👤 John F Staropoli

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. Show less

Neuronal ceroid lipofuscinosis (NCL) is a genetically heterogeneous group of lysosomal diseases that collectively compose the most common Mendelian form of childhood-onset neurodegeneration. It is estimated that ∼8% of individuals diagnosed with NCL by conservative clinical and histopathologic criteria have been ruled out for mutations in the nine known NCL-associated genes, suggesting that additional genes remain unidentified. To further understand the genetic underpinnings of the NCLs, we performed whole-exome sequencing on DNA samples from a Mexican family affected by a molecularly undefined form of NCL characterized by infantile-onset progressive myoclonic epilepsy (PME), vision loss, cognitive and motor regression, premature death, and prominent NCL-type storage material. Using a recessive model to filter the identified variants, we found a single homozygous variant, c.550C>T in KCTD7, that causes a p.Arg184Cys missense change in potassium channel tetramerization domain-containing protein 7 (KCTD7) in the affected individuals. The mutation was predicted to be deleterious and was absent in over 6,000 controls. The identified variant altered the localization pattern of KCTD7 and abrogated interaction with cullin-3, a ubiquitin-ligase component and known KCTD7 interactor. Intriguingly, murine cerebellar cells derived from a juvenile NCL model (CLN3) showed enrichment of endogenous KCTD7. Whereas KCTD7 mutations have previously been linked to PME without lysosomal storage, this study clearly demonstrates that KCTD7 mutations also cause a rare, infantile-onset NCL subtype designated as CLN14. Show less

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development. Show less

Loss-of-function mutations in CLN3 are responsible for juvenile-onset neuronal ceroid lipofuscinosis (JNCL), or Batten disease, which is an incurable lysosomal disease that manifests with vision loss, followed by seizures and progressive neurodegeneration, robbing children of motor skills, speech and cognition, and eventually leading to death in the second or third decade of life. Emerging clinical evidence points to JNCL pathology outside of the CNS, including the cardiovascular system. The CLN3 gene encodes an unusual transmembrane protein, CLN3 or battenin, whose elusive function has been the subject of intense study for more than 10 years. Owing to the detailed characterization of a large number of disease models, our knowledge of CLN3 protein function is finally coming into focus. This review will describe the most current understanding of CLN3 structure, function and dysfunction in JNCL. Show less

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf) cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8) cerebellar cells. CbCln6(nclf/nclf) cells and CbCln3(Δex7/8/Δex7/8) cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf) cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf) and CbCln3(Δex7/8/Δex7/8) cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8) and Cln6(nclf) mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival. Show less