Márcia Almeida Liz

ApoA-I cleaved by transthyretin has reduced ability to promote cholesterol efflux and increased amyloidogenicity

A fraction of plasma transthyretin (TTR) circulates in HDL through binding to apolipoprotein A-I (apoA-I). Moreover, TTR is able to cleave the C terminus of lipid-free apoA-I. In this study, we addressed the relevance of apoA-I cleavage by TTR in lipoprotein metabolism and in the formation of apoA-I amyloid fibrils. We determined that TTR may also cleave lipidated apoA-I, with cleavage being more effective in the lipid-poor preβ-HDL subpopulation. Upon TTR cleavage, discoidal HDL particles displayed a reduced capacity to promote cholesterol efflux from cholesterol-loaded THP-1 macrophages. In similar assays, TTR-containing HDL from mice expressing human TTR in a TTR knockout background had a decreased ability to perform reverse cholesterol transport compared with similar particles from TTR knockout mice, reinforcing the notion that cleavage by TTR reduces the ability of apoA-I to promote cholesterol efflux. As amyloid deposits composed of N-terminal apoA-I fragments are common in the atherosclerotic intima, we assessed the impact of TTR cleavage on apoA-I aggregation and fibrillar growth. We determined that TTR-cleaved apoA-I has a high propensity to form aggregated particles and that it formed fibrils faster than full-length apoA-I, as assessed by electron microscopy. Our results show that apoA-I cleavage by TTR may affect HDL biology and the development of atherosclerosis by reducing cholesterol efflux and increasing the apoA-I amyloidogenic potential.

Substrate specificity of transthyretin: identification of natural substrates in the nervous system.

Besides functioning as the plasma transporter of retinol and thyroxine, TTR (transthyretin) is a protease, cleaving apoA-I (apolipoprotein A-I) after a phenylalanine residue. In the present study, we further investigated TTR substrate specificity. By using both P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a lysine residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homoeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We investigated whether TTR proteolytic activity is involved in these functions. Both wild-type TTR and TTR(prot-) (proteolytically inactive TTR) had a similar effect in the expression of peptidylglycine alpha-amidating mono-oxygenase, the rate-limiting enzyme in neuropeptide amidation, excluding the involvement of TTR proteolytic activity in neuropeptide maturation. However, TTR was able to cleave amidated NPY (neuropeptide Y), probably contributing to the increased NPY levels reported in TTR-knockout mice. To assess the involvement of TTR proteolytic activity in axonal regeneration, neurite outgrowth of cells cultivated with wild-type TTR or TTR(prot-), was measured. Cells grown with TTR(prot-) displayed decreased neurite length, thereby suggesting that TTR proteolytic activity is important for its function as a regeneration enhancer. By showing that TTR is able to cleave NPY and that its proteolytic activity affects axonal growth, the present study shows that TTR has natural substrates in the nervous system, establishing further its relevance in neurobiology.

Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2

BackgroundIn the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity.ResultsFollowing a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3β (GSK3β) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3β regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3β activity in neurons. Following spinal cord injury, reduced GSK3β levels or complete neuronal deletion of GSK3β led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3β substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3β-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3β-CRMP-2 relevance during axon regeneration.ConclusionsOur work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3β-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.

Neuropeptide Y and osteoblast differentiation - the balance between the neuro-osteogenic network and local control

Accumulating evidence has contributed to a novel view in bone biology: bone remodeling, specifically osteoblast differentiation, is under the tight control of the central and peripheral nervous systems. Among other players in this neuro‐osteogenic network, the neuropeptide Y (NPY) system has attracted particular attention. At the central nervous system level, NPY exerts its function in bone homeostasis through the hypothalamic Y2 receptor. Locally in the bone, NPY action is mediated by its Y1 receptor. Besides the presence of Y1, a complex network exists locally: not only there is input of the peripheral nervous system, as the bone is directly innervated by NPY‐containing fibers, but there is also input from non‐neuronal cells, including bone cells capable of NPY expression. The interaction of these distinct players to achieve a multilevel control system of bone homeostasis is still under debate. In this review, we will integrate the current knowledge on the impact of the NPY system in bone biology, and discuss the mechanisms through which the balance between central and the peripheral NPY action might be achieved.

Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe's disease.

In Krabbes disease, a demyelinating disorder, add‐on strategies targeting the peripheral nervous system (PNS) are needed, as it is not corrected by bone‐marrow (BM) transplantation. To circumvent this limitation of BM transplantation, we assessed whether i.v. delivery of immortalized EGFP+ BM‐derived murine mesenchymal stromal cells (BM‐MSCTERT‐EGFP) targets the PNS of a Krabbes disease model, the Twitcher mouse. In vitro, BM‐MSCTERT‐EGFP retained the phenotype of primary BM‐MSC and did not originate tumors upon transplantation in nude mice. In vivo, undifferentiated EGFP+ cells grafted the Twitcher sciatic nerve where an increase in Schwann cell precursors and axonal number was detected. The same effect was observed on BM‐MSCTERT‐EGFP i.v. delivery following sciatic nerve crush, a model of axonal regeneration. Reiterating the in vivo findings, in a coculture system, BM‐MSCTERT‐EGFP induced the proliferation of Twitcher‐derived Schwann cells and the neurite outgrowth of both Twitcher‐derived neurons and wild‐type neurons grown in the presence of psychosine, the toxic substrate that accumulates in Krabbes disease. In vitro, this neuritogenic effect was blocked by K252a, an antagonist of Trk receptors, and by antibody blockage of brain derived neurotrophic factor, a neurotrophin secreted by BM‐MSCTERT‐EGFP and induced in neighboring Schwann cells. In vivo, BM‐MSCTERT‐EGFP surmounted the effect of K252a, indicating their ability to act through a neurotrophin‐independent mechanism. In summary, i.v. delivery of BM‐MSCTERT‐EGFP exerts a multilevel effect targeting neurons and Schwann cells, coordinately diminishing neuropathology. Therefore, to specifically target the PNS, MSC should be considered an add‐on option to BM transplantation in Krabbes disease and in other disorders where peripheral axonal loss occurs. STEM CELLS 2011;29:1738–1751

Neuropeptide Y expression and function during osteoblast differentiation – insights from transthyretin knockout mice

To better understand the role of neuropeptide Y (NPY) in bone homeostasis, as its function in the regulation of bone mass is unclear, we assessed its expression in this tissue. By immunohistochemistry, we demonstrated, both at embryonic stages and in the adult, that NPY is synthesized by osteoblasts, osteocytes, and chondrocytes. Moreover, peptidylglycine α‐amidating monooxygenase, the enzyme responsible for NPY activation by amidation, was also expressed in these cell types. Using transthyretin (TTR) KO mice as a model of augmented NPY levels, we showed that this strain has increased NPY content in the bone, further validating the expression of this neuropeptide by bone cells. Moreover, the higher amidated neuropeptide levels in TTR KO mice were related to increased bone mineral density and trabecular volume. Additionally, RT‐PCR analysis established that NPY is not only expressed in MC3T3‐E1 osteoblastic cells and bone marrow stromal cells (BMSCs), but is also detectable by RIA in BMSCs undergoing osteoblastic differentiation. In agreement with our in vivo observations, in vitro, TTR KO BMSCs differentiated in osteoblasts had increased NPY levels and exhibited enhanced competence in undergoing osteoblastic differentiation. In summary, this work contributes to a better understanding of the role of NPY in the regulation of bone formation by showing that this neuropeptide is expressed in bone cells and that increased amidated neuropeptide content is related to increased bone mass.

The cytoskeleton as a novel therapeutic target for old neurodegenerative disorders

Cytoskeleton defects, including alterations in microtubule stability, in axonal transport as well as in actin dynamics, have been characterized in several unrelated neurodegenerative conditions. These observations suggest that defects of cytoskeleton organization may be a common feature contributing to neurodegeneration. In line with this hypothesis, drugs targeting the cytoskeleton are currently being tested in animal models and in human clinical trials, showing promising effects. Drugs that modulate microtubule stability, inhibitors of posttranslational modifications of cytoskeletal components, specifically compounds affecting the levels of tubulin acetylation, and compounds targeting signaling molecules which regulate cytoskeleton dynamics, constitute the mostly addressed therapeutic interventions aiming at preventing cytoskeleton damage in neurodegenerative disorders. In this review, we will discuss in a critical perspective the current knowledge on cytoskeleton damage pathways as well as therapeutic strategies designed to revert cytoskeleton-related defects mainly focusing on the following neurodegenerative disorders: Alzheimers Disease, Parkinsons Disease, Huntingtons Disease, Amyotrophic Lateral Sclerosis and Charcot-Marie-Tooth Disease.

Aboard transthyretin: From transport to cleavage.

Transthyretin (TTR) is a plasma and cerebrospinal fluid protein mainly recognized as the transporter of thyroxine (T4) and retinol. Mutated TTR leads to familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR amyloid deposition particularly in peripheral nerves. Beside its transport activities, TTR is a cryptic protease and participates in the biology of the nervous system. Several studies have been directed at finding new ligands of TTR to further explore the biology of the protein. From the identified ligands, some were in fact TTR protease substrates. In this review, we will discuss the existent information concerning TTR ligands/substrates.

Primary bone marrow mesenchymal stromal cells rescue the axonal phenotype of Twitcher mice.

Krabbes disease (KD) is a demyelinating disorder caused by the deficiency of lysosomal galactocerebrosi-dase (GALC), affecting both the central (CNS) and the peripheral nervous system (PNS). A current therapy, hematopoietic stem cell transplantation (HSCT), is ineffective at correcting the PNS pathology. We have previously shown that systemic delivery of immortalized bone marrow-derived murine mesenchymal stromal cells (BM-MSCs) diminishes the neuropathology of transplanted Twitcher mice, a murine model of KD. In this study, to move one step closer to clinical application, the effectiveness of a systematic delivery of primary BM-MSCs to promote recovery of the Twitcher PNS was assessed. Primary BM-MSCsgrafted to the Twitcher sciatic nerve led to increased GALC activity that was not correlated to decreased psychosine (the toxic GALC substrate) accumulation. Nevertheless, BM-MSC transplantation rescued the axonal phenotype of Twitcher mice in the sciatic nerve, with an increased density of both myelinated and unmyelinated axons in transplanted animals. Whereas no increase in myelination was observed, upon transplantation an increased proliferation of Schwann cell precursors occurred. Supporting these findings, in vitro, BM-MSCs promoted neurite outgrowth of Twitcher sensory neurons and proliferation of Twitcher Schwann cells. Moreover, BM-MSCs expressed nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and promoted increased BDNF synthesis by neighboring Schwann cells. Besides their action in neurons and glia, BM-MSCs led to macrophage activation in Twitcher sciatic nerves. In summary, primary BM-MSCs diminish the neuropathology of Twitcher sciatic nerves by coordinately affecting neurons, glia, and macrophages.

Transthyretin Null Mice as a Model to Study the Involvement of Transthyretin in Neurobiology: From Neuropeptide Processing to Nerve Regeneration

Physiologically, TTR is mainly acknowledged for being the plasma transporter of thyroxine (T4) and retinol. Under pathological conditions, several mutations in TTR are associated with familial amyloid polyneuropathy (FAP), a neurodegenerative disorder characterized by deposition of TTR amyloid fibrils, particularly in the peripheral nervous system (PNS), where it leads to axonal loss and neuronal death. Although it is well established that TTR synthesis occurs in the liver and in the choroid plexus (the sources of TTR in the plasma and cerebrospinal fluid –CSF, respectively), the origin of TTR deposited in the PNS of FAP patients is unknown. Under physiological conditions TTR has access to the nerve both through the blood and CSF. Additionally, a function for TTR in nerve biology could explain its preferential deposition, when mutated, in the PNS. In this respect, several studies using TTR knockout (KO) mice revealed new TTR functions specifically related to the nervous system: (1) the absence of TTR is associated with reduced signs of depressive-like behavior and with memory impairment; (2) TTR participates in sensorimotor performance; (3) TTR regulates neuropeptide maturation and, (4) TTR enhances nerve regeneration. In the following pages, these novel TTR functions related to the nervous system, as well as the use of TTR KO mice as a means to study them, will be discussed.