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.

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.

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.

Single chain variable fragment antibodies directed against SOD1 ameliorate disease in mutant SOD1 transgenic mice.

Mutations in Cu/Zn superoxide dismutase (SOD1) are the cause of ~20% of cases of familial ALS (FALS), which comprise ~10% of the overall total number of cases of ALS. Mutant (mt) SOD1 is thought to cause FALS through a gain and not loss in function, perhaps as a result of the mutant protein's misfolding and aggregation. Previously we used a phage display library to raise single chain variable fragment antibodies (scFvs) against SOD1, which were found to decrease aggregation of mtSOD1 and toxicity in vitro. In the present study, we show that two scFvs directed against SOD1 ameliorate disease in G93A mtSOD1 transgenic mice and also decrease motor neuron loss, microgliosis, astrocytosis, as well as SOD1 burden and aggregation. The results suggest that the use of antibodies or antibody mimetics directed against SOD1 may be a useful therapeutic direction in mtSOD1-induced FALS. Since studies suggest that wild type SOD1 may be misfolded similar to that seen with mtSOD1, this therapeutic direction may be effective in sporadic as well as FALS.

Translation of dipeptide repeat proteins from the C9ORF72 expanded repeat is associated with cellular stress.

Expansion of a hexanucleotide repeat (HRE), GGGGCC, in the C9ORF72 gene is recognized as the most common cause of familial amyotrophic lateral sclerosis (FALS), frontotemporal dementia (FTD) and ALS-FTD, as well as 5-10% of sporadic ALS. Despite the location of the HRE in the non-coding region (with respect to the main C9ORF72 gene product), dipeptide repeat proteins (DPRs) that are thought to be toxic are translated from the HRE in all three reading frames from both the sense and antisense transcript. Here, we identified a CUG that has a good Kozak consensus sequence as the translation initiation codon. Mutation of this CTG significantly suppressed polyglycine-alanine (GA) translation. GA was translated when the G4C2 construct was placed as the second cistron in a bicistronic construct. CRISPR/Cas9-induced knockout of a non-canonical translation initiation factor, eIF2A, impaired GA translation. Transfection of G4C2 constructs induced an integrated stress response (ISR), while triggering the ISR led to a continuation of translation of GA with a decline in conventional cap-dependent translation. These in vitro observations were confirmed in chick embryo neural cells. The findings suggest that DPRs translated from an HRE in C9ORF72 aggregate and lead to an ISR that then leads to continuing DPR production and aggregation, thereby creating a continuing pathogenic cycle.

Poloxamer 188 decreases membrane toxicity of mutant SOD1 and ameliorates pathology observed in SOD1 mouse model for ALS.

Here we report a gain in function for mutant (mt) superoxide dismutase I (SOD1), a cause of familial amyotrophic lateral sclerosis (FALS), wherein small soluble oligomers of mtSOD1 acquire a membrane toxicity. Phosphatidylglycerol (PG) lipid domains are selectively targeted, which could result in membrane damage or "toxic channels" becoming active in the bilayer. This PG-selective SOD1-mediated membrane toxicity is largely reversible in vitro by a widely-available FDA-approved surfactant and membrane-stabilizer P188. Treatment of G93ASOD1 transgenic mice with P188 significantly delayed symptoms onset, extended survival and decreased motoneuron death. The use of P188 or an analogue, which targets mtSOD1 misfolding-induced membrane toxicity, may provide a new direction for ALS treatment.

Evidence for an early innate immune response in the motor cortex of ALS.

BACKGROUND: Recent evidence indicates the importance of innate immunity and neuroinflammation with microgliosis in amyotrophic lateral sclerosis (ALS) pathology. The MCP1 (monocyte chemoattractant protein-1) and CCR2 (CC chemokine receptor 2) signaling system has been strongly associated with the innate immune responses observed in ALS patients, but the motor cortex has not been studied in detail. METHODS: After revealing the presence of MCP1 and CCR2 in the motor cortex of ALS patients, to elucidate, visualize, and define the timing, location and the extent of immune response in relation to upper motor neuron vulnerability and progressive degeneration in ALS, we developed MCP1-CCR2-hSOD1G93A mice, an ALS reporter line, in which cells expressing MCP1 and CCR2 are genetically labeled by monomeric red fluorescent protein-1 and enhanced green fluorescent protein, respectively. RESULTS: In the motor cortex of MCP1-CCR2-hSOD1G93A mice, unlike in the spinal cord, there was an early increase in the numbers of MCP1+ cells, which displayed microglial morphology and selectively expressed microglia markers. Even though fewer CCR2+ cells were present throughout the motor cortex, they were mainly infiltrating monocytes. Interestingly, MCP1+ cells were found in close proximity to the apical dendrites and cell bodies of corticospinal motor neurons (CSMN), further implicating the importance of their cellular interaction to neuronal pathology. Similar findings were observed in the motor cortex of ALS patients, where MCP1+ microglia were especially in close proximity to the degenerating apical dendrites of Betz cells. CONCLUSIONS: Our findings reveal that the intricate cellular interplay between immune cells and upper motor neurons observed in the motor cortex of ALS mice is indeed recapitulated in ALS patients. We generated and characterized a novel model system, to study the cellular and molecular basis of this close cellular interaction and how that relates to motor neuron vulnerability and progressive degeneration in ALS.

Intermuscular coherence in amyotrophic lateral sclerosis: A preliminary assessment.

INTRODUCTION: Simple laboratory tests of upper motor neuron involvement in amyotrophic lateral sclerosis (ALS) are not available. Intermuscular coherence has been shown to distinguish patients with primary lateral sclerosis, a pure upper motor neuron disorder, from normal subjects, suggesting it could be useful for assessing ALS. We aimed to determine whether intermuscular coherence can distinguish ALS patients from normal subjects. METHODS: We measured biceps brachii and brachioradialis activity using surface electromyography while subjects held the elbow at flexion and the forearm in semipronation. Intermuscular coherence was calculated at between 20 and 40 Hz in 15 ALS patients and 15 normal subjects. RESULTS: On average, intermuscular coherence was 3.8-fold greater in normal subjects than in ALS patients (P < 0.01), and it distinguished ALS patients from normal subjects with a sensitivity of 87% and specificity of 87%. CONCLUSION: Intermuscular coherence measurement is a rapid, painless method that may detect upper motor neuron dysfunction in ALS. Muscle Nerve 55: 862-868, 2017.

An enhanced integrated stress response ameliorates mutant SOD1-induced ALS.

Varied stresses to cells can lead to a repression in translation by triggering phosphorylation of eukaryotic translation initiator factor 2α (eIF2α), which is central to a process known as the integrated stress response (ISR). PKR-like ER-localized eIF2 kinase (PERK), one of the kinases that phosphorylates eIF2α and coordinates the ISR, is activated by stress occurring from the accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER). Mutant Cu/Zn superoxide dismutase (mtSOD1) is thought to cause familial amyotrophic lateral sclerosis (FALS) because it misfolds and aggregates. Published studies have suggested that ER stress is involved in FALS pathogenesis since mtSOD1 accumulates inside the ER and activates PERK leading to phosphorylated eIF2α (p-eIF2α). We previously used a genetic approach to show that haploinsufficiency of PERK significantly accelerates disease onset and shortens survival of G85R mtSOD1 FALS transgenic mice. We now show that G85R mice that express reduced levels of active GADD34, which normally dephosphorylates p-eIF2α and allows recovery from the global suppression of protein synthesis, markedly ameliorates disease. These studies emphasize the importance of the ISR, and specifically the PERK pathway, in the pathogenesis of mtSOD1-induced FALS and as a target for treatment. Furthermore, the ISR may be an appropriate therapeutic target for sporadic ALS and other neurodegenerative diseases since misfolded proteins have been implicated in these disorders.

Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function.

Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1(nm3419) (UCHL1(-/-)) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1(-/-) mice as a tool to study CSMN pathology.

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.

Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis.

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) cases are caused by mutant superoxide dismutase type 1 (mtSOD1). Although the mechanisms of mtSOD1-induced toxicity remain poorly understood, evidence suggests that accumulation of misfolded SOD1 is fundamental to its toxicity and the death of motor neurons. Misfolded mtSOD1 can accumulate inside the endoplasmic reticulum (ER), leading to ER stress, with activation of the unfolded protein response (UPR). We have previously carried out genetic studies focused on PERK (which is an eIF2α kinase that is rapidly activated in response to ER stress and leads to a repression in translation) and GADD34 (which participates in the dephosphorylation of eIF2α). We reported that mtSOD1 transgenic mice that are haploinsufficient for PERK have a significantly accelerated ALS disease, while mtSOD1 mice that are mutated for GADD34 have a remarkably ameliorated disease. Guanabenz, a centrally acting oral drug approved for the treatment of hypertension, enhances the PERK pathway by selectively inhibiting GADD34-mediated dephosphorylation of eIF2α. We have now treated G93A mtSOD1 transgenic mice with guanabenz and found a significant amelioration of disease with a delay in the onset and prolongation of the early phase of disease and survival. Guanabenz-treated G93A mice have less accumulation of mtSOD1 and an enhanced phosphorylation of eIF2α at endstage. This study further emphasizes the importance of the PERK pathway in the pathogenesis of FALS and as a therapeutic target in ALS, and identifies guanabenz as a candidate drug for the treatment of ALS patients.

Venous thromboembolism after diaphragm pacing in amyotrophic lateral sclerosis.

INTRODUCTION: Patients with amyotrophic lateral sclerosis (ALS) are prone to venous thromboembolism (VTE) and secondary complications. Because there is an increased incidence of VTE after surgical procedures, placement of a Diaphragm Pacing System (DPS) in ALS patients as treatment for respiratory muscle weakness could potentially increase the incidence of VTE, especially in patients with limited mobility. METHODS: We implanted a DPS in 10 ALS patients who met the criteria for this procedure. They underwent a preoperative evaluation as recommended by the guidelines. RESULTS: We report 2 patients with no symptoms of deep vein thrombosis (DVT) before the surgical procedure who then developed perioperative VTE. CONCLUSIONS: These patients highlight the need to consider preoperative screening for DVT and postoperative thromboprophylaxis in high-risk ALS patients who undergo DPS placement.

Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity.

Astrocyte loss of mutant SOD1 delays ALS disease onset and progression in G85R transgenic mice.

Approximately 10% of patients with amyotrophic lateral sclerosis (ALS) have familial ALS (FALS), and 20% of FALS are caused by mutations of superoxide dismutase type 1 (MTSOD1). The fact that some MTSOD1s that cause FALS have full dismutase activity (e.g. G37R) and others no dismutase activity (e.g. G85R) suggests that MTSOD1 causes FALS due to toxicity of the protein rather than a loss in enzymatic function. Compelling data have demonstrated that motor neuron (MN) degeneration can result from a non-cell autonomous effect of the MTSOD1. In order to clarify the role of astrocytes in FALS, we deleted MTSOD1 in astrocytes of G85R transgenic mice. In contrast to a similar study using G37R mice in which astrocyte MTSOD1 loss affected only the late phase of ALS disease, we found that astrocyte MTSOD1 loss in G85R mice delayed disease onset and prolonged the early phase of disease progression, without affecting the late phase. In addition, astrocyte G85R knockdown resulted in decreased microgliosis, decreased SOD1-immunoreactive inclusions and preservation of GLT-1 transporter expression. The differential effects of astrocyte G85R versus G37R knockdown on MN death demonstrate SOD1 mutation-specific effects on ALS pathogenesis; these differences may be a result of the different dismutase activities of the two mutants. The effect of the knockdown of G85R expression in astrocytes on onset as well as disease duration highlights the importance of this cell type in FALS.

The unfolded protein response in familial amyotrophic lateral sclerosis.

Mutant superoxide dismutase type 1 (MTSOD1) is thought to cause ∼20% of cases of familial amyotrophic lateral sclerosis (FALS) because it misfolds and aggregates. Previous studies have shown that MTSOD1 accumulates inside the endoplasmic reticulum (ER) and activates the unfolded protein response (UPR), suggesting that ER stress is involved in the pathogenesis of FALS. We used a genetic approach to investigate the role of the UPR in FALS. We crossed G85RSOD1 transgenic mice with pancreatic ER kinase haploinsufficient (PERK(+/-)) mice to obtain G85R/PERK(+/-) mice. PERK(+/-) mice carry a loss of function mutation of PERK, which is the most rapidly activated UPR pathway, but have no abnormal phenotype. Compared with G85R transgenic mice, G85R/PERK(+/-) mice had a dramatically accelerated disease onset as well as shortened disease duration and lifespan. There was also acceleration of the pathology and earlier MTSOD1 aggregation. A diminished PERK response accelerated disease and pathology in G85R transgenic mice presumably because the mice had a reduced capacity to turn down synthesis of misfolded SOD1, leading to an early overloading of the UPR. The results indicate that the UPR has a significant influence on FALS, and suggest that enhancing the UPR may be effective in treating ALS.

Translation of dipeptide repeat proteins in C9ORF72 ALS/FTD through unique and redundant AUG initiation codons.

A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Translation of sense DPRs, such as poly-GA and poly-GR, depends on non-canonical (non-AUG) initiation codons. Here, we provide evidence for canonical AUG-dependent translation of two antisense DPRs, poly-PR and poly-PG. A single AUG is required for synthesis of poly-PR, one of the most toxic DPRs. Unexpectedly, we found redundancy between three AUG codons necessary for poly-PG translation. Further, the eukaryotic translation initiation factor 2D (EIF2D), which was previously implicated in sense DPR synthesis, is not required for AUG-dependent poly-PR or poly-PG translation, suggesting that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings on DPR synthesis from the C9ORF72 locus may be broadly applicable to many other nucleotide repeat expansion disorders.

Selective knockdown of mutant SOD1 in Schwann cells ameliorates disease in G85R mutant SOD1 transgenic mice.

Mutants of superoxide dismutase type 1 (mtSOD1) that have full dismutase activity (e.g., G37R) as well as none (e.g., G85R) cause familial amyotrophic lateral sclerosis (FALS), indicating that mtSOD1-induced FALS results from a toxicity rather than loss in SOD1 enzymatic activity. Still, it has remained unclear whether mtSOD1 dismutase activity can influence disease. A previous study demonstrated that Cre-mediated knockdown of G37R expression in Schwann cells (SCs) of G37R transgenic mice shortened the late phase of disease and survival. These results suggested that the neuroprotective effect of G37R expressed in SCs was greater than its toxicity, presumably because its dismutase activity counteracted reactive oxygen species (ROS). In order to further investigate this, we knocked down G85R in SCs by crossing G85R(flox) mice with myelin-protein-zero (P(0)):Cre mice, which express Cre recombinase in SCs. Knockdown of G85R in SCs of G85R mice delayed disease onset and extended survival indicating that G85R expression in SCs is neurotoxic. These results demonstrate differences in the effect on disease of dismutase active vs. inactive mtSOD1 suggesting that both a loss as well as gain in function of mtSOD1 influence FALS pathogenesis. The results suggest that mtSOD1-induced FALS treatment may have to be adjusted depending on the cell type targeted and particular mtSOD1 involved.

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.