Mechanisms of central axon and myelin injury using advanced morphological and biochemical characterization

In the adult nervous system, white matter connecting brain, spinal, and peripheral regions is essential for neuron communication. In the white matter, the axon plays an essential role in transmitting nerve impulses, while myelin facilitates conduction through rapid propagation and optimal energy-consumption. White matter injury in the central nervous system, leading to the degeneration of axons and myelin, is commonly found in multiple sclerosis, anoxia, ischemic stroke, and traumatic brain and spinal cord injury. These lead to cognitive impairment, sensorimotor disability or death. During white matter injury, axonal spheroid formation (ASF) and demyelination are the pathological hallmarks of white matter degeneration. However, the mechanism by which this occurs is unknown. I hypothesize that focal spheroids are induced by activation of glutamate receptors and lead to local calcium overload in axons. To test this hypothesis, I established an ex vivo imaging model of central white matter dorsal column using two-photon microscopy to investigate this phenomenon. Ex vivo imaging of the white matter dorsal column with high-spatial resolution was achieved, which allowed the observation of morphology of axon and myelin before and during injury. The dynamic imaging of live myelinated axon response to injury suggests that axons and myelin are not as passive as previously thought. The data presented in this thesis support the notion of over-activation of glutamate receptors inducing ASF and a calcium rise leading to axonal transection. In addition, I established a novel method using solvatochromic dyes to investigate the biochemical property of myelin in which changes in the lipid composition served as a health indicator of myelin. The study of live myelin with solvatochromic dyes to stain myelin lipids could report different stages ranging from development, maturity, injury, and repair of myelin

Restriction of Feline Immunodeficiency Virus by Ref1, Lv1, and Primate TRIM5α Proteins

The Ref1 and Lv1 postentry restrictions in human and monkey cells have been analyzed for lentiviruses in the primate and ungulate groups, but no data exist for the third (feline) group. We compared feline immunodeficiency virus (FIV) to other restricted (human immunodeficiency virus type 1 [HIV-1], equine infectious anemia virus [EIAV]) and unrestricted (NB-tropic murine leukemia virus [NB-MLV]) retroviruses across wide ranges of viral inputs in cells from multiple primate and nonprimate species. We also characterized restrictions conferred to permissive feline and canine cells engineered to express rhesus and human TRIM5α proteins and performed RNA interference (RNAi) against endogenous TRIM5α. We find that expression of rhesus or human TRIM5α proteins in feline cells restricts FIV, impairing pseudotyped vector transduction and viral replication, but rhesus TRIM5α is more restricting than human TRIM5α. Notably, however, canine cells did not support restriction by human TRIM5α and supported minimal restriction by rhesus TRIM5α, suggesting that these proteins may not function autonomously or that a canine factor interferes. Stable RNAi knockdown of endogenous rhesus TRIM5α resulted in marked increases in FIV and HIV-1 infectivities while having no effect on NB-MLV. A panel of nonprimate cell lines varied widely in susceptibility to lentiviral vector transduction, but normalized FIV and HIV-1 vectors varied concordantly. In contrast, in human and monkey cells, relative restriction of FIV compared to HIV-1 varied from none to substantial, with the greatest relative infectivity deficit for FIV vectors observed in human T-cell lines. Endogenous and introduced TRIM5α restrictions of FIV could be titrated by coinfections with FIV, HIV-1, or EIAV virus-like particles. Arsenic trioxide had complex and TRIM5α-independent enhancing effects on lentiviral but not NB-MLV infection. Implications for human gene therapy are discussed

Nile Red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity

The molecular composition of myelin membranes determines their structure and function. Even minute changes to the biochemical balance can have profound consequences for axonal conduction and the synchronicity of neural networks. Hypothesizing that the earliest indication of myelin injury involves changes in the composition and/or polarity of its constituent lipids, we developed a sensitive spectroscopic technique for defining the chemical polarity of myelin lipids in fixed frozen tissue sections from rodent and human. The method uses a simple staining procedure involving the lipophilic dye Nile Red, whose fluorescence spectrum varies according to the chemical polarity of the microenvironment into which the dye embeds. Nile Red spectroscopy identified histologically intact yet biochemically altered myelin in prelesioned tissues, including mouse white matter following subdemyelinating cuprizone intoxication, as well as normal-appearing white matter in multiple sclerosis brain. Nile Red spectroscopy offers a relatively simple yet highly sensitive technique for detecting subtle myelin changes

Perfusion of His-Tagged Eukaryotic Myocilin Increases Outflow Resistance in Human Anterior Segments in the Presence of Aqueous Humor

PURPOSE. A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. METHODS. A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 g/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. RESULTS. Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% Ϯ 68% [mean Ϯ SD] versus 18% Ϯ 31% in fellow control eye; n ϭ 9, P ϭ 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for Ͼ3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% Ϯ 77% when preincubated in DMEM containing porcine aqueous humor versus 13% Ϯ 15% when preincubated with DMEM alone, n ϭ 6, P ϭ 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. CONCLUSIONS. Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork. (Invest Ophthalmol Vis Sci

Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy

For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC-demyelination in mice. We found that LPC nonspecifically disrupted myelin lipids. LPC integrated into cellular membranes and rapidly induced cell membrane permeability; in mice, LPC injury was phenocopied by other lipid disrupting agents. Interestingly, following its injection into white matter, LPC was cleared within 24 hr but by five days there was an elevation of endogenous LPC that was not associated with damage. This elevation of LPC in the absence of injury raises the possibility that the brain has mechanisms to buffer LPC. In support, LPC injury in culture was significantly ameliorated by albumin buffering. These results shed light on the mechanisms of LPC injury and homeostasis