Alon Monsonego

The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.

In this study we present a method to achieve a complete transection of optic nerve axons in adult rat, while preserving the vasculature and retaining the continuity of the meninges. Under deep anesthesia, the optic nerve of adult rat is exposed. Using specially designed instruments built from disposable glass microsampling pipettes, a small opening is created in the meninges of the optic nerve, 2-3 mm behind the eye globe. A glass dissector is introduced through the opening and is used to cut all the axons through the whole width of the nerve. Complete transection of the optic nerve axons was achieved, while retaining the continuity of the meninges and avoiding damage to the nerve's vascular supply. Transection was confirmed by transillumination showing a complete gap in the continuity of the nerve axons, and by both morphological and electrophysiological criteria. Nerve transection performed by the conventional technique leads to neuroma formation and hampers regeneration. Crush injury may cause nerve ischemia, which is detrimental to axonal recovery. Both of these disadvantages are avoided by the method of transection presented here. The opening created in the 'meningeal tube' can be used to inject substances that may be of benefit in recovery, rescue and/or regeneration of the injured axons. The model is particularly suitable for in vivo studies on nerve regeneration, and especially for screening of putative therapeutic agents.

GTP and Ca2+, two well-known modulators of intracellular signaling pathways, control a structural/functional switch between two vital and mutually exclusive activities, cross-linking and Galpha activity, in the same enzyme. The enzyme, a brain-derived tissue-type transglutaminase (TGase), was recently cloned by us in two forms, one of which (s-TGN) lacks a C-terminal region that is present in the other (l-TGN). Immunoreaction with antibodies directed against a peptide present in the C-terminus of l-TGN but missing in s-TGN suggested that this site, which is located in the C-terminal fourth domain, undergoes conformational changes as a result of interaction between l-TGN and GTP. Site-directed mutagenesis suggested that the third domain is involved in mediating the inhibition of the cross-linking activity. These results were supported by molecular modeling, which further suggested that domains III and IV both participate in conformational changes leading to the functional switch between the Ca2+-dependent cross-linking activity and the Galpha activity.

Neuroinflammation is critically involved in numerous neurodegenerative diseases, and key signaling steps of innate immune activation hence represent promising therapeutic targets. This mini review series originated from the 4(th) Venusberg Meeting on Neuroinflammation held in Bonn, Germany, 7-9(th) May 2015, presenting updates on innate immunity in acute brain injury and chronic neurodegenerative disorders, such as traumatic brain injury and Alzheimer's disease, on the role of astrocytes and microglia, as well as technical developments that may help elucidate neuroinflammatory mechanisms and establish clinical relevance. In this meeting report, a brief overview of physiological and pathological microglia morphology is followed by a synopsis on PGE2 receptors, insights into the role of arginine metabolism and further relevant aspects of neuroinflammation in various clinical settings, concluded by a presentation of technical challenges and solutions when working with microglia and...