Ataxia with giant axonopathy in Acbd5-deficient mice halted by adeno-associated virus gene therapy.

Acyl-CoA binding domain containing 5 (ACBD5) is a critical player in handling very long chain fatty acids (VLCFA) en route for peroxisomal ß-oxidation. Mutations in ACBD5 lead to the accumulation of VLCFA and patients present retinal dystrophy, ataxia, psychomotor delay and a severe leukodystrophy. Using CRISPR/Cas9, we generated and characterized an Acbd5 Gly357* mutant allele. Gly357* mutant mice recapitulated key features of the human disorder, including reduced survival, impaired locomotion and reflexes, loss of photoreceptors, and demyelination. The ataxic presentation of Gly357* mice involved the loss of cerebellar Purkinje cells and a giant axonopathy throughout the CNS. Lipidomic studies provided evidence for the extensive lipid dysregulation caused by VLCFA accumulation. Following a proteomic survey, functional studies in neurons treated with VLCFA unravelled a deregulated cytoskeleton with reduced actin dynamics and increased neuronal filopodia. We also show that an adeno-associated virus-mediated gene delivery ameliorated the gait phenotypes and the giant axonopathy, also improving myelination and astrocyte reactivity. Collectively, we established a mouse model with significance for VLCFA-related disorders. The development of relevant neuropathological outcomes enabled the understanding of mechanisms modulated by VLCFA and the evaluation of the efficacy of preclinical therapeutic interventions.

C57BL/6J and B6129F1 Embryo Transfer: Unilateral and Bilateral Transfer, Embryo Number and Recipient Female Background Control for the Optimization of Embryo Survival and Litter Size.

Embryo transfer (ET) is a common procedure in rodent facilities. Optimizing this technique may help to reduce the number of animals, but little information is available regarding wild type strains and the conditions that affect embryo transfer. To explore this theme, 2-cell C57BL/6J embryos were transferred after overnight culture of freshly collected zygotes using different conditions: unilateral transfers using a total of 6, 8, 12, 15, 20 and 25 embryos were performed initially; then, this strain was also used for bilateral transfers using a total of 6, 12 and 20 embryos equally divided by the two oviducts. Groups of 25 embryos were not tested for the bilateral technique, since this condition produced the lower success rate when using the unilateral technique and 20 embryos would still represent a large number of embryos. A group of 2-cell B6129F1 embryos was also transferred using unilateral and bilateral ET with 6, 12 and 20 embryos. Crl:CD1(ICR) were used as recipient females for non-reciprocal transfers and C57BL/6J were used to test reciprocal transfers (only tested for six C57BL/6J unilateral transfers). Unilateral transfers using C57BL/6J mice produced higher success rates using six embryos, compared to the other groups transferred unilaterally (p-values between 0.0001 and 0.0267), but the mean number of pups per litter was not different among groups. Bilateral transfer produced higher number of pups when 20 embryos were divided by the two oviducts compared to six (p = 0.0012) or 12 (p = 0.0148) embryos, but with no differences in success rates. No statistical differences were found between the groups of B6129F1, but better results were obtained on bilateral transfers using a total of six embryos. For the strain tested (C57BL/6J), the uterine environment (Crl:CD1(ICR) or C57BL/6J recipient) does not impact the outcome of the technique. These results complement previous work published using genetically engineered mice strains and show that unilateral transfers using low number of embryos (6), produce better outcomes when compared to bilateral or unilateral transfers using more embryos. It also highlights differences between the outcome of bilateral transfers in the two strains tested. A set of historical data of genetically engineered mice at a C57BL/6J background was also included, confirming that lower embryo numbers are related to higher success rates. Together, the outcome of these experiments can be important to reduce the number of recipient and donor females, optimize embryo transfers and improve animal welfare discouraging the use of a more invasive technique.

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.

The Dyslexia-susceptibility Protein KIAA0319 Inhibits Axon Growth Through Smad2 Signaling.

KIAA0319 is a transmembrane protein associated with dyslexia with a presumed role in neuronal migration. Here we show that KIAA0319 expression is not restricted to the brain but also occurs in sensory and spinal cord neurons, increasing from early postnatal stages to adulthood and being downregulated by injury. This suggested that KIAA0319 participates in functions unrelated to neuronal migration. Supporting this hypothesis, overexpression of KIAA0319 repressed axon growth in hippocampal and dorsal root ganglia neurons; the intracellular domain of KIAA0319 was sufficient to elicit this effect. A similar inhibitory effect was observed in vivo as axon regeneration was impaired after transduction of sensory neurons with KIAA0319. Conversely, the deletion of Kiaa0319 in neurons increased neurite outgrowth in vitro and improved axon regeneration in vivo. At the mechanistic level, KIAA0319 engaged the JAK2-SH2B1 pathway to activate Smad2, which played a central role in KIAA0319-mediated repression of axon growth. In summary, we establish KIAA0319 as a novel player in axon growth and regeneration with the ability to repress the intrinsic growth potential of axons. This study describes a novel regulatory mechanism operating during peripheral nervous system and central nervous system axon growth, and offers novel targets for the development of effective therapies to promote axon regeneration.

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

In Krabbe's 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-MSC(TERT-EGFP) ) targets the PNS of a Krabbe's disease model, the Twitcher mouse. In vitro, BM-MSC(TERT-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-MSC(TERT-EGFP) i.v. delivery following sciatic nerve crush, a model of axonal regeneration. Reiterating the in vivo findings, in a coculture system, BM-MSC(TERT-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 Krabbe's 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-MSC(TERT-EGFP) and induced in neighboring Schwann cells. In vivo, BM-MSC(TERT-EGFP) surmounted the effect of K252a, indicating their ability to act through a neurotrophin-independent mechanism. In summary, i.v. delivery of BM-MSC(TERT-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 Krabbe's disease and in other disorders where peripheral axonal loss occurs.

Histological and stereological characterization of brown trout (Salmo trutta f. fario) trunk kidney.

The large variability in kidney morphology among fish makes it difficult to build a "universal" model on its function and structure. Therefore, a morphological study of brown trout trunk kidney was performed, considering potential seasonal and sex effects. Three-year-old specimens of both sexes were collected at four stages of their reproductive cycle. Kidney was processed for light and electron microscopy. The relative volumes of renal components, such as renal corpuscles and different nephron tubules, were estimated by stereological methods. Qualitatively, the general nephron structure of brown trout was similar to that described for other glomerular teleost species. Quantitatively, however, differences in the relative volume of some renal components were detected between sexes and among seasons. Particularly, highest values of vacuolized tubules and new growing tubules were observed after spawning, being more relevant in females. Despite seasonal changes, more linear correlations were found between those parameters and the reno-somatic index than the gonado-somatic index. Thus, we verified that some brown trout renal components undergo sex dependent seasonal variations, suggesting a morphological adaptation of the components to accomplish physiological needs. These findings constitute a baseline for launching studies to know which factors govern the morphological variations and their functional consequences.

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.

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.

Transthyretin knockout mice display decreased susceptibility to AMPA-induced neurodegeneration.

Transthyretin (TTR) has been regarded as a neuroprotective protein given that TTR knockout (KO) mice display increased susceptibility for amyloid beta deposition and memory deficits during aging. In parallel, TTR KO mice have increased levels of neuropeptide Y (NPY), which promotes neuroprotection and neuroproliferation. In this work, we aimed at evaluating TTR neuroprotective effect against an excitotoxic insult that is known to be prevented by NPY action. We show that despite a putative neuroprotective role of TTR, hippocampal slice cultures from TTR KO mice display a decreased susceptibility to AMPA-induced neurodegeneration. We also suggest that increased NPY levels in TTR KO mice are not associated with increased cell proliferation in the dentate gyrus or subventricular zone. In summary, the alleged neuroprotective role of TTR in the nervous system should be regarded with caution and should not be generalized to all types of insults.

Transthyretin internalization by sensory neurons is megalin mediated and necessary for its neuritogenic activity.

Mutated transthyretin (TTR) causes familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR deposition in the peripheral nervous system (PNS). The origin/reason for TTR deposition in the nerve is unknown. Here we demonstrate that both endogenous mouse TTR and TTR injected intravenously have access to the mouse sciatic nerve. We previously determined that in the absence of TTR, both neurite outgrowth in vitro and nerve regeneration in vivo were impaired. Reinforcing this finding, we now show that local TTR delivery to the crushed sciatic nerve rescues the regeneration phenotype of TTR knock-out (KO) mice. As the absence of TTR was unrelated to neuronal survival, we further evaluated the Schwann cell and inflammatory response to injury, as well as axonal retrograde transport, in the presence/absence of TTR. Only retrograde transport was impaired in TTR KO mice which, in addition to the neurite outgrowth impairment, might account for the decreased regeneration in this strain. Moreover, we show that in vitro, in dorsal root ganglia neurons, clathrin-dependent megalin-mediated TTR internalization is needed for TTR neuritogenic activity. Supporting this observation, we demonstrate that in vivo, decreased levels of megalin lead to decreased nerve regeneration and that megalin's action as a regeneration enhancer is dependent on TTR. In conclusion, our work unravels the mechanism of TTR action during nerve regeneration. Additionally, TTR presence in the nerve, as is here shown, may underlie its preferential deposition in the PNS of familial amyloid polyneuropathy patients.