Tuesday, 29 July 2014

Clearing up debris to help repair

Rajbhandari L, Tegenge MA, Shrestha S, Ganesh Kumar N, Malik A, Mithal A, Hosmane S, Venkatesan A. Toll-like receptor 4 deficiency impairs microglial phagocytosis of degenerating axons.
Glia. 2014 Jul 8. doi: 10.1002/glia.22719. [Epub ahead of print]

Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial-axon co-culture to explore the role of Toll-like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type-1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4-mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration

Toll-like receptor 4 is a is a toll-like receptor. It detects lipopolysaccharide from Gram-negative bacteria and is thus important in the activation of the innate immune system. TLR 4 has also been designated as CD284 (cluster of differentiation 284). The protein encoded by this gene is a member of the Toll-like receptor (TLR) family, which plays a fundamental role in pathogen recognition. In this study they report that TLR4 is involved in triggering microglia to remove myelin debris, which allows remyelination to occur. P2X purinoceptor 7 is a protein that in humans is encoded by the P2RX7 gene. The product of this gene belongs to the family of purinoceptors for ATP (cellular energy molecule. The receptor is found in the central and peripheral nervous systems, in microglia, in macrophages, P2X7 receptors have been implicated in ATP-mediated cell death, regulation of receptor trafficking, and inflammation. P2X7 signalling can influence TLR4 which recognise the pathogen-associated molecular patterns (PAMPs) that are expressed by infectious agents. There are many agents that act on P2X7 receptors can they influence myelination in MS?

lentiviral anti-lingo supports remyelination

Wang CJ, Qu CQ, Zhang J, Fu PC, Guo SG, Tang RH. Lingo-1 Inhibited by RNA Interference Promotes Functional Recovery of Experimental Autoimmune Encephalomyelitis. Anat Rec (Hoboken). 2014 Jul . doi: 10.1002/ar.22988. [Epub ahead of print]

Lingo-1 is a negative regulator of myelination. Repairment of demyelinating diseases, such as multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE), requires activation of the myelination program. In this study, we observed the effect of RNA interference on Lingo-1 expression, and the impact of Lingo-1 suppression on functional recovery and myelination/remyelination in EAE mice. Lentiviral vectors encoding Lingo-1 short hairpin RNA (LV/Lingo-1-shRNA) were constructed to inhibit Lingo-1 expression. LV/Lingo-1-shRNA of different titress were transferred into myelin oligodendrocyte glycoprotein-induced EAE mice by intracerebroventricular (ICV) injection. Meanwhile, lentiviral vectors carrying nonsense gene sequence (LVCON053) were used as negative control. The Lingo-1 expression was detected and locomotor function was evaluated at different time points (on days 1,3,7,14,21, and 30 after ICV injection). Myelination was investigated by luxol fast blue (LFB) staining. LV/Lingo-1-shRNA administration via ICV injection could efficiently down-regulate the Lingo-1 mRNA and protein expression in EAE mice on days 7,14,21, and 30 (P < 0.01), especially in the 5 × 108 TU/mL and 5 × 109 TU/mL LV/Lingo-1-shRNA groups. The locomotor function score in the LV/Lingo-1-shRNA treated groups were significantly lower than the untreated or LVCON053 group from day 7 on. The 5 × 108 TU/mL LV/Lingo-1-shRNA group achieved the best functional improvement (0.87 ± 0.11 vs. 3.05 ± 0.13, P < 0.001). Enhanced myelination/remyelination was observed with 50 million to 5 billion Units of virus/mL LV/Lingo-1-shRNA groups by LFB staining (P < 0.05, P < 0.01, and P < 0.05).The data showed that administering LV/Lingo-1-shRNA by ICV injection could efficiently knockdown Lingo-1 expression in vivo, improve functional recovery and enhance myelination/remyelination. Antagonism of Lingo-1 by RNA interference is, therefore, a promising approach for the treatment of demyelinating diseases, such as MS/EAE.

Anti-Lingo-1 antibody can promote remyelination and is clinical trial. However the problem with antibodies is that they do not readily get into the CNS so about 99.9% of that delivered will probably not get there. In the early trials they gave it intravenously wonder if there would be a better effect of intrathecal delivery so more gets in the brain.

 This study uses gene therapy to deliver the therapy to block Lingo-1 and inject a genetically engineered lentiviral vector. The virus delivers something that blocks message of Lingo-1 and this blocks a non-remyelination signal.

Would you be willing to have a viral particle put into your brain?
This is a perceived problem with this type of therapy.

However it provides additional evidence that Lingo-1 blockage is useful.  In MS how will an anti-Lingo, be used given every few weeks forever or as a pulse therapy. With alemtuzumab you are being introduced to an £50,000 short course of antibody.

The phase II trial will be occurring soon.

MRI of tumefactive lesions

Did you present with tumefactive MS? #MSBlog #MSResearch

"In recent years I have become interested in the science and philosophy of how we define a disease. This may seem esoteric to you, but it is not and it has many practical implications. For example, saying someone has MS, or not, may affect their ability to get life-insurance or a pay-out from a critical illness policy."

"The paper below tackles the thorny issue of whether someone who presents with a tumefactive lesion that turns out on biopsy not to be a tumour, but an inflammatory demyelinating lesion. Another term for these lesions is pseudotumoural; i.e. the lesion mimics a tumour. The paper is trying to better define the so called clinico-pathological correlate, or more specifically the radiological-pathological correlate; i.e. what features on MRI can be used to predict the underlying pathology? Seven out of 9 of the cases went onto develop MS, i.e. they developed new lesions that fulfilled the McDonald diagnostic criteria for dissemination in time and space and no other aetiology could explain the presentation. One case was labelled as having acute hemorrhagic leukoencephalitis (AHLE) and another acute disseminated encephalomyelitis (ADEM). Unfortunately, the MRI findings were not specific enough to identify these latter two cases; hence there is no specific radiological-pathological correlate that can be used to help make a diagnosis. In short, this paper tells us that the MRI findings are non-specific when it comes to MS vs. ADEM/AHLE. The number of cases studied is small and hence the field may benefit from an International effort to pool cases."

"What does this mean for those of you reading this blog who presented with tumefactive MS? Unfortunately, nothing regarding your management or prognosis. What this paper highlights is that we academics like to describe and classify things. Sometimes we do this without a sound philosophical perspective. I would have designed this study differently and come at it from a different perspective. For one, I would have include a large number of cases who turned out to have tumours as a comparative group; the real question that needs answering is can we define any imaging features specific to mono-focal tumefactive MS that suggest the lesion is not a tumour? This may prevent unnecessary biopsies and hence has a clinical application. The second question is to differentiate those who will go onto to develop MS from those with ADEM or AHLE, which are monophasic MS. The latter will only Be answered by increasing the number of cases studied and by possibly looking at other biomarkers, e.g. CSF findings. "

J Neurol. 2014 Jul.

Background: Tumefactive demyelinating lesions (TDLs) can mimic brain tumors on radiological images. TDLs are often referred to as tumefactive multiple sclerosis (TMS), but the heterogeneous nature and monophasic course of TDLs do not fulfill clinical and magnetic resonance imaging (MRI) criteria for multiple sclerosis. 

Objective: Redefining TDLs, TMS and other inflammatory brain lesions is essential for the accurate clinical diagnosis of extensive demyelinating brain lesions. 

Methods: We retrospectively analyzed MRI from nine TDL cases that underwent brain biopsy. Patterns of gadolinium enhancement on MRI were categorized as homogenous, inhomogeneous, patchy and diffuse, open ring or irregular rim, and were compared with pathological hallmarks including demyelination, central necrosis, macrophage infiltration, angiogenesis and perivascular lymphocytic cuffing. 

Results: All cases had coexistence of demyelinating features and axonal loss. Open-ring and irregular rim patterns of gadolinium enhancement were associated with macrophage infiltrations and angiogenesis at the inflammatory border. An inhomogeneous pattern of gadolinium enhancement was associated with perivascular lymphocytic cuffing. Central necrosis was seen in cases of severe multiple sclerosis and hemorrhagic leukoencephalopathy. 

Conclusions: These results suggest that the radiological features of TDLs may be related to different pathological processes, and indicate that MRI may be useful in understanding their pathophysiology. Further investigation is needed to determine the precise disease entity of these inflammatory demyelinating brain lesions.