ABSTRACT
A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington's disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.
Subject(s)
ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Cognitive Dysfunction/enzymology , Huntington Disease/enzymology , Membrane Proteins/metabolism , Post-Synaptic Density/enzymology , ADAM10 Protein/genetics , Adult , Aged , Amyloid Precursor Protein Secretases/genetics , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cadherins/genetics , Cadherins/metabolism , Cognitive Dysfunction/genetics , Cognitive Dysfunction/pathology , Disease Models, Animal , Female , HEK293 Cells , Humans , Huntington Disease/genetics , Huntington Disease/pathology , Male , Membrane Proteins/genetics , Mice, Transgenic , Middle Aged , Post-Synaptic Density/genetics , Post-Synaptic Density/pathologyABSTRACT
INTRODUCTION: Microglia are highly dynamic immune cells that play a key role in the development, hemostasis and inflammatory response of the central nervous system. These cells could be a valid therapeutic target because of their involvement in the inflammatory scenario in many neuropathological diseases. AREA COVERED: Many attempts have aimed to act on microglial cells through different approaches, for instance as viral carriers to transfer genetic material, anti-inflammatory drugs to polarize and shift microglia from M1 toward an M2 phenotype, and stem cell therapy. EXPERT OPINION: The challenge remains to find ways to act selectively on this population in the inflammatory site. Original approaches are genetic targeting or pharmacological therapies that exploit some nanomaterials to deliver promising compounds. These results strongly encourage work aimed to modulate activated microglia, laying the base for treating many neurological diseases.
Subject(s)
Microglia/immunology , Molecular Targeted Therapy , Nervous System Diseases/therapy , Animals , Drug Design , Genetic Therapy/methods , Humans , Inflammation/drug therapy , Inflammation/immunology , Inflammation/pathology , Microglia/metabolism , Nanostructures , Nervous System Diseases/immunology , Nervous System Diseases/physiopathology , Patents as Topic , Stem Cell Transplantation/methodsABSTRACT
Many efforts have been performed in order to understand the role of recruited macrophages in the progression of spinal cord injury (SCI). Different studies revealed a pleiotropic effect played by these cells associated to distinct phenotypes (M1 and M2), showing a predictable spatial and temporal distribution in the injured site after SCI. Differently, the role of activated microglia in injury progression has been poorly investigated, mainly because of the challenges to target and selectively modulate them in situ. A delivery nanovector tool (poly-ε-caprolactone-based nanoparticles) able to selectively treat/target microglia has been developed and used here to clarify the temporal and spatial involvement of the pro-inflammatory response associated to microglial cells in SCI. We show that a treatment with nanoparticles loaded with minocycline, the latter a well-known anti-inflammatory drug, when administered acutely in a SCI mouse model is able to efficiently modulate the resident microglial cells reducing the pro-inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, by using this selective delivery tool we demonstrate a mechanistic link between early microglia activation and M1 macrophages recruitment to the injured site via CCL2 chemokine, revealing a detrimental contribution of pro-inflammatory macrophages to injury progression after SCI.
Subject(s)
Inflammation/pathology , Microglia/pathology , Minocycline/therapeutic use , Nanoparticles/chemistry , Spinal Cord Injuries/drug therapy , Animals , Behavior, Animal/drug effects , Cell Movement/drug effects , Chemokine CCL2/metabolism , Disease Models, Animal , Disease Progression , Macrophages/drug effects , Mice, Inbred C57BL , Microglia/drug effects , Models, Biological , Nerve Regeneration/drug effects , Phenotype , Polyesters/chemistry , Spinal Cord Injuries/pathology , Spinal Cord Injuries/physiopathologyABSTRACT
Stem cell therapy with human mesenchymal stem cells (hMSCs) represents a promising strategy in spinal cord injury (SCI). However, both systemic and parenchymal hMSCs administrations show significant drawbacks as a limited number and viability of stem cells in situ. Biomaterials able to encapsulate and sustain hMSCs represent a viable approach to overcome these limitations potentially improving the stem cell therapy. In this study, we evaluate a new agarose/carbomer based hydrogel which combines different strategies to optimize hMSCs viability, density and delivery of paracrine factors. Specifically, we evaluate a new loading procedure on a lyophilized scaffold (soaked up effect) that reduces mechanical stress in encapsulating hMSCs into the hydrogel. In addition, we combine arginine-glycine-aspartic acid (RGD) tripeptide and 3D extracellular matrix deposition to increase the capacity to attach and maintain healthy hMSCs within the hydrogel over time. Furthermore, the fluidic diffusion from the hydrogel toward the injury site is improved by using a cling film that oriented efficaciously the delivery of paracrine factors in vivo. Finally, we demonstrate that an improved combination as here proposed of hMSCs and biomimetic hydrogel is able to immunomodulate significantly the pro-inflammatory environment in a SCI mouse model, increasing M2 macrophagic population and promoting a pro-regenerative environment in situ.
Subject(s)
Biomimetic Materials/pharmacology , Hydrogel, Polyethylene Glycol Dimethacrylate/pharmacology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Spinal Cord Injuries/therapy , Animals , Cell Adhesion/drug effects , Cell Count , Cell Survival/drug effects , Extracellular Matrix/metabolism , Female , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Inflammation/pathology , Macrophages/drug effects , Macrophages/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/ultrastructure , Mice, Inbred C57BL , Microfluidics , Oligopeptides/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal disease due to motoneuron degeneration. Magnetic resonance imaging (MRI) is becoming a promising non-invasive approach to monitor the disease course but a direct correlation with neuropathology is not feasible in human. Therefore in this study we aimed to examine MRI changes in relation to histopathology in two mouse models of ALS (C57BL6/J and 129S2/SvHsd SOD1G93A mice) with different disease onset and progression. A longitudinal in vivo analysis of T2 maps, compared to ex vivo histological changes, was performed on cranial motor nuclei. An increased T2 value was associated with a significant tissue vacuolization that occurred prior to motoneuron loss in the cranial nuclei of C57 SOD1G93A mice. Conversely, in 129Sv SOD1G93A mice, which exhibit a more severe phenotype, MRI detected a milder increase of T2 value, associated with a milder vacuolization. This suggests that alteration within brainstem nuclei is not predictive of a more severe phenotype in the SOD1G93A mouse model. Using an ex vivo paradigm, Diffusion Tensor Imaging was also applied to study white matter spinal cord degeneration. In contrast to degeneration of cranial nuclei, alterations in white matter and axons loss reflected the different disease phenotype of SOD1G93A mice. The correspondence between MRI and histology further highlights the potential of MRI to monitor progressive motoneuron and axonal degeneration non-invasively in vivo. The identification of prognostic markers of the disease nevertheless requires validation in multiple models of ALS to ensure that these are not merely model-specific. Eventually this approach has the potential to lead to the development of robust and validated non-invasive imaging biomarkers in ALS patients, which may help to monitor the efficacy of therapies.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Brain Stem/pathology , Magnetic Resonance Imaging/methods , Spinal Cord/pathology , Amino Acid Substitution , Amyotrophic Lateral Sclerosis/genetics , Animals , Axons/pathology , Diffusion Tensor Imaging , Disease Progression , Hand Strength , Humans , Lumbosacral Region , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Motor Neurons/pathology , Mutation, Missense , Point Mutation , Random Allocation , Recombinant Proteins/genetics , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Vacuoles/ultrastructure , White Matter/pathologyABSTRACT
Spinal cord injury (SCI) is the result of a traumatic primary event followed by a so-called secondary injury, which is characterized by a large spectrum of biochemical cellular pathways able to spread the lesion, worsening neurologic recovery. A growing number of potential therapeutic interventions to counteract different neurodegenerative mechanisms of SCI have been proposed, but they did not show relevant efficacy when translated as clinical treatments. Different reasons could explain these disappointing results: on one side the multifactorial evolution of SCI after the primary injury that limits the beneficial effect of just one targeted treatment and, on the other, the restricted access of pharmacological therapies to the spinal cord. For these reasons, recently, a growing interest has been shown in the development of alternative delivery strategies to administer drugs and/or biological/cellular therapies into the spine (hydrogel and nanoparticles).
Subject(s)
Drug Delivery Systems , Nanoparticles , Spinal Cord Injuries/therapy , Animals , Cell- and Tissue-Based Therapy , Cells, Cultured , Disease Models, Animal , Humans , Neuroprotective Agents/therapeutic useABSTRACT
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease caused by the degeneration of motor neurons. The transgenic mouse model carrying the human SOD1G93A mutant gene (hSOD1G93A mouse) represents one of the most reliable and widely used model of this pathology. In the present work, the innovative technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was applied in the study of pathological alterations at the level of small brain regions such as facial and trigeminal nuclei, which in rodents are extremely small and would be difficult to analyze with classical proteomics approaches. Comparing slices from three mice groups (transgenic hSOD1G93A, transgenic hSOD1WT, and nontransgenic, Ntg), this technique allowed us to evidence the accumulation of hSOD1G93A in the facial and trigeminal nuclei, where it generates aggregates. This phenomenon is likely to be correlated to the degeneration observed in these regions. Moreover, a statistical analysis allowed us to highlight other proteins as differentially expressed among the three mice groups analyzed. Some of them were identified by reverse-phase HPLC fractionation of extracted proteins and mass spectrometric analysis before and after trypsin digestion. In particular, the 40S ribosomal protein S19 (RPS19) was upregulated in the parenkyma and reactive glial cells in facial nuclei of hSOD1G93A mice when compared to transgenic hSOD1WT and nontransgenic ones.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Brain Chemistry/genetics , Molecular Imaging/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Superoxide Dismutase/metabolism , Animals , Brain/metabolism , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Tissue DistributionABSTRACT
The possibility to control the fate of the cells responsible for secondary mechanisms following spinal cord injury (SCI) is one of the most relevant challenges to reduce the post traumatic degeneration of the spinal cord. In particular, microglia/macrophages associated inflammation appears to be a self-propelling mechanism which leads to progressive neurodegeneration and development of persisting pain state. In this study we analyzed the interactions between poly(methyl methacrylate) nanoparticles (PMMA-NPs) and microglia/macrophages in vitro and in vivo, characterizing the features that influence their internalization and ability to deliver drugs. The uptake mechanisms of PMMA-NPs were in-depth investigated, together with their possible toxic effects on microglia/macrophages. In addition, the possibility to deliver a mimetic drug within microglia/macrophages was characterized in vitro and in vivo. Drug-loaded polymeric NPs resulted to be a promising tool for the selective administration of pharmacological compounds in activated microglia/macrophages and thus potentially able to counteract relevant secondary inflammatory events in SCI.
Subject(s)
Drug Carriers/administration & dosage , Microglia/metabolism , Nanoparticles/administration & dosage , Polymethyl Methacrylate/administration & dosage , Spinal Cord Injuries/metabolism , Animals , Behavior, Animal/drug effects , Carbocyanines/administration & dosage , Carbocyanines/chemistry , Cell Survival/drug effects , Cells, Cultured , Coloring Agents/administration & dosage , Coloring Agents/chemistry , Drug Carriers/chemistry , Female , Hydrogels , Lipopolysaccharides , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Microglia/drug effects , Nanoparticles/chemistry , Polymethyl Methacrylate/chemistry , Spinal Cord/metabolismABSTRACT
Much evidence shows that acute and chronic inflammation in spinal cord injury (SCI), characterized by immune cell infiltration and release of inflammatory mediators, is implicated in development of the secondary injury phase that occurs after spinal cord trauma and in the worsening of damage. Activation of microglia/macrophages and the associated inflammatory response appears to be a self-propelling mechanism that leads to progressive neurodegeneration and development of persisting pain state. Recent advances in polymer science have provided a huge amount of innovations leading to increased interest for polymeric nanoparticles (NPs) as drug delivery tools to treat SCI. In this study, we tested and evaluated in vitro and in vivo a new drug delivery nanocarrier: minocycline loaded in NPs composed by a polymer based on poly-ε-caprolactone and polyethylene glycol. These NPs are able to selectively target and modulate, specifically, the activated proinflammatory microglia/macrophages in subacute progression of the secondary injury in SCI mouse model. After minocycline-NPs treatment, we demonstrate a reduced activation and proliferation of microglia/macrophages around the lesion site and a reduction of cells with round shape phagocytic-like phenotype in favor of a more arborized resting-like phenotype with low CD68 staining. Treatment here proposed limits, up to 15 days tested, the proinflammatory stimulus associated with microglia/macrophage activation. This was demonstrated by reduced expression of proinflammatory cytokine IL-6 and persistent reduced expression of CD68 in traumatized site. The nanocarrier drug delivery tool developed here shows potential advantages over the conventionally administered anti-inflammatory therapy, maximizing therapeutic efficiency and reducing side effects.
Subject(s)
Macrophages/pathology , Microglia/pathology , Minocycline/administration & dosage , Nanomedicine/methods , Spinal Cord Injuries/therapy , Animals , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Biocompatible Materials/chemistry , Cell Survival , Coculture Techniques , Disease Models, Animal , Drug Delivery Systems , Enzyme-Linked Immunosorbent Assay , Hydrogels/chemistry , Inflammation , Interleukin-6/blood , Mice , Mice, Inbred C57BL , Nanoparticles/chemistry , Polyesters/chemistry , Polyethylene Glycols/chemistry , Polymers/chemistry , Quantum Dots , Rhodamines/chemistry , Spinal Cord/pathologyABSTRACT
It has been shown that chronic treatment with lithium carbonate (Li(2)CO(3)) in presymptomatic SOD1G93A transgenic male mice, a model of ALS, was able to remarkably increase their lifespan through the activation of autophagy and the promotion of mitochondriogenesis and neurogenesis. This prompted us to test the lithium effect also in female SOD1G93A mice with two phenotypes of different disease severity. Female SOD1G93A mice of C57BL/6J or 129S2/Sv genetic background were treated daily with Li(2)CO(3) 37 mg/kg (1 mEq/kg) i.p. starting from age 75 days until death. Grip strength, latency to fall on rotarod and body weight were monitored twice weekly. At the time of death the spinal cord was removed to assess the number of motor neurons and to measure the expression of a marker of autophagy (LCII) and the activity of mitochondrial complex IV. We observed a significant anticipation of the onset and reduced survival in 129Sv/G93A and no effect in C57/G93A mice treated with lithium compared to vehicle treated mice. Moreover, lithium neither exerted neuroprotective effects nor increased the expression of LCII and the activity of mitochondrial complex IV in the spinal cord. The present study does not identify any therapeutic or neuroprotective effect of lithium in SOD1G93A female mice.