Carole Ho

Lack of Enhanced Spinal Regeneration in Nogo-Deficient Mice

The failure of regeneration of severed axons in the adult mammalian central nervous system is thought to be due partly to the presence of endogenous inhibitors of axon regeneration. The nogo gene encodes three proteins (Nogo-A, -B, and -C) that have been proposed to contribute to this inhibition. To determine whether deletion of nogo enhances regenerative ability, we generated two lines of mutant mice, one lacking Nogo-A and -B but not -C (Nogo-A/B mutant), and one deficient in all three isoforms (Nogo-A/B/C mutant). Although Nogo-A/B-deficient myelin has reduced inhibitory activity in a neurite outgrowth assay in vitro, tracing of corticospinal tract fibers after dorsal hemisection of the spinal cord did not reveal an obvious increase in regeneration or sprouting of these fibers in either mouse line, suggesting that elimination of Nogo alone is not sufficient to induce extensive axon regeneration.

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

ONE approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed β-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles – they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with β-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Dishevelled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

An Effector-Reduced Anti-β-Amyloid (Aβ) Antibody with Unique Aβ Binding Properties Promotes Neuroprotection and Glial Engulfment of Aβ

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimers disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1–42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP(V717I)/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Axotomy-induced Smad1 activation promotes axonal growth in adult sensory neurons.

Mature neurons have diminished intrinsic regenerative capacity. Axotomy of the peripheral branch of adult dorsal root ganglia (a “conditioning” lesion) triggers a transcription-dependent axon growth program. Here, we show that this growth program requires the function of the transcription factor Smad1. After peripheral axotomy, neuronal Smad1 is upregulated, and phosphorylated Smad1 accumulates in the nucleus. Both events precede the onset of axonal extension. Reducing Smad1 by RNA interference in vitro impairs axonal growth, and the continued presence of Smad1 is required to maintain the growth program. Furthermore, intraganglionic injection of BMP2 or 4, which activates Smad1, markedly enhances axonal growth capacity, mimicking the effect of a conditioning lesion. Thus, activation of Smad1 by axotomy is a key component of the transcriptional switch that promotes an enhanced growth state of adult sensory neurons.

A Plasmablast Biomarker for Nonresponse to Antibody Therapy to CD20 in Rheumatoid Arthritis

Plasmablast biomarkers predict whether rheumatoid arthritis patients will respond to therapeutic antibodies to CD20. A Molecular Magic Eight Ball Ever wish you could predict the future? From children’s toys to psychic consultants, there’s an entire industry devoted to providing people with insight into upcoming events. This desire for precognition extends to clinical medicine—both doctors and patients wish they could predict whether a treatment will work for a particular disease in a particular patient. Thus, the search for biomarkers was born. However, many studies that claim to identify “biomarkers” have as little experimental validation as a late-night TV psychic, making the truly validated biomarker a rare gem. Owczarczyk et al. now develop such a predictor for nonresponsiveness to anti-CD20 antibody therapy for rheumatoid arthritis. Rituximab and ocrelizumab are therapeutic antibodies that bind to CD20 on the surface of effector and memory B cells, causing them to be depleted from the circulation. These antibodies can be helpful to rheumatoid arthritis patients who don’t fare well with more general antirheumatic drugs, such as nonsteroidal anti-inflammatory drugs, and disease-modifying antirheumatic drugs, such as hydroxychloroquine, sulfasalazine, leflunomide, or methotrexate. But not all patients respond to these expensive targeted biologics. Owczarczyk et al. observed that rheumatoid arthritis patients who don’t respond to anti-CD20 antibodies had elevated amounts of IgJ mRNA, a marker for antibody-secreting plasmablasts. They then performed prospective testing of IgJ mRNA concentrations in one ocrelizumab and two rituximab patient cohorts and found that this marker could predict nonresponse to anti-CD20 antibody therapy. Moreover, a combination mRNA biomarker, IgJhiFCRL5lo, improved test performance over IgJhi alone. Will these biomarkers also be useful in stratifying response rates in other diseases in which anti-CD20 antibody therapy has shown clinical activity such as relapsing-remitting multiple sclerosis and ANCA-associated vasculitis? Cannot predict now. Ask again later. An important goal for personalized health care is the identification of biomarkers that predict the likelihood of treatment responses. Here, we tested the hypothesis that quantitative mRNA assays for B lineage cells in blood could serve as baseline predictors of therapeutic response to B cell depletion therapy in subjects with rheumatoid arthritis (RA). In samples from the REFLEX trial of rituximab in inadequate responders to antibodies to tumor necrosis factor–α, a 25% subgroup of treated subjects with elevated baseline mRNA levels of IgJ, a marker for antibody-secreting plasmablasts, showed reduced clinical response rates. There were no significant efficacy differences in the placebo arm subjects stratified by this marker. Prospective testing of the IgJ biomarker in the DANCER and SERENE rituximab clinical trial cohorts and the SCRIPT ocrelizumab cohort confirmed the utility of this marker to predict nonresponse to anti-CD20 therapy. A combination mRNA biomarker, IgJ hiFCRL5lo, showed improved test performance over IgJ hi alone. This study demonstrates that baseline blood levels of molecular markers for late-stage B lineage plasmablasts identify a ~20% subgroup of active RA subjects who are unlikely to gain substantial clinical benefit from anti-CD20 B cell depletion therapy.

The dorsolateral corticospinal tract in mice: An alternative route for corticospinal input to caudal segments following dorsal column lesions

In rodents, the main contingent of corticospinal tract (CST) axons descends in the ventral part of the dorsal column. There is, however, a contingent of CST axons that descends in the dorsolateral column (the “dorsolateral corticospinal tract,” or DLCST). Here, we define some of the features of the DLCST by tracing CST projections following injections of biotinylated dextran amine into the sensorimotor cortex, assessing the distribution of DLCST axons and terminal arborizations in intact mice and in mice in which the main contingent of CST axons in the dorsal column had been transected. Axons of the DLCST diverge from the main tract at the pyramidal decussation, gather in fascicles in the dorsolateral gray matter below the spinomedullary junction, and project in a gradual trajectory laterally toward the dorsolateral column over the first few cervical segments. DLCST axons then project along the dorsolateral column to sacral levels, giving rise to collaterals that project into the gray matter. Labeled DLCST axons were most abundant in cervical segments, where they were often collected in fascicles, and progressively decreased in number in more caudal segments. Tracing of DLCST axons in mice with selective lesions of the dorsal column revealed that DLCST axons arborize extensively throughout the dorsal and ventral horns and that the overall territory that the DLCST axons invade is similar to the territory innervated by the CST axons in the main tract. Some DLCST axon arbors with varicosities are seen near large neurons in the ventral horn (presumed motoneurons). Substantial numbers of DLCST axons project across the midline to the gray matter on the contralateral side. Thus, the DLCST provides an alternate route for CST input to caudal segments, which is of particular relevance for studies of CST distribution and function following partial spinal cord injuries. J. Comp. Neurol. 472:463–477, 2004.

Reassessment of Corticospinal Tract Regeneration in Nogo-Deficient Mice

The myelin-derived neurite growth inhibitor Nogo has been proposed to play a major role in blocking axon regeneration in the CNS after injuries. However, past studies have produced mixed results regarding the regenerative phenotype of various Nogo-deficient mouse lines after experimental spinal cord injury. Two lines did not display enhanced corticospinal tract (CST) regeneration, and one displayed modest regeneration. A fourth line, a Nogo-A,B gene-trap mutant, was instead reported to exhibit extensive CST regeneration, but the results were later found to be inadvertently confounded with an axon labeling artifact. Of the four Nogo mutant lines studied so far, three continue to express some isoform(s) of Nogo, leaving open the question whether any remaining Nogo protein contributes to the modest regenerative phenotype reported in some. The remaining Nogo mutant line studied was confounded by the unexplained rescue of embryonic lethality associated with this mutation. To gain a better understanding of the contribution of Nogo as an inhibitor of regeneration of CNS axons, and particularly CST axons, we reanalyzed the Nogo-A,B gene-trap mutant line and analyzed a novel, fully viable Nogo deletion mutant line that is null for all known isoforms of Nogo. Our analyses failed to reveal any enhanced CST regeneration after experimental spinal cord injury in either line. These results indicate that Nogo alone does not account for lack of CST regeneration and have implications for current therapeutic development for spinal cord injury in humans by targeting Nogo.

Targeting factor D of the alternative complement pathway reduces geographic atrophy progression secondary to age-related macular degeneration

The phase 2 MAHALO clinical trial shows that lampalizumab reduces geographic atrophy secondary to age-related macular degeneration and implicates complement dysregulation in disease pathogenesis. Illuminating a new treatment for macular degeneration Geographic atrophy secondary to age-related macular degeneration is a major cause of vision loss for which there is no treatment. Yaspan et al. now report the results of the MAHALO phase 2 randomized, controlled trial that evaluated lampalizumab in patients with geographic atrophy secondary to age-related macular degeneration. Lampalizumab is a specific inhibitor of complement factor D, a pivotal regulator of the alternative complement pathway. The MAHALO study met its primary efficacy endpoint with a 20% reduction in lesion area progression compared to sham control with monthly lampalizumab treatment. Moreover, lampalizumab showed an acceptable safety profile. A more substantial monthly treatment benefit of 44% reduction in geographic atrophy progression versus sham control was observed in a subgroup of patients who were complement factor I risk-allele carriers. Geographic atrophy is an advanced form of age-related macular degeneration (AMD) and a leading cause of vision loss for which there are no approved treatments. Genetic studies in AMD patients have implicated dysregulation of the alternative complement pathway in the pathogenesis of geographic atrophy. Lampalizumab is a potential therapeutic that targets complement factor D, a pivotal activator of the alternative complement pathway. The MAHALO phase 2 clinical trial was a multicenter, randomized, controlled study that evaluated lampalizumab administered by intravitreal injection monthly (n = 42) and every other month (n = 41) versus sham control (n = 40) in patients with geographic atrophy secondary to AMD. The primary endpoint was the mean change in lesion area from baseline to month 18 as measured by fundus autofluorescence. Specific AMD-associated genetic polymorphisms were also analyzed. The MAHALO study met its primary efficacy endpoint with an acceptable safety profile; monthly lampalizumab treatment demonstrated a 20% reduction in lesion area progression versus sham control [80% confidence interval (CI), 4 to 37%]. A more substantial monthly treatment benefit of 44% reduction in geographic atrophy area progression versus sham control (95% CI, 15 to 73%) was observed in a subgroup of complement factor I (CFI) risk-allele carriers (57% of the patients analyzed were CFI risk-allele carriers). The MAHALO study shows a potential treatment effect in patients with geographic atrophy and supports therapeutic targeting of the alternative complement pathway for treating AMD pathogenesis.

Parallel Discovery of Alzheimer’s Therapeutics

Government support for parallel development of AD therapeutics. As the prevalence of Alzheimer’s disease (AD) grows, so do the costs it imposes on society. Scientific, clinical, and financial interests have focused current drug discovery efforts largely on the single biological pathway that leads to amyloid deposition. This effort has resulted in slow progress and disappointing outcomes. Here, we describe a “portfolio approach” in which multiple distinct drug development projects are undertaken simultaneously. Although a greater upfront investment is required, the probability of at least one success should be higher with “multiple shots on goal,” increasing the efficiency of this undertaking. However, our portfolio simulations show that the risk-adjusted return on investment of parallel discovery is insufficient to attract private-sector funding. Nevertheless, the future cost savings of an effective AD therapy to Medicare and Medicaid far exceed this investment, suggesting that government funding is both essential and financially beneficial.

Homozygosity of the autosomal dominant Alzheimer disease presenilin 1 E280A mutation

We identified several families in Antioquia, Colombia, with early-onset Alzheimer disease (AD) due to the mendelian autosomal dominant inheritance of a PSEN1 E280A gene mutation. Extended family members were interviewed and parish baptism certificates in Antioquian municipalities examined.1 The size of these extended families (including carriers and noncarriers) approaches 5,000 individuals. Full genomes in carriers proved a single founder.2 To support an AD prevention clinical trial, we established a registry in 2010 of all family members over age 8 years.3 Since then we genotyped 3,407 family members and identified 823 (24%) carriers of the PSEN1 E280A mutation. The Comite de Bioetica de la Sede de Investigacion Universitaria, SIU Universidad de Antioquia, approved this study. All participants provided written informed consent. Despite the size of this exceptionally large family and frequent consanguinity, homozygosity at this gene locus had not been reported. The apparent absence of homozygous PSEN1 mutations led to the speculation that E280A homozygosity could be lethal. Generally, homozygous dominant mutations are more severely affected than heterozygotes in both humans and model systems.4 However, human cases in which dominant point mutations are homozygous are rare.