Methods in Molecular Biology

Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals

Centronuclear myopathy in labrador retrievers: a recent founder mutation in the PTPLA gene has rapidly disseminated worldwide

Centronuclear myopathies (CNM) are inherited congenital disorders characterized by an excessive number of internalized nuclei. In humans, CNM results from ~70 mutations in three major genes from the myotubularin, dynamin and amphiphysin families. Analysis of animal models with altered expression of these genes revealed common defects in all forms of CNM, paving the way for unified pathogenic and therapeutic mechanisms. Despite these efforts, some CNM cases remain genetically unresolved. We previously identified an autosomal recessive form of CNM in French Labrador retrievers from an experimental pedigree, and showed that a loss-of-function mutation in the protein tyrosine phosphatase-like A (PTPLA) gene segregated with CNM. Around the world, client-owned Labrador retrievers with a similar clinical presentation and histopathological changes in muscle biopsies have been described. We hypothesized that these Labradors share the same PTPLA<sup>cnm</sup> mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLA<sup>cnm</sup> carriers in 13 countries. Haplotype analysis demonstrated that the PTPLA<sup>cnm</sup> allele resulted from a single and recent mutational event that may have rapidly disseminated through the extensive use of popular sires. PTPLA-deficient Labradors will help define the integrated role of PTPLA in the existing CNM gene network. They will be valuable complementary large animal models to test innovative therapies in CNM

Microtubule and actin differentially regulate synaptic vesicle cycling to maintain high-frequency neurotransmission

Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission

STOP Proteins are Responsible for the High Degree of Microtubule Stabilization Observed in Neuronal Cells

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation

Development of 3D culture scaffolds for directional neuronal growth using 2-photon lithography

Conventional applications of transplant technology, applied to severe traumatic injuries of the nervous system, have met limited success in the clinics due to the complexity of restoring function to the damaged tissue. Neural tissue engineering aims to deploy scaffolds mimicking the physiological properties of the extracellular matrix to facilitate the elongation of axons and the repair of damaged nerves. However, the fabrication of ideal scaffolds with precisely controlled thickness, texture, porosity, alignment, and with the required mechanical strength, features needed for effective clinical applications, remains technically challenging. We took advantage of state-of-the-art 2-photon photolithography to fabricate highly ordered and biocompatible 3D nanogrid structures to enhance neuronal directional growth. First, we characterized the physical and chemical properties and proved the biocompatibility of said scaffolds by successfully culturing primary sensory and motor neurons on their surface. Interestingly, axons extended along the fibers with a high degree of alignment to the pattern of the nanogrid, as opposed to the lack of directionality observed on flat glass or polymeric surfaces, and could grow in 3D between different layers of the scaffold. The axonal growth pattern observed is highly desirable for the treatment of traumatic nerve damage occurring during peripheral and spinal cord injuries. Thus, our findings provide a proof of concept and explore the possibility of deploying aligned fibrous 3D scaffold/implants for the directed growth of axons, and could be used in the design of scaffolds targeted towards the restoration and repair of lost neuronal connections

Anterograde Axonal Transport in Neuronal Homeostasis and Disease

Neurons are highly polarized cells with an elongated axon that extends far away from the cell body. To maintain their homeostasis, neurons rely extensively on axonal transport of membranous organelles and other molecular complexes. Axonal transport allows for spatio-temporal activation and modulation of numerous molecular cascades, thus playing a central role in the establishment of neuronal polarity, axonal growth and stabilization, and synapses formation. Anterograde and retrograde axonal transport are supported by various molecular motors, such as kinesins and dynein, and a complex microtubule network. In this review article, we will primarily discuss the molecular mechanisms underlying anterograde axonal transport and its role in neuronal development and maturation,including the establishment of functional synaptic connections.We will then provide an overview of the molecular and cellular perturbations that affect axonal transport and are often associated with axonal degeneration. Lastly, we will relate our current understanding of the role of axonal trafficking concerning anterograde trafficking of mRNA and its involvement in the maintenance of the axonal compartment and disease

Bmcc1s, a Novel Brain-Isoform of Bmcc1, Affects Cell Morphology by Regulating MAP6/STOP Functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology

Biotensegrity of the Extracellular Matrix: Physiology, Dynamic Mechanical Balance, and Implications in Oncology and Mechanotherapy

Cells have the capacity to convert mechanical stimuli into chemical changes. This process is based on the tensegrity principle, a mechanism of tensional integrity. To date, this principle has been demonstrated to act in physiological processes such as mechanotransduction and mechanosensing at different scales (from cell sensing through integrins to molecular mechanical interventions or even localized massage). The process involves intra- and extracellular components, including the participation of extracellular matrix (ECM) and microtubules that act as compression structures, and actin filaments which act as tension structures. The nucleus itself has its own tensegrity system which is implicated in cell proliferation, differentiation, and apoptosis. Despite present advances, only the tip of the iceberg has so far been uncovered regarding the role of ECM compounds in influencing biotensegrity in pathological processes. Groups of cells, together with the surrounding ground substance, are subject to different and specific forces that certainly influence biological processes. In this paper, we review the current knowledge on the role of ECM elements in determining biotensegrity in malignant processes and describe their implication in therapeutic response, resistance to chemo- and radiotherapy, and subsequent tumor progression. Original data based on the study of neuroblastic tumors will be provided

Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10-20), ER-negative BC (P=1.1 × 10-13), BRCA1-associated BC (P=7.7 × 10-16) and triple negative BC (P-diff=2 × 10-5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10-3) and ABHD8 (P<2 × 10-3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk