bioRxiv (Cold Spring Harbor Laboratory), Mar 4, 2024
We previously reported that in the absence of Prostaglandin D2 synthase (L-PGDS) peripheral nerve... more We previously reported that in the absence of Prostaglandin D2 synthase (L-PGDS) peripheral nerves are hypomyelinated in development and that with aging they present aberrant myelin sheaths. We now demonstrate that L-PGDS expressed in Schwann cells is part of a coordinated program aiming at preserving myelin integrity. In vivo and in vitro lipidomic, metabolomic and transcriptomic analyses confirmed that myelin lipids composition, Schwann cells energetic metabolism and key enzymes controlling these processes are altered in the absence of L-PGDS. Moreover, Schwann cells undergo a metabolic rewiring and turn to acetate as the main energetic source. Further, they produce ketone bodies to ensure glial cell and neuronal survival. Importantly, we demonstrate that all these changes correlate with morphological myelin alterations and describe the first physiological pathway implicated in preserving PNS myelin. Collectively, we posit that myelin lipids serve as a reservoir to provide ketone bodies, which together with acetate represent the adaptive substrates Schwann cells can rely on to sustain the axo-glial unit and preserve the integrity of the PNS. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RESULTS Loss of l-pgds alters Schwann cell lipid homeostasis and energetic metabolism. We previously showed that in the absence of the PGD2 prostanoid, sciatic nerves are hypomyelinated and characterized by aberrant myelin in aged mutant mice lacking hand l-pgds expression. We also reported that ablation of l-pgds alone is sufficient to cause hypomyelination 16. To define the molecular components at the basis of this altered myelin structure, we analyzed myelin protein expression as well as lipid and metabolism composition in sciatic nerves of null l-pgds and littermate controls at different ages. Quantitative RT-PCR (Fig. 1a-b, and 1d-e) and Western Blotting analyses (Fig. 1c and 1f) on sciatic nerves of 1-and 8-months old l-pgds-/mice showed no differences in Myelin Basic Protein (MBP) and Protein Zero (P0) protein expression, indicating that myelin lipid profile rather than proteins might be affected in l-pgds-/mice. Hence, we performed lipidomic and metabolomic analyses on sciatic nerves of 4-, 6-and 8-months old l-pgds-/mutants. Principal component analyses (PCA) (Fig. 1g) and heat map analyses of the top 50 dysregulated metabolites, (Supplementary Fig. 1ac) showed that both at 4 and at 6 months l-pgds-/mice cluster separately from littermate controls, a difference less pronounced at 8 months (Fig. 1g), indicating that L-PGDS likely participates in lipid/metabolic homeostasis in sciatic nerves. Myelin alterations increase with age in sciatic nerves l-pgds-/mice. Next, we assessed whether these changes correlated with the previously reported altered morphology 16. Thus, we quantified the number of myelin alterations in sciatic nerves in 4-and 8-months old l-pgds-/mice and we observed that while at 8 months myelin aberrations are significantly increased in l-pgds-/sciatic nerves, at 4 months, albeit augmented in mutant mice, they did not reach a significant difference (Fig. 2a-d). These in vivo results strongly suggest an important role for L-PGDS in myelin maintenance 16. Further, they indicate a trend in accumulating myelin alterations in adulthood. Myelin alterations accumulate in vivo in the absence of glial L-PGDS. Since L-PGDS is expressed both in neurons and in Schwann cells and neuronal L-PGDS contributes to myelin formation 16 , we next sought to determine the cell specific effect of l-pgds in adulthood. Thus, we crossed l-pgds flx/flx mice 19 with mutant mice driving Cre recombinase expression either in motor neurons (Chat-Cre) 20 or in myelinating Schwann cells (P0-Cre) 21. We first confirmed specific recombination in ventral spinal cord (Chat-Cre) (Supplementary Fig. 2a) and in sciatic nerves (P0-Cre)
Animal models provide an important tool to investigate the pathogenesis of neuromuscular disorder... more Animal models provide an important tool to investigate the pathogenesis of neuromuscular disorders. In the present study, we analyze fiber composition of the brachial plexus branches to the pectoral muscles: the medial anterior thoracic nerve (MATN) and the lateral anterior thoracic nerve (LATN). The morphological and morphometric characteristics and the percentage of motor fibers within each nerve are here reported, adding information to microscopic anatomy knowledge of the rat brachial plexus. As control, we employed the quadriceps nerve, commonly used for the evaluation of motor fibers at hindlimbs. We demonstrated that the MATN and the LATN are predominantly composed of large motor fibers and therefore could be employed to evaluate the peripheral nervous system (PNS) involvement at forelimbs in neurological diseases models, predominantly affecting the motor fiber compartment.
were mainly associated with ribosomal proteins (RP) and olfactory receptors (OR) signaling pathwa... more were mainly associated with ribosomal proteins (RP) and olfactory receptors (OR) signaling pathways, respectively. Among the top up-regulated genes were actin alpha 1 skeletal muscle (ACTA1) and desmin (DES). Similarly, in NL nerves ACTA1, DES and several RPs were highly expressed, associated with endothelial cells and pericytes abnormalities. Peripheral nerve involvement may be due to conversion towards a more aggressive phenotype, potentially explaining the poor prognosis. The candidate genes reported in this study may be a source of clinical biomarkers for NL.
sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease... more sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.
Journal of The Peripheral Nervous System, Feb 1, 2003
Charcot-Marie-Tooth neuropathies and Dejerine-Sottas Syndrome are associated with mutations in th... more Charcot-Marie-Tooth neuropathies and Dejerine-Sottas Syndrome are associated with mutations in the gene encoding Myelin Protein Zero. Here we report the characterization of two lines of transgenic mice expressing, in addition to the wild type protein, different point mutations in the extracellular domain of P0: the first is a deletion of Ser63 (DSer63), described in Charcot-Marie-Tooth 1B patients, whereas the second is a substitution of Ser 63 with a Cys (Ser63Cys) reported in a Dejerine-Sottas patient. By crossing the transgenic mice with P0 null mice, we produced animals that expressed only the mutated P0. We then investigated in vivo the expression and the effect of the two mutated proteins by morphological, biochemical and immunohistochemical analyses. Sciatic nerves of mice expressing P0DSer63 show severe hypomyelination, with the presence of onion bulbs and some uncompacted myelin sheaths. P0DSer63 does not reach the myelin sheaths and it accumulates in the cytoplasm, where it is probably degraded. In contrast, sciatic nerves of mice expressing P0Ser63Cys present a milder hypomyelination with rare onion bulbs and, surprisingly, normal myelin sheaths. The mutated protein is a full-length protein that, presumably, is integrated into the myelin sheaths. This study demonstrates that different mutations in P0 glycoprotein, leading to disorders of different severity in human, have different fates in the Schwann cells.
Although inflammation appears to play a role in neurolymphomatosis (NL), the mechanisms leading t... more Although inflammation appears to play a role in neurolymphomatosis (NL), the mechanisms leading to degeneration in the peripheral nervous system are poorly understood. The purpose of this exploratory study was to identify molecular pathways underlying NL pathogenesis, combining clinical and neuropathological investigation with gene expression (GE) studies. We characterized the clinical and pathological features of eight patients with NL. We further analysed GE changes in sural nerve biopsies obtained from a subgroup of NL patients (n=3) and thirteen patients with inflammatory neuropathies as neuropathic controls. Based on the neuropathic symptoms and signs, NL patients were classified into three forms of neuropathy: chronic symmetrical sensorimotor polyneuropathy (SMPN, n=3), multiple mononeuropathy (MN, n=4) and acute motor-sensory axonal neuropathy (AMSAN, n=1). Predominantly diffuse malignant cells infiltration of epineurium was present in chronic SMPN, whereas endoneurial periva...
Figure S1. MHCI depletion affect the number of CD3+ / CD4+ T cells but not their extent of infilt... more Figure S1. MHCI depletion affect the number of CD3+ / CD4+ T cells but not their extent of infiltration in the spinal cord during the disease progression. Figure S2. MHCI depletion reduces the impairment of the cervical motor neurons in mSOD1 mice. Figure S3. MHCI depletion lowered the CD68 mRNA levels in the spinal cord of mSOD1 mice. Figure S4. MHCI depletion did not affect the extent of astrocytosis in the cervical and the lumbar spinal cord of G93A+/+ mice. Figure S5. MHCI expression is lower in the cervical than in the lumbar spinal cord of G93A+/+ mice. Figure S6. MHCI depletion accelerates denervation of hindlimb muscles in mSOD1mice. Figure S7. MHCI depletion inhibits the proliferation of the terminal Schwann cells and the size of AChR clusters in SOD1 mutant mice. Figure S8. MHCI depletion accelerates the atrophy of hindlimbs muscles in SOD1 mutant mice. Figure S9. MHCI depletion preserves the diaphragm innervation in SOD1 mutant mice. Figure S10. GFAP and phospho-ERK expre...
Abstract: We investigated the cell association and intracellular pathway to the nucleus of comple... more Abstract: We investigated the cell association and intracellular pathway to the nucleus of complexes formed between DNA and cationic lipid DOTAP (lipoplexes) or cationic polymer polyethylenimine (polyplexes). Flow cytometry and confocal microscope analysis showed that lipoplexes presented higher affinity for cell membrane than polyplexes. Electron microscopy demonstrated that both types of complexes followed an endocytic pathway and were metabolized but did not enter the nucleus. However, by in situ PCR and FISH it was possible to show that the plasmid localized to the nuclei, indicating that DNA must be dissociated from the vectors to be delivered to the nucleus. Our results identify a different behaviour in the interaction of polyplexes and lipoplexes with the cell, indicating that a combination of advantageous properties of the two kinds of cationic molecules could further ameliorate efficiency of nonviral gene transfer vectors.
Proceedings of the National Academy of Sciences, 2021
Significance This study describes an astrocyte-dependent mechanism contributing to CNS demyelinat... more Significance This study describes an astrocyte-dependent mechanism contributing to CNS demyelination. The key molecular and functional checkpoint is the neurotrophin receptor TrkB, whose ligands are absent in chronic demyelinated MS lesions. Here, we show that TrkB can be transactivated by inflammatory and toxic signals and is critical for the generation of glial calcium flux and the up-regulation of the copper transporter CTR1. This process enhances copper distribution by the astrocyte, thereby fostering myelin and oligodendrocyte loss.
The cellular barriers to efficient gene transfer into the airways are still poorly understood. Ra... more The cellular barriers to efficient gene transfer into the airways are still poorly understood. Rat tracheal epithelial (RTE) cells faithfully reproduce the mucociliary, pseudostratified characteristics of the in vivo human airway epithelium and are a suitable model for studying major pathways of airway cell differentiation. We studied the efficiency of various non-viral cationic vectors in the primary culture of RTE cells grown in different conditions. When we grew RTE cells on collagenated plastic dishes for 4 days in basal conditions (25 ng/ml EGF), a poorly-differentiated (PD) phenotype appeared, whether or not retinoic acid (RA) was present in culture medium. The cells were transfected with a plasmid encoding a luciferase reporter gene under the control of the cytomegalovirus promoter. The highest luciferase levels were obtained with a cationic lipid (DOTAP) and a cationic polymer (PEI). When cells were grown for 12 days on collagen-coated inserts (Transwells ® ) at the air-liqu...
Phosphorylated TDP-43 (pTDP-43) aggregates in the cytoplasm of motor neurons and neuroglia in the... more Phosphorylated TDP-43 (pTDP-43) aggregates in the cytoplasm of motor neurons and neuroglia in the brain are one of the pathological hallmarks of amyotrophic lateral sclerosis. Although the axons exceed the total volume of motor neuron soma by several orders of magnitude, systematic studies investigating the presence and distribution of pTDP-43 aggregates within motor nerves are still lacking. The aim of this study is to define the TDP-43/pTDP-43 pathology in diagnostic motor nerve biopsies performed on a large cohort of patients presenting with a lower motor neuron syndrome and to assess whether this might be a discriminating tissue biomarker for amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases. We retrospectively evaluated 102 lower motor neuron syndrome patients referred to our centre for a diagnostic motor nerve biopsy. Histopathological criteria of motor neuron disease and motor neuropathy were applied by two independent evaluators, who were blind to cli...
Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia... more Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease of the upper motoneuron with still undefined pathomechanism. The intermittent mitochondrial permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain mitochondrial homeostasis by reducing intra-matrix Ca 2+ and reactive oxygen species (ROS) concentration, and is critical for efficient synaptic function. Methods: We use a fluorescence-based approach to measure mPTP flickering in living cells and biochemical and molecular biology techniques to dissect the pathogenic mechanism of SPG7. In the SPG7 animal model we evaluate the potential improvement of the motor defect, neuroinflammation and neurodegeneration by means of an mPTP inducer, the benzodiazepine Bz-423. Findings: We demonstrate that paraplegin is required for efficient transient opening of the mPTP, that is impaired in both SPG7 patients-derived fibroblasts and primary neurons from Spg7 À/À mice. We show that dysregulation of mPTP opening at the pre-synaptic terminal impairs neurotransmitter release leading to ineffective synaptic transmission. Lack of paraplegin impairs mPTP flickering by a mechanism involving increased expression and activity of sirtuin3, which promotes deacetylation of cyclophilin D, thus hampering mPTP opening. Pharmacological treatment with Bz-423, which bypasses the activity of CypD, normalizes synaptic transmission and rescues the motor impairment of the SPG7 mouse model. Interpretation: mPTP targeting opens a new avenue for the potential therapy of this form of spastic paraplegia.
Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alph... more Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.
Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient pr... more Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient propagation of the electric impulse in the nervous system and to maintain neuronal integrity. In the central nervous system (CNS), the development of oligodendrocytes and the process of myelination are regulated by the coordinated action of several positive and negative cell‐extrinsic factors. We and others previously showed that secretases regulate the activity of proteins essential for myelination. We now report that the neuronal α‐secretase ADAM17 controls oligodendrocyte differentiation and myelin formation in the CNS. Ablation of Adam17 in neurons impairs in vivo and in vitro oligodendrocyte differentiation, delays myelin formation throughout development and results in hypomyelination. Furthermore, we show that this developmental defect is, in part, the result of altered Notch/Jagged 1 signaling. Surprisingly, in vivo conditional loss of Adam17 in immature oligodendrocytes has no effe...
The major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononuclea... more The major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord microglia and motor axons of transgenic SOD1 mice. To assess the role of MHCI in the disease, we examined transgenic SOD1 mice crossbred with β2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8 T cells. The lack of MHCI and CD8 T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8 T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1 mice. We provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the...
Increasing evidence suggests that the immune system has a beneficial role in the progression of a... more Increasing evidence suggests that the immune system has a beneficial role in the progression of amyotrophic lateral sclerosis (ALS) although the mechanism remains unclear. Recently, we demonstrated that motor neurons (MNs) of C57SOD1(G93A) mice with slow disease progression activate molecules classically involved in the cross-talk with the immune system. This happens a lot less in 129SvSOD1(G93A) mice which, while expressing the same amount of transgene, had faster disease progression and earlier axonal damage. The present study investigated whether and how the immune response is involved in the preservation of motor axons in the mouse model of familial ALS with a more benign disease course. First, the extent of axonal damage, Schwann cell proliferation, and neuromuscular junction (NMJ) denervation were compared between the two ALS mouse models at the disease onset. Then, we compared the expression levels of different immune molecules, the morphology of myelin sheaths, and the prese...
sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease... more sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.
bioRxiv (Cold Spring Harbor Laboratory), Mar 4, 2024
We previously reported that in the absence of Prostaglandin D2 synthase (L-PGDS) peripheral nerve... more We previously reported that in the absence of Prostaglandin D2 synthase (L-PGDS) peripheral nerves are hypomyelinated in development and that with aging they present aberrant myelin sheaths. We now demonstrate that L-PGDS expressed in Schwann cells is part of a coordinated program aiming at preserving myelin integrity. In vivo and in vitro lipidomic, metabolomic and transcriptomic analyses confirmed that myelin lipids composition, Schwann cells energetic metabolism and key enzymes controlling these processes are altered in the absence of L-PGDS. Moreover, Schwann cells undergo a metabolic rewiring and turn to acetate as the main energetic source. Further, they produce ketone bodies to ensure glial cell and neuronal survival. Importantly, we demonstrate that all these changes correlate with morphological myelin alterations and describe the first physiological pathway implicated in preserving PNS myelin. Collectively, we posit that myelin lipids serve as a reservoir to provide ketone bodies, which together with acetate represent the adaptive substrates Schwann cells can rely on to sustain the axo-glial unit and preserve the integrity of the PNS. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RESULTS Loss of l-pgds alters Schwann cell lipid homeostasis and energetic metabolism. We previously showed that in the absence of the PGD2 prostanoid, sciatic nerves are hypomyelinated and characterized by aberrant myelin in aged mutant mice lacking hand l-pgds expression. We also reported that ablation of l-pgds alone is sufficient to cause hypomyelination 16. To define the molecular components at the basis of this altered myelin structure, we analyzed myelin protein expression as well as lipid and metabolism composition in sciatic nerves of null l-pgds and littermate controls at different ages. Quantitative RT-PCR (Fig. 1a-b, and 1d-e) and Western Blotting analyses (Fig. 1c and 1f) on sciatic nerves of 1-and 8-months old l-pgds-/mice showed no differences in Myelin Basic Protein (MBP) and Protein Zero (P0) protein expression, indicating that myelin lipid profile rather than proteins might be affected in l-pgds-/mice. Hence, we performed lipidomic and metabolomic analyses on sciatic nerves of 4-, 6-and 8-months old l-pgds-/mutants. Principal component analyses (PCA) (Fig. 1g) and heat map analyses of the top 50 dysregulated metabolites, (Supplementary Fig. 1ac) showed that both at 4 and at 6 months l-pgds-/mice cluster separately from littermate controls, a difference less pronounced at 8 months (Fig. 1g), indicating that L-PGDS likely participates in lipid/metabolic homeostasis in sciatic nerves. Myelin alterations increase with age in sciatic nerves l-pgds-/mice. Next, we assessed whether these changes correlated with the previously reported altered morphology 16. Thus, we quantified the number of myelin alterations in sciatic nerves in 4-and 8-months old l-pgds-/mice and we observed that while at 8 months myelin aberrations are significantly increased in l-pgds-/sciatic nerves, at 4 months, albeit augmented in mutant mice, they did not reach a significant difference (Fig. 2a-d). These in vivo results strongly suggest an important role for L-PGDS in myelin maintenance 16. Further, they indicate a trend in accumulating myelin alterations in adulthood. Myelin alterations accumulate in vivo in the absence of glial L-PGDS. Since L-PGDS is expressed both in neurons and in Schwann cells and neuronal L-PGDS contributes to myelin formation 16 , we next sought to determine the cell specific effect of l-pgds in adulthood. Thus, we crossed l-pgds flx/flx mice 19 with mutant mice driving Cre recombinase expression either in motor neurons (Chat-Cre) 20 or in myelinating Schwann cells (P0-Cre) 21. We first confirmed specific recombination in ventral spinal cord (Chat-Cre) (Supplementary Fig. 2a) and in sciatic nerves (P0-Cre)
Animal models provide an important tool to investigate the pathogenesis of neuromuscular disorder... more Animal models provide an important tool to investigate the pathogenesis of neuromuscular disorders. In the present study, we analyze fiber composition of the brachial plexus branches to the pectoral muscles: the medial anterior thoracic nerve (MATN) and the lateral anterior thoracic nerve (LATN). The morphological and morphometric characteristics and the percentage of motor fibers within each nerve are here reported, adding information to microscopic anatomy knowledge of the rat brachial plexus. As control, we employed the quadriceps nerve, commonly used for the evaluation of motor fibers at hindlimbs. We demonstrated that the MATN and the LATN are predominantly composed of large motor fibers and therefore could be employed to evaluate the peripheral nervous system (PNS) involvement at forelimbs in neurological diseases models, predominantly affecting the motor fiber compartment.
were mainly associated with ribosomal proteins (RP) and olfactory receptors (OR) signaling pathwa... more were mainly associated with ribosomal proteins (RP) and olfactory receptors (OR) signaling pathways, respectively. Among the top up-regulated genes were actin alpha 1 skeletal muscle (ACTA1) and desmin (DES). Similarly, in NL nerves ACTA1, DES and several RPs were highly expressed, associated with endothelial cells and pericytes abnormalities. Peripheral nerve involvement may be due to conversion towards a more aggressive phenotype, potentially explaining the poor prognosis. The candidate genes reported in this study may be a source of clinical biomarkers for NL.
sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease... more sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.
Journal of The Peripheral Nervous System, Feb 1, 2003
Charcot-Marie-Tooth neuropathies and Dejerine-Sottas Syndrome are associated with mutations in th... more Charcot-Marie-Tooth neuropathies and Dejerine-Sottas Syndrome are associated with mutations in the gene encoding Myelin Protein Zero. Here we report the characterization of two lines of transgenic mice expressing, in addition to the wild type protein, different point mutations in the extracellular domain of P0: the first is a deletion of Ser63 (DSer63), described in Charcot-Marie-Tooth 1B patients, whereas the second is a substitution of Ser 63 with a Cys (Ser63Cys) reported in a Dejerine-Sottas patient. By crossing the transgenic mice with P0 null mice, we produced animals that expressed only the mutated P0. We then investigated in vivo the expression and the effect of the two mutated proteins by morphological, biochemical and immunohistochemical analyses. Sciatic nerves of mice expressing P0DSer63 show severe hypomyelination, with the presence of onion bulbs and some uncompacted myelin sheaths. P0DSer63 does not reach the myelin sheaths and it accumulates in the cytoplasm, where it is probably degraded. In contrast, sciatic nerves of mice expressing P0Ser63Cys present a milder hypomyelination with rare onion bulbs and, surprisingly, normal myelin sheaths. The mutated protein is a full-length protein that, presumably, is integrated into the myelin sheaths. This study demonstrates that different mutations in P0 glycoprotein, leading to disorders of different severity in human, have different fates in the Schwann cells.
Although inflammation appears to play a role in neurolymphomatosis (NL), the mechanisms leading t... more Although inflammation appears to play a role in neurolymphomatosis (NL), the mechanisms leading to degeneration in the peripheral nervous system are poorly understood. The purpose of this exploratory study was to identify molecular pathways underlying NL pathogenesis, combining clinical and neuropathological investigation with gene expression (GE) studies. We characterized the clinical and pathological features of eight patients with NL. We further analysed GE changes in sural nerve biopsies obtained from a subgroup of NL patients (n=3) and thirteen patients with inflammatory neuropathies as neuropathic controls. Based on the neuropathic symptoms and signs, NL patients were classified into three forms of neuropathy: chronic symmetrical sensorimotor polyneuropathy (SMPN, n=3), multiple mononeuropathy (MN, n=4) and acute motor-sensory axonal neuropathy (AMSAN, n=1). Predominantly diffuse malignant cells infiltration of epineurium was present in chronic SMPN, whereas endoneurial periva...
Figure S1. MHCI depletion affect the number of CD3+ / CD4+ T cells but not their extent of infilt... more Figure S1. MHCI depletion affect the number of CD3+ / CD4+ T cells but not their extent of infiltration in the spinal cord during the disease progression. Figure S2. MHCI depletion reduces the impairment of the cervical motor neurons in mSOD1 mice. Figure S3. MHCI depletion lowered the CD68 mRNA levels in the spinal cord of mSOD1 mice. Figure S4. MHCI depletion did not affect the extent of astrocytosis in the cervical and the lumbar spinal cord of G93A+/+ mice. Figure S5. MHCI expression is lower in the cervical than in the lumbar spinal cord of G93A+/+ mice. Figure S6. MHCI depletion accelerates denervation of hindlimb muscles in mSOD1mice. Figure S7. MHCI depletion inhibits the proliferation of the terminal Schwann cells and the size of AChR clusters in SOD1 mutant mice. Figure S8. MHCI depletion accelerates the atrophy of hindlimbs muscles in SOD1 mutant mice. Figure S9. MHCI depletion preserves the diaphragm innervation in SOD1 mutant mice. Figure S10. GFAP and phospho-ERK expre...
Abstract: We investigated the cell association and intracellular pathway to the nucleus of comple... more Abstract: We investigated the cell association and intracellular pathway to the nucleus of complexes formed between DNA and cationic lipid DOTAP (lipoplexes) or cationic polymer polyethylenimine (polyplexes). Flow cytometry and confocal microscope analysis showed that lipoplexes presented higher affinity for cell membrane than polyplexes. Electron microscopy demonstrated that both types of complexes followed an endocytic pathway and were metabolized but did not enter the nucleus. However, by in situ PCR and FISH it was possible to show that the plasmid localized to the nuclei, indicating that DNA must be dissociated from the vectors to be delivered to the nucleus. Our results identify a different behaviour in the interaction of polyplexes and lipoplexes with the cell, indicating that a combination of advantageous properties of the two kinds of cationic molecules could further ameliorate efficiency of nonviral gene transfer vectors.
Proceedings of the National Academy of Sciences, 2021
Significance This study describes an astrocyte-dependent mechanism contributing to CNS demyelinat... more Significance This study describes an astrocyte-dependent mechanism contributing to CNS demyelination. The key molecular and functional checkpoint is the neurotrophin receptor TrkB, whose ligands are absent in chronic demyelinated MS lesions. Here, we show that TrkB can be transactivated by inflammatory and toxic signals and is critical for the generation of glial calcium flux and the up-regulation of the copper transporter CTR1. This process enhances copper distribution by the astrocyte, thereby fostering myelin and oligodendrocyte loss.
The cellular barriers to efficient gene transfer into the airways are still poorly understood. Ra... more The cellular barriers to efficient gene transfer into the airways are still poorly understood. Rat tracheal epithelial (RTE) cells faithfully reproduce the mucociliary, pseudostratified characteristics of the in vivo human airway epithelium and are a suitable model for studying major pathways of airway cell differentiation. We studied the efficiency of various non-viral cationic vectors in the primary culture of RTE cells grown in different conditions. When we grew RTE cells on collagenated plastic dishes for 4 days in basal conditions (25 ng/ml EGF), a poorly-differentiated (PD) phenotype appeared, whether or not retinoic acid (RA) was present in culture medium. The cells were transfected with a plasmid encoding a luciferase reporter gene under the control of the cytomegalovirus promoter. The highest luciferase levels were obtained with a cationic lipid (DOTAP) and a cationic polymer (PEI). When cells were grown for 12 days on collagen-coated inserts (Transwells ® ) at the air-liqu...
Phosphorylated TDP-43 (pTDP-43) aggregates in the cytoplasm of motor neurons and neuroglia in the... more Phosphorylated TDP-43 (pTDP-43) aggregates in the cytoplasm of motor neurons and neuroglia in the brain are one of the pathological hallmarks of amyotrophic lateral sclerosis. Although the axons exceed the total volume of motor neuron soma by several orders of magnitude, systematic studies investigating the presence and distribution of pTDP-43 aggregates within motor nerves are still lacking. The aim of this study is to define the TDP-43/pTDP-43 pathology in diagnostic motor nerve biopsies performed on a large cohort of patients presenting with a lower motor neuron syndrome and to assess whether this might be a discriminating tissue biomarker for amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases. We retrospectively evaluated 102 lower motor neuron syndrome patients referred to our centre for a diagnostic motor nerve biopsy. Histopathological criteria of motor neuron disease and motor neuropathy were applied by two independent evaluators, who were blind to cli...
Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia... more Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease of the upper motoneuron with still undefined pathomechanism. The intermittent mitochondrial permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain mitochondrial homeostasis by reducing intra-matrix Ca 2+ and reactive oxygen species (ROS) concentration, and is critical for efficient synaptic function. Methods: We use a fluorescence-based approach to measure mPTP flickering in living cells and biochemical and molecular biology techniques to dissect the pathogenic mechanism of SPG7. In the SPG7 animal model we evaluate the potential improvement of the motor defect, neuroinflammation and neurodegeneration by means of an mPTP inducer, the benzodiazepine Bz-423. Findings: We demonstrate that paraplegin is required for efficient transient opening of the mPTP, that is impaired in both SPG7 patients-derived fibroblasts and primary neurons from Spg7 À/À mice. We show that dysregulation of mPTP opening at the pre-synaptic terminal impairs neurotransmitter release leading to ineffective synaptic transmission. Lack of paraplegin impairs mPTP flickering by a mechanism involving increased expression and activity of sirtuin3, which promotes deacetylation of cyclophilin D, thus hampering mPTP opening. Pharmacological treatment with Bz-423, which bypasses the activity of CypD, normalizes synaptic transmission and rescues the motor impairment of the SPG7 mouse model. Interpretation: mPTP targeting opens a new avenue for the potential therapy of this form of spastic paraplegia.
Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alph... more Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.
Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient pr... more Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient propagation of the electric impulse in the nervous system and to maintain neuronal integrity. In the central nervous system (CNS), the development of oligodendrocytes and the process of myelination are regulated by the coordinated action of several positive and negative cell‐extrinsic factors. We and others previously showed that secretases regulate the activity of proteins essential for myelination. We now report that the neuronal α‐secretase ADAM17 controls oligodendrocyte differentiation and myelin formation in the CNS. Ablation of Adam17 in neurons impairs in vivo and in vitro oligodendrocyte differentiation, delays myelin formation throughout development and results in hypomyelination. Furthermore, we show that this developmental defect is, in part, the result of altered Notch/Jagged 1 signaling. Surprisingly, in vivo conditional loss of Adam17 in immature oligodendrocytes has no effe...
The major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononuclea... more The major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord microglia and motor axons of transgenic SOD1 mice. To assess the role of MHCI in the disease, we examined transgenic SOD1 mice crossbred with β2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8 T cells. The lack of MHCI and CD8 T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8 T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1 mice. We provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the...
Increasing evidence suggests that the immune system has a beneficial role in the progression of a... more Increasing evidence suggests that the immune system has a beneficial role in the progression of amyotrophic lateral sclerosis (ALS) although the mechanism remains unclear. Recently, we demonstrated that motor neurons (MNs) of C57SOD1(G93A) mice with slow disease progression activate molecules classically involved in the cross-talk with the immune system. This happens a lot less in 129SvSOD1(G93A) mice which, while expressing the same amount of transgene, had faster disease progression and earlier axonal damage. The present study investigated whether and how the immune response is involved in the preservation of motor axons in the mouse model of familial ALS with a more benign disease course. First, the extent of axonal damage, Schwann cell proliferation, and neuromuscular junction (NMJ) denervation were compared between the two ALS mouse models at the disease onset. Then, we compared the expression levels of different immune molecules, the morphology of myelin sheaths, and the prese...
sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease... more sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.
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Papers by Giorgia Dina