Saida Ortolano

New insights in the amyloid-Beta interaction with mitochondria.

Biochemical and morphological alterations of mitochondria may play an important role in the pathogenesis of Alzheimers disease (AD). Particularly, mitochondrial dysfunction is a hallmark of amyloid-beta-induced neuronal toxicity in Alzheimers disease. The recent emphasis on the intracellular biology of amyloid-beta and its precursor protein (APP) has led researchers to consider the possibility that mitochondria-associated and mitochondrial amyloid-beta may directly cause neurotoxicity. Both proteins are known to localize to mitochondrial membranes, block the transport of nuclear-encoded mitochondrial proteins to mitochondria, interact with mitochondrial proteins, disrupt the electron transport chain, increase reactive oxygen species production, cause mitochondrial damage, and prevent neurons from functioning normally. In this paper, we will outline current knowledge of the intracellular localization of amyloid-beta. Moreover, we summarize evidence from AD postmortem brain as well as animal AD models showing that amyloid-beta triggers mitochondrial dysfunction through a number of pathways such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. Thus, this paper supports the Alzheimer cascade mitochondrial hypothesis such as the most important early events in this disease, and probably one of the future strategies on the therapy of this neurodegenerative disease.

A novel MYH7 mutation links congenital fiber type disproportion and myosin storage myopathy

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patients biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy.

LRP-1 and LRP-2 receptors function in the membrane neuron. Trafficking mechanisms and proteolytic processing in Alzheimer's disease

Low density lipoprotein receptor-related protein (LRP) belongs to the low-density lipoprotein receptor family, generally recognized as cell surface endocytic receptors, which bind and internalize extracellular ligands for degradation in lysosomes. Neurons require cholesterol to function and keep the membrane rafts stable. Cholesterol uptake into the neuron is carried out by ApoE via LRPs receptors on the cell surface. In neurons the most important are LRP-1 and LRP-2, even it is thought that a causal factor in Alzheimers disease (AD) is the malfunction of this process which cause impairment intracellular signaling as well as storage and/or release of nutrients and toxic compounds. Both receptors are multifunctional cell surface receptors that are widely expressed in several tissues including neurons and astrocytes. LRPs are constituted by an intracellular (ICD) and extracellular domain (ECD). Through its ECD, LRPs bind at least 40 different ligands ranging from lipoprotein and protease inhibitor complex to growth factors and extracellular matrix proteins. These receptors has also been shown to interact with scaffolding and signaling proteins via its ICD in a phosphorylation-dependent manner and to function as a co-receptor partnering with other cell surface or integral membrane proteins. Thus, LRPs are implicated in two major physiological processes: endocytosis and regulation of signaling pathways, which are both involved in diverse biological roles including lipid metabolism, cell growth processes, degradation of proteases, and tissue invasion. Interestingly, LRPs were also localized in neurons in different stages, suggesting that both receptors could be implicated in signal transduction during embryonic development, neuronal outgrowth or in the pathogenesis of AD.

Treatment of Lysosomal Storage Diseases: Recent Patents and Future Strategies

Lysosomal storage diseases (LSDs) are a group of rare genetic multisystemic disorders, resulting in deficient lysosomal activity. These pathologies are characterized by progressive accumulation of storage material within the lysosomes, ultimately leading to organ dysfunctions. LSDs patients clinical outcomes have significantly improved, since the advent of enzyme replacement therapy (ERT). ERT is approved worldwide for 6 LSDs: Gaucher disease, Fabry disease, Mucopolysaccharidosis types I, II, and VI, and Pompe disease. The efficacy and safety of ERT for LSDs has been confirmed by extensive clinical trials, however therapy with infused protein is life-long and disease progression is still observed in treated patients. Obstacles to successful ERT, such as immune reactions against the infused enzyme, miss-targeting of recombinant enzymes, and difficult delivery to crucial tissues (i.e. brain and bone), determine the need for further research, in order to ameliorate therapeutic strategies. Viral gene therapy, stem cell based therapy, pharmacological chaperones and could be considered essential tools for future improvement of recombinant enzyme trafficking and targeting. This review will discuss recent patents and new strategic approaches for enzyme delivery to highlight the most relevant aspects, concerning next generation LSDs treatment.

Present and Future of Adeno Associated Virus Based Gene Therapy Approaches

During the last 20 years, transgenic constructs based on adeno associated virus (AAV) have been tested in disease models and proved their efficacy to revert a wide range of pathologies without major side effects. Based on these results, up to 20 clinical trials have been set up to prove therapeutic effect of AAV vectors on neurodegenerative diseases, retinopathies and neuromuscular diseases, among others. It has been shown that AAV vectors support localized long-term, gene expression in the central nervous system, and that restoration of visual function can be achieved in Lebers congenital amaurosis retinopathy. The clinical trials also highlighted new challenges for AAV mediated gene transfer, such as the circumvention of T-cell response to transduced cells. Currently, miniaturized and codon-optimized transgenes, exon skipping cassettes, novel tissue-specific promoters and vector chimeras with tissue-selective tropism are being tested to improve the efficiency and safety of transgene delivery, as required to meet pharmaceutical industry standards. The aim of this review is to revise the latest patents and news on AAV vectors, in order to summarize the state of the art and the potential issues that still need to be faced by pharmaceutical companies for successful gene transfer and commercialization of AAV-based drugs.

Molecular and clinical study of McArdle's disease in a cohort of 123 European patients. Identification of 20 novel mutations.

McArdles disease is the most common muscle glycogenosis. It is caused by the deficiency of myophosphorylase, encoded by the PYGM gene. We studied 123 patients previously diagnosed with McArdles disease and we identified 20 novel mutations (10 missense and 3 nonsense mutations, 3 small deletions, 2 gross deletions and 2 small insertions). Most patients of this cohort belong to Spanish and French populations. This allowed us to determine the differences between the allelic frequencies of the common mutations R50X and G205S of these populations. The R50X has an allelic frequency in this cohort of about 61.7%, being 68.5% in French and 53.7% in Spanish patients. The G205S had a higher allelic frequency in the Spanish (10.2%) than in the French population (3.2%). Moreover, a clinical study of 91 patients was performed to establish both genotype-phenotype correlation and gender influence in the severity of the disease. We conclude that no genotype-phenotype correlation is evident and that no gender effect is related to the phenotype.

Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene

In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy 1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion (c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/arginine-rich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and is absent in genomic sequence databases and a set of >200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of inherited myopathies.

tPA in the central nervous system: relations between tPA and cell surface LRPs.

A growing body of evidence has implicated the plasminogen activating system in various aspects of neurophysiology and pathophysiology. In ischemic stroke, blood-brain barrier (BBB) regulations, typically involving matrix metalloproteinases (MMPs), inhibitors tissue inhibitors of metalloproteinases (TIMPs) and the low density lipoprotein receptor- related protein/alpha 2-macroglobulin receptor (LRPs) as mediators became interesting since tissue plasminogen activator (tPA)-related BBB breakdown with risk of secondary hemorrhage was considered to involve these mediators too. The mechanism by which tPA implements its actions within the central nervous system (CNS) has been the topic of much controversy. Binding of plasminogen to surfaces is of crucial importance in regulating the function of this system. tPA can modulate permeability of the neurovascular unit in physiological conditions and pathological events exacerbating injury in ischemic stroke, vascular dementia, traumatic brain injury or neurotoxic events. The plasminogen activating enzyme system is widely appreciated for its role in fibrinolysis and thrombolysis and in other areas related to remodelling of the extracellular matrix. However, this enzyme system also has a major impact in the central nervous system under pathological circumstances. The aim of this review is to revise the last patents and news to understand the mechanism by which t-PA modulates BBB permeability.

Frailty in mouse ageing: A conceptual approach

Human life expectancy has increased dramatically in the last century and as a result also the prevalence of a variety of age-related diseases and syndromes. One such syndrome is frailty, which is defined as a combination of organ dysfunctions leading to increased vulnerability to adverse health outcomes. In humans, frailty is associated with various biomarkers of ageing and predicts relevant outcomes such as responses to therapies and progression of health status and mortality. Moreover, it is relatively easy to assess. To foster translation of mechanistic understanding of the ageing process and, importantly, of interventions that may extend healthy lifespan, frailty scales have been reverse translated into mice in recent years. We will review these approaches with a view to identify what is known and what is not known at present about their validity, reproducibility and reliability with a focus on the potential for further improvement.

Lafora Progressive Myoclonus Epilepsy: Recent Insights into Cell Degeneration

Lafora disease (LD) is a fatal autosomal recessive form of progressive myoclonus epilepsy. Patients manifest myoclonus and tonic-clonic seizures, visual hallucinations, intellectual, and progressive neurologic deterioration beginning in adolescence. The two genes known to be involved in Lafora disease are EPM2A and NHLRC1 (EPM2B). The EPM2A gene encodes laforin, a dual-specificity protein phosphatase, and the NHLRC1 gene encodes malin, an E3-ubiquitin ligase. The two proteins interact with each other and, as a complex, are thought to regulate glycogen synthesis. It may also be considered as a disorder of carbohydrate metabolism because of the formation of polyglucosan inclusion bodies in neural and other tissues due to abnormalities of the proteins laforin or malin. The review also outlines important patents related to Lafora disease.