regeneration

Cartilage targeting exosomes for OA gene therapy and pain treatment

Project Summary Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address this, we have developed surface-modified cartilage-targeting MSC exosomes as non-viral carriers for gene therapy. MSC derived exosomes have intrinsic therapeutic potential as they can induce cartilage repair and are non-immunogenic, making them desirable for gene delivery. We have engineered charge-reversed cationic exosomes by anchoring cartilage targeting optimally charged arginine-rich cationic peptide (CPC) motifs into the anionic exosome bilayer (Exo-CPC) by using buffer pH as a charge-reversal switch. Exo-CPC use charge interactions to penetrate through the full thickness of arthritic cartilage (close to tidemark) and deliver the packaged genetic material cargo to chondrocytes residing in the deep tissue layers while native anionic exosomes cannot. They can also bind within the synovial joint, making them effective for OA pain relief gene therapy. Here we will engineer charge-reversed Exo-CPC for delivery of IL-1RA (receptor antagonist of interleukin-1) mRNA and NaV1.8 (voltage gated sodium channel 1.8) inhibitor siRNA to stimulate both disease modifying response and long-term pain relief with a one-time intra-articular dose. IL-1RA mRNA targets are in the chondrocytes and synovium cells; Nav1.8 expressing nerves innervate into synovium and subchondral bone in OA – sites that Exo-CPC can readily target. Aim 1 will engineer cartilage targeting Exo-CPC for delivery of IL- 1RA mRNA and Nav1.8 inhibitor siRNA. Their ability to deliver IL-1RA mRNA to chondrocytes and IL-1RA protein translation efficiency will be evaluated in-vitro. Exo-CPC-Na v1.8’s ability to reduce NaV1.8 bioactivity of sensory nerves will also be evaluated. In Aim 2, their distribution intra-articular (proximity to NaV1.8-positive nerves), extra-articular, and DRG and spinal cord using partial meniscectomy NaV1.8-tdTomato reporter mice OA models will be evaluated. Additionally, their dose dependent reduction on MMP activity, neuronal excitability and pain- related behaviors, and any immunogenicity will be assessed. Aim 3 will use the determined functional doses to study the long-term disease modifying and pain-relief effects of mono and combination therapy with Exo-CPC- IL-1RA and Exo-CPC-Nav1.8 in rescuing injury induced tissue structural damage as well as in reducing pain (weight bearing asymmetry) for up to one month following IA administration in early vs. late stages (intervention at 2 vs 6 weeks) of MMT (medial meniscectomy) induced OA rats. The project paves way for utilizing the intrinsic therapeutic potential of MSC Exosomes as viral-free, non-immunogenic carriers for OA gene therapy by employing cartilage as a drug depot. Cationic exosomes can be used to deliver other OA gene targets, and can be widely used for targeting other negatively charged tissues like meniscus, ligaments, discs, fracture callus etc.

Intrinsic and extrinsic mechanisms underlying trigeminal nerve deficits in familial dysautonomia

PROJECT SUMMARY Rare diseases impose a significant burden on the US healthcare system, accounting for nearly half of all expenditures for their treatment. This statistic alone supports the need to invest in research to develop therapeutic interventions for rare diseases since the economic benefit outweighs the continued expense of financial resources. Familial dysautonomia (FD) is a rare, hereditary disease that arises from a splice site mutation in Elongator acetyltransferase complex subunit 1 (ELP1) and impacts the nervous system. To date, FD patients continue to face life-threatening complications involving basic involuntary functions like swallowing and somatosensation because there is no cure for this ultimately fatal neuropathy. FD patients exhibit symptoms due to defects in their somatosensory trigeminal nerves, whose cell bodies reside in the trigeminal ganglion (TG) and are derived from neural crest and placode cells. Recent studies from our lab using an FD mouse model (Elp1 deleted from neural crest cells) revealed TG axon outgrowth and target tissue innervation deficits, recapitulating phenotypes observed in FD patients. However, the mechanisms by which Elp1 mediates normal TG development, and how this goes awry in FD, remain largely elusive. To gain insight into Elp1 function, we performed mass spectrometry to evaluate the TG proteome of normal and FD mouse embryos. Our results uncovered statistically significant increases in extracellular matrix (ECM) and ECM binding proteins, pointing to altered TG biomechanical properties and, more broadly, changes in mechanotransduction, the process by which cells translate extrinsic cues into intrinsic signaling pathways that modulate gene expression. Importantly, proper axon outgrowth relies upon mechanotransduction as growth cones on axons sense and respond to their environment. In the head, this environment consists of ECM and cranial mesenchyme cells, but the impact of Elp1 loss from the latter is not known, including the potential for altered tissue biomechanics that could influence TG axon outgrowth. We hypothesize that loss of Elp1 induces changes in the biomechanical properties of both the TG/nerves and ECM/cranial mesenchyme, modifying mechanotransduction and leading to TG defects in FD, which we will interrogate in the following Specific Aims: 1) define the biomechanical properties of the TG/nerves and ECM/cranial mesenchyme and 2) determine the role of cranial mesenchyme Elp1 in mediating proper TG axon outgrowth. Our innovative research proposal takes a systems-level, multidisciplinary approach involving embryology, biomechanics, and high-resolution microscopy, with the goal of integrating molecular, cellular, and tissue data. These results will significantly advance our knowledge of the molecular mechanisms underscoring TG development and, collectively, inform treatment strategies for birth defects or disorders like FD with TG dysfunction, as well as nerve repair and/or regeneration after injury or disease.

SWEBAGS conference 2024: The basal ganglia in action

Clonal analysis at single cell level helps to understand neural crest development

Recent research on the neural crest has revealed the multipotency and plasticity of nerve-associated Schwann cell precursors, which can differentiate into diverse cell types, including parasympathetic neurons, neuroendocrine cells, and mesenchymal stem cells. These findings challenge the traditional view of peripheral nerves, highlighting their role as niches for migratory progenitor cells that contribute to tissue formation and regeneration.

Gut/Body interactions in health and disease

The adult intestine is a major barrier epithelium and coordinator of multi-organ functions. Stem cells constantly repair the intestinal epithelium by adjusting their proliferation and differentiation to tissue intrinsic as well as micro- and macro-environmental signals. How these signals integrate to control intestinal and whole-body homeostasis is largely unknown. Addressing this gap in knowledge is central to an improved understanding of intestinal pathophysiology and its systemic consequences. Combining Drosophila and mammalian model systems my laboratory has discovered fundamental mechanisms driving intestinal regeneration and tumourigenesis and outlined complex inter-organ signaling regulating health and disease. During my talk, I will discuss inter-related areas of research from my lab, including:1- Interactions between the intestine and its microenvironment influencing intestinal regeneration and tumourigenesis. 2- Long-range signals from the intestine impacting whole-body in health and disease.

Pro-regenerative functions of microglia in demyelinating diseases

Our goal is to understand why myelin repair fails in multiple sclerosis and to develop regenerative medicines for the nervous system. A central obstacle for progress in this area has been the complex biology underlying the response to CNS injury. Acute CNS damage is followed by a multicellular response that encompasses different cell types and spans different scales. Currently, we do not understand which factors determines lesion recovery. Failure of inflammation to resolve is a key underlying reason of poor regeneration, and one focus is therefore on the biology of microglia during de- and remyelination, and their cross talk to other cells, in particular oligodendrocytes and the progenitor cells. In addition, we are exploring the link between lipid metabolism and inflammation, and its role in the regulation of regeneration. I will report about our recent progress in our understanding of how microglia promote regeneration in the CNS.

Mechanisms of Axon Growth and Regeneration

Almost everybody that has seen neurons under a microscope for the first time is fascinated by their beauty and their complex shape. Early on during development, however, there are hardly any signs of their future complexity, but the neurons look round and simple. How do neurons develop their sophisticated structure? How do they initially generate domains that later have distinct function within neuronal circuits, such as the axon? And, can a better understanding of the underlying developmental mechanisms help us in pathological conditions, such as a spinal cord injury, to induce axons to regenerate? Here, I will talk about the cytoskeleton as a driving force for neuronal polarization. We will then explore how cytoskeletal changes help to reactivate the growth program of injured CNS axons to elicit axon regeneration after a spinal cord injury. Finally, we will discuss whether axon growth and synapse formation may be processes in neurons that might exclude each other. Following this developmental hypothesis, it will help us to generate a novel perspective on regeneration failure in the adult CNS, and how we can overcome this failure to induce axon regeneration. Thus, this talk will describe how we can exploit developmental mechanisms to induce axon regeneration after a spinal cord injury.

Mechanisms to medicines in neurodegeneration

Dysregulation of protein synthesis both globally and locally in neurons and astrocytes is a key feature of neurodegenerative diseases. Aberrant signalling through the Unfolded Protein Response (UPR) and related Integrated Stress Response (ISR) have become major targets for neuroprotection in these disorders. In addition, other homeostatic mechanisms and stress responses, including the cold shock response, appear to regulate local translation and RNA splicing to control synapse maintenance and regeneration and can also be targeted therapeutically for neuroprotection. We have defined the role of UPR/ISR and the cold-shock response in neurodegenerative disorders and have developed translational strategies targeting them for new treatments for dementia.

Keeping axons alive after injury: Inhibiting programmed axon death

Activation of pro-degenerative protein SARM1 in response to diverse physical and disease-relevant injuries triggers programmed axon death. Original studies indicated substantially decreased levels of SARM1 were required for neuroprotection. However, we demonstrate that lowering SARM1 levels by 50% in Sarm1 haploinsufficient mice delays axon degeneration in vivo (after sciatic nerve transection), in vitro (in response to diverse traumatic, neurotoxic, and genetic triggers), and partially prevents neurite outgrowth defects in mice lacking pro-survival factor NMNAT2. We also demonstrate the capacity for Sarm1 antisense oligonucleotides to decrease SARM1 levels by more than 50% which delays or prevents programmed axon degeneration in vitro. Combining Sarm1 haploinsufficiency with antisense oligonucleotides further decreases SARM1 levels and prolongs protection after neurotoxic injuries. These data demonstrate that axon protection occurs in a Sarm1 gene-dose responsive manner and that SARM1 lowering agents have therapeutic potential. Thus, antisense oligonucleotide targeting of Sarm1 is a promising therapeutic strategy against diverse triggers of axon degeneration.

Activity dependent myelination: a mechanism for learning and regeneration?

The CNS is responsive to an ever-changing environment. Until recently, studies of neural plasticity focused almost exclusively on functional and structural changes of neuronal synapses. In recent years, myelin plasticity has emerged as a potential modulator of neural networks. Myelination of previously unmyelinated axons, and changes in the structure on already-myelinated axons, can have large effects on network function. The heterogeneity of the extent of how axons in the CNS are myelinated offers diverse scope for dynamic myelin changes to fine-tune neural circuits. The traditionally held view of myelin as a passive insulator of axons is now changing to one of lifelong changes in myelin, modulated by neuronal activity and experience. Myelin, produced by oligodendrocytes (OLs), is essential for normal brain function, as it provides fast signal transmission, promotes synchronization of neuronal signals and helps to maintain neuronal function. OLs differentiate from oligodendrocyte precursor cells (OPCs), which are distributed throughout the adult brain, and myelination continues into late adulthood. OPCs can sense neuronal activity as they receive synaptic inputs from neurons and express voltage-gated ion channels and neurotransmitter receptors, and differentiate into myelinating OLs in response to changes in neuronal activity. This lecture will explore to what extent myelin plasticity occurs in adult animals, whether myelin changes occur in non-motor learning tasks, especially in learning and memory, and questions whether myelin plasticity and myelin regeneration are two sides of the same coin.

Microbiota in the health of the nervous system and the response to stress

Microbes have shaped the evolution of eukaryotes and contribute significantly to the physiology and behavior of animals. Some of these traits are inherited by the progenies. Despite the vast importance of microbe-host communication, we still do not know how bacteria change short term traits or long-term decisions in individuals or communities. In this seminar I will present our work on how commensal and pathogenic bacteria impact specific neuronal phenotypes and decision making. The traits we specifically study are the degeneration and regeneration of neurons and survival behaviors in animals. We use the nematode Caenorhabditis elegans and its dietary bacteria as model organisms. Both nematode and bacteria are genetically tractable, simplifying the detection of specific molecules and their effect on measurable characteristics. To identify these molecules we analyze their genomes, transcriptomes and metabolomes, followed by functional in vivo validation. We found that specific bacterial RNAs and bacterially produced neurotransmitters are key to trigger a survival behavioral and neuronal protection respectively. While RNAs cause responses that lasts for many generations we are still investigating whether bacterial metabolites are capable of inducing long lasting phenotypic changes.

Regenerative Neuroimmunology - a stem cell perspective

There are currently no approved therapies to slow down the accumulation of neurological disability that occurs independently of relapses in multiple sclerosis (MS). International agencies are engaging to expedite the development of novel strategies capable of modifying disease progression, abrogating persistent CNS inflammation, and support degenerating axons in people with progressive MS. Understanding why regeneration fails in the progressive MS brain and developing new regenerative approaches is a key priority for the Pluchino Lab. In particular, we aim to elucidate how the immune system, in particular its cells called myeloid cells, affects brain structure and function under normal healthy conditions and in disease. Our objective is to find how myeloid cells communicate with the central nervous system and affect tissue healing and functional recovery by stimulating mechanisms of brain plasticity mechanisms such as the generation of new nerve cells and the reduction of scar formation. Applying combination of state-of-the-art omic technologies, and molecular approaches to study murine and human disease models of inflammation and neurodegeneration, we aim to develop experimental molecular medicines, including those with stem cells and gene therapy vectors, which slow down the accumulation of irreversible disabilities and improve functional recovery after progressive multiple sclerosis, stroke and traumatic injuries. By understanding the mechanisms of intercellular (neuro-immune) signalling, diseases of the brain and spinal cord may be treated more effectively, and significant neuroprotection may be achieved with new tailored molecular therapeutics.

Brain Awareness Week @ IITGN

Traumatic injury in the nervous system leads to devastating consequences such as paralysis. The regenerative capacity of the nervous system is limited in adulthood. In this talk, Dr. Anindya would be sharing how the simple nematode C. elegans with its known connectome can inform us about the biology of nervous system repair.

Beyond energy - an unconventional role of mitochondria in cone photoreceptors

The long-term goal of my research is to study the mammalian retina as a model for the central nervous system (CNS) -- to understand how it functions in physiological conditions, how it is formed, how it breaks down in pathological conditions, and how it can be repaired. I have focused on two research themes: 1) Photoreceptor structure, synapse, circuits, and development, 2) Hibernation and metabolic adaptations in the retina and beyond. As the first neuron of the visual system, photoreceptors are vital for photoreception and transmission of visual signals. I am particularly interested in cone photoreceptors, as they mediate our daylight vision with high resolution color information. Diseases affecting cone photoreceptors compromise visual functions in the central macular area of the human retina and are thus most detrimental to our vision. However, because cones are much less abundant compared to rods in most mammals, they are less well studied. We have used the ground squirrel (GS) as a model system to study cone vision, taking advantage of their unique cone-dominant retina. In particular, we have focused on short-wavelength sensitive cones (S-cones), which are not only essential for color vision, but are also an important origin of signals for biological rhythm, mood and cognitive functions, and the growth of the eye during development. We are studying critical cone synaptic structures – synaptic ribbons, the synaptic connections of S-cones, and the development of S-cones with regard to their specific connections. These works will provide knowledge of normal retinal development and function, which can also be extended to the rest of CNS; for example, the mechanisms of synaptic targeting during development. In addition, such knowledge will benefit the development of optimal therapeutic strategies for regeneration and repair in cases of retinal degenerative disease. Many neurodegenerative diseases, including retinal diseases, are rooted in metabolic stress in neurons and/or glial cells. Using the same GS model, we aim to learn from this hibernating mammal, which possesses an amazing capability to adapt to the extreme metabolic conditions during hibernation. By exploring the mechanisms of such adaptation, we hope to discover novel therapeutic tactics for neurodegenerative diseases.

Microenvironment role in axonal regeneration- looking beyond the neurons

After an injury in the adult mammalian central nervous system, lesioned axons fail to regenerate. This failure to regenerate contrasts with the remarkable potential of axons to grow during embryonic development and after an injury in the peripheral nervous system. Peripheral sensory neurons with cell soma in dorsal root ganglia (DRG) switch to a regenerative state after nerve injury to enable axon regeneration and functional recovery. Decades of research have focused on the signaling pathways elicited by injury in sensory neurons and in Schwann cells that insulate axons as central mechanisms regulating nerve repair. However, neuronal microenvironment is far more complex and is composed of multiple cell types including endothelial, immune and glial cells. Whether the microenvironment surrounding neuronal soma contribute to the poor regenerative outcomes following central injuries remains largely unexplored. To answer this question, we performed a single cell transcriptional profiling of the DRG neuronal microenvironment response to peripheral and central injuries. In dissecting the roles of the microenvironment contribution, we have focused on a poorly studied population of Satellite Glial Cells (SGC) surrounding the neuronal cell soma. This study has uncovered a previously unknown role for SGC in nerve regeneration and defined SGC as transcriptionally distinct from Schwann cells while sharing similarities with astrocytes. Upon a peripheral injury, SGC contribute to axon regeneration via Fatty acid synthase (Fasn)-PPARα signaling pathway. Through repurposing fenofibrate, an FDA- approved PPARα agonist used for dyslipidemia treatment, we were able to rescue the impaired regeneration in mice lacking Fasn in SGC. Our analysis reveals that in response to central injuries, SGC do not activate the PPAR signaling pathway. However, induction of this pathway with fenofibrate treatment, rescued axon regeneration following an injury to the central nerves. Collectively, our results uncovered a previously unappreciated role of the neuronal microenvironment differential response in central and peripheral injuries.

3D-microchannalled scaffold for peripheral nerve regeneration

AAV-mediated gene therapy for sensory regeneration after spinal cord injury

Activation of regeneration-associated pathways in neurons following photocapacitive stimulation

Development and regeneration of cornea innervation

Effects of Olfactory Mucosa Stem/Stromal Cell and Olfactory Ensheating Cells Secretome on Peripheral Nerve Regeneration

Improving nerve regeneration with chitosan blended micro-grooved membranes

Inhibition of Chondroitin Sulfate Proteoglycans (CSPGs) to Promote Regeneration in the CNS in Multiple Sclerosis

A novel hyaluronic acid (HLA) based hydrogel for the enhancement of peripheral nerve regeneration

A novel model for multiplexed, tridimensional analysis of cortical regeneration upon brain damage in vivo

Peripheral nerve regeneration: in vitro model

The RSK protein promote central nervous system regeneration and functional recovery via translational control

Resident neural stem cells guarantee the regeneration promoted by bulbar olfactory ensheathing cell transplantation after spinal cord injury

Selective translation controls axon regeneration in the central nervous system

Spontaneous regeneration of cholecystokinergic descending axons after a complete spinal cord injury in lampreys

Transcriptomic analysis’ of axon regeneration-inducing manipulations discover distinct molecular programs defining a cell’s fate to die, survive or regenerate after an injury

Adaptive plasticity in adult brain circuitry during naturally occurring regeneration of sensory inputs

FENS Forum 2024

Adeno-associated viruses to study formation and regeneration of neuronal circuits in the axolotl brain

FENS Forum 2024

Delivery of PTEN inhibitory peptide PAP2 to promote regeneration in a mouse model of traumatic brain injury

FENS Forum 2024

Developing an adaptable visual behavior assay to functionally characterize brain regeneration in the axolotl (Ambystoma mexicanum)

FENS Forum 2024

Sex differences in nociceptor regeneration after burn injury

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Differential roles for dysregulated microRNAs after a peripheral nerve injury in neuropathic pain or nerve regeneration

FENS Forum 2024

miR-145 depletion promotes myelin regeneration in a novel mouse model of multiple sclerosis

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Neuronal regeneration to restore hand and arm functions after paralysis

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Olfactory ensheathing cells and fibroblasts: Dynamic partners in nervous system repair and regeneration

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Pharmacological treatment targeting angiotensin receptor type 2 after severe spinal cord injury improves axonal and myelin regeneration

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REMaST®: A novel immune cell therapy for neural tissue regeneration

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Role of adenosine A1 receptors in oligodendrocyte regeneration

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The role of MAP1B protein in axonal regeneration

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The role of MAP6 in axonal regeneration

FENS Forum 2024

Subacute inhibition of plasma kallikrein stabilizes the blood-brain barrier and promotes regeneration after ischemic stroke

FENS Forum 2024

In vitro studying of the role of miRNAs in neuronal injury and regeneration

FENS Forum 2024