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 CbCln6nclf/nclf cerebellar cells and compared them to wild-type and CbCln3Δex7/8/Δex7/8 cerebellar cells. CbCln6nclf/nclf cells and CbCln3Δex7/8/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 CbCln6nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6nclf/nclf and CbCln3Δex7/8/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 Cln3Δex7/8 and Cln6nclf 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

Influence of species and anatomical location on chondrocyte expansion

<p>Abstract</p> <p>Background</p> <p>Bovine articular cartilage is often used to study chondrocytes <it>in vitro</it>. It is difficult to correlate <it>in vitro </it>studies using bovine chondrocytes with <it>in vivo </it>studies using other species such as rabbits and sheep. The aim of this investigation was to study the effect of species, anatomical location and exogenous growth factors on chondrocyte proliferation <it>in vitro</it>.</p> <p>Methods</p> <p>Equine (EQ), bovine (BO) and ovine (OV) articular chondrocytes from metacarpophalangeal (fetlock (F)), shoulder (S) and knee (K) joints were cultured in tissue culture flasks. Growth factors (rh-FGFb: 10 ng/ml; rh-TGFβ: 5 ng/ml) were added to the cultures at days 2 and 4. On day 6, cells were counted and flow cytometry analysis was performed to determine cell size and granularity. A three factor ANOVA with paired Tukey's correction was used for statistical analysis.</p> <p>Results</p> <p>After 6 days in culture, cell numbers had increased in control groups of EQ-F, OV-S, OV-F and BO-F chondrocytes. The addition of rh-FGFb led to the highest increase in cell numbers in the BO-F, followed by EQ-F and OV-S chondrocytes. The addition of rh-TGFβ increased cell numbers in EQ-S and EQ-F chondrocytes, but showed nearly no effect on EQ-K, OV-K, OV-S, OV-F and BO-F chondrocytes. There was an overall difference with the addition of growth factors between the different species and joints.</p> <p>Conclusion</p> <p>Different proliferation profiles of chondrocytes from the various joints were found. Therefore, we recommend performing <it>in vitro </it>studies using the species and site where subsequent <it>in vivo </it>studies are planned.</p