Moving towards a new era of genomics in the neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCL) are a group of disorders defined by shared clinical and pathological features, including seizures and progressive decline in vision, neurocognition, and motor functioning, as well as accumulation of autofluorescent lysosomal storage material, or 'ceroid lipofuscin'. Research has revealed thirteen distinct genetic subtypes. Precisely how the gene mutations lead to the clinical phenotype is still incompletely understood, but recent research progress is starting to shed light on disease mechanisms, in both gene-specific and shared pathways. As the application of new sequencing technologies to genetic disease diagnosis has grown, so too has the spectrum of clinical phenotypes caused by mutations in the NCL genes. Most genes causing NCL have probably been identified, underscoring the need for a shift towards applying genomics approaches to achieve a deeper understanding of the molecular basis of the NCLs and related disorders. Here, we summarize the current understanding of the thirteen identified NCL genes and the proteins they encode, touching upon the spectrum of clinical manifestations linked to each of the genes, and we highlight recent progress leading to a broader understanding of key pathways involved in NCL disease pathogenesis and commonalities with other neurodegenerative diseases.

An Autophagy Modifier Screen Identifies Small Molecules Capable of Reducing Autophagosome Accumulation in a Model of CLN3-Mediated Neurodegeneration.

Alterations in the autophagosomal-lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal-lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.

The CLN3 gene and protein: What we know.

BACKGROUND: One of the most important steps taken by Beyond Batten Disease Foundation in our quest to cure juvenile Batten (CLN3) disease is to understand the State of the Science. We believe that a strong understanding of where we are in our experimental understanding of the CLN3 gene, its regulation, gene product, protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency, lays the groundwork for determining therapeutic action plans. OBJECTIVES: To present an unbiased comprehensive reference tool of the experimental understanding of the CLN3 gene and gene product of the same name. METHODS: BBDF compiled all of the available CLN3 gene and protein data from biological databases, repositories of federally and privately funded projects, patent and trademark offices, science and technology journals, industrial drug and pipeline reports as well as clinical trial reports and with painstaking precision, validated the information together with experts in Batten disease, lysosomal storage disease, lysosome/endosome biology. RESULTS: The finished product is an indexed review of the CLN3 gene and protein which is not limited in page size or number of references, references all available primary experiments, and does not draw conclusions for the reader. CONCLUSIONS: Revisiting the experimental history of a target gene and its product ensures that inaccuracies and contradictions come to light, long-held beliefs and assumptions continue to be challenged, and information that was previously deemed inconsequential gets a second look. Compiling the information into one manuscript with all appropriate primary references provides quick clues to which studies have been completed under which conditions and what information has been reported. This compendium does not seek to replace original articles or subtopic reviews but provides an historical roadmap to completed works.

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function.

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca(2+)-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca(2+) chelation. Interrogation of intracellular Ca(2+) handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca(2+) pools and in store-operated Ca(2+) uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca(2+) handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.

Human iPSC models of neuronal ceroid lipofuscinosis capture distinct effects of TPP1 and CLN3 mutations on the endocytic pathway.

Neuronal ceroid lipofuscinosis (NCL) comprises ∼13 genetically distinct lysosomal disorders primarily affecting the central nervous system. Here we report successful reprograming of patient fibroblasts into induced pluripotent stem cells (iPSCs) for the two most common NCL subtypes: classic late-infantile NCL, caused by TPP1(CLN2) mutation, and juvenile NCL, caused by CLN3 mutation. CLN2/TPP1- and CLN3-iPSCs displayed overlapping but distinct biochemical and morphological abnormalities within the endosomal-lysosomal system. In neuronal derivatives, further abnormalities were observed in mitochondria, Golgi and endoplasmic reticulum. While lysosomal storage was undetectable in iPSCs, progressive disease subtype-specific storage material was evident upon neural differentiation and was rescued by reintroducing the non-mutated NCL proteins. In proof-of-concept studies, we further documented differential effects of potential small molecule TPP1 activity inducers. Fenofibrate and gemfibrozil, previously reported to induce TPP1 activity in control cells, failed to increase TPP1 activity in patient iPSC-derived neural progenitor cells. Conversely, nonsense suppression by PTC124 resulted in both an increase of TPP1 activity and attenuation of neuropathology in patient iPSC-derived neural progenitor cells. This study therefore documents the high value of this powerful new set of tools for improved drug screening and for investigating early mechanisms driving NCL pathogenesis.

Scintillation proximity assay for total p53 protein as an alternative to ELISA.

Measurement of the level of a specific protein can be an important parameter to discern as that can change and reflect disease status. A number of methods have been developed to quantitate the level of a protein, some amenable to high throughput screening. A method is described to measure the total level of the tumor suppressor p53 using scintillation proximity assay (SPA) beads and radiolabeled streptavidin. Three different cell extracts were used, with one used to develop the standard curve for the amount of p53. This method allows the specific detection of p53 in the range of 50 to 300 pg in 10 microl of an extract. While this detection is less than what can be detected by commercially available enzyme linked immunosorbent assay (ELISA) kits, the SPA compares favorably on time required and cost. This new assay also has the potential to be coupled with measurements for p53 DNA binding, a unique aspect of this approach.

Three assays show differences in binding of wild-type and mutant p53 to unique gene sequences.

Cancer-associated mutations in the p53 gene often change amino acids in the protein's DNA binding domain. We used three different binding assays specifically gel shift, DNA binding scintillation proximity assay and a streptavidin magnetic bead assay to analyze the DNA binding of the tumor suppressor p53 from 4 human cell lines with different DNA sequences from the mdm2, p21 and cyclin G genes and a mutant form of the cyclin G sequence. Treatment of MCF-7 cells having wild-type p53 with hydrogen peroxide increased the binding of p53 to DNA as detected using all three assays, but to different extents. The p53 proteins from the thyroid cancer cell lines with different p53 mutations (ARO, WRO and NPA) have comparable binding reactions in the three assays, but show different specificities for the sequences. Here we show that multiple different binding assays allow us to generate a more complete picture of the function of DNA transcription factors in diseases such as cancer.

Assembly-disassembly of DNAs and gold nanoparticles: a strategy of intervention based on oligonucleotides and restriction enzymes.

The ability to manipulate and intervene in the processes of assembly and disassembly of DNAs and nanoparticles is important for the exploitation of nanoparticles in medical diagnostics and drug delivery. This report describes the results of an investigation of a strategy to intervene in the assembly and disassembly processes of DNAs and gold nanoparticles based on two approaches. The first approach explores the viability of molecular intervention to the assembly-disassembly-reassembly process. The temperature-induced assembly and disassembly processes of DNAs and gold nanoparticles were studied as a model system to illustrate this approach. The introduction of a molecular recognition probe leads to intervention in the assembly-disassembly process depending on its specific biorecognition. This process was detected by monitoring the change in the optical properties of gold nanoparticles and their DNA assemblies. The second approach involves the disassembly of the DNA-linked assembly of nanoparticles using restriction enzymes (e.g., MspI). The presence of the double stranded DNAs in the nanoparticle assembly was also substantiated by a Southern blot. Implications of the results to exploration of the molecular intervention for fine-tuning interfacial reactivities in DNA-based bioassays are also discussed.