Richard Mead

Guidelines for preclinical animal research in ALS/MND: A consensus meeting

The development of therapeutics for ALS/MND is largely based on work in experimental animals carrying human SOD mutations. However, translation of apparent therapeutic successes from in vivo to the human disease has proven difficult and a considerable amount of financial resources has been apparently wasted. Standard operating procedures (SOPs) for preclinical animal research in ALS/MND are urgently required. Such SOPs will help to establish SOPs for translational research for other neurological diseases within the next few years. To identify the challenges and to improve the research methodology, the European ALS/MND group held a meeting in 2006 and published guidelines in 2007 (1). A second international conference to improve the guidelines was held in 2009. These second and improved guidelines are dedicated to the memory of Sean F. Scott.

Systemic Delivery of scAAV9 Expressing SMN Prolongs Survival in a Model of Spinal Muscular Atrophy

An adeno-associated virus vector expressing the survival motor neuron protein rescues mice with spinal muscular atrophy. Enough Protein to Reverse Spinal Muscular Atrophy A common neuromuscular disease, spinal muscular atrophy (SMA) causes ever-worsening muscle weakness, usually in babies or young children, almost always resulting in early death. The culprit is a defective gene—survival motor neuron (SMN)—that must be inherited from both parents for the child to be affected. A second SMN gene is usually incorrectly spliced and so is nonfunctional. To treat this disease, researchers have set their sights on delivering a replacement SMN to the motor neurons. The hope has been that gene therapy methods could be used to generate enough normal SMN protein to restore neuronal innervation of muscles in the affected children. Valori et al. have now improved on previous attempts to implement such a treatment in mice with an SMN-like disease. Their new gene therapy vector makes enough functional SMN protein to improve the agility of the affected animals and markedly increase their survival. To find gene therapy methods for SMA that work in animals before trying these treatments in humans, researchers had created mice with the disease. The mouse SMN gene was deleted (mice have only one copy) and replaced with the two human genes—the defective disease-causing form and its poorly spliced sibling. Valori et al. then treated these animals with a gene therapy vector that they had tested in fibroblasts. They coupled a fast, efficient virus (a modified self-complementary adeno-associated virus) to the coding sequence for the SMN protein, optimized for efficient codon usage. A single injection of this vector to new born mice with SMA improved their ability to move and perform physical tests. These mice, which usually die at 2 weeks of age, also showed markedly increased survival times. Subsequent immunohistochemistry showed that the introduced gene was expressed all over the body of the animals but particularly in the lumbar spinal cord, muscle, and liver. These results bring us one step closer to a successful gene therapy treatment for patients with SMA. The vector used here produces large amounts of replacement SMN protein, a goal that had not been achieved with previous approaches. Another noteworthy feature of this vector is that it increases SMN expression in numerous cell types, not just motor neurons, which may be a clue that the critical defects in SMA may be located in more than one kind of cell. Spinal muscular atrophy is one of the most common genetic causes of death in childhood, and there is currently no effective treatment. The disease is caused by mutations in the survival motor neuron gene. Gene therapy aimed at restoring the protein encoded by this gene is a rational therapeutic approach to ameliorate the disease phenotype. We previously reported that intramuscular delivery of a lentiviral vector expressing survival motor neuron increased the life expectancy of transgenic mice with spinal muscular atrophy. The marginal efficacy of this therapeutic approach, however, prompted us to explore different strategies for gene therapy delivery to motor neurons to achieve a more clinically relevant effect. Here, we report that a single injection of self-complementary adeno-associated virus serotype 9 expressing green fluorescent protein or of a codon-optimized version of the survival motor neuron protein into the facial vein 1 day after birth in mice carrying a defective survival motor neuron gene led to widespread gene transfer. Furthermore, this gene therapy resulted in a substantial extension of life span in these animals. These data demonstrate a significant increase in survival in a mouse model of spinal muscular atrophy and provide evidence for effective therapy.

The Membrane Attack Complex of Complement Causes Severe Demyelination Associated with Acute Axonal Injury

Complement is implicated in pathology in the human demyelinating disease multiple sclerosis and in animal models that mimic the demyelination seen in multiple sclerosis. However, the components of the complement system responsible for demyelination in vivo remain unidentified. In this study, we show that C6-deficient (C6−) PVG/c rats, unable to form the membrane attack complex (MAC), exhibit no demyelination and significantly reduced clinical score in the Ab-mediated experimental autoimmune encephalomyelitis model when compared with matched C6-sufficient (C6+) rats. In C6+ rats, perivenous demyelination appeared, accompanied by abundant mononuclear cell infiltration and axonal injury. Neither demyelination nor axonal damage was seen in C6− rats, whereas levels of mononuclear cell infiltration were equivalent to those seen in C6+ rats. Reconstitution of C6 to C6− rats yielded pathology and clinical disease indistinguishable from that in C6+ rats. We conclude that demyelination and axonal damage occur in the presence of Ab and require activation of the entire complement cascade, including MAC deposition. In the absence of MAC deposition, complement activation leading to opsonization and generation of the anaphylatoxins C5a and C3a is insufficient to initiate demyelination.

Deficiency of the complement regulator CD59a enhances disease severity, demyelination and axonal injury in murine acute experimental allergic encephalomyelitis

There is a growing body of evidence implicating complement and, in particular, the terminal pathway (membrane attack complex; MAC) in inducing demyelination in multiple sclerosis and experimental allergic encephalomyelitis. In this paper, we examined the disease course and pathological changes in mice deficient in the major regulator of MAC assembly, CD59a, during the course of acute experimental allergic encephalomyelitis induced by immunisation with recombinant myelin oligodendrocyte glycoprotein. Disease incidence and severity were significantly increased in CD59a-deficient mice. The extent of inflammation, demyelination and axonal injury were assessed in spinal cord cross-sections from CD59a-deficient and control mice, and all these parameters were enhanced in the absence of CD59a. Areas of myelin loss and axonal damage in CD59a-deficient mice were associated with deposits of MAC, firmly implicating MAC as a cause of the observed injury. These findings are relevant to some types of human demyelination, where abundant deposits of MAC are found in association with pathology.

An in vitro screening cascade to identify neuroprotective antioxidants in ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

Optimised and rapid pre-clinical screening in the SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS).

The human SOD1G93A transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS). In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3–4 months) is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) SOD1G93A transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved.

Molecular cloning, expression and characterization of the rat analogue of human membrane cofactor protein (MCP/CD46)

In humans, host cells are protected from homologous complement by membrane proteins encoded in the regulators of complement activation (RCA) gene cluster. These include complement receptor 1 (CR1), decay‐accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46). In mouse and rat a single membrane inhibitor, Crry, appeared to perform the functions of both DAF and MCP and was proposed to be the functional analogue of both. Recently, however, murine homologues of DAF and MCP have been identified, prompting a search for the rat counterparts. We have described the identification of rat DAF and here describe the cloning of rat MCP from cDNA and genomic libraries, using a probe based on the mouse MCP cDNA sequence. The domain structure for rat MCP was identical to that of mouse MCP with four short consensus repeats (SCRs) followed by a STP domain, transmembrane segment and cytoplasmic tail. Overall identity of rat and mouse MCP was 77% at the amino acid level and 88% at the nucleotide level. Northern blot analysis from a range of tissues indicated that high‐level expression was limited to the testis, although expression in other tissues was detected using reverse transcription–polymerase chain reaction. Rat MCP mRNA localized to Sertoli cells and spermatogonia in seminiferous tubules by in situ hybridization, but was absent in mature sperm. In cofactor assays utilizing human factor I, a recombinant soluble form of rat MCP catalysed cleavage of human C3ma.

Mannose-binding lectin alleles in a prospectively recruited UK population

Vol 349 • June 7, 1997 1669 lymphnode was easily found upon commencing axillary dissection (figure). With a handheld gamma probe to measure radioactive uptake, these blue-stained sentinel nodes were shown to correspond to the scintigraphic foci visible in 30 cases; they also contained radioactivity in the remaining three patients with negative scintigraphy. There was 100% concordance in delineation of the sentinel node with intradermal blue dye and intramammary Tc-labelled albumin. Breast tumour location and size were irrelevant. Eight patients refused to undergo formal lymphadenectomy and requested that only the sentinel node be removed. In 25 patients complete ALND was done, with an average of 16 lymphnodes harvested. On pathological examination the (blue and radioactive) sentinel node revealed metastases in 14 patients (56%): in nine cases these were the only axillary nodes to contain tumour. In the remaining 11 patients (44%) both the sentinel node and ALND were free of metastases—ie, there were no falsenegative sentinel nodes. This pilot study confirms our hypothesis that the lymphatics of the overlying skin drain to the same axillary sentinel node as the underlying glandular breast tissue. This new approach simplifies the technique of localising the sentinel node during operation, allowing identification in all of our patients. By combining lymphoscintigraphy and gamma-probe detection with the visual guidance of intradermal blue dye, sentinel-node biopsy may be a feasible alternative to routine ALND.

S[+] Apomorphine is a CNS penetrating activator of the Nrf2-ARE pathway with activity in mouse and patient fibroblast models of amyotrophic lateral sclerosis

Compelling evidence indicates that oxidative stress contributes to motor neuron injury in amyotrophic lateral sclerosis (ALS), but antioxidant therapies have not yet achieved therapeutic benefit in the clinic. The nuclear erythroid 2-related-factor 2 (Nrf2) transcription factor is a key regulator of an important neuroprotective response by driving the expression of multiple cytoprotective genes via its interaction with the antioxidant response element (ARE). Dysregulation of the Nrf2-ARE system has been identified in ALS models and human disease. Taking the Nrf2-ARE pathway as an attractive therapeutic target for neuroprotection in ALS, we aimed to identify CNS penetrating, small molecule activators of Nrf2-mediated transcription in a library of 2000 drugs and natural products. Compounds were screened extensively for Nrf2 activation, and antioxidant and neuroprotective properties in vitro. S[+]-Apomorphine, a receptor-inactive enantiomer of the clinically approved dopamine-receptor agonist (R[–]-apomorphine), was identified as a nontoxic Nrf2 activating molecule. In vivo S[+]-apomorphine demonstrated CNS penetrance, Nrf2 induction, and significant attenuation of motor dysfunction in the SOD1G93A transgenic mouse model of ALS. S[+]-apomorphine also reduced pathological oxidative stress and improved survival following an oxidative insult in fibroblasts from ALS patients. This molecule emerges as a promising candidate for evaluation as a potential neuroprotective agent in ALS patients in the clinic.

TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD

Amyotrophic lateral sclerosis–frontotemporal dementia (ALS-FTD) constitutes a devastating disease spectrum characterized by 43-kDa TAR DNA-binding protein (TDP-43) pathology. Understanding how TDP-43 contributes to neurodegeneration will help direct therapeutic efforts. Here we have created a TDP-43 knock-in mouse with a human-equivalent mutation in the endogenous mouse Tardbp gene. TDP-43Q331K mice demonstrate cognitive dysfunction and a paucity of parvalbumin interneurons. Critically, TDP-43 autoregulation is perturbed, leading to a gain of TDP-43 function and altered splicing of Mapt, another pivotal dementia-associated gene. Furthermore, a new approach to stratify transcriptomic data by phenotype in differentially affected mutant mice revealed 471 changes linked with improved behavior. These changes included downregulation of two known modifiers of neurodegeneration, Atxn2 and Arid4a, and upregulation of myelination and translation genes. With one base change in murine Tardbp, this study identifies TDP-43 misregulation as a pathogenic mechanism that may underpin ALS-FTD and exploits phenotypic heterogeneity to yield candidate suppressors of neurodegenerative disease.TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD, leading to aberrant Mapt splicing and a paucity of parvalbumin interneurons. Phenotypic heterogeneity is exploited to yield modifiers of disease.