Function of COX-2 and prostaglandins in neurological disease.

Induction of COX-2 expression and enzymatic activity promotes neuronal injury in a number of models of neurological disease. Inhibition of COX-2 activity, either genetically or pharmacologically, has been shown to be neuroprotective in rodent models of stroke, Parkinson's disease, and amyotrophic lateral sclerosis. Inhibition of COX activity with nonsteroidal anti-inflammatory drugs (NSAIDs) reduces inflammation and amyloid accumulation in murine transgenic models of Familial Alzheimer's disease, and the use of NSAIDs decreases the risk of developing Alzheimer's disease in healthy aging populations.

PGE2 receptors rescue motor neurons in a model of amyotrophic lateral sclerosis.

Recent studies suggest that the inducible isoform of cyclooxygenase, COX-2, promotes motor neuron loss in rodent models of ALS. We investigated the effects of PGE2, a principal downstream prostaglandin product of COX-2 activity, on motor neuron survival in an organotypic culture model of ALS. We find that PGE2 paradoxically protects motor neurons at physiological concentrations in this model.

NAALADase inhibition protects motor neurons against chronic glutamate toxicity.

Glutamate toxicity is implicated in the pathogenesis of amyotrophic lateral sclerosis. The neuropeptide N-acetyl-aspartyl glutamate (NAAG) appears to function both as a storage form for glutamate and as a neuromodulator at glutamatergic synapses. N-acetylated-alpha-linked acidic dipeptidase (NAALADase; also termed glutamate carboxypeptidase II) yields N-acetyl aspartate (NAA) and glutamate.

Exocytotic "constipation" is a mechanism of tubulin/lysosomal interaction in colchicine myopathy.

Colchicine, a known microtubule disrupting agent, produces a human myopathy, characterized by accumulation of lysosomes. We have created a reliable animal model of colchicine myopathy that replicates the subacute myopathy seen in humans, reproducing the chronic proximal weakness and vacuolar changes in nonnecrotic myofibers. If a microtubule network plays a role in lysosomal function in muscle, disturbance of it could alter degradation of intrinsic membrane receptors, presumably at some intracellular processing site or at exocytosis.

Intracellular fibroblast growth factor produces effects different from those of extracellular application on development of avian cochleovestibular ganglion cells in vitro.

In an avian coculture system, the neuronal precursors of the cochleovestibular ganglion typically migrated from the otocyst and differentiated in response to soluble fibroblast growth factor (FGF-2), which had free access to FGF receptors on the cell surface. Free FGF-2 switched cells from a proliferation mode to migration, accompanied by increases in process outgrowth, fasciculation, and polysialic acid expression. Microsphere-bound FGF-2 had some of the same effects, but in addition it increased proliferation and decreased fasciculation and polysialic acid.

Identification of the neuroprotective molecular region of pigment epithelium-derived factor and its binding sites on motor neurons.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) family, is a survival factor for various types of neurons. We studied the mechanisms by which human PEDF protects motor neurons from degeneration, with the goal of eventually conducting human clinical trials. We first searched for a molecular region of human PEDF essential to motor neuron protection.

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9.

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease.

Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis.

Pigment epithelium-derived factor (PEDF), a recently defined retinal trophic factor and anti-angiogenic factor for the eye, is also present in the CNS and is a motor neuron protectant. We asked whether PEDF levels in CSF are altered in patients with amyotrophic lateral sclerosis (ALS). Pigment epithelium-derived factor protein was detected by quantitative western blot analysis with a PEDF-specific antiserum.

Additivity and potentiation of IGF-I and GDNF in the complete rescue of postnatal motor neurons.

Background: Both growth and survival of motor neurons may depend on multiple neurotrophic factors. Individually, insulin-like growth factor I (IGF-I) and glial cell line-derived neurotrophic factor (GDNF) are potent neurotrophic/survival factors for postnatal motor neurons.

Methods: We used an organotypic spinal cord model of glutamatergic degeneration in ALS to investigate whether IGF-I and GDNF interact to enhance motor neuron survival, their trophic effect on choline acetyltransferase (ChAT) activity, and their effect on neurite outgrowth.

Delayed application of IGF-I and GDNF can rescue already injured postnatal motor neurons.

IGF-I, GDNF, and other neurotrophic factors, when applied at the time of injury, can protect postnatal motor neurons from slow glutamate injury in organotypic spinal cord. However, in human spinal cord diseases, motor neuron injury is already established when treatment could begin. We tested whether neurotrophic factors can protect already-injured motor neurons, and whether combinations of factors can further lengthen the therapeutic time window.

Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration.

Many neurotrophic factors have been shown to enhance survival of embryonic motor neurons or affect their response to injury. Few studies have investigated the potential effects of neurotrophic factors on more mature motor neurons that might be relevant for neurodegenerative diseases. Using organotypic spinal cord cultures from postnatal rats, we have demonstrated that insulin-like growth factor-I (IGF-I) and glial-derived neurotrophic factor (GDNF) significantly increase choline acetyltransferase (ChAT) activity, but brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) do not.

Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration.

Although pigment epithelium-derived factor (PEDF) is a neurotrophic factor that may aid the development, differentiation, and survival of adjacent neural retinae, the wider distribution of PEDF mRNA in the central nervous system suggested to us that this factor could have pleiotropic neurotrophic and neuroprotective effects on nonretinal neurons. We examined the distribution of PEDF mRNA and its transcript in the spinal cord. By immunohistochemistry and western blot analysis using an antihuman PEDF antiserum of known specificity, we found that PEDF protein is present in spinal cord, cerebrospinal fluid, and skeletal muscle and that its mRNA appears concentrated in motor neurons of the human spinal cord.

Neuroprotective utility and neurotrophic action of neurturin in postnatal motor neurons: comparison with GDNF and persephin.

Neurturin and persephin are recently discovered homologs of glial cell line-derived neurotrophic factor (GDNF). Here, we report that neurturin, like GDNF, increases the choline acetyltransferase activity of normal postnatal motor neurons, induces neurite outgrowth in spinal cord, and potently protects motor neurons from chronic glutamate-mediated degeneration. Persephin, in contrast, does not appear to have neurotrophic or neurite-promoting effects on mature motor neurons and may instead worsen the glutamate injury of motor neurons.

Multiple roles of neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1 adhesion molecules during sensory innervation of the otic epithelium in vitro.

To explore the role of cell adhesion molecules in the innervation of the inner ear, antibody perturbation was used on histotypic co-cultures of the ganglionic and epithelial anlagen derived from the otocyst. When unperturbed, these tissues survived and differentiated in this culture system with outgrowth of fasciculated neuronal fibers which expressed neural cell adhesion molecule and L1. The fibers exhibited target choice and penetration, then branching and spreading within the otic epithelium as individual axons.

Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle.

We examined specific properties of highly purified synemin (230 kDa), recently identified as a novel intermediate filament (IF) protein, from avian smooth muscle. Soluble synemin in 10 mM Tris-HCl, pH 8.5, appears as approximately 11-nm-diameter globular structures by negative-stain and low-angle shadow electron microscopy.

New growth of axons in the cochlear nucleus of adult chinchillas after acoustic trauma.

This study determined the effect of acoustic overstimulation of the adult cochlea on axons in the cochlear nucleus. Chinchillas were exposed to an octave-band noise centered at 4 kHz at 108 dB sound pressure level for 1.75 h.

Differential expression of N-methyl-D-aspartate receptor in the cochlear nucleus of the mouse.

Glutamate is used in the cochlear nucleus as a neurotransmitter by cochlear nerve synapses and by local circuits of granule cell axons. In the present study, immunocytochemistry and in situ hybridization were used to identify different types of neurons expressing N-methyl-D-aspartate receptor subunit I (NMDAR1) in the mouse cochlear nucleus. N-Methyl-D-aspartate receptor subunit 1 was expressed in most neuronal types, but granule cells in the dorsal cochlear nucleus had little, if any, expression, unlike their heavily labeled counterparts in the small cell shell and cerebellum.

Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.

Sporadic inclusion-body myositis (s-IBM) and the hereditary inclusion-body myopathies (h-IBMs) are severe and progressive muscle diseases, characterized pathologically by vacuolated muscle fibers containing paired-helical filaments (PHFs). An interesting feature of the s- and h-IBM muscle phenotype is its striking similarity to Alzheimer-disease (AD) brain. We immunostained muscle biopsies of 9 s-IBM patients, 9 autosomal-recessive h-IBM patients, 1 autosomal-dominant h-IBM patients, and 18 normal and disease-controls with several antibodies known to react with the hyperphosphorylated tau of AD-PHFs.

NMDA receptor expression in the mouse cerebellar cortex.

A detailed, light microscopic study on the distribution of the N-methyl- D-aspartate receptor subunit 1 (NMDAR1) was carried out with immunohistochemistry and in situ hybridization on the cerebellar cortex of the mouse. With a monoclonal antibody, labeling of Purkinje cell bodies varied from intense to negative, while heavy dendritic staining was limited to the proximal dendrites (unlike the rat, which also had heavily stained distal dendrites). In the granular layer, the cell bodies and and the dendritic shafts of Golgi II cells were only moderately stained, but very intense labeling was associated with granule cell bodies, and with their dendrites and dendritic endings in the glomeruli.

Human muscle macrophages express beta-amyloid precursor and prion proteins and their mRNAs.

Well-characterized antibodies against beta-amyloid precursor protein (beta APP) and prion protein (PrP), and specific cRNA probes, were used to localize beta APP and PrP and their mRNAs in human muscle macrophages. Macrophages present in muscle biopsies of 51 patients with various neuromuscular disorders showed accumulation of beta APP and PrP, and strongly expressed beta APP and PrP mRNAs. These were present in all muscle macrophages unrelated to their localization within the muscle tissue or diagnosis.

Alpha 1-antichymotrypsin is strongly immunolocalized at normal human and rat neuromuscular junctions.

alpha 1-Antichymotrypsin (alpha 1-ACT) is an early-stage acute-phase plasma protein and a serpin that preferentially inactivates chymotrypsin, cathepsin G, and chymase. Using immunofluorescence with four rabbit polyclonal and two monoclonal specific antibodies against human alpha 1-ACT, we have localized alpha 1-ACT at human and rat neuromuscular junctions (NMJs). Strong alpha 1-ACT immunoreactivity (IR) was present at all NMJs identified by bound alpha-bungarotoxin (alpha-BT).

Twisted tubulofilaments of inclusion body myositis muscle resemble paired helical filaments of Alzheimer brain and contain hyperphosphorylated tau.

We immunostained muscle biopsies of 8 patients with sporadic inclusion body myositis (S-IBM), 7 patients with autosomal recessive hereditary inclusion body myopathy (H-IBM) (both diseases being characterized by similar muscle fiber vacuoles containing inclusions), and 11 normal and disease controls. We used the following well-characterized antibodies against tau protein: Tau-1, Alz-50, and anti-paired helical filament (PHF) antiserum. By light microscopy, in all S-IBM muscle biopsies virtually all vacuoles immunoreactive for ubiquitin and beta-amyloid protein also contained inclusions immunoreactive with Alz-50 and anti-PHF antiserum.

Tropomodulin is highly concentrated at the postsynaptic domain of human and rat neuromuscular junctions.

Tropomodulin is expressed in skeletal muscle and recent studies suggest that tropomodulin is associated with synaptic membranes. Therefore, we have examined neuromuscular junctions by immunofluorescence analysis for tropomodulin localization. Anti-tropomodulin antibodies generated against either the whole protein or a 15-amino acid peptide fragment label human neuromuscular junctions as demonstrated by colocalization with alpha-bungarotoxin label.

Prion protein is abnormally accumulated in inclusion-body myositis.

In muscle biopsies of 8 sporadic inclusion-body myositis (S-IBM) and 4 hereditary inclusion-body myopathy (H-IBM) patients, vacuolated muscle fibers contained within their vacuoles strongly immunoreactive inclusions with 2 polyclonal and 1 monoclonal antibodies against prion protein (PrP). By light-microscopy, PrP deposits co-localized with beta-amyloid protein (A beta) and ubiquitin (Ub). By immuno-electronmicroscopy, both PrP and A beta were present on amorphous material and on 6-10 nm amyloid-like fibrils; and PrP and Ub co-localized on cytoplasmic twisted tubulofilaments (TTFs) and on amorphous material.

Prion protein is strongly immunolocalized at the postsynaptic domain of human normal neuromuscular junctions.

Using three well-characterized polyclonal and monoclonal antibodies against prion protein (PrP), we demonstrated a strong concentration of PrP at human neuromuscular junctions (NMJs). Applying double and triple fluorescence-labeling, we found that PrP immunoreactivity exactly co-localized with alpha-bungarotoxin (alpha-BT) identified acetylcholine receptors, as well as with the high junctional concentrations of beta-amyloid precursor protein, beta-amyloid protein, desmin, ubiquitin and dystrophin. Therefore, PrP was considered to be located on the postsynaptic muscle membrane.