Gamma Interferon Is Critical for Neuronal Viral Clearance and Protection in a Susceptible Mouse Strain following Early Intracranial Theiler's Murine Encephalomyelitis Virus Infection

We evaluated the role of gamma interferon (IFN-γ) in protecting neurons from virus-induced injury following central nervous system infection. IFN-γ(−/−) and IFN-γ(+/+) mice of the resistant major histocompatibility complex (MHC) H-2(b) haplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-γ(+/+) H-2(b) mice also cleared virus from the spinal cord white matter, whereas IFN-γ(−/−) H-2(b) mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-γ(−/−) mice of the susceptible H-2(q) haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-γ(−/−) H-2(q) mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-γ(−/−) H-2(q) mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-γ(−/−) H-2(q) mice. IFN-γ deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-γ(−/−) H-2(q) mice was lower than those in IFN-γ(+/+) H-2(q) mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-γ exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-γ plays a critical role in protecting spinal cord neurons from persistent infection and death

A natural human IgM that binds to gangliosides is therapeutic in murine models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating, fatal neurological disease that primarily affects spinal cord anterior horn cells and their axons for which there is no treatment. Here we report the use of a recombinant natural human IgM that binds to the surface of neurons and supports neurite extension, rHIgM12, as a therapeutic strategy in murine models of human ALS. A single 200 µg intraperitoneal dose of rHIgM12 increases survival in two independent genetic-based mutant SOD1 mouse strains (SOD1G86R and SOD1G93A) by 8 and 10 days, delays the onset of neurological deficits by 16 days, delays the onset of weight loss by 5 days, and preserves spinal cord axons and anterior horn neurons. Immuno-overlay of thin layer chromatography and surface plasmon resonance show that rHIgM12 binds with high affinity to the complex gangliosides GD1a and GT1b. Addition of rHIgM12 to neurons in culture increases α-tubulin tyrosination levels, suggesting an alteration of microtubule dynamics. We previously reported that a single peripheral dose of rHIgM12 preserved neurological function in a murine model of demyelination with axon loss. Because rHIgM12 improves three different models of neurological disease, we propose that the IgM might act late in the cascade of neuronal stress and/or death by a broad mechanism

Abbreviated Exposure to Hypoxia Is Sufficient to Induce CNS Dysmyelination, Modulate Spinal Motor Neuron Composition, and Impair Motor Development in Neonatal Mice

<div><p>Neonatal white matter injury (nWMI) is an increasingly common cause of cerebral palsy that results predominantly from hypoxic injury to progenitor cells including those of the oligodendrocyte lineage. Existing mouse models of nWMI utilize prolonged periods of hypoxia during the neonatal period, require complex cross-fostering and exhibit poor growth and high mortality rates. Abnormal CNS myelin composition serves as the major explanation for persistent neuro-motor deficits. Here we developed a simplified model of nWMI with low mortality rates and improved growth without cross-fostering. Neonatal mice are exposed to low oxygen from postnatal day (P) 3 to P7, which roughly corresponds to the period of human brain development between gestational weeks 32 and 36. CNS hypomyelination is detectable for 2–3 weeks post injury and strongly correlates with levels of body and brain weight loss. Immediately following hypoxia treatment, cell death was evident in multiple brain regions, most notably in superficial and deep cortical layers as well as the subventricular zone progenitor compartment. PDGFαR, Nkx2.2, and Olig2 positive oligodendrocyte progenitor cell were significantly reduced until postnatal day 27. In addition to CNS dysmyelination we identified a novel pathological marker for adult hypoxic animals that strongly correlates with life-long neuro-motor deficits. Mice reared under hypoxia reveal an abnormal spinal neuron composition with increased small and medium diameter axons and decreased large diameter axons in thoracic lateral and anterior funiculi. Differences were particularly pronounced in white matter motor tracts left and right of the anterior median fissure. Our findings suggest that 4 days of exposure to hypoxia are sufficient to induce experimental nWMI in CD1 mice, thus providing a model to test new therapeutics. Pathological hallmarks of this model include early cell death, decreased OPCs and hypomyelination in early postnatal life, followed by dysmyelination, abnormal spinal neuron composition, and neuro-motor deficits in adulthood.</p></div

Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity

<div><p>For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or <i>a priori</i> knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (<i>Rag1</i>, <i>Mda5</i>, <i>Mavs</i>, <i>Ifnar1</i>, <i>Ifngr1</i>, and <i>Tlr3)</i> established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.</p></div

Comparison of growth and survival in models of neonatal hypoxic injury.

<p>A: Experimental timeline showing rearing strategies of neonatal CD1 mice under abbreviated hypoxia (red) (P3 → P7) followed by normoxia (gray), or long-duration hypoxia (green) (P3 <b>→</b> P12). B: Body weight comparison under abbreviated or long hypoxia (P3 <b>→</b> P7 or P3 <b>→</b> P12) (normoxia: n = 102; 10d hypoxia: n = 20; 4d hypoxia: n = 102). C: Survival rate of neonatal CD1 mice at P12 under abbreviated (red bar, n = 102) or long hypoxia (green bar), n = 20. D, E: Body (D) and (E) brain weight development of neonatal mice after hypoxia (P3 <b>→</b> P7) or normoxia (P3, n = 73 normoxic, 78 hypoxic; P7, n = 108 normoxic, 119 hypoxic; P13, n = 84 normoxic, 96 hypoxic; P27, n = 25 normoxic, 34 hypoxic; P43, n = 25 normoxic, 34 hypoxic; P80, n = 24 normoxic, 23 hypoxic mice). Litter sizes in A-E were 12 neonatal mice per dam. F-I: Body weight (F-H) and body weight ratios (I) in cross-fostered (F, G) and non-cross-fostered (H) normoxic and hypoxic (P3 <b>→</b> P7) CD1 and C57/Bl6 mice. Litter sizes in F were 6 neonatal mice per dam (non-cross-fostered CD1 mice: P7, n = 23 normoxic, 18 hypoxic; P13, n = 18 normoxic, 12 hypoxic mice; cross-fostered CD1 mice by C57/bl6 dam: n = 12 normoxic and 12 hypoxic mice for P7, P13 and P27; cross-fostered C57/Bl6 mice by CD1 dam: n = 12 normoxic and 12 hypoxic mice for P7, P13 and P27). Data are shown as mean ± std.-dev. *** p < 0.001; ** p < 0.01; * p < 0.05.</p

Fold-change in gene transcripts at P13, as detected by RT-PCR.

<p>Fold-change in gene transcripts at P13, as detected by RT-PCR.</p

Abbreviated hypoxia (P3 → P7) does not increase levels of OPCs but causes substantial apoptosis throughout cortical layers, hippocampus and SVZ.

<p>A: Immunohistochemistry and stereologic analysis of mouse cerebra at P13 using OL markers Olig-2 (OPCs, immature OLs, mature OLs), MBP (mature OLs) and NKX2.2 (OPCs). B: Immunohistochemical staining of level matched hypoxic and control cerebra at P7 showing anti-CC3 (red) and nuclear marker DAPI (green arrows indicate specific regions SVZ, DG and CA1/3 field; yellow arrows mark levels of high apoptotic intensity in hypoxic mice). C: Representative Western blots using total brain homogenates from hypoxic and control CD1 mice at P7 using apoptosis marker CC3 and β-actin as a loading control. Densitometric analysis of Western blots from 3 independent experiments showing brain levels of CC3 at P7 in hypoxic and control mice with *** equals p < 0.001; ** equals p < 0.01; * equals p < 0.05.. SVZ, subventricular zone; DG, dentate gyrus; CA1-3, hippocampal CA fields; Ctx, cortex, I-VI = cortical layers 1–6. (n = 6 hypoxic + 6 normoxic animals for immunohistochemistry and Western blotting (each)).</p

Abbreviated hypoxia is sufficient to induce persistent motor-deficits in mice.

<p>Mice reared under hypoxia (P3 <b>→</b> P7) or room air were tested at weaning age (P21), early adulthood and adulthood (P43, P80) for motor coordination and strength (D-F), strength (A-C), balance and endurance (G-I), front limb grip strength (L), body composition (E, F), global nocturnal activity in groups of three mice per box (J, K) and global nocturnal activity of single mice per box (I, J). A-C: hanging wire-single test at P21 (A), P43 (B), P80 (C); D-F: hanging wire-mesh test at P21 (D), P43 (E), P80 (F); G-I: Rotarod test at P21 (G), P43 (H), P80 (I). J, K: Global nocturnal activity of grouped mice (3 per activity box) showing horizontal hourly beambreaks (J) and vertical hourly beambreaks (K) for 5 nights from P91-P96. L: Grip strength meter test at P90. N per test and time-point >34 animals for hanging wire tests, Rotarod and grouped nocturnal activity; N = 14 per group for grip strength meter test with *** equals p < 0.001; ** equals p < 0.01; * equals p < 0.05.</p

Long hypoxia vs abbreviated hypoxia.

<p>Long hypoxia vs abbreviated hypoxia.</p

Behavioral motor testing—Male mice.

<p>Behavioral motor testing—Male mice.</p