Kozak Similarity Score Algorithm Identifies Alternative Translation Initiation Codons Implicated in Cancers.

Ribosome profiling and mass spectroscopy have identified canonical and noncanonical translation initiation codons (TICs) that are upstream of the main translation initiation site and used to translate oncogenic proteins. There have previously been conflicting reports about the patterns of nucleotides that surround noncanonical TICs. Here, we use a Kozak Similarity Score algorithm to find that nearly all of these TICs have flanking nucleotides closely matching the Kozak sequence. Remarkably, the nucleotides flanking alternative noncanonical TICs are frequently closer to the Kozak sequence than the nucleotides flanking TICs used to translate the gene's main protein. Of note, the 5' untranslated region (5'UTR) of cancer-associated genes with an upstream TIC tend to be significantly longer than the same region in genes not associated with cancer. The presence of a longer-than-typical 5'UTR increases the likelihood of ribosome binding to upstream noncanonical TICs, and may be a distinguishing feature of a number of genes overexpressed in cancer. Noncanonical TICs that are located in the 5'UTR, although thought by some to be disadvantageous and suppressed by evolution, may translate oncogenic proteins because of their flanking nucleotides.

RNA-binding protein altered expression and mislocalization in MS.

OBJECTIVE: To determine whether there are nuclear depletion and cellular mislocalization of RNA-binding proteins (RBPs) transactivation response DNA-binding protein of 43 kDa (TDP-43), fused in sarcoma (FUS), and polypyrimidine tract-binding protein (PTB) in MS, as is the case in amyotrophic lateral sclerosis (ALS) and oligodendrocytes infected with Theiler murine encephalomyelitis virus (TMEV), we examined MS lesions and in vitro cultured primary human brain-derived oligodendrocytes. METHODS: Nuclear depletion and mislocalization of TDP-43, FUS, and PTB are thought to contribute to the pathogenesis of ALS and TMEV demyelination. The latter findings prompted us to investigate these RBPs in the demyelinated lesions of MS and in in vitro cultured human brain-derived oligodendrocytes under metabolic stress conditions. RESULTS: We found (1) mislocalized TDP-43 in oligodendrocytes in active lesions in some patients with MS; (2) decreased PTB1 expression in oligodendrocytes in mixed active/inactive demyelinating lesions; (3) decreased nuclear expression of PTB2 in neurons in cortical demyelinating lesions; and (4) nuclear depletion of TDP-43 in oligodendrocytes under metabolic stress induced by low glucose/low nutrient conditions compared with optimal culture conditions. CONCLUSION: TDP-43 has been found to have a key role in oligodendrocyte function and viability, whereas PTB is important in neuronal differentiation, suggesting that altered expression and mislocalization of these RBPs in MS lesions may contribute to the pathogenesis of demyelination and neurodegeneration. Our findings also identify nucleocytoplasmic transport as a target for treatment.

Knockdown of GADD34 in neonatal mutant SOD1 mice ameliorates ALS.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause ~20% of familial ALS (FALS), which comprises 10% of total ALS cases. In mutant SOD1- (mtSOD1-) induced ALS, misfolded aggregates of SOD1 lead to activation of the unfolded protein response/integrated stress response (UPR/ISR). Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a kinase that phosphorylates eukaryotic translation initiator factor 2α (p-eIF2α), coordinates the response by causing a global suppression of protein synthesis. Growth arrest and DNA damage 34 (GADD34) dephosphorylates p-eIF2α, allowing protein synthesis to return to normal. If the UPR/ISR is overwhelmed by the amount of misfolded protein, CCAAT/enhancer-binding homologous protein (CHOP) is activated leading to apoptosis. In the current study we investigated the effect of knocking down CHOP and GADD34 on disease of G93A and G85R mtSOD1 mice. Although a CHOP antisense oligonucleotide had no effect on survival, an intravenous injection of GADD34 shRNA encoded in adeno-associated virus 9 (AAV9) into neonatal G93A as well as neonatal G85R mtSOD1 mice led to a significantly increased survival. G85R mtSOD1 mice had a reduction in SOD1 aggregates/load, astrocytosis, and microgliosis. In contrast, there was no change in disease phenotype when GADD34 shRNA was delivered to older G93A mtSOD1 mice. Our current study shows that GADD34 shRNA is effective in ameliorating disease when administered to neonatal mtSOD1 mice. Targeting the UPR/ISR may be beneficial in mtSOD1-induced ALS as well as other neurodegenerative diseases in which misfolded proteins and ER stress have been implicated.

TDP-43 proteinopathy in Theiler's murine encephalomyelitis virus infection.

TDP-43, an RNA-binding protein that is primarily nuclear and important in splicing and RNA metabolism, is mislocalized from the nucleus to the cytoplasm of neural cells in amyotrophic lateral sclerosis (ALS), and contributes to disease. We sought to investigate whether TDP-43 is mislocalized in infections with the acute neuronal GDVII strain and the persistent demyelinating DA strain of Theiler's virus murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus of Picornaviridae because: i) L protein of both strains is known to disrupt nucleocytoplasmic transport, including transport of polypyrimidine tract binding protein, an RNA-binding protein, ii) motor neurons and oligodendrocytes are targeted in both TMEV infection and ALS. TDP-43 phosphorylation, cleavage, and cytoplasmic mislocalization to an aggresome were observed in wild type TMEV-infected cultured cells, with predicted splicing abnormalities. In contrast, cells infected with DA and GDVII strains that have L deletion had rare TDP-43 mislocalization and no aggresome formation. TDP-43 mislocalization was also present in neural cells of TMEV acutely-infected mice. Of note, TDP-43 was mislocalized six weeks after DA infection to the cytoplasm of oligodendrocytes and other glial cells in demyelinating lesions of spinal white matter. A recent study showed that TDP-43 knock down in oligodendrocytes in mice led to demyelination and death of this neural cell [1], suggesting that TMEV infection mislocalization of TDP-43 and other RNA-binding proteins is predicted to disrupt key cellular processes and contribute to the pathogenesis of TMEV-induced diseases. Drugs that inhibit nuclear export may have a role in antiviral therapy.

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.7 × 10-6). Postmortem neuropathology of five TIA1 mutations carriers showed a consistent pathological signature with numerous round, hyaline, TAR DNA-binding protein 43 (TDP-43)-positive inclusions. TIA1 mutations significantly increased the propensity of TIA1 protein to undergo phase transition. In live cells, TIA1 mutations delayed stress granule (SG) disassembly and promoted the accumulation of non-dynamic SGs that harbored TDP-43. Moreover, TDP-43 in SGs became less mobile and insoluble. The identification of TIA1 mutations in ALS/FTD reinforces the importance of RNA metabolism and SG dynamics in ALS/FTD pathogenesis.

S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects.

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord.

Neoplastic cauda equina syndrome: a neuroimaging-based review.

Cauda equina syndrome refers to dysfunction of the cauda equina, the collection of ventral and dorsal lumbar, sacral and coccygeal nerve roots that surround the filum terminale. This most commonly occurs as a result of compression by a herniated lumbosacral disc. However, the syndrome may also complicate metastatic cancer or a primary neoplasm within or infiltrating the spinal canal. An accurate and timely diagnosis is critical to avoid irreversible loss of neurological function. The clinician and radiologist must therefore be aware of the many possible causes to guide timely management. Here we review the diverse neoplastic causes affecting the cauda equina nerve roots from a neuroimaging-based perspective. We divide them by location into intramedullary neoplasms at the conus (such as astrocytoma), intradural-extramedullary neoplasms (such as schwannoma and leptomeningeal metastases) and extradural neoplasms (such as spinal metastases from systemic neoplasms). We also discuss the clinical features associated with cauda equina tumours, with special focus on cauda equina syndrome.

Palmitoylation of superoxide dismutase 1 (SOD1) is increased for familial amyotrophic lateral sclerosis-linked SOD1 mutants.

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.

Mutant SOD1 forms ion channel: implications for ALS pathophysiology.

Point mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) impart a gain-of-function to this protein that underlies 20-25% of all familial amyotrophic lateral sclerosis (FALS) cases. However, the specific mechanism of mutant SOD1 toxicity has remained elusive. Using the complementary techniques of atomic force microscopy (AFM), electrophysiology, and cell and molecular biology, here we examine the structure and activity of A4VSOD1, a mutant SOD1. AFM of A4VSOD1 reconstituted in lipid membrane shows discrete tetrameric pore-like structure with outer and inner diameters 12.2 and 3.0nm respectively. Electrophysiological recordings show distinct ionic conductances across bilayer for A4VSOD1 and none for wildtype SOD1. Mouse neuroblastoma cells exposed to A4VSOD1 undergo membrane depolarization and increases in intracellular calcium. These results provide compelling new evidence that a mutant SOD1 is capable of disrupting cellular homeostasis via an unregulated ion channel mechanism. Such a "toxic channel" mechanism presents a new therapeutic direction for ALS research.

Theiler's murine encephalomyelitis virus L* amino acid position 93 is important for virus persistence and virus-induced demyelination.

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection associated with an inflammatory white matter demyelinating disease. TO subgroup strains synthesize an 18-kDa protein, L*, out of frame with the polyprotein from an initiation codon 13 nucleotides downstream from the polyprotein's AUG codon. We previously generated a mutant virus from our infectious DA full-length clone that has a change of the L* AUG codon to ACG (with no change in the polyprotein's amino acid sequence). Studies of this mutant virus showed that L* was key to the TO subgroup phenotype because the mutant had a decreased ability to persist and demyelinate. This work was initially called into question because a similar mutant derived from a different full-length DA infectious clone persisted and demyelinated similarly to wild-type DA virus (O. van Eyll and T. Michiels, J. Virol. 74:9071-9077, 2000). We now report that (i) the sequence of the L* coding region differs in the two infectious clones, resulting in a Ser or Leu as the predicted amino acid at position 93 of L* (with no change in the polyprotein's amino acid sequence), (ii) the difference in this amino acid is key to the phenotypic differences between the two mutants, and (iii) the change in amino acid 93 may affect L* phosphorylation. It is of interest that this amino acid only appears critical in determining the virus phenotype when L* is present in a significantly reduced amount (i.e., following translation from an ACG initiating codon).

The effect of mutant SOD1 dismutase activity on non-cell autonomous degeneration in familial amyotrophic lateral sclerosis.

Mutant superoxide dismutase type 1 (MTSOD1), the most common known cause of familial amyotrophic lateral sclerosis (FALS), is believed to cause FALS as a result of a toxicity of the protein. MTSOD1s with full dismutase enzymatic activity (e.g., G37R) and without any enzymatic activity (e.g., G85R) cause FALS, demonstrating that the ability of MTSOD1 to cause FALS is not dependent on the dismutase activity; however, it remains unclear whether MTSOD1 dismutase activity can influence disease phenotype. In the present study, we selectively knocked down G85R expression in particular cell types of G85R mice. Results following knockdown of G85R in motor neurons (MNs)/interneurons of G85R mice were similar to results from a published study involving knockdown of G37R in G37R mice; however, G85R knockdown in microglia/macrophages induced a prolonged early and late disease phase while G37R knockdown in the same cells only affected late phase. These results show that: (i) MN as well as non-MN expression of G85R, like G37R, has a significant effect on disease in transgenic mice - indicating the role of non-cell autonomous degeneration in both dismutase-active and inactive MTSOD1s. (ii) The effect of MTSOD1 expression in microglia/macrophages varies with different mutants, and may be influenced by the MTSOD1's dismutase activity.

Molecular mechanisms of attenuation of the Sabin strain of poliovirus type 3.

Mutations critical for the central nervous system (CNS) attenuation of the Sabin vaccine strains of poliovirus (PV) are located within the viral internal ribosome entry site (IRES). We examined the interaction of the IRESs of PV type 3 (PV3) and Sabin type 3 (Sabin3) with polypyrimidine tract-binding protein (PTB) and a neural cell-specific homologue, nPTB. PTB and nPTB were found to bind to a site directly adjacent to the attenuating mutation, and binding at this site was less efficient on the Sabin3 IRES than on the PV3 IRES. Translation mediated by the PV3 and Sabin3 IRESs in neurons of the chicken embryo spinal cord demonstrated a translation deficit for the Sabin3 IRES that could be rescued by increasing PTB expression in the CNS. These data suggest that the low levels of PTB available in the CNS, coupled to a reduced binding of PTB on the Sabin3 IRES, leads to its CNS-specific attenuation. This study also demonstrates the use of the chicken embryo to easily investigate translation of RNA within a neuron in the CNS of an intact living organism.

Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells.

The neuropathology of Parkinson's Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.

Glutamate carboxypeptidase II inhibition protects motor neurons from death in familial amyotrophic lateral sclerosis models.

Approximately 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and approximately 20% of these cases of familial ALS (FALS) are caused by mutations of copper/zinc superoxide dismutase type 1. Glutamate excitotoxicity has been implicated as a mechanism of MN death in both ALS and FALS. In this study, we tested whether a neuroprotective strategy involving potent and selective inhibitors of glutamate carboxypeptidase II (GCPII), which converts the abundant neuropeptide N-acetylaspartylglutamate to glutamate, could protect MNs in an in vitro and animal model of FALS. Data suggest that the GCPII inhibitors prevented MN cell death in both of these systems because of the resultant decrease in glutamate levels. GCPII inhibition may represent a new therapeutic target for the treatment of ALS.

Membrane lymphotoxin is required for resistance to Theiler's virus infection.

Lymphotoxin (LT) and tumor necrosis factor (TNF) are important in immune system development and function. LT consists of soluble LT-alpha(3), which binds to TNF-R1 and TNF-R2, and membrane LT-alpha(1)beta(2), which binds to LT-beta-R. We investigated the role of LT and TNF in disease induced by Daniel's (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) since the immune response is believed to be important in both resistance to DA infection as well as mediation of virus-induced demyelination. DA persisted and induced inflammatory demyelination in LT-alpha(-/-) (but not TNF(-/-)) weanling mice of a normally resistant haplotype (C57BL/6), suggesting that LT, but not TNF, is critical for resistance to DA infection. This activity of LT depends on membrane LT-alpha(1)beta(2) and not soluble LT-alpha(3), since DA virus persisted and induced inflammatory demyelination in LT-beta-R(-/-), but not TNF-R1(-/-) or TNF-R2(-/-), mice. The LT-alpha(-/-) and LT-beta-R(-/-) mice failed to mount a virus-specific cytotoxic T cell response. Treatment of weanling C57BL/6 mice with LT-beta-R-Ig, which blocks membrane LT activity, failed to increase susceptibility, suggesting that the LT effect is related to its action on immune system development which is fixed by 3 weeks of age. Our data suggest that membrane LT is important in resistance to DA infection (possibly through interference with CD8+ T cell development and function). There was relatively little demyelination associated with inflammation in LT-alpha(-/-) and LT-beta-R(-/-) mice compared to susceptible SJL mice, suggesting the possibility that LT plays a role in mediating demyelination.