Advances, challenges and future directions for stem cell therapy in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative condition where loss of motor neurons within the brain and spinal cord leads to muscle atrophy, weakness, paralysis and ultimately death within 3–5 years from onset of symptoms. The specific molecular mechanisms underlying the disease pathology are not fully understood and neuroprotective treatment options are minimally effective. In recent years, stem cell transplantation as a new therapy for ALS patients has been extensively investigated, becoming an intense and debated field of study. In several preclinical studies using the SOD1G93A mouse model of ALS, stem cells were demonstrated to be neuroprotective, effectively delayed disease onset and extended survival. Despite substantial improvements in stem cell technology and promising results in preclinical studies, several questions still remain unanswered, such as the identification of the most suitable and beneficial cell source, cell dose, route of delivery and therapeutic mechanisms. This review will cover publications in this field and comprehensively discuss advances, challenges and future direction regarding the therapeutic potential of stem cells in ALS, with a focus on mesenchymal stem cells. In summary, given their high proliferation activity, immunomodulation, multi-differentiation potential, and the capacity to secrete neuroprotective factors, adult mesenchymal stem cells represent a promising candidate for clinical translation. However, technical hurdles such as optimal dose, differentiation state, route of administration, and the underlying potential therapeutic mechanisms still need to be assessed

Direct visualization of the arterial wall water permeability barrier using CARS microscopy.

The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies

CAP defines a second signalling pathway required for insulin-stimulated glucose transport

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl protooncogene product(1). Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP(2). Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction(3). Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62940/1/407202a0.pd

Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin. </jats:p

Glucose transporter 4: cycling, compartments and controversies: Third in the Cycles Review Series

Insulin promotes glucose uptake into muscle and adipose tissues through glucose transporter 4 (GLUT4). In unstimulated cells, rapid endocytosis, slow exocytosis and dynamic or static retention cause GLUT4 to concentrate in early recycling endosomes, the trans-Golgi network and vesicle-associated protein 2-containing vesicles. The coordinated action of phosphatidylinositol 3-kinase effectors, protein kinase Akt, atypical protein kinase C (aPKC) and Akt substrate of 160-kDa (AS160), regulates the GLUT4 cycle by affecting its translocation, fusion with the plasma membrane, internalization and sorting. We review the evidence that supports such cycling, evaluate current models proposing static or dynamic retention, and highlight how distinct steps of GLUT4 transport are regulated by insulin signals. In particular, fusion seems to be regulated by aPKC (via munc18) and Akt (via syntaxin4-interacting protein (synip)). AS160 participates in GLUT4 intracellular retention, and possibly fusion, through candidate ras-related GTP-binding protein (Rab)2, Rab8, Rab10 and/or Rab14. The localization of the insulin-sensitive GLUT4 compartment and the precise target of insulin-derived signals remain open for future investigation