Erika Lorenzetto

Genetic perturbation of postsynaptic activity regulates synapse elimination in developing cerebellum

In many parts of the vertebrate nervous system, synaptic connections are remodeled during early postnatal life. Neural activity plays an important role in regulating one such rearrangement, synapse elimination, in the developing neuromuscular system, but there is little direct evidence on roles of pre- or postsynaptic activity in regulating synapse elimination in the developing brain. To address this issue, we expressed a chloride channel-yellow fluorescent protein fusion in cerebellar Purkinje cells (PCs) of transgenic mice to decrease their excitability. We then assessed elimination of supernumerary climbing fiber inputs to PCs. Individual PCs are innervated by multiple climbing fibers at birth; all but one are eliminated during the first three postnatal weeks in wild-type mice, but multiple innervation persists for at least three months in the transgenic mice. The normal redistribution of climbing fiber synapses from PC somata to proximal dendrites was also blunted in transgenics. These results show that normal electrical activity of the postsynaptic cell is required for it to attain a mature innervation pattern.

Reelin is transiently expressed in the peripheral nerve during development and is upregulated following nerve crush

Reelin is an extracellular matrix protein which is critical for the positioning of migrating post-mitotic neurons and the laminar organization of several brain structures during development. We investigated the expression and localization of Reelin in the rodent peripheral nerve during postnatal development and following crush injury in the adult stage. As shown with Western blotting, immunocytochemistry and RT-PCR, Schwann cells in the developing peripheral nerve and in primary cultures from neonatal nerves produce and secrete Reelin. While Reelin levels are downregulated in adult stages, they are again induced following sciatic nerve injury. A morphometric analysis of sciatic nerve sections of reeler mice suggests that Reelin is not essential for axonal ensheathment by Schwann cells, however, it influences the caliber of myelinated axons and the absolute number of fibers per unit area. This indicates that Reelin may play a role in peripheral nervous system development and repair by regulating Schwann cell-axon interactions.

Rac1 Selective Activation Improves Retina Ganglion Cell Survival and Regeneration

In adult mammals, after optic nerve injury, retinal ganglion cells (RGCs) do not regenerate their axons and most of them die by apoptosis within a few days. Recently, several strategies that activate neuronal intracellular pathways were proposed to prevent such degenerative processes. The rho-related small GTPase Rac1 is part of a complex, still not fully understood, intracellular signaling network, mediating in neurons many effects, including axon growth and cell survival. However, its role in neuronal survival and regeneration in vivo has not yet been properly investigated. To address this point we intravitreally injected selective cell-penetrating Rac1 mutants after optic nerve crush and studied the effect on RGC survival and axonal regeneration. We injected two well-characterized L61 constitutively active Tat-Rac1 fusion protein mutants, in which a second F37A or Y40C mutation confers selectivity in downstream signaling pathways. Results showed that, 15 days after crush, both mutants were able to improve survival and to prevent dendrite degeneration, while the one harboring the F37A mutation also improved axonal regeneration. The treatment with F37A mutant for one month did not improve the axonal elongation respect to 15 days. Furthermore, we found an increase of Pak1 T212 phosphorylation and ERK1/2 expression in RGCs after F37A treatment, whereas ERK1/2 was more activated in glial cells after Y40C administration. Our data suggest that the selective activation of distinct Rac1-dependent pathways could represent a therapeutic strategy to counteract neuronal degenerative processes in the retina.

Rho GTPase-dependent plasticity of dendritic spines in the adult brain

Brain activity is associated with structural changes in the neural connections. However, in vivo imaging of the outer cortical layers has shown that dendritic spines, on which most excitatory synapses insist, are predominantly stable in adulthood. Changes in dendritic spines are governed by small GTPases of the Rho family through modulation of the actin cytoskeleton. Yet, while there are abundant data about this functional effect of Rho GTPases in vitro, there is limited evidence that Rho GTPase signaling in the brain is associated with changes in neuronal morphology. In the present work, both chronic in vivo two-photon imaging and Golgi staining reveal that the activation of Rho GTPases in the adult mouse brain is associated with little change of dendritic spines in the apical dendrites of primary visual cortex pyramidal neurons. On the contrary, considerable increase in spine density is observed (i) in the basal dendrites of the same neurons (ii) in both basal and apical dendrites of the hippocampal CA1 pyramidal cells. While confirming that Rho GTPase-dependent increase in spine density can be substantial, the study indicates region and dendrite selectivity with relative stability of superficial cortical circuits.

Impaired nerve regeneration in reeler mice after peripheral nerve injury

Reelin, an extracellular matrix protein, plays an important role in the regulation of neuronal migration and cortical lamination in the developing brain. Little is known, however, about the role of this protein in axonal regeneration. We have previously shown that Reelin is secreted by Schwann cells in the peripheral nerve compartment during postnatal development and that it is up‐regulated following nerve injury in adult mice. In this work, we generated mice deficient in Reelin (reeler) that express yellow fluorescent protein (YFP) in a subset of neurons and examined the axonal regeneration following nerve crush. We found that axonal regeneration was significantly altered compared with wild‐type mice. By contrast, retrograde tracing with Fluorogold dye after sciatic nerve crush was unaffected in these mutants, being comparable with normal axonal transport observed in wild‐type. These results indicate that the absence of Reelin impairs axonal regeneration following injury and support a role for this protein in the process of peripheral nerve regeneration.

Astrocytes Regulate the Expression of Insulin-Like Growth Factor 1 Receptor (IGF1-R) in Primary Cortical Neurons During In Vitro Senescence

The role of insulin-like growth factor 1 (IGF1) pathway as regulator of aging and age-related diseases is increasingly recognized. Recent evidence has been provided that neuronal IGF1-R increases during aging leading to activation of a signaling pathway that causes an increased production of amyloid β-peptide, the principal event in the pathogenesis of Alzheimer’s disease. Here, by using long-term neuronal cultures as a model of aging, we show that astroglial cells are required to upregulate the expression of IGF1-R in neurons during in vitro senescence. Moreover, evidence is provided that the cross-talk between astrocytes and neurons is independent of cell-to-cell contact, and it is mediated by low molecular weight soluble factor(s) released by astrocytes in culture medium. These results suggest that astrocytes could play an important role in aging and age-related pathological processes.

The Potential Role of Rho GTPases in Alzheimer's Disease Pathogenesis

Alzheimers disease (AD) is characterized by a wide loss of synapses and dendritic spines. Despite extensive efforts, the molecular mechanisms driving this detrimental alteration have not yet been determined. Among the factors potentially mediating this loss of neuronal connectivity, the contribution of Rho GTPases is of particular interest. This family of proteins is classically considered a key regulator of actin cytoskeleton remodeling and dendritic spine maintenance, but new insights into the complex dynamics of its regulation have recently determined how its signaling cascade is still largely unknown, both in physiological and pathological conditions. Here, we review the growing evidence supporting the potential involvement of Rho GTPases in spine loss, which is a unanimously recognized hallmark of early AD pathogenesis. We also discuss some new insights into Rho GTPase signaling framework that might explain several controversial results that have been published. The study of the connection between AD and Rho GTPases represents a quite unchartered avenue that holds therapeutic potential.

β-Amyloid-aluminum complex alters cytoskeletal stability and increases ROS production in cortical neurons.

Several lines of evidence have supported the potential involvement of metal ions in the etiology of Alzheimers Disease (AD). However, the molecular mechanisms underlying this interaction are still partially unknown. Previous work from our laboratory has shown that β-amyloid peptide (Aβ) aggregation was strongly influenced by the conjugation of the peptide with few metal ions (aluminum, copper, zinc, and iron) that are found in high concentrations in the senile plaque core. The binding of aluminum (Al) to Aβ specifically stabilized the peptide in an oligomeric conformation. Here, we show that the aggregation of Aβ-Al was boosted by sodium dodecyl sulfate, a detergent that mimics some characteristics of biological membrane, suggesting a potential role for membrane components in the Aβ aggregation process. Notably, we also found that Aβ-Al caused mitochondrial dysfunction and reactive oxygen species production in primary cortical neurons. Aβ-Al strongly promoted also alterations in cytoskeleton network as shown by the increased F-actin expression and the occurrence of neuritic beading. Interestingly, the neurotoxic effect of this metal complex was associated with a decreased mRNA expression of ubiquitin thiolesterase, an ubiquitin-dependent protein involved in catabolic process, and by the increased expression of glutaminyl cyclase, responsible for pathological post-translational modification of Aβ. These results suggest that, in neuronal cells, Aβ-Al can induce relevant detrimental changes that resemble pathological hallmarks of AD.

Synapse formation and elimination : Role of activity studied in different models of adult muscle reinnervation

Synapse competition and elimination are a general developmental process both in central and in peripheral nervous systems that is strongly activity dependent. Some common features regulate synapse competition, and one of these is an application to development of the Hebbs postulate of learning: repeated coincident spike activity in competing presynaptic inputs on the same target cell inhibits competition, whereas noncoincident activity promotes weakening of some of the inputs and ultimately their elimination. Here we report experiments that indicate that the development of muscle innervation (initial polyneuronal innervation and subsequent synapse elimination) follows the Hebbs paradigm. We utilized two different models of muscle reinnervation in the adult rat: 1) we crushed nerves going to soleus or extensor digitorum longus muscles, to activate regeneration of the presynaptic component of the neuromuscular junctions (NMJ), or 2) we injected the soleus muscle with Marcaine (a myotoxic agent) to activate regeneration of the postsynaptic component, the muscle fiber. A condition of transient polyneuronal innervation occurs during NMJ regeneration in both cases, although the two models differ insofar as the relative strength of the competing inputs is concerned. During the period of competition (a few days or weeks, in Marcaine or crush experiments, respectively), we imposed a synchronous firing pattern on the competing inputs by stimulating motor axons distal to a chronic conduction block and demonstrated that this procedure strongly inhibits synapse elimination, with respect to control muscles in which regeneration occurs under natural impulse activity of motoneurons.

Sos1 Regulates Macrophage Podosome Assembly and Macrophage Invasive Capacity

Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation.