Lorne Zinman

A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD

The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal-dominant genes underlying these common neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK, and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one-third of familial ALS cases of outbred European descent, making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.

IV immunoglobulin in patients with myasthenia gravis : A randomized controlled trial

Objective: We aimed to determine the effectiveness of IV immunoglobulin (IVIG) in the treatment of patients with myasthenia gravis (MG) and worsening weakness in a randomized, placebo-controlled, masked study. Methods: Fifty-one patients with worsening weakness due to MG were randomized to infusion with 2 g/kg of IVIG or an equivalent volume of IV dextrose 5% in water. The Quantitative Myasthenia Gravis (QMG) Score for Disease Severity, a validated clinical composite scale, was calculated by a masked observer at baseline and days 14 and 28. Results: In IVIG-treated patients, a clinically meaningful improvement in QMG Score for Disease Severity was observed at day 14 and persisted at day 28. The greatest improvement occurred in patients with more severe disease as defined by a QMG Score for Disease Severity greater than 10.5. Conclusion: This study provides level 1 evidence for the effectiveness of IV immunoglobulin in patients with worsening weakness due to myasthenia gravis.

Safety and efficacy of lithium in combination with riluzole for treatment of amyotrophic lateral sclerosis: a randomised, double-blind, placebo-controlled trial

BACKGROUND In a pilot study, lithium treatment slowed progression of amyotrophic lateral sclerosis (ALS). We aimed to confirm or disprove these findings by assessing the safety and efficacy of lithium in combination with riluzole in patients with ALS. METHODS We did a double-blind, placebo-controlled trial with a time-to-event design. Between January and June, 2009, patients with ALS who were taking a stable dose of riluzole for at least 30 days were randomly assigned (1:1) by a centralised computer to receive either lithium or placebo. Patients, caregivers, investigators, and all site study staff with the exception of site pharmacists were masked to treatment assignment. The primary endpoint was the time to an event, defined as a decrease of at least six points on the revised ALS functional rating scale score or death. Interim analyses were planned for when 84 patients had been allocated treatment, 6 months later or after 55 events, and after 100 events. Analysis was by intention to treat. The stopping boundary for futility at the first interim analysis was a p value of at least 0.68. We used a log-rank test to compare the distributions of the time to an event between the lithium and placebo groups. This trial is registered with ClinicalTrials.gov, NCT00818389. FINDINGS At the first interim analysis, 22 of 40 patients in the lithium group had an event compared with 20 of 44 patients in the placebo group (log rank p=0.51). The hazard ratio of reaching the primary endpoint was 1.13 (95% CI 0.61-2.07). The study was stopped at the first interim analysis because criterion for futility was met (p=0.78). The difference in mean decline in the ALS functional rating scale score between the lithium group and the placebo group was 0.15 (95% CI -0.43 to 0.73, p=0.61). There were no major safety concerns. Falls (p=0.04) and back pain (p=0.05) were more common in the lithium group than in the placebo group. INTERPRETATION We found no evidence that lithium in combination with riluzole slows progression of ALS more than riluzole alone. The time-to-event endpoint and use of prespecified interim analyses enabled a clear result to be obtained rapidly. This design should be considered for future trials testing the therapeutic efficacy of drugs that are easily accessible to people with ALS. FUNDING National Institute of Neurological Disorders and Stroke, ALS Association, and ALS Society of Canada.

Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form

Amyotrophic lateral sclerosis (ALS) is a conformational disease in which misfolding and aggregation of proteins such as SOD1 (familial ALS) and TDP-43 (sporadic ALS) are central features. The conformations adopted by such proteins within motor neurons in affected patients are not well known. We have developed a novel conformation-specific antibody (USOD) targeted against SOD1 residues 42–48 that specifically recognizes SOD1 in which the beta barrel is unfolded. Use of this antibody, in conjunction with the previously described SEDI antibody that recognizes the SOD1 dimer interface, allows a detailed investigation of the in vivo conformation of SOD1 at the residue-specific level. USOD and SEDI immunohistochemistry of spinal cord sections from ALS cases resulting from SOD1 mutations (A4V and ΔG27/P28) shows that inclusions within remaining motor neurons contain SOD1 with both an unfolded beta barrel and a disrupted dimer interface. Misfolded SOD1 can also be immunoprecipitated from spinal cord extracts of these cases using USOD. However, in ten cases of sporadic ALS, misfolded SOD1 is not detected by either immunohistochemistry or immunoprecipitation. Using the amyloid-specific dyes, Congo Red and Thioflavin S, we find that SOD1-positive inclusions in familial ALS, as well as TDP-43- and ubiquitin-positive inclusions in sporadic ALS, contain non-amyloid protein deposits. We conclude that SOD1 misfolding is not a feature of sporadic ALS, and that both SOD1-ALS and sporadic ALS, rather than being amyloid diseases, are conformational diseases that involve amorphous aggregation of misfolded protein. This knowledge will provide new insights into subcellular events that cause misfolding, aggregation and toxicity.

RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS

TDP-43 is a predominantly nuclear DNA/RNA binding protein involved in transcriptional regulation and RNA processing. TDP-43 is also a component of the cytoplasmic inclusion bodies characteristic of amyotrophic lateral sclerosis (ALS) and of frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). We have investigated the premise that abnormalities of TDP-43 in disease would be reflected by changes in processing of its target RNAs. To this end, we have firstly identified RNA targets of TDP-43 using UV-Cross-Linking and Immunoprecipitation (UV-CLIP) of SHSY5Y cells, a human neuroblastoma cell line. We used conventional cloning strategies to identify, after quality control steps, 127 targets. Results show that TDP-43 binds mainly to introns at UG/TG repeat motifs (49%) and polypyrimidine rich sequences (17.65%). To determine if the identified RNA targets of TDP-43 were abnormally processed in ALS versus control lumbar spinal cord RNA, we performed RT-PCR using primers designed according to the location of TDP-43 binding within the gene, and prior evidence of alternative splicing of exons adjacent to this site. Of eight genes meeting these criteria, five were differentially spliced in ALS versus control. This supports the premise that abnormalities of TDP-43 in ALS are reflected in changes of RNA processing.

Pathological heterogeneity in amyotrophic lateral sclerosis with FUS mutations: two distinct patterns correlating with disease severity and mutation.

Mutations in the gene encoding the fused in sarcoma (FUS) protein are responsible for ~3% of familial amyotrophic lateral sclerosis (ALS) and <1% of sporadic ALS (ALS-FUS). Descriptions of the associated neuropathology are few and largely restricted to individual case reports. To better define the neuropathology associated with FUS mutations, we have undertaken a detailed comparative analysis of six cases of ALS-FUS that include sporadic and familial cases, with both juvenile and adult onset, and with four different FUS mutations. We found significant pathological heterogeneity among our cases, with two distinct patterns that correlated with the disease severity and the specific mutation. Frequent basophilic inclusions and round FUS-immunoreactive (FUS-ir) neuronal cytoplasmic inclusions (NCI) were a consistent feature of our early-onset cases, including two with the p.P525L mutation. In contrast, our late-onset cases that included two with the p.R521C mutation had tangle-like NCI and numerous FUS-ir glial cytoplasmic inclusions. Double-labeling experiments demonstrated that many of the glial inclusions were in oligodendrocytes. Comparison with the neuropathology of cases of frontotemporal lobar degeneration with FUS-ir pathology showed significant differences and suggests that FUS mutations are associated with a distinct pathobiology.

Emerging targets and treatments in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease that is currently untreatable. Many compounds have been tested in laboratory-based models and in patients with ALS, but so far only one drug, riluzole, has shown efficacy, yet it only slightly slows disease progression. Several new insights into the causes of motor neuron death have led to the identification of some important novel targets for intervention. At no time have studies involved such a wide range of innovations and such advanced technologies. Many promising studies are underway to test potential targets that will hopefully translate into meaningful therapeutics for patients with ALS.

Investigation of C9orf72 in 4 Neurodegenerative Disorders

OBJECTIVE To estimate the allele frequency of C9orf72 (G4C2) repeats in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Alzheimer disease (AD), and Parkinson disease (PD). DESIGN The number of repeats was estimated by a 2-step genotyping strategy. For expansion carriers, we sequenced the repeat flanking regions and obtained APOE genotypes and MAPT H1/H2 haplotypes. SETTING Hospitals specializing in neurodegenerative disorders. SUBJECTS We analyzed 520 patients with FTLD, 389 patients with ALS, 424 patients with AD, 289 patients with PD, 602 controls, 18 families, and 29 patients with PD with the LRRK2 G2019S mutation. MAIN OUTCOME MEASURE The expansion frequency. RESULTS Based on a prior cutoff (>30 repeats), the expansion was detected in 9.3% of patients with ALS, 5.2% of patients with FTLD, and 0.7% of patients with PD but not in controls or patients with AD. It was significantly associated with family history of ALS or FTLD and age at onset of FTLD. Phenotype variation (ALS vs FTLD) was not associated with MAPT, APOE, or variability in the repeat flanking regions. Two patients with PD were carriers of 39 and 32 repeats with questionable pathological significance, since the 39-repeat allele does not segregate with PD. No expansion or intermediate alleles (20-29 repeats) were found among the G2019S carriers and AD cases with TAR DNA-binding protein 43-positive inclusions. Surprisingly, the frequency of the 10-repeat allele was marginally increased in all 4 neurodegenerative diseases compared with controls, indicating the presence of an unknown risk variation in the C9orf72 locus. CONCLUSIONS The C9orf72 expansion is a common cause of ALS and FTLD, but not of AD or PD. Our study raises concern about a reliable cutoff for the pathological repeat number, which is important in the utility of genetic screening.

Safety and efficacy of ceftriaxone for amyotrophic lateral sclerosis: A multi-stage, randomised, double-blind, placebo-controlled trial

BACKGROUND Glutamate excitotoxicity might contribute to the pathophysiology of amyotrophic lateral sclerosis. In animal models, decreased excitatory aminoacid transporter 2 (EAAT2) overexpression delays disease onset and prolongs survival, and ceftriaxone increases EAAT2 activity. We aimed to assess the safety and efficacy of ceftriaxone for amyotrophic lateral sclerosis in a combined phase 1, 2, and 3 clinical trial. METHODS This three-stage randomised, double-blind, placebo-controlled study was done at 59 clinical sites in the USA and Canada between Sept 4, 2006, and July 30, 2012. Eligible adult patients had amyotrophic lateral sclerosis, a vital capacity of more than 60% of that predicted for age and height, and symptom duration of less than 3 years. In stages 1 (pharmacokinetics) and 2 (safety), participants were randomly allocated (2:1) to ceftriaxone (2 g or 4 g per day) or placebo. In stage 3 (efficacy), participants assigned to ceftriaxone in stage 2 received 4 g ceftriaxone, participants assigned to placebo in stage 2 received placebo, and new participants were randomly assigned (2:1) to 4 g ceftriaxone or placebo. Participants, family members, and site staff were masked to treatment assignment. Randomisation was done by a computerised randomisation sequence with permuted blocks of 3. Participants received 2 g ceftriaxone or placebo twice daily through a central venous catheter administered at home by a trained caregiver. To minimise biliary side-effects, participants assigned to ceftriaxone also received 300 mg ursodeoxycholic acid twice daily and those assigned to placebo received matched placebo capsules. The coprimary efficacy outcomes were survival and functional decline, measured as the slope of Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) scores. Analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00349622. FINDINGS Stage 3 included 66 participants from stages 1 and 2 and 448 new participants. In total, 340 participants were randomly allocated to ceftriaxone and 173 to placebo. During stages 1 and 2, mean ALSFRS-R declined more slowly in participants who received 4 g ceftriaxone than in those on placebo (difference 0·51 units per month, 95% CI 0·02 to 1·00; p=0·0416), but in stage 3 functional decline between the treatment groups did not differ (0·09, -0·06 to 0·24; p=0·2370). No significant differences in survival between the groups were recorded in stage 3 (HR 0·90, 95% CI 0·71 to 1·15; p=0·4146). Gastrointestinal adverse events and hepatobiliary adverse events were more common in the ceftriaxone group than in the placebo group (gastrointestinal, 245 of 340 [72%] ceftriaxone vs 97 of 173 [56%] placebo, p=0·0004; hepatobiliary, 211 [62%] vs 19 [11%], p<0·0001). Significantly more participants who received ceftriaxone had serious hepatobiliary serious adverse events (41 participants [12%]) than did those who received placebo (0 participants). INTERPRETATION Despite promising stage 2 data, stage 3 of this trial of ceftriaxone in amyotrophic lateral sclerosis did not show clinical efficacy. The adaptive design allowed for seamless transition from one phase to another, and central venous catheter use in the home setting was shown to be feasible. FUNDING National Institute of Neurological Disorders and Stroke.

Lack of evidence of monomer/misfolded superoxide dismutase-1 in sporadic amyotrophic lateral sclerosis

In familial amyotrophic lateral sclerosis (fALS) harboring superoxide dismutase (SOD1) mutations (fALS1), SOD1 toxicity has been linked to its propensity to misfold and aggregate. It has recently been proposed that misfolded SOD1 may be causative of all types of ALS, including sporadic cases (sALS). In the present study, we have used a specific antibody to test for the presence of monomer/misfolded SOD1 in sALS.