Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and copper/zinc superoxide dismutase (SOD1) interact to modulate NFL mRNA stability. Implications for altered RNA processing in amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive motor neuron degeneration in association with neurofilament (NF) aggregate formation. This process is accompanied by an alteration in the stoichiometry of NF subunit protein expression such that the steady state levels of the low molecular weight NF (NFL) mRNA levels are selectively suppressed. We have previously shown that each of TDP-43, 14-3-3 and mutant SOD1 can function as NFL mRNA 3'UTR binding proteins that directly affect the stability of NFL transcripts. In this study, we demonstrate that the interaction of TDP-43 with the NFL mRNA 3' UTR involves ribonucleotide (UG) motifs present on stem loops of the 3'UTR as well as the RRM1 and RRM2 motifs of TDP-43. Ex vivo, TDP-43, 14-3-3 and SOD1 proteins interact to modulate NFL mRNA stability, although in vivo, only TDP-43 and either mutant or wild-type SOD1 co-localize in ALS motor neurons. TDP-43 was observed to co-localize to RNA transport granules (Staufen immunoreactive) in both control and ALS spinal motor neurons. In contrast, both stress granules (TIA-1 immunoreactive) and processing bodies (P-bodies; XRN-1 immunoreactive) were more prevalent in ALS motor neurons than in controls and demonstrated strong co-localization with TDP-43. Using RNA-IP-PCR, we further demonstrate that NFL mRNA is preferentially sequestered to both stress granules and P-bodies in ALS. These data suggest that NFL mRNA processing is fundamentally altered in ALS spinal motor neurons to favour compartmentalization within both stress granules and P-bodies, and that TDP-43 plays a fundamental role in this process.

Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases.

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.

Proteomic profiling: a novel method for differential diagnosis?

Tumors with identical phenotype can have markedly different biologic behavior. The differentiation between hemangioblastoma, a benign vascular brain tumor, and renal cell carcinoma (RCC), a malignant tumor that can metastasize to the brain, is a well-known pathological quandary. We report a 59-year-old female with von Hippel-Lindau disease and known RCC. She presented with a cystic cerebellar tumor that had typical MRI appearance of a hemangioblastoma. Progressive symptoms led to surgical intervention. The histology included features consistent with either hemangioblastoma with epitheloid phenotype or atypical RCC metastasis mimicking hemangioblastoma. The discrepancy between the benign clinical course and the histologic phenotype could not be clearly resolved using conventional clinical techniques. We used proteomic profiling to clarify this diagnostic dilemma. We compared the proteomic expression pattern of the unknown lesion with the patterns of three typical hemangioblastomas and three typical RCCs by using selective tissue microdissection combined with two-dimensional proteomic profiling and protein sequencing. The results clearly indicated a protein profile consistent with RCC. Proteomics may complement pathological evaluation and characterization of tumors in the future. Furthermore, these results show that metastasis of malignant RCC is not necessarily equivalent to aggressive growth patterns.