Advanced glycation endproduct changes to Bruch's membrane promotes lipoprotein retention by lipoprotein lipase.

Lipoprotein particles accumulate in Bruch's membrane before the development of basal deposits and drusen, two histopathologic lesions that define age-related macular degeneration (AMD). We therefore, sought to determine which molecules could participate in lipoprotein retention. Wild-type or lipoprotein lipase-deficient mice were injected with low-dose D-galactose or PBS subcutaneously for 8 weeks to induce advanced glycation endproduct (AGE) formation. Some mice were also injected with the AGE breaker phenacylphiazolium bromide and D-galactose. Rhodamine-labeled low-density lipoproteins were injected into mice, and the fluorescence was measured up to 72 hours later. AGEs, proteoglycans, and other lipid-retaining molecules were evaluated by IHC. Lipoprotein lipase distribution was assessed in AMD samples by IHC. D-galactose-treated mice retained lipoproteins in the retinal pigment epithelial and Bruch's membrane to a greater extent than either PBS- or phenacylphiazolium bromide/D-galactose-treated mice at 24 and 72 hours after injection (P ≤ 0.04). Immunolabeling for carboxymethyllysine, biglycan, and lipoprotein lipase was found in D-galactose-treated mice only. Mice deficient for lipoprotein lipase treated with D-galactose did not retain lipoproteins to any measureable extent. Human AMD samples had lipoprotein lipase labeling within drusen, basal deposits, and the choroid. Mice treated with D-galactose to induce AGE formation in Bruch's membrane retain intravenously injected lipoproteins. Our results suggest that lipoprotein retention in Bruch's membrane is mediated by lipoprotein lipase.

BAK alters neuronal excitability and can switch from anti- to pro-death function during postnatal development.

BAK is a pro-apoptotic BCL-2 family protein that localizes to mitochondria. Here we evaluate the function of BAK in several mouse models of neuronal injury including neuronotropic Sindbis virus infection, Parkinson's disease, ischemia/stroke, and seizure. BAK promotes or inhibits neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. BAK protects neurons from excitotoxicity and virus infection in the hippocampus. As mice mature, BAK is converted from anti- to pro-death function in virus-infected spinal cord neurons. In addition to regulating cell death, BAK also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. BAK can alter neurotransmitter release in a direction consistent with its protective effects on neurons and mice. These findings suggest that BAK inhibits cell death by modifying neuronal excitability.

IL-6 induces regionally selective spinal cord injury in patients with the neuroinflammatory disorder transverse myelitis.

Transverse myelitis (TM) is an immune-mediated spinal cord disorder associated with inflammation, demyelination, and axonal damage. We investigated the soluble immune derangements present in TM patients and found that IL-6 levels were selectively and dramatically elevated in the cerebrospinal fluid and directly correlated with markers of tissue injury and sustained clinical disability. IL-6 was necessary and sufficient to mediate cellular injury in spinal cord organotypic tissue culture sections through activation of the JAK/STAT pathway, resulting in increased activity of iNOS and poly(ADP-ribose) polymerase (PARP). Rats intrathecally infused with IL-6 developed progressive weakness and spinal cord inflammation, demyelination, and axonal damage, which were blocked by PARP inhibition. Addition of IL-6 to brain organotypic cultures or into the cerebral ventricles of adult rats did not activate the JAK/STAT pathway, which is potentially due to increased expression of soluble IL-6 receptor in the brain relative to the spinal cord that may antagonize IL-6 signaling in this context. The spatially distinct responses to IL-6 may underlie regional vulnerability of different parts of the CNS to inflammatory injury. The elucidation of this pathway identifies specific therapeutic targets in the management of CNS autoimmune conditions.

High resolution diffusion tensor imaging of axonal damage in focal inflammatory and demyelinating lesions in rat spinal cord.

Inflammation, demyelination, gliosis and axonal degeneration are pathological hallmarks of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis. Axonal damage is thought to contribute to irreversible damage and functional impairment, but is difficult to quantify. Conventional MRI has been used to assess the inflammatory and demyelinating aspects of MS lesions, but more sensitive and specific methods are needed to identify axonal damage to monitor disease progression and to determine efficacy of putative neuroprotective agents. We used high resolution diffusion tensor imaging (DTI) and fibre tracking to examine the spinal cord in rats with focal dorsal column inflammatory or demyelinating lesions to determine whether DTI measures can be used to detect pathology at the site of the focal lesion and to measure axonal damage in tracts distal to the focal lesion. Distant from the focal lesion, total axon counts, degenerating axon counts and SMI-31 staining, but not Luxol fast blue staining, were significantly correlated with fractional anisotropy, axial diffusivity and radial diffusivity, all of which are derived from the DTI data. These data suggest that high resolution DTI may be a more sensitive method than conventional imaging for detecting axonal damage at sites distant from inflammation.

Viral-induced spinal motor neuron death is non-cell-autonomous and involves glutamate excitotoxicity.

Neuroadapted Sindbis virus (NSV) is a neurotropic virus capable of inducing the death of spinal motor neurons in mice and rats. In this study we investigated the mechanisms that underlie NSV-induced motor neuron death. We found that many degenerating spinal motor neurons were not infected directly with NSV, suggesting that bystander cell death occurs. An excitotoxic mechanism was confirmed when blockade of calcium-permeable AMPA receptors attenuated motor neuron death both in vitro and in vivo. Blockade of astroglial glutamate reuptake potentiated NSV-induced motor neuron loss in vivo, suggesting that astrocyte-mediated removal of perisynaptic glutamate is important in limiting NSV-induced excitotoxic injury. Astroglial glutamate transport was reduced markedly in the spinal cord during NSV infection, in advance of motor neuron injury in susceptible mice. In contrast, we found 5.6-fold elevated glutamate uptake in the spinal cords of mice resistant to NSV-induced paralysis. Likewise, minocycline markedly increased spinal cord glutamate transport and protected mice from NSV-induced motor neuron death. These studies suggest that NSV infection triggers a cascade of events in the spinal cord resulting in impaired astrocytic glutamate transport and excitotoxic injury of motor neurons mediated via calcium-permeable AMPA receptors. Similar changes may occur in other motor neuron disorders such as amyotrophic lateral sclerosis or West Nile Virus-induced poliomyelitis, suggesting a common tissue injury pathway.

Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury.

We have investigated the potential of human pluripotent cells to restore function in rats paralyzed with a virus-induced motor neuronopathy. Cells derived from embryonic germ cells, termed embryoid body-derived (EBD) cells, introduced into the CSF were distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchyma in paralyzed, but not uninjured, animals. Some of the transplanted human cells expressed the neuroglial progenitor marker nestin, whereas others expressed immunohistochemical markers characteristic of astrocytes or mature neurons. Rare transplanted cells developed immunoreactivity to choline acetyltransferase (ChAT) and sent axons into the sciatic nerve as detected by retrograde labeling. Paralyzed animals transplanted with EBD cells partially recovered motor function 12 and 24 weeks after transplantation, whereas control animals remained paralyzed. Semi-quantitative analysis revealed that the efficiency of neuronal differentiation and extension of neurites could not account for the functional recovery. Rather, transplanted EBD cells protected host neurons from death and facilitated reafferentation of motor neuron cell bodies. In vitro, EBD cells secrete transforming growth factor-alpha (TGF-alpha) and brain-derived neurotrophic factor (BDNF). Neutralizing antibodies to TGF-alpha and to BDNF abrogated the ability of EBD-conditioned media to sustain motor neuron survival in culture, whereas neutralizing antibodies to BDNF eliminated the axonal outgrowth from spinal organotypics observed with direct coculture of EBD cells. We conclude that cells derived from human pluripotent stem cells have the capacity to restore neurologic function in animals with diffuse motor neuron disease via enhancement of host neuron survival and function.

n-Dodecyl-ß-D-maltoside inhibits aggregation of human interferon-ß-1b and reduces its immunogenicity.

The development of neutralizing antibodies to the protein drug interferon-ß is a significant impediment to its use in the treatment of multiple sclerosis. Neutralizing antibodies to interferon-ß arise from aggregation of the peptide during manufacturing and storage. We tested the ability of dodecylmaltoside, a nontoxic alkylsaccharide surfactant, to reduce aggregation of interferon-ß in vitro and to reduce its immunogenicity in vivo. Interferon-ß, in solution with and without dodecylmaltoside, was periodically evaluated for aggregation by light scatter for 1 month. Interferon-ß, with and without dodecylmaltoside, was given 3 days/week for 1 month to mice; the sera of these mice were analyzed for anti-interferon-ß antibodies by ELISA. Dodecylmaltoside reduces the aggregation of interferon-ß in vitro and its immunogenicity in vivo. Our positive findings warrant additional tests of dodecylmaltoside as a therapeutic adjuvant in rodent models of multiple sclerosis.

Recovery from paralysis in adult rats using embryonic stem cells.

OBJECTIVE: We explored the potential of embryonic stem cell-derived motor neurons to functionally replace those cells destroyed in paralyzed adult rats. METHODS: We administered a phosphodiesterase type 4 inhibitor and dibutyryl cyclic adenosine monophosphate to overcome myelin-mediated repulsion and provided glial cell-derived neurotrophic factor within the sciatic nerve to attract transplanted embryonic stem cell-derived axons toward skeletal muscle targets. RESULTS: We found that these strategies significantly increased the success of transplanted axons extending out of the spinal cord into ventral roots. Furthermore, transplant-derived axons reached muscle, formed neuromuscular junctions, were physiologically active, and mediated partial recovery from paralysis. INTERPRETATION: We conclude that restoration of functional motor units by embryonic stem cells is possible and represents a potential therapeutic strategy for patients with paralysis. To our knowledge, this is the first report of the anatomical and functional replacement of a motor neuron circuit within the adult mammalian host.