ALS-Related Mutant FUS Protein Is Mislocalized to Cytoplasm and Is Recruited into Stress Granules of Fibroblasts from Asymptomatic FUS P525L Mutation Carriers.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) shows a strong genetic basis, with SOD1, FUS, TARDBP, and C9ORF72 being the genes most frequently involved. This has allowed identification of asymptomatic mutation carriers, which may be of help in understanding the molecular changes preceding disease onset. OBJECTIVES: We studied the cellular expression of FUS protein and the effect of heat-shock- and dithiothreitol-induced stress in fibroblasts from FUS P525L mutation carriers, healthy controls, and patients with sporadic ALS. METHODS: Western blots and immunocytochemistry were performed to study the subcellular localization of FUS protein. Control and stressed cells were double stained with FUS and the stress marker TIA-R. RESULTS: Fibroblasts from healthy controls and sporadic ALS cases showed a prominent nuclear FUS expression. In the 2 FUS P525L mutation carriers, instead, most cells showed a protein localization in both nucleus and cytoplasm, or exclusively in the cytoplasm. Stress prompted the formation of cytoplasmic granules in all subjects and in sporadic ALS FUS mislocalization to the cytoplasm. Cytoplasmic FUS was recruited into stress granules, which showed a time-dependent decrease in all subjects. However, in the FUS P525L fibroblasts, the granules persisted longer, and they were more numerous than those detected in the cells from controls and sporadic ALS patients. CONCLUSIONS: We show that in fibroblasts of FUS P525L mutation carriers, FUS mislocalized to the cytoplasm where it redistributed into stress granules with likely a dose effect, i.e. a higher number of cells with granules, which persist longer, than in controls and ALS cases. These data represent an early molecular change occurring before ALS onset, suggesting a transient preaggregative state.

Hsp60 response in experimental and human temporal lobe epilepsy.

The mitochondrial chaperonin Hsp60 is a ubiquitous molecule with multiple roles, constitutively expressed and inducible by oxidative stress. In the brain, Hsp60 is widely distributed and has been implicated in neurological disorders, including epilepsy. A role for mitochondria and oxidative stress has been proposed in epileptogenesis of temporal lobe epilepsy (TLE). Here, we investigated the involvement of Hsp60 in TLE using animal and human samples. Hsp60 immunoreactivity in the hippocampus, measured by Western blotting and immunohistochemistry, was increased in a rat model of TLE. Hsp60 was also increased in the hippocampal dentate gyrus neurons somata and neuropil and hippocampus proper (CA3, CA1) of the epileptic rats. We also determined the circulating levels of Hsp60 in epileptic animals and TLE patients using ELISA. The epileptic rats showed circulating levels of Hsp60 higher than controls. Likewise, plasma post-seizure Hsp60 levels in patients were higher than before the seizure and those of controls. These results demonstrate that Hsp60 is increased in both animals and patients with TLE in affected tissues, and in plasma in response to epileptic seizures, and point to it as biomarker of hippocampal stress potentially useful for diagnosis and patient management.

Comparative analysis of Hsp10 and Hsp90 expression in healthy mucosa and adenocarcinoma of the large bowel.

BACKGROUND: Heat shock proteins (Hsps) assist other proteins in their folding and drive the degradation of defective proteins. During evolution, these proteins have also acquired other roles. Hsp10 is involved in immunomodulation and tumor progression. Hsp90 stabilizes a range of "client" proteins involved in cell signaling. The present study evaluated the expression levels of Hsp10 and Hsp90 in normal mucosa and adenocarcinoma samples of human large bowel. MATERIALS AND METHODS: Samples of normal mucosa and adenocarcinoma were collected and Reverse transcriptase-polymerase chain reaction RT-PCR, western blotting (WB) analyses, as well as immunohistochemistry were performed to evaluate the expression levels of Hsp10 and Hsp90. RESULTS: RT-PCR showed a higher gene expression of Hsp10 and Hsp90 in adenocarcinoma samples compared to healthy mucosa. WB results confirmed these findings. Immunohistochemistry revealed higher levels of Hsp10 in adenocarcinoma in both the epithelium and the lamina propria, while Hsp90 expression was higher in the adenocarcinoma samples only in the lamina propria. CONCLUSION: Hsp10 and Hsp90 may be involved in large bowel carcinogenesis.