Recombinant adeno-associated virus mediated gene delivery in the extracranial nervous system of adult mice by direct nerve immersion.

This protocol outlines a minimally invasive and quickly performed approach for transgene delivery in the extracranial nervous system of adult mice using recombinant adeno-associated virus (AAV). The technique, named Sciatic Nerve Direct Immersion (SciNDi), relies on the direct bilateral immersion of the exposed sciatic nerve with AAV. We show that in comparison with intramuscular AAV delivery, SciNDi results in widespread transduction in connected neuroanatomical tracts both in the sciatic nerve trunk and the lumbar spinal cord. For complete details on the use and execution of this protocol, please refer to Jan et al. (2019) and Richner et al. (2011, 2017).

The Prion-Like Spreading of Alpha-Synuclein in Parkinson's Disease: Update on Models and Hypotheses.

The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson's disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn accumulation in the CNS are not fully understood, growing evidence suggests that de novo α-syn misfolding and/or neuronal internalization of aggregated α-syn facilitates conformational templating of endogenous α-syn monomers in a mechanism reminiscent of prions. A refined understanding of the biochemical and cellular factors mediating the pathological neuron-to-neuron propagation of misfolded α-syn will potentially elucidate the etiology of PD and unravel novel targets for therapeutic intervention. Here, we discuss recent developments on the hypothesis regarding trans-synaptic propagation of α-syn pathology in the context of neuronal vulnerability and highlight the potential utility of novel experimental models of synucleinopathies.

Trans-synaptic spreading of alpha-synuclein pathology through sensory afferents leads to sensory nerve degeneration and neuropathic pain.

Pain is a common non-motor symptom of Parkinson's disease (PD), with current limited knowledge of its pathophysiology. Here, we show that peripheral inoculation of mouse alpha-synuclein (α-Syn) pre-formed fibrils, in a transgenic mouse model of PD, elicited retrograde trans-synaptic spreading of α-Syn pathology (pSer129) across sensory neurons and dorsal nerve roots, reaching central pain processing regions, including the spinal dorsal horn and the projections of the anterolateral system in the central nervous system (CNS). Pathological peripheral to CNS propagation of α-Syn aggregates along interconnected neuronal populations within sensory afferents, was concomitant with impaired nociceptive response, reflected by mechanical allodynia, reduced nerve conduction velocities (sensory and motor) and degeneration of small- and medium-sized myelinated fibers. Our findings show a link between the transneuronal propagation of α-Syn pathology with sensory neuron dysfunction and neuropathic impairment, suggesting promising avenues of investigation into the mechanisms underlying pain in PD.

Peripheral Glial Cells in the Development of Diabetic Neuropathy.

The global prevalence of diabetes is rapidly increasing, affecting more than half a billion individuals within the next few years. As diabetes negatively affects several physiological systems, this dramatic increase represents not only impaired quality of life on the individual level but also a huge socioeconomic challenge. One of the physiological consequences affecting up to half of diabetic patients is the progressive deterioration of the peripheral nervous system, resulting in spontaneous pain and eventually loss of sensory function, motor weakness, and organ dysfunctions. Despite intense research on the consequences of hyperglycemia on nerve functions, the biological mechanisms underlying diabetic neuropathy are still largely unknown, and treatment options lacking. Research has mainly focused directly on the neuronal component, presumably from the perspective that this is the functional signal-transmitting unit of the nerve. However, it is noteworthy that each single peripheral sensory neuron is intimately associated with numerous glial cells; the neuronal soma is completely enclosed by satellite glial cells and the length of the longest axons covered by at least 1,000 Schwann cells. The glial cells are vital for the neuron, but very little is still known about these cells in general and especially how they respond to diabetes in terms of altered neuronal support. We will discuss current knowledge of peripheral glial cells and argue that increased research in these cells is imperative for a better understanding of the mechanisms underlying diabetic neuropathy.

MMP-14 overexpression correlates with the neurodegenerative process in familial amyloidotic polyneuropathy.

Levels of matrix metalloproteases (MMPs) can be differentially regulated in response to injury or neurological diseases. For instance, it is known that selective and short-term inhibition of MMP-14, a membrane-type 1 MMP, accelerates axon regeneration. Because axon growth and regeneration is impaired in familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder characterized by misfolding and deposition of mutant transthyretin (TTR) in the peripheral nervous system (PNS), we presently investigated the expression levels and the potential role for MMP-14 in this condition. By using cell culture studies, a mouse model of disease and human clinical samples, we observed that MMP-14: (i) is overexpressed in FAP nerves, correlating with TTR deposition; (ii) is upregulated in sciatic nerves from a preclinical transgenic mouse model, increasing with TTR deposition; (iii) levels in the PNS and plasma are rescued upon treatment of mice with anakinra or TTR siRNA, drugs acting over the IL-1 signaling pathway or TTR liver synthesis, respectively; (iv) increases in Schwann cells upon incubation with amyloid-like aggregates; and, finally, (v) is increased in plasma of FAP patients, correlating with disease progression. These results highlight the relevance of MMP-14 in the pathophysiology of FAP, suggesting not only a potential role for this molecule as a novel biomarker for therapy follow up, but also as a new potential therapeutic target.

Differential expression of Cathepsin E in transthyretin amyloidosis: from neuropathology to the immune system.

BACKGROUND: Increasing evidence supports a key role for inflammation in the neurodegenerative process of familial amyloidotic polyneuropathy (FAP). While there seems to be an overactivation of the neuronal interleukin-1 signaling pathway, the immune response is apparently compromised in FAP. Accordingly, little immune cell infiltration is observed around pre-fibrillar or fibrillar amyloid deposits, with the underlying mechanism for this phenomenon remaining poorly understood. Cathepsin E (CtsE) is an important intermediate for antigen presentation and chemotaxis, but its role in the pathogenesis of FAP disease remains unknown. METHODS: In this study, we used both mouse primary macrophages and in vivo studies based on transgenic models of FAP and human samples to characterize CtsE expression in different physiological systems. RESULTS: We show that CtsE is critically decreased in bone marrow-derived macrophages from a FAP mouse model, possibly contributing for cell function impairment. Compromised levels of CtsE were also found in injured nerves of transgenic mice and, most importantly, in naïve peripheral nerves, sensory ganglia, murine stomach, and sural nerve biopsies derived from FAP patients. Expression of CtsE in tissues was associated with transthyretin (TTR) deposition and differentially regulated accordingly with the physiological system under study. Preventing deposition with a TTR small interfering RNA rescued CtsE in the peripheral nervous system (PNS). In contrast, the expression of CtsE increased in splenic cells (mainly monocytes) or peritoneal macrophages, indicating a differential macrophage phenotype. CONCLUSION: Altogether, our data highlights the potential of CtsE as a novel FAP biomarker and a possible modulator for innate immune cell chemotaxis to the disease most affected tissues-the peripheral nerve and the gastrointestinal tract.

Tissue remodeling after interference RNA mediated knockdown of transthyretin in a familial amyloidotic polyneuropathy mouse model.

Transthyretin (TTR) deposition in the peripheral nervous system is the hallmark of familial amyloidotic polyneuropathy (FAP). Currently, liver transplantation is the only available treatment to halt the progression of clinical symptoms; however, due to the limitations of this procedure, development of alternative therapeutic strategies is of utmost importance. In this regard, interference RNA (RNAi) targeting TTR is currently in phase III clinical development. To dissect molecular changes occurring in dorsal root ganglia (DRG) upon RNAi-mediated knockdown of TTR, we treated both chronically and acutely an FAP mouse model, in different stages of disease. Our data show that inhibition of TTR expression by the liver with RNAi reverse TTR deposition in DRG, decrease matrix metalloproteinase-2 (MMP-2) protein levels in plasma, inhibit Mmp-2 gene expression and downregulate MMP-9 activity in DRG, indicating extracellular matrix remodeling. Furthermore, protein levels of MMP-2 were found upregulated in plasma samples from FAP patients indicating that MMP-2 might be a novel potential biomarker for FAP diagnosis. Collectively, our data show that silencing TTR liver synthesis in vivo can modulate TTR-induced pathology in the peripheral nervous system and highlight the potential of MMP-2 as a novel disease biomarker.

Protective role of anakinra against transthyretin-mediated axonal loss and cell death in a mouse model of familial amyloidotic polyneuropathy.

Familial amyloidotic polyneuropathy (FAP) is characterized by a length-dependent axonal loss in the peripheral nervous system that results from deposition of extracellular prefibrillar transthyretin (TTR) and amyloid fibrils. We have previously shown that an inflammatory stimulus in the peripheral nerve in a mouse model of FAP triggers local TTR expression and deposition, leading to poor regeneration. We also demonstrated that blocking interleukin-1 (IL-1) signaling by the IL-1 receptor antagonist anakinra is beneficial in preventing nerve TTR deposition and associated toxicity. Here, we investigated whether IL-1 signaling influences TTR biology after an injury stimulus in a V30M FAP mouse model. Animals were treated with anakinra 48 hours before sciatic nerve ligation; the nerves were analyzed 7 days postlesion. Anakinra decreased TTR expression by Schwann cells and TTR extracellular deposition after nerve injury, which resulted in improved regeneration. Moreover, treated mice had less apoptotic cell death. In wild-type mice, inflammation is important for regeneration but, in the FAP model mice, an altered threshold of the inflammatory response differentially regulates TTR. Taken together, our results show that anakinra administration before injury can modulate TTR-induced peripheral nervous system pathology, thereby corroborating the protective interference of this drug in a FAP preclinical model.

Interleukin-1 signaling pathway as a therapeutic target in transthyretin amyloidosis.

INTRODUCTION: Inflammation is a key pathological hallmark of several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and familial amyloidotic polyneuropathy (FAP). Among all inflammatory cytokines associated with FAP, IL-1ß, in particular, has been implicated in playing a key pathogenic role. In the present study, we sought to investigate whether blocking IL-1ß signaling provides disease-modifying benefits in an FAP mouse model. METHODS: We assessed the effect of chronic administration of Anakinra, an IL-1 antagonist, on FAP pathogenesis in vivo, using real-time polymerase chain reaction (qPCR), semi-quantitative immunohistochemistry (SQ-IHC), western blot and nerve morphometric analyses. RESULTS: We found that treatment with Anakinra prevents transthyretin (TTR) extracellular deposition in sciatic nerve, protecting unmyelinated nerve fibers from aggregate-induced degeneration. Moreover, Anakinra administration significantly suppressed IL-1 signaling pathway and inhibited apoptosis and nitrative stress. CONCLUSIONS: The present work highlights the relevance of the IL-1 signaling pathway in the pathophysiology of FAP. Our results bring to light the importance of non-amyloid targets in the therapeutic strategies for this disorder. Thus, we propose the use of Anakinra as a potential therapeutic agent for TTR-related amyloidosis.

The inflammatory response to sciatic nerve injury in a familial amyloidotic polyneuropathy mouse model.

Inflammation is a hallmark of several neurodegenerative disorders including familial amyloidotic polyneuropathy (FAP). FAP is associated with extracellular deposition of mutant transthyretin (TTR), leading to degeneration of cells and tissues, particularly in the peripheral nervous system (PNS). With this work, our goal was to characterize the expression/deposition of TTR and the associated inflammatory immune response, induced by nerve injury, in WT mice and in a mouse model carrying the most common TTR mutation in FAP (V30M). Our results indicate that upon nerve injury TTR is significantly produced by Schwann cells and is dynamically regulated over time in V30M mice, accompanying a peak of inflammation. Strikingly, V30M TTR deposition in nerve tissue occurred, suggesting that inflammation contributes to TTR polymerization. In response to nerve injury, V30M mice display a downregulated innate immune response when compared to WT mice. More specifically, we saw decreased expression of cytokines and chemokines important for the recruitment of immune cells like macrophages and neutrophils, known to be important for the tissue regenerative process which was found impaired in V30M mice. In conclusion, with this work we were able to characterize the biology of TTR both in WT and V30M animals, upon nerve injury, and found that V30M TTR impairs the inflammatory response necessary for nerve regeneration. Taken together, our findings suggest that inflammation is an important target to be considered in therapeutic strategies for FAP.

A novel nanoparticle delivery system for in vivo targeting of the sciatic nerve: impact on regeneration.

AIM: Innovative solutions in the development of drug delivery systems targeting the nerve tissue are awaited. In this regard, a novel system for the delivery of drugs to the sciatic nerve was created using nanomedical principles. MATERIALS & METHODS: Chitosan was the vehicle material used in the experiment. Heparin bound to growth factors has been administered to enhance peripheral nerve regeneration, and since heparin possesses the appropriate charge to be able to form nanoparticles with chitosan, it appears to be a good candidate to base this new delivery system on. RESULTS: Maximal absorption took place throughout the extracellular matrix at day 15. No major inflammatory response was observed, indicating that this is a safe and biocompatible system for drug delivery to nerves. Sensorimotor performance and nerve regeneration of mice receiving these nanoparticles were superior as compared with controls. CONCLUSION: Our work demonstrates a versatile nanoparticle delivery system that successfully targets drugs 'in vivo' to the sciatic nerve, opening novel avenues in the field of nanomedicine to the design of therapeutic strategies that enhance axonal regeneration.