Coronal brain atlas in stereotaxic coordinates of the African spiny mouse, Acomys cahirinus.

The African spiny mouse (Acomys cahirinus) is an emerging model of mammalian epimorphic regeneration that has aroused the interest of the scientific community in the last decade. To date, studies on brain repair have been hindered by the lack of knowledge on the neuroanatomy of this species. Here, we present a coronal brain atlas in stereotaxic coordinates, which allows for three-dimensional identification and localization of the brain structures of this species. The brain of 12-week-old spiny mice was mapped in stereotaxic coordinates using cresyl violet-stained brain sections obtained from coronal cryosectioning of the brain after transcardial perfusion with fixative. The atlas is presented in 42 plates representing sections spaced 240 µm apart. Stereotaxic coordinates were validated using both a model of Parkinsonian lesion of the striatum with 6-hydroxydopamine and labeling of the corticospinal tract in the spiny mouse spinal cord using AAV1/2-GFP intracortical injections. This work presents a new tool in A. cahirinus neurobiology and opens new avenues of research for the investigation of the regenerative ability of A. cahirinus in models of brain disorders.

Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration.

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.

Effects of early intravesical administration of resiniferatoxin to spinal cord-injured rats in neurogenic detrusor overactivity.

OBJECTIVES: To investigate if intravesical administration during spinal shock of resiniferatoxin (RTX), an ultrapotent desensitizing agonist of transient receptor potential vanilloid-1 (TRPV1), would silence TRPV1-expressing bladder afferents at an early stage of disease progression and modulate neurogenic detrusor overactivity (NDO) emergence. MATERIALS AND METHODS: Rats submitted to largely incomplete spinal cord transection at T8/9 spinal segment were treated with intravesical RTX (50 nM) or its vehicle during spinal shock. Four weeks after spinal lesion, bladder-reflex activity was evaluated by cystometry under urethane anesthesia, after which the bladder, spinal cord, and dorsal root ganglia were collected and processed. RESULTS: We found improvements on bladder function several weeks after early intravesical RTX administration, including a marked decrease of intravesical pressures and amplitude of bladder contractions. Such strong long-lasting urodynamic effects resulted from the very potent desensitizing activity of RTX on peripheral terminals of sensory afferents, an effect restricted to the bladder. CONCLUSION: Our results support that an early intervention with RTX could potentially attenuate NDO development and ensuing urinary incontinence, with a dramatic impact on the quality of life of spinal cord injury patients.

Fibrin functionalization with synthetic adhesive ligands interacting with α6ß1 integrin receptor enhance neurite outgrowth of embryonic stem cell-derived neural stem/progenitors.

To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor cells (NSPCs) and its capacity to support NSPC migration and neurite extension, we explored the tethering of synthetic peptides engaging integrin α6ß1, a cell receptor enriched in NSPCs. Six α6ß1 integrin ligands were tested for their ability to support integrin α6ß1-mediated adhesion of embryonic stem cell-derived NSPCs (ES-NSPs) and sustain ES-NSPC viability, migration, and neuronal differentiation. Due to their better performance, peptides T1, HYD1, and A5G81 were immobilized into fibrin and functionalized gels characterized in terms of peptide binding efficiency, structure and viscoelastic properties. Tethering of T1 or HYD1 successfully enhanced cell outgrowth from ES-NSPC neurospheres (up to 2.4-fold increase), which exhibited a biphasic response to peptide concentration. Inhibition assays evidenced the involvement of α6ß1 and α3ß1 integrins in mediating radial outgrowth on T1-/HYD1-functionalized gels. Fibrin functionalization also promoted neurite extension of single ES-NSPCs in fibrin, without affecting cell proliferation and neuronal differentiation. Finally, HYD1-functionalized gels were found to provide a permissive environment for axonal regeneration, leading up to a 2.0-fold increase in neurite extension from rat dorsal root ganglia explants as compared to unmodified fibrin, and to significant improved locomotor function after spinal cord injury (complete transection), along with a trend toward a higher area positive for growth associated protein 43 (marker for axonal growth cone formation). Our results suggest that conjugation of α6ß1 integrin-binding motifs is of interest to increase the biofunctionality of hydrogels used in 3D platforms for ES-NSPC culture and potentially, in matrix-assisted ES-NSPC transplantation. STATEMENT OF SIGNIFICANCE: Impact statement: The transplantation of NSPCs derived from pluripotent stem cells holds much promise for the treatment of central nervous system disorders. Moreover, the combinatorial use of biodegradable hydrogels with NSPCs was shown to contribute to the establishment of a more permissive environment for survival and integration of transplanted cells. In this study, fibrin hydrogels functionalized with a synthetic peptide engaging integrin α6ß1 (HYD1) were shown to promote neurite extension of ES-NSPCs, which is fundamental for the formation of functional neuronal relay circuits after NSPC transplantation. Notably, HYD1-functionalized fibrin per se led to enhanced axonal growth ex vivo and to an improvement in locomotor function after implantation in a rat model of spinal cord injury. Conjugation of α6ß1 integrin-binding motifs may therefore be of interest to confer bioactivity to NSPC hydrogel vehicles.

Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons.

Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague-Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration.

Inhibitory Injury Signaling Represses Axon Regeneration After Dorsal Root Injury.

Following injury to peripheral axons, besides increased cyclic adenosine monophosphate (cAMP), the positive injury signals extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT-3) are locally activated and retrogradely transported to the cell body, where they induce a pro-regenerative program. Here, to further understand the importance of injury signaling for successful axon regeneration, we used dorsal root ganglia (DRG) neurons that have a central branch without regenerative capacity and a peripheral branch that regrows after lesion. Although injury to the DRG central branch (dorsal root injury (DRI)) activated ERK, JNK, and STAT-3 and increased cAMP levels, it did not elicit gain of intrinsic growth capacity nor the ability to overcome myelin inhibition, as occurred after peripheral branch injury (sciatic nerve injury (SNI)). Besides, gain of growth capacity after SNI was independent of ERK and cAMP. Antibody microarrays of dynein-immunoprecipitated axoplasm from rats with either DRI or SNI revealed a broad differential activation and transport of signals after each injury type and further supported that ERK, JNK, STAT-3, and cAMP signaling pathways are minor contributors to the differential intrinsic axon growth capacity of both injury models. Increased levels of inhibitory injury signals including GSK3ß and ROCKII were identified after DRI, not only in axons but also in DRG cell bodies. In summary, our work shows that activation and transport of positive injury signals are not sufficient to promote increased axon growth capacity and that differential modulation of inhibitory molecules may contribute to limited regenerative response.

CNS axons globally increase axonal transport after peripheral conditioning.

Despite the inability of CNS axons to regenerate, an increased regenerative capacity can be elicited following conditioning lesion to the peripheral branch of dorsal root ganglia neurons (DRGs). By in vivo radiolabeling of rat DRGs, coupled to mass spectrometry and kinesin immunoprecipitation of spinal cord extracts, we determined that the anterograde transport of cytoskeleton components, metabolic enzymes and axonal regeneration enhancers, was increased in the central branch of DRGs following a peripheral conditioning lesion. Axonal transport of mitochondria was also increased in the central branch of Thy1-MitoCFP mice following a peripheral injury. This effect was generalized and included augmented transport of lysosomes and synaptophysin- and APP-carrying vesicles. Changes in axonal transport were only elicited by a peripheral lesion and not by spinal cord injury. In mice, elevated levels of motors and of polyglutamylated and tyrosinated tubulin were present following a peripheral lesion and can explain the increase in axonal transport induced by conditioning. In summary, our work shows that a peripheral injury induces a global increase in axonal transport that is not restricted to the peripheral branch, and that, by extending to the central branch, allows a rapid and sustained support of regenerating central axons.

Peripheral nervous system plasmalogens regulate Schwann cell differentiation and myelination.

Rhizomelic chondrodysplasia punctata (RCDP) is a developmental disorder characterized by hypotonia, cataracts, abnormal ossification, impaired motor development, and intellectual disability. The underlying etiology of RCDP is a deficiency in the biosynthesis of ether phospholipids, of which plasmalogens are the most abundant form in nervous tissue and myelin; however, the role of plasmalogens in the peripheral nervous system is poorly defined. Here, we used mouse models of RCDP and analyzed the consequence of plasmalogen deficiency in peripheral nerves. We determined that plasmalogens are crucial for Schwann cell development and differentiation and that plasmalogen defects impaired radial sorting, myelination, and myelin structure. Plasmalogen insufficiency resulted in defective protein kinase B (AKT) phosphorylation and subsequent signaling, causing overt activation of glycogen synthase kinase 3ß (GSK3ß) in nerves of mutant mice. Treatment with GSK3ß inhibitors, lithium, or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) restored Schwann cell defects, effectively bypassing plasmalogen deficiency. Our results demonstrate the requirement of plasmalogens for the correct and timely differentiation of Schwann cells and for the process of myelination. In addition, these studies identify a mechanism by which the lack of a membrane phospholipid causes neuropathology, implicating plasmalogens as regulators of membrane and cell signaling.

Aboard transthyretin: From transport to cleavage.

Transthyretin (TTR) is a plasma and cerebrospinal fluid protein mainly recognized as the transporter of thyroxine (T(4)) 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.

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 alpha-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.

Chapter 17: Transthyretin: an enhancer of nerve regeneration.

Transthyretin (TTR), a plasma and cerebrospinal fluid protein secreted by the liver and choroid plexus, is mainly known as the physiological carrier of thyroxine (T(4)) and retinol. Under pathological conditions, various TTR mutations are related to familial amyloid polyneuropathy (FAP), a neurodegenerative disorder characterized by deposition of TTR amyloid fibrils, particularly in the peripheral nervous system (PNS), leading to axonal loss and neuronal death. Recently, a number of TTR functions in neurobiology have been described; these may explain the preferential TTR deposition, when mutated, in the PNS of FAP patients. In this respect, and with a particular relevance in the PNS, TTR has been shown to have the ability to enhance neurite outgrowth in vitro and nerve regeneration following injury, in vivo. In the following pages, this novel TTR function, as well as its importance in nerve biology and repair will be discussed.

NPY revealed as a critical modulator of osteoblast function in vitro: new insights into the role of Y1 and Y2 receptors.

Neuropeptide Y (NPY) has recently emerged as a potential regulator of bone homeostasis. However, the relevance of NPY's role in osteoblast activity and the biological functions involving NPY receptors in bone homeostasis remain to be clarified. Here we report that chronically elevated NPY levels leaded to a modulation of the level of Y2 receptor expression marked with a transient down and upregulation according to the stage of osteoblast differentiation. We also show that NPY is a negative regulator of Y1 receptor expression. The pharmacological activation of Y2 receptor with its agonist resulted in similar effect. Functional analysis also revealed the osteogenic potential of NPY with osteoblast phenotype markers being significantly enhanced in osteoprogenitor cells stimulated by NPY, probably due to the down-regulation of Y1 receptor. In contrasts, these cells exhibit a reduction in calcium deposition in extracellular matrix most likely mediated via Y2 receptor signalling. Furthermore, we show that NPY modulates receptor activator of nuclear factor kB (NF-kB) (RANK) ligand and osteoprotegerin, two key factors regulating bone remodelling. Specifically, NPY inhibits the transcriptional activity of RANKL promoter in osteoprogenitor cells and enhances OPG expression in osteoblasts at early stages of differentiation. However, NPY effect on OPG seemed to be unrelated to Y2 receptor activation. Taken together the present data supported the contribution of NPY pathway in bone homeostasis via a direct action on osteoblasts cells.

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.

Increase in ghrelin levels after weight loss in obese Zucker rats is prevented by gastric banding.

BACKGROUND: Gastric banding is thought to decrease appetite in addition to the mechanical effects of food restriction, although this has been difficult to demonstrate in human studies. Our aim was to investigate the changes in orexigenic signals in the obese Zucker rat after gastric banding. METHODS: Obese Zucker rats (fa/fa) were submitted to gastric banding (GBP), sham gastric banding fed ad libitum (sham), or sham operation with food restriction, pair-fed to the gastric banding group (sham-PF). Lean Zucker rats (fa/+) were used as additional controls. Body weight and food intake were daily recorded for 21 days after surgery when epididymal fat was weighed and fasting ghrelin and hypothalamic NPY mRNA expression were measured. RESULTS: Gastric banding in obese Zucker rats resulted in a significant decrease of cumulative body weight gain and food intake. Furthermore, gastric banded rats were leaner than Sham-PF, as expressed by a significantly lower epididymal fat weight. Ghrelin levels of gastric banded rats were not increased when compared to sham-operated animals fed ad libitum and were significantly lower than the levels of weight matched sham-PF rats (1116.9 +/- 103.3 g GBP vs 963.2 +/- 54.3 g sham, 3,079.5 +/- 221.6 sham-PF and 2,969.9 +/- 150.9 g lean rats, p < 0.001); hypothalamic NPY mRNA expression was not increased in GBP when compared to sham-operated rats. CONCLUSION: In obese Zucker rats, GBP prevents the increase in orexigenic signals that occur during caloric deprivation. Our data support the hypothesis that sustained weight loss observed after gastric banding does not depend solely on food restriction.

Transthyretin enhances nerve regeneration.

Mutations in transthyretin (TTR) are associated with familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR deposition in the PNS. The aim of this study was to unravel whether TTR has a role in nerve physiology that could account for its preferential accumulation in the PNS, when mutated. The sensorimotor performance of wild-type and TTR knockout (KO) littermate mice was compared and showed impairment in mice lacking TTR. Given the possibility that, upon regeneration, the consequences arising from TTR absence might be exacerbated, nerve crush was performed in both strains. TTR KO mice presented delayed functional recovery resulting from decreased number of myelinated and unmyelinated fibers. Moreover, in transgenic mice in a TTR KO background, expressing human TTR in neurons, this phenotype was rescued, reinforcing that TTR enhances nerve regeneration. In vitro assays showed that neurite outgrowth and extension were decreased in the absence of TTR, probably underlying the decreased number of regenerating axons in TTR KO mice. Our findings demonstrate that TTR participates in nerve physiology and that it enhances nerve regeneration. Moreover, the assignment of a TTR function in nerve biology and repair, may explain its preferential deposition, when mutated, in the PNS of familial amyloid polyneuropathy patients.

Deposition and passage of transthyretin through the blood-nerve barrier in recipients of familial amyloid polyneuropathy livers.

Familial amyloid polyneuropathy (FAP) is characterized by deposition of mutated transthyretin (TTR) in the peripheral nervous system. Prior to amyloid fibrils, nonfibrillar TTR aggregates are deposited inducing oxidative stress with increased nitration (3-NT). As the major source of TTR is the liver, liver transplantation (LT) is used to halt FAP. Given the shortage of liver donors, domino LT (DLT) using FAP livers is performed. The correlation between TTR deposition in the skin and nerve was tested in biopsies from normal individuals, asymptomatic carriers (FAP 0) and FAP patients; in FAP 0, nonfibrillar TTR was observed both in the skin and nerve in the same individuals; in patients, amyloid was detected in both tissues. The occurrence of amyloidosis in recipients of FAP livers was evaluated 1-7 years after DLT: TTR deposition occurred in the skin 3 years after transplantation either as amyloid or aggregates; in one of the recipients, fibrillar TTR was present in the epineurium 6 years after DLT. Deposits were scarce and 3-NT immunostaining was irrelevant. Nerve biopsies from DLT recipients had no FAP-related neuropathy. Our findings suggest that TTR amyloid formation occurs faster than predicted and that TTR of liver origin can cross the blood-nerve barrier. Recipients of FAP livers should be under surveillance for TTR deposition and tissue damage.