Fabiola Puentes

Inflammation in neurodegenerative diseases

Summary Neurodegeneration, the slow and progressive dysfunction and loss of neurons and axons in the central nervous system, is the primary pathological feature of acute and chronic neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury and multiple sclerosis. Despite different triggering events, a common feature is chronic immune activation, in particular of microglia, the resident macrophages of the central nervous system. Apart from the pathogenic role of immune responses, emerging evidence indicates that immune responses are also critical for neuroregeneration. Here, we review the impact of innate and adaptive immune responses on the central nervous system in autoimmune, viral and other neurodegenerative disorders, and discuss their contribution to either damage or repair. We also discuss potential therapies aimed at the immune responses within the central nervous system. A better understanding of the interaction between the immune and nervous systems will be crucial to either target pathogenic responses, or augment the beneficial effects of immune responses as a strategy to intervene in chronic neurodegenerative diseases.

In vitro and in vivo models of multiple sclerosis

Multiple sclerosis (MS) is widely considered to be the result of an aggressive autoreactive T cell attack on myelin. How these autoimmune responses arise in MS is unclear, but they could result from virus infections. Thus, viral and autoimmune diseases in animals have been used to investigate the possible pathogenic mechanisms operating in MS. The autoimmune model, experimental autoimmune encephalomyelitis, is the most widely-used animal model and has greatly influenced therapeutic approaches targeting autoimmune responses. To investigate demyelination and remyelination in the absence of the adaptive immune response, toxin-induced demyelination models are used. These include using cuprizone, ethidium bromide and lysolecithin to induce myelin damage, which rapidly lead to remyelination when the toxins are withdrawn. The virus models include natural and experimental infections such as canine distemper, visna infection of sheep, and infection of non-human primates. The most commonly used viral models in rodents are Semliki Forest virus and Theilers murine encephalomyelitis virus. The viral and experimental autoimmune encephalomyelitis models have been instrumental in the understanding of how viruses trigger inflammation, demyelination and neurodegeneration in the central nervous system. However, due to complexity of the animal models, pathological mechanisms are also examined in central nervous system cell culture systems including co-cultures, aggregate cultures and brain slice cultures. Here we critically review in vitro and in vivo models used to investigate MS. Since knowledge gained from these models forms the basis for the development of new therapeutic approaches for MS, we address the applicability of the models. Finally, we provide guidance for using and reporting animal studies with the aim of improving translational studies to the clinic.

Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis.

Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood‐borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well‐characterized antibodies to axonal antigens, that axonal damage is associated with HLA‐DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA‐DR+ cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF‐L), present in white and gray matter, was observed in human microglia. The number of NF‐L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF‐L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF‐L is taken up and degraded by the cell. In vivo, NF‐L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA‐DR+ cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens.

Disease origin and progression in amyotrophic lateral sclerosis: an immunology perspective

The immune system is inextricably linked with many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a devastating neuromuscular disorder affecting motor cell function with an average survival of 3 years from symptoms onset. In ALS, there is a dynamic interplay between the resident innate immune cells, that is, microglia and astrocytes, which may become progressively harmful to motor neurons. Although innate and adaptive immune responses are associated with progressive neurodegeneration, in the early stages of ALS immune activation pathways are primarily considered to be beneficial promoting neuronal repair of the damaged tissues, though a harmful effect of T cells at this stage of disease has also been observed. In addition, although auto-antibodies against neuronal antigens are present in ALS, it is unclear whether these arise as a primary or secondary event to neuronal damage, and whether the auto-antibodies are indeed pathogenic. Understanding how the immune system contributes to the fate of motor cells in ALS may shed light on the triggers of disease as well as on the mechanisms contributing to the propagation of the pathology. Immune markers may also act as biomarkers while pathways involved in immune action may be targets of new therapeutic strategies. Here, we review the modalities by which the immune system senses the core pathological process in motor neuron disorders, focusing on tissue-specific immune responses in the neuromuscular junction and in the neuroaxis observed in affected individuals and in animal models of ALS. We elaborate on existing data on the immunological fingerprint of ALS that could be used to identify clues on the disease origin and patterns of progression.

Experimental in vivo and in vitro models of multiple sclerosis: EAE and beyond

Although the primary cause of multiple sclerosis (MS) is unknown, the widely accepted view is that aberrant (auto)immune responses possibly arising following infection(s) are responsible for the destructive inflammatory demyelination and neurodegeneration in the central nervous system (CNS). This notion, and the limited access of human brain tissue early in the course of MS, has led to the development of autoimmune, viral and toxin-induced demyelination animal models as well as the development of human CNS cell and organotypic brain slice cultures in an attempt to understand events in MS. The autoimmune models, collectively known as experimental autoimmune encephalomyelitis (EAE), and viral models have shaped ideas of how environmental factors may trigger inflammation, demyelination and neurodegeneration in the CNS. Understandably, these models have also heavily influenced the development of therapies targeting the inflammatory aspect of MS. Demyelination and remyelination in the absence of overt inflammation are better studied in toxin-induced demyelination models using cuprizone and lysolecithin. The paradigm shift of MS as an autoimmune disease of myelin to a neurodegenerative disease has required more appropriate models reflecting the axonal and neuronal damage. Thus, secondary progressive EAE and spastic models have been crucial to develop neuroprotective approaches. In this review the current in vivo and in vitro experimental models to examine pathological mechanisms involved in inflammation, demyelination and neuronal degeneration, as well as remyelination and repair in MS are discussed. Since this knowledge is the basis for the development of new therapeutic approaches for MS, we particularly address whether the currently available models truly reflect the human disease, and discuss perspectives to further optimise and develop more suitable experimental models to study MS.

Immune reactivity to neurofilament proteins in the clinical staging of amyotrophic lateral sclerosis

Background Neurofilament (NF) proteins detection in biological fluids as a by-product of axonal loss is technically challenging and to date relies mostly on cerebrospinal fluid (CSF) measurements. Plasma antibodies against NF proteins and particularly to their soluble light chain (NF-L) could be a more practical surrogate marker for disease staging in amyotrophic lateral sclerosis (ALS), an invariably fatal and clinically heterogeneous neuromuscular disorder. Methodology We have used a recombinant neurofilament light chain (NF-L) protein for the ELISA detection of antibodies against NF proteins in plasma samples from a well-characterised cohort of ALS individuals (n:73). The use of an established functional rating scale and of a recently proposed staging of disease progression allowed stratification of the ALS cohort based on disease stage, site of onset, survival and speed of disease progression. Results Antibody levels to NF proteins in plasma were significantly higher in ALS individuals compared to healthy controls (p<0.001). Higher NF plasma immunoreactivity was seen in advanced ALS cases (stage IVA-B) compared to earlier phases of the disease (p<0.05). There was no difference in anti-NF plasma antibodies between ALS individuals treated with riluzole and untreated patients; although riluzole-treated ALS cases with an earlier age of onset and with a shorter diagnostic delay displayed higher anti-NFL antibody levels compared to untreated ALS patients with similar features. Conclusions Immunoreactivity to plasma NF-L and homologous NF proteins is informative of the stage of disease progression in ALS. The determination of NF antibody levels in plasma could be added to the growing panel of disease-monitoring biomarkers in ALS targeting cytoskeletal antigens.

Non-neuronal Cells in ALS: Role of Glial, Immune cells and Blood-CNS Barriers.

Neurological dysfunction and motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is strongly associated with neuroinflammation reflected by activated microglia and astrocytes in the CNS. In ALS endogenous triggers in the CNS such as aggregated protein and misfolded proteins activate a pathogenic response by innate immune cells. However, there is also strong evidence for a neuroprotective immune response in ALS. Emerging evidence also reveals changes in the peripheral adaptive immune responses as well as alterations in the blood brain barrier that may aid traffic of lymphocytes and antibodies into the CNS. Understanding the triggers of neuroinflammation is key to controlling neuronal loss. Here, we review the current knowledge regarding the roles of non‐neuronal cells as well as the innate and adaptive immune responses in ALS. Existing ALS animal models, in particular genetic rodent models, are very useful to study the underlying pathogenic mechanisms of motor neuron degeneration. We also discuss the approaches used to target the pathogenic immune responses and boost the neuroprotective immune pathways as novel immunotherapies for ALS.

Selective Inhibition of the Mitochondrial Permeability Transition Pore Protects against Neurodegeneration in Experimental Multiple Sclerosis

The mitochondrial permeability transition pore is a recognized drug target for neurodegenerative conditions such as multiple sclerosis and for ischemia-reperfusion injury in the brain and heart. The peptidylprolyl isomerase, cyclophilin D (CypD, PPIF), is a positive regulator of the pore, and genetic down-regulation or knock-out improves outcomes in disease models. Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and exhibit significant off-target effects, immunosuppression, and toxicity. We therefore designed and synthesized a new mitochondrially targeted CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine. X-ray analysis was used to validate the design concept, and biological evaluation revealed selective cellular inhibition of CypD and the permeability transition pore with reduced cellular toxicity compared with cyclosporine. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple sclerosis, JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium as a mitochondrial targeting group for in vivo use.

Neurofilament light antibodies in serum reflect response to natalizumab treatment in multiple sclerosis

Background: Increased levels of antibodies to neurofilament light protein (NF-L) in biological fluids have been found to reflect neuroinflammatory responses and neurodegeneration in multiple sclerosis (MS). Objective: To evaluate whether levels of serum antibodies against NF-L correlate with clinical variants and treatment response in MS. Methods: The autoantibody reactivity to NF-L protein was tested in serum samples from patients with relapsing–remitting MS (RRMS) (n=22) and secondary progressive MS (SPMS) (n=26). Two other cohorts of RRMS patients under treatment with natalizumab were analysed cross-sectionally (n=16) and longitudinally (n=24). The follow-up samples were taken at 6, 12, 18 and 24 months after treatment, and the NF-L antibody levels were compared against baseline levels. Results: NF-L antibodies were higher in MS clinical groups than healthy controls and in RRMS compared to SPMS patients (p<0.001). NF-L antibody levels were lower in natalizumab treated than in untreated patients (p<0.001). In the longitudinal series, NF-L antibody levels decreased over time and a significant difference was found following 24 months of treatment compared with baseline measurements (p=0.001). Conclusions: Drug efficacy in MS treatment indicates the potential use of monitoring the content of antibodies against the NF-L chain as a predictive biomarker of treatment response in MS.

The epigenetics of multiple sclerosis and other related disorders

Multiple Sclerosis (MS) is a demyelinating disease characterized by chronic inflammation of the central nervous system (CNS) gray and white matter. Although the cause of MS is unknown, it is widely appreciated that innate and adaptive immune processes contribute to its pathogenesis. These include microglia/macrophage activation, pro-inflammatory T-cell (Th1) responses and humoral responses. Additionally, there is evidence indicating that MS has a neurodegenerative component since neuronal and axonal loss occurs even in the absence of overt inflammation. These aspects also form the rationale for clinical management of the disease. However, the currently available therapies to control the disease are only partially effective at best indicating that more effective therapeutic solutions are urgently needed. It is appreciated that in the immune-driven and neurodegenerative processes MS-specific deregulation of gene expressions and resulting protein dysfunction are thought to play a central role. These deviations in gene expression patterns contribute to the inflammatory response in the CNS, and to neuronal or axonal loss. Epigenetic mechanisms control transcription of most, if not all genes, in nucleated cells including cells of the CNS and in haematopoietic cells. MS-specific alterations in epigenetic regulation of gene expression may therefore lie at the heart of the deregulation of gene expression in MS. As such, epigenetic mechanisms most likely play an important role in disease pathogenesis. In this review we discuss a role for MS-specific deregulation of epigenetic features that control gene expression in the CNS and in the periphery. Furthermore, we discuss the application of small molecule inhibitors that target the epigenetic machinery to ameliorate disease in experimental animal models, indicating that such approaches may be applicable to MS patients.