MD, PhD
  • Anil Mukherjee, MD, PhD, Head, Section on Developmental Genetics
  • Zhongjian (Gary) Zhang, MD, PhD, Staff Scientist
  • Sondra W. Levin, MD, Adjunct Scientist
  • Maria B. Bagh, PhD, Visiting Fellow
  • Goutam Chandra, PhD, Visiting Fellow
  • Shiyong Peng, PhD, Visiting Fellow
  • Chinmoy Sarkar, PhD, Visiting Fellow
  • Ashleigh Bouchelion, BS, Medical Student

The Section on Developmental Genetics conducts laboratory and clinical investigations into hereditary neurodegenerative lysosomal storage disorders that mostly affect children. Our current laboratory research focuses on understanding the molecular mechanism(s) of pathogenesis of a group of hereditary childhood neurodegenerative lysosomal storage disorders (LSDs) called neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease. Mutations in at least 13 genes cause various types of NCLs. Currently, there are no effective treatments for any of the NCL types. The infantile NCL (INCL) is an autosomal recessive disease caused by mutations in the CLN1 gene, which encodes a lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes the cleavage of thioester linkage in palmitoylated (S-acylated) proteins (constituent of ceroid), facilitating their degradation in lysosomes. Thus, PPT1 deficiency causes accumulation of ceroid in lysosomes, leading to INCL pathogenesis. Children afflicted with INCL are normal at birth but, by 11 to 18 months of age, exhibit signs of psychomotor retardation. By two years of age, they are completely blind due to retinal degeneration and, by the age 4, manifest no brain activity and remain in a vegetative state for 6 to 8 years before eventual death. These grim outcomes underscore the urgent need for the development of rational and effective therapeutic strategies not only for INCL but also for all NCLs.

One of the major aims of our laboratory is to apply the knowledge gained from these investigations to develop novel therapeutic strategies for INCL and possibly for other Batten diseases. The results of our earlier investigations led to a bench-to-bedside clinical trial that is currently under way. Using CNL1-knockout (Ppt1-KO) 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. We also delineated a mechanism by which PPT1 deficiency disrupts the recycling of the synaptic vesicles (SVs), causing progressive loss of SV pool size, required for maintaining uninterrupted neurotransmission at the nerve terminals (1). We also demonstrated that PPT1 deficiency disrupts adaptive energy metabolism and elevates ribosomal p-S6K1 levels, thus contributing to neuropathology in INCL. Moreover, we found that resveratrol, a naturally occurring anti-oxidant polyphenol, ameliorates these abnormalities. During the past year, we developed a non-invasive method with which the progression of neurodegeneration in Ppt1-KO mice can be assessed by MRI and MRS, allowing repeated evaluations of potential therapeutic agents on treated animals. We also found that the blood-brain barrier is disrupted in Ppt1-KO mice and that this pathology is ameliorated by treatment with resveratrol. A major advance during the past year was the identification of a nucleophilic small molecule with antioxidant property that arrests neuropathology in Ppt1-KO mice and markedly extends their lifespan. A patent application has been filed for this invention. In a clinical investigation, we continue to evaluate the effects of a combination therapy with cystagon and mucomyst on INCL patients. Our ongoing laboratory and clinical investigations continue to advance our knowledge of INCL, paving the way for the application of our laboratory findings to develop novel therapeutic approaches not only for INCL but also for other NCLs.

Dynamic palmitoylation links cytosol-membrane shuttling of APT1 and APT2 with that of H-Ras and GAP-43.

Acyl-protein thioesterase-1 (APT1) and APT2 are cytosolic enzymes that catalyze de-palmitoylation of membrane-anchored, palmitoylated (S-acylated)-H-Ras and growth-associated protein-43 (GAP-43), respectively. However, the mechanism(s) by which these thioesterases shuttle between cytosol and membrane, essential for catalyzing depalmitoylation of their substrates, H-Ras and GAP-43, respectively, remained largely unknown. We found that both APT1 and APT2 undergo palmitoylation on Cys-2. Moreover, blocking palmitoylation adversely affected membrane-localization of APT1 and APT2 as well as that of their substrates. Unexpectedly, we also found that APT1 not only catalyzed its own depalmitoylation but also that of APT2, promoting dynamic palmitoylation (palmitoylation-depalmitoylation) of both cytosolic thioesterases. However, APT2 did not catalyze depalmitoylation of APT1. Further, shRNA suppression of APT1 expression or inhibition of its thioesterase activity markedly increased membrane localization of APT2 whereas shRNA suppression of APT2 had virtually no effect on membrane localization of APT1. Our findings provide insight into a novel mechanism by which dynamic palmitoylation links cytosol-membrane trafficking of APT1 and APT2 with that of their substrates, facilitating steady-state membrane localization and function of both thioesterases.

Development of a non-invasive method for evaluating disease progression in INCL mouse model by MRI and MRS

Mutations in eight different genes cause various forms of NCLs. INCL, the most lethal disease, is caused by inactivating mutations in the PPT1 gene. The availability of Ppt1-knockout (Ppt1-KO) mice, which recapitulate virtually all clinical and pathological features of INCL, provides an opportunity to test the effectiveness of novel therapeutic strategies in vivo. However, such studies would require noninvasive methods that can be used to perform serial evaluations of the same animal receiving an experimental therapy and are thus urgently needed. We evaluated the progression of neurodegeneration in Ppt1-KO mice starting at 3 months of age by MRI and MR spectroscopy (MRS) and repeated the tests using the same mice at 4, 5, and 6 months of age. Our results showed progressive cerebral atrophy, which was associated with histological loss of neuronal content and an increase in astroglia. Remarkably, while the brain volumes in Ppt1-KO mice progressively declined with advancing age, the MRS signals, which were significantly lower than those of their wild-type littermates, remained virtually unchanged from 3 to 6 months of age. In addition, our results also showed an abnormality in cerebral blood flow in the mice, which progressed with age. Our findings provide methods to serially examine the brains of mouse models of neurodegenerative diseases (e.g., Ppt1-KO mice) using noninvasive and nonlethal procedures such as MRI and MRS. The methods may be useful to understand the progression of neuropathology in animal models of neurodegenerative diseases, as repeated evaluations can be performed in the same animal in which an experimental therapy is tested.

A thioesterase-mimetic small molecule arrests neuropathology and markedly extends lifespan in the INCL mouse model.

Despite the fact that in the majority of more than 50 lysosomal storage disorders (LSDs), neurodegeneration is a frequent and devastating manifestation, the development of effective therapies remains challenging. PPT1-deficiency causes lysosomal ceroid accumulation, leading to INCL pathogenesis. PPT1 cleaves thioester linkage in palmitoylated (S-acylated) proteins (constituents of ceroid), facilitating degradation by lysosomal hydrolases. Because the thioester linkage is labile and is disrupted by nucleophilic attacks, we hypothesized that nucleophilic compounds may have therapeutic potential for INCL. Hydroxylamine (HA), a naturally occurring nucleophilic small molecule, cleaves thioester linkage with high specificity and functionally mimics PPT1. However, clinical application of HA is precluded because it stimulates production of methemoglobin, which unlike hemoglobin, does not transport oxygen to the tissues. While some HA-derivatives are non-toxic, it is unknown whether such compounds cleave thioester linkage in palmitoylated proteins and can mediate ceroid depletion in INCL. Thus, we first screened a panel of HA-derivatives for their ability to cleave thioester linkage in [14C]palmitoyl-CoA, a model thioester substrate of PPT1. We identied an HA-derivative, N-tert-butyl HA (NtBuHA), which efficiently cleaves thioester linkage in both [14C]palmitoyl-CoA and, in palmitoylated proteins in cultured cells from INCL patients, mediates ceroid depletion, inhibits apoptosis, and is non-toxic. These beneficial effects in cellulo are completely reproducible in Ppt1-knockout mice, which recapitulate virtually all clinical and pathological features of INCL. Moreover, NtBuHA treatment of these mice depletes ceroid, suppresses neuronal apoptosis, ameliorates neurological deterioration, does not stimulate methemoglobin production, crosses the blood-brain barrier, and markedly extends their lifespan. Our findings provide the proof of principle that nucleophilic small molecules such as NtBuHA have therapeutic potential for INCL as well as for other as yet unrecognized thioesterase-deficiency diseases.

Impaired adaptive energy metabolism and elevated p-S6K1 contribute to neuropathology in the INCL mouse model.

We previously reported that ER and oxidative stress contribute to neuropathology in INCL and in Ppt1-KO mice, a reliable animal model of INCL. Given that mitochondria play critical roles in maintaining cellular energy homeostasis, we hypothesized that oxidative stress-mediated disruption of energy metabolism and homeostasis may contribute to INCL pathogenesis. In cultured INCL fibroblasts and in the brain tissues of Ppt1-KO mice, we observed marked down-regulation of the NAD+/NADH ratio and levels of phosphorylated AMPK (p-AMPK), peroxisome proliferator-activated receptor-gamma (PPARgamma), coactivator-1alpha (PGC-1alpha), and Silent Information Regulator T1 (SIRT1), suggesting an abnormality in the AMPK/SIRT1/PGC-1alpha signaling pathway of energy metabolism. Moreover, we found that, in INCL fibroblasts and in the Ppt1-KO mice, phosphorylated-S6K-1 (p-S6K1) levels, which inversely correlate with lifespan, are markedly elevated. Most important, resveratrol, an antioxidant polyphenol, elevated the NAD+/NADH ratio and levels of ATP, p-AMPK, PGC-1alpha, and SIRT1 while decreasing the level of p-S6K1 in both INCL fibroblasts and Ppt1-KO mice, which showed a modest increase in lifespan (2). Our results indicate that oxidative stress-induced disruption of adaptive energy metabolism and increased levels of p-S6K1 are contributing factors to INCL pathogenesis and that small molecules such as resveratrol, which alleviate these abnormalities, may have therapeutic potential.

T-helper 17 (TH17) lymphocytes mediate blood-brain barrier disruption in INCL mouse model: amelioration by resveratrol.

The disruption of the blood-brain barrier (BBB) is a serious complication in neurodegenerative disorders; however, the molecular mechanism(s) remains unclear, and the development of effective therapy remains challenging. Recent evidence indicates that CD4+ T-helper 17 (TH17) lymphocytes expressing the retinoic acid receptor-related orphan receptor γt (RORγt) cause BBB disruption and neuroinflammation. However, the mechanism by which these immune cells disrupt the BBB remains unknown. Using Ppt1-KO mice, we tested the hypothesis that interleukin-17A (IL-17A), produced by TH17 lymphocytes, stimulates production of matrix metalloproteinases (MMPs), which are known to degrade the tight-junction proteins between adjacent endothelial cells, junctions that are essential for maintaining BBB integrity. We found that, in Ppt1-KO mice, the BBB disruption coincided with elevated levels of RORt (an orphan nuclear receptor) positive TH17 lymphocytes and interleukin-17A (IL-17A). Moreover, the mice showed increased levels of MMPs. Furthermore, IL-17A treatment of cultured brain endothelial cells (constituent of the BBB) stimulated production of MMPs. Importantly, dietary supplementation with resveratrol (RSV), an antioxidant/anti-inflammatory polyphenol, in Ppt1-KO mice, which manifest oxidative stress and neuroinflammation, markedly reduced IL-17A and MMPs and elevated levels of tight-junction proteins (3). Intriguingly, RSV suppressed the differentiation of CD4+ T lymphocytes to IL-17A–positive TH17 cells. We propose that, via MMPs, TH17 lymphocytes cause BBB disruption, and we suggest that anti-oxidative and anti-inflammatory small molecules that suppress TH17-differentiation have therapeutic potential for neurodegenerative disorders such as INCL.

Impaired adaptive energy metabolism disrupts thermoregulation in a mouse model of INCL.

We previously showed that children with INCL are at increased risk of hypothermia during anesthesia and that PPT1-deficiency in mice is associated with disruption of adaptive energy metabolism, downregulation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and mitochondrial dysfunction. We hypothesized that Ppt1-KO mice, a well-studied model of INCL that shows many of the neurologic manifestations of the disease, would recapitulate the impairment in thermoregulation observed in children with INCL. We also posited that when exposed to cold, Ppt1-KO mice would be unable to maintain body temperature, given that, in mice, thermogenesis requires upregulation of Pgc-1α and uncoupling protein 1 (Ucp-1) in brown adipose tissue. We found that the Ppt1-KO mice had lower basal body temperature as they aged and developed hypothermia during cold exposure (4). Surprisingly, the inability to maintain body temperature during cold exposure in Ppt1-KO mice was associated with adequate upregulation of Pgc-1α and Ucp-1 but with lower levels of sympathetic neurotransmitters in brown adipose tissue. In addition, during baseline conditions, brown adipose tissue of Ppt1-KO mice displayed less vacuolization (lipid droplets) than did wild-type (WT)animals. After cold stress, WT animals exhibited significant decreases whereas in Ppt1-KO mice we observed insignificant changes in lipid droplets compared with baseline measurements, suggesting that in Ppt1-KO mice lipolysis was reduced in response to cold stress. The results uncover a previously unknown phenotype associated with PPT1-deficiency, that of altered thermoregulation, which is associated with impaired lipolysis and neurotransmitter release to brown adipose tissue during cold exposure. The findings suggest that INCL should be added to the list of neurodegenerative diseases that are linked to alterations in peripheral metabolic processes. In addition, extrapolating these findings clinically, impaired thermoregulation and hypothermia are potential risks for patients with INCL.

Combination CystagonTM and Mucomyst© treatment of patients with INCL: a bench-to-bedside protocol

Given that PPT1 catalyzes the cleavage of thioester linkages in palmitoylated (S-acylated) proteins (see above), its deficiency impairs degradation of these lipid-modified proteins by lysosomal proteases. Consequently, accumulation of these modified proteins (ceroids) in lysosomes leads to INCL pathogenesis. Given the suceptibility of thioester linkages to nucleophilic attack, drugs that can cleave such linkages may have therapeutic potential for INCL. The results of our laboratory study showed that two drugs, phosphocysteamine and N-acetylcysteine, cleave thioester linkages in [14C] palmitoyl-CoA, a model substrate of PPT1, in lymphoblasts and fibroblasts from INCL patients. The drugs also facilitated the depletion of lysosomal ceroids and inhibited apoptosis. The results prompted us to initiate a bench-to-bedside clinical protocol to determine whether a combination of CystagonTM (cysteamine bitartrate) and Mucomyst© (N-acetylcysteine) is beneficial for patients with INCL. Initially, the NICHD-IRB approved our protocol for the treatment of 20 INCL patients; the study has now been extended to include 40 INCL patients carrying any two mutations in the CNL1 gene. So far, we have treated 10 INCL patients; the study is currently recruiting patients and will continue until the approved number of patients is treated. The protocol admits INCL patients who are 6 months to 3 years of age. Patients may be referred to Dr. Mukherjee at

Group photo

Click image to enlarge.

Members of the Section on Developmental Genetics, PDEGEN.
Sitting from left to right: Ashleigh Bouchemion, Sondra W. Levin, Maria B. Bagh; standing from left to right: Shiyong (Sam) Peng, Goutam Chandra, Anil Mukherjee and Zhongjian (Gary) Zhang

Additional Funding

  • Batten Disease Support and Research Association (BDSRA)


  • Kim SJ, Zhang Z, Sarkar C, Tsai PC, Lee YC, Dye L, Mukherjee AB. Palmitoyl protein thioesterase-1 deficiency impairs synaptic vesicle recycling at nerve terminals, contributing to neuropathology in humans and mice. J Clin Invest 2008;118:3075-3086.
  • Wei H, Zhang Z, Saha A, Peng S, Chandra G, Quezado Z, Mukherjee AB. Disruption of adaptive energy metabolism and elevated ribosomal p-S6K1 levels contribute to INCL pathogenesis: partial rescue by resveratrol. Mum Mol Genet 2011;20:1111-1121.
  • Saha A, Sarkar C, Singh SP, Zhang Z, Munasinghe J, Peng S, Chandra G, Kong E, Mukherjee AB. The blood-brain barrier is disrupted in a mouse model of infantile neuronal ceroid lipofuscinosis: amelioration by resveratrol. Hum Mol Genet 2012;21:2233-2244.
  • Khaibullina A, Kenyon N, Guptill V, Quezado MM, Koziol D, Wesley R, Moya PR, Zhang Z, Saha A, Mukherjee AB, Quezado ZMN. In a model of Batten disease, palmitoyl protein thioesterase-1 deficiency is associated with brown adipose tissue and thermoregulation abnormalities. PLoS One 2012;in press.
  • Mukherjee AB, Zhang Z. Allergic asthma: influence of genetic and environmental factors. J Biol Chem 2011;286:32883-32889.


  • Eva Baker, MD, PhD, Clinical Center, NIH, Bethesda, MD
  • Andrea Gropman, MD, Children's National Medical Center, Center for Neuroscience Research (CNR), Washington, D.C.
  • Christian Hedberg, PhD, Max Planck Institute of Molecular Physiology, Dortmund, Germany
  • Jeeva Munasinghe, PhD, NMR Center, NINDS, Bethesda, MD
  • Zenaide Quezado, MD, Children's National Medical Center, Washington, D.C.
  • Satya P. Singh, PhD, Laboratory of Molecular Immunology, NIAID, Bethesda, MD
  • Herbert Waldmann, PhD, Max Planck Institute of Molecular Physiology, Dortmund, Germany
  • Yan Xu, PhD, Indiana University Medical School, Indianapolis, IN


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