Large-scale phenotyping of an accurate genetic mouse model of JNCL identifies novel early pathology outside the central nervous system.

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.

Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells.

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.

Sensory regulation of neuroligins and neurexin I in the honeybee brain.

BACKGROUND: Neurexins and neuroligins, which have recently been associated with neurological disorders such as autism in humans, are highly conserved adhesive proteins found on synaptic membranes of neurons. These binding partners produce a trans-synaptic bridge that facilitates maturation and specification of synapses. It is believed that there exists an optimal spatio-temporal code of neurexin and neuroligin interactions that guide synapse formation in the postnatal developing brain. Therefore, we investigated whether neuroligins and neurexin are differentially regulated by sensory input using a behavioural model system with an advanced capacity for sensory processing, learning and memory, the honeybee. METHODOLOGY/PRINCIPAL FINDINGS: Whole brain expression levels of neuroligin 1-5 (NLG1-5) and neurexin I (NrxI) were estimated by qRT-PCR analysis in three different behavioural paradigms: sensory deprivation, associative scent learning, and lateralised sensory input. Sensory deprived bees had a lower level of NLG1 expression, but a generally increased level of NLG2-5 and NrxI expression compared to hive bees. Bees that had undergone associative scent training had significantly increased levels of NrxI, NLG1 and NLG3 expression compared to untrained control bees. Bees that had lateralised sensory input after antennal amputation showed a specific increase in NLG1 expression compared to control bees, which only happened over time. CONCLUSIONS/SIGNIFICANCE: Our results suggest that (1) there is a lack of synaptic pruning during sensory deprivation; (2) NLG1 expression increases with sensory stimulation; (3) concomitant changes in gene expression suggests NrxI interacts with all neuroligins; (4) there is evidence for synaptic compensation after lateralised injury.

Bridging the synaptic gap: neuroligins and neurexin I in Apis mellifera.

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31-246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate alpha- and beta-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3' region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3' splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.