M. N. Zakharova

Gene therapy of amyotrophic lateral sclerosis

Two-year experiments were performed to evaluate the neurotrophic effect of hypoxia-inducible factors (vascular endothelial growth factor and angiogenin) expressed in recombinant human adenoviruses in amyotrophic lateral sclerosis. Randomized placebo-controlled trial demonstrated safety and good tolerability of the recombinant antiviral drugs. The life span of patients under conditions of hypoxia increased after treatment with the test drug, which was probably related to improved resistance of motoneurons. The presence of virus-neutralizing antibodies decreases the effectiveness of adenoviral vectors, which necessitates differential approach to the selection of patients and continuous monitoring of gene therapy.

Genetic studies of Russian patients with amyotrophic lateral sclerosis.

Abstract Our objective was to search for mutations in genes SOD1, TARDBP, C9orf72, ANG, ATXN2 and VEGF in Russian patients with amyotrophic lateral sclerosis (ALS). A group of 208 Russian patients with ALS was examined. Molecular genetic analysis was conducted using direct sequencing, fragment analysis, and real-time PCR. We found eight different point mutations in the SOD1 gene, with the frequency of mutations being 50% in familial ALS and 3% in sporadic ALS. No mutations were found in exon 6 of the TARDBP gene; however, deletion c.715-126delG in intron 5 of TARDBP was over-represented in ALS patients compared to controls (38% vs. 26.6%; χ2 = 13.17; p = 0.002). Hexanucleotide repeat expansion of the C9orf72 gene was revealed in 2.5% of sporadic ALS patients. Mutations in the ANG gene were identified in 1.5% of sporadic ALS patients. The presence of an intermediate number (28–33) of GAC repeats in the ATXN2 gene was observed significantly more often in the study group compared to the control group (5% vs. 1.7%; χ2 = 3.89; p = 0.0486). In the cohort examined, we found an association between the disease and the risk A-allele and the A/A genotype at the −2578С/А locus of the VEGF gene. In conclusion, we determined for the first time the genetic basis of ALS in a Russian population.

[Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors].

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg(SOD1*G93A)dl1 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1G93A. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1G93A mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1G93A mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2–3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + AGF can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.

Enzyme immunoassay for detection of protein-bound nitrotyrosine in brain tissue and cerebrospinal fluid: Methodological issues

Protein-bound nitrotyrosine is a standard marker of nitrosative stress in vivo, its levels being associated with severity of nitrosative stress in neurological diseases. Using two commercially available antibodies we have developed protocols for an indirect competitive enzyme-linked immunosorbent assay (ELISA) aimed at detecting protein-bound nitrotyrosine. The working concentration range of the assay is 0.094–4 μg/mL, the lowest limit of detection 0.063 μg/mL, IC50 0.538 μg/mL. In a pilot study protein-bound nitrotyrosine levels in cerebrospinal fluid of acute stroke patients and in brain tissue of rats after focal ischemia were evaluated.

Disruption of blood-brain barrier in amyotrophic lateral sclerosis: an update

Amyotrophic lateral sclerosis (ALS) is a continuously progressing neurodegenerative disease that is characterized by selective damage to motoneurons in the neocortex, brainstem, and spinal cord. The general opinion is that sporadic ALS is a multifactor and multigene disease. Realization of its genetic predisposition is determined by environmental factors. The blood-brain barrier (BBB) is the border for the neurons of the brain and spinal cord, where they interact with environmental factors. Current knowledge on BBB alterations during ALS helps to reevaluate views on the pathogenesis of the disease, as well as on the development of therapeutic means and methods of their delivery. It has been found that in ALS all BBB components, which separate CNS neurons from endogenous and exogenous damaging factors and participate in the communication between cells of nerve and immune systems, are involved in the pathological process. Currently, there are no data that show that BBB disruption triggers the death of neurons; however, it is an important mechanism of ALS pathogenesis. This should be considered during studies of this disease and development of new methods of treatment. Keeping in mind alterations of the BBB, it is possible to consider CNS cells, BBB cells, and peripheral immune cells as targets of new drugs for ALS treatment.

Modulators of cystein proteases and cell-death markers in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective damage of motor neurons in the brain and spinal cord. The aim of the current study was to reveal new specific markers of neurodegeneration in the cerebrospinal fluid (CSF) of ALS patients. We measured the activities of substances that modulate the activity of cysteine proteases (calpain, caspase, and cathepsin B) in the CSF of ALS patients. The CSF of ALS patients, in contrast to the CSF of patients with multiple sclerosis, inhibits calpain and cathepsin B significantly more strongly, as compared to the control CSF. The LDH activity and the concentration of neuron-specific enolase (NSE) in the CSF of control patients and ALS patients did not differ. The concentration of neurofilament heavy chains in the CSF was significantly higher in the ALS patients as compared to the control group. The albumin index increased in 50% of all ALS patients. We also have shown a correlation between the cathepsin inhibitor activity, the concentration of neurofilament heavy chains, and the albumin index and the clinical findings of the disease. According to our data, the concentration of neurofilament heavy chains is a sensitive marker of neurodegenerative process during ALS. An increase in the CSF inhibiting activity with respect to cathepsin B and calpain is specific for ALS and may confirm the role of an imbalance of proteolytic systems in the pathogenesis of this disease.

The role of vascular endothelial growth factor in the progression of amytrophic lateral sclerosis

We investigated the content of vascular endothelial growth factor (VEGF) in the blood plasma of patients suffering from the sporadic form of amyotrophic lateral sclerosis (ALS). Two groups of patients were selected for the study: in the first group, the disease started before the age of 40 and, in the second group, after the age of 40. In the older patients, the decrease in the content of VEGF correlated with the age and duration of the disease. This demonstrates the involvement of VEGF in the mechanisms of motor neuron death. In patients with early onset of ALS, the paradoxical reaction of VEGF under hypoxia conditions was revealed. This may be one of the mechanisms which determine the progression and severity of the disease in this group of patients. Our data on the impairment of VEGF regulation in the patients with sporadic form of ALS are the basis for the development of new methods of therapy with the use of neurotrophic factors.

Calpain-like and lactate dehydrogenase activities in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is neurodegenerative disease accompanied by the death of motor neurons. To clarify the role of calpain in the ALS-induced death of neurons, we measured the calpainlike and lactate dehydrogenase (LDH) activities in the cerebrospinal fluid (CSF) of ALS patients and patients of a control group. The LDH activity measured in the CSF of patients with ALS was significantly higher than that in the control which, presumably, reflected the death of CNS neurons during ALS. At pH 7.4 the calpainlike activity (CLA) of the ALS patients did not differ from that of the control groups, but at pH 5.5 CLA was significantly higher in the subjects with ALS. Moreover, we found a significant correlation between CLA at pH 5.5 and the LDH activity in patients with ALS, which suggests that this calpain-like activity may be associated with neuronal death. We also present data on the dependence of the LDH and calpain-like activities on ALS type and duration and the progression rate of the disease.

Role of superoxide dismutase in the pathogenesis of amyotrophic lateral sclerosis

Activity of cytosolic Cu2+/Zn2+-superoxide dismutase and total superoxide dismutase activity of the spinal fluid of patients with sporadic amyotrophic lateral sclerosis were measured. These parameters correlates with the form of this disease, its duration, and the severity of neurological disorders. Our findings indicate that free-radical processes are involved in the damage to motor neurons in this disease.

[Neuromyelitis optica and aquaporin-associated syndromes].

Identification of aquaporin-4 antibodies in neuromyelitis optica (NMO) is highly important to provide early diagnosis for starting pathogenetic treatment, and for differential diagnosis in neuromyelitis optica spectrum disorders (NMO-SD). In this paper, we review current pathogenetic and clinical aspects of NMO and NMO NMO-SD. We present our data on the identification of aquaporin-4 antibodies in CNS disorders. Aquaporin-4 antibodies were detected in 86.36% of patients with neuromyelitis optica, in 12.5% of patients with partial syndromes and in 100% of patients with systemic lupus erythematosus with longitudinally extensive transverse myelitis or optic neuritis. There was the correlation between the extent of lesion in the spinal cord and positive aquaporin-4 antibodies.