Mechano growth factor interacts with nucleolin to protect against cisplatin-induced neurotoxicity.

Mechano growth factor (MGF) is an alternatively spliced form of insulin-like growth factor-1 (IGF-1) that has shown to be neuroprotective against 6-hydroxydopamine toxicity and ischemic injury in the brain. MGF also induces neural stem cell proliferation in the hippocampus and preserves olfactory function in aging mice. Cisplatin is a chemotherapy drug that induces peripheral neuropathy in 30-40% of treated patients. Our studies were designed to see if MGF would protect dorsal root ganglion (DRG) neurons from cisplatin-induced neurotoxicity and to identify potential mechanisms that may be involved. Expression of endogenous MGF in adult DRG neurons in vivo ameliorated cisplatin-induced thermal hyperalgesia. Exogenous MGF and MGF with a cysteine added to the N-terminus (CMGF) also protected embryonic DRG neurons from cisplatin-induced cell death in vitro. Mass spectroscopy analysis of proteins bound to MGF showed that nucleolin is a key-binding partner. Antibodies against nucleolin prevented the neuroprotective effect of MGF and CMGF in culture. Both nucleolin and MGF are located in the nucleolus of DRG neurons. RNAseq of RNA associated with MGF indicated that MGF may be involved in RNA processing, protein targeting and transcription/translation. Nucleolin is an RNA binding protein that is readily shuttled between the nucleus, cytoplasm and plasma membrane. Nucleolin and MGF may work together to prevent cisplatin-induced neurotoxicity. Exploring the known mechanisms of nucleolin may help us better understand the mechanisms of cisplatin toxicity and how MGF protects DRG neurons.

Dynamic role of kallikrein 6 in traumatic spinal cord injury.

Kallikrein 6 (K6) is a member of the kallikrein gene family that comprises 15 structurally and functionally related serine proteases. In prior studies we showed that, while this trypsin-like enzyme is preferentially expressed in neurons and oligodendroglia of the adult central nervous system (CNS), it is up-regulated at sites of injury due to expression by infiltrating immune and resident CNS cells. Given this background we hypothesized that K6 is a key contributor to the pathophysiology of traumatic spinal cord injury (SCI), influencing neural repair and regeneration. Examination of K6 expression following contusion injury to the adult rat cord, and in cases of human traumatic SCI, indicated significant elevations at acute and chronic time points, not only at the injury site but also in cord segments above and below. Elevations in K6 were particularly prominent in macrophages, microglia and reactive astrocytes. To determine potential effects of elevated K6 on the regeneration environment, the ability of neurons to adhere to and extend processes on substrata which had been exposed to recombinant K6 was examined. Limited (1 h) or excess (24 h) K6-mediated proteolytic digestion of a growth-facilitatory substrate, laminin, significantly decreased neurite outgrowth. By contrast, similar hydrolysis of a growth-inhibitory substrate, aggrecan, significantly increased neurite extension and cell adherence. These data support the hypothesis that K6 enzymatic cascades mediate events secondary to spinal cord trauma, including dynamic modification of the capacity for axon outgrowth.

Animal models of spinal cord injury for evaluation of tissue engineering treatment strategies.

Tissue engineering approaches to spinal cord injury (SCI) treatment are attractive because they allow for manipulation of native regeneration processes involved in restoration of the integrity and function of damaged tissue. A clinically relevant spinal cord regeneration animal model requires that the model mimics specific pathologic processes that occur in human SCI. This manuscript discusses issues related to preclinical testing of tissue engineering spinal cord regeneration strategies from a number of perspectives. This discussion includes diverse causes, pathology and functional consequences of human SCI, general and species related considerations, technical and animal care considerations, and data analysis methods.

Inflammation and neuropathic attacks in hereditary brachial plexus neuropathy.

OBJECTIVE: To study the role of mechanical, infectious, and inflammatory factors inducing neuropathic attacks in hereditary brachial plexus neuropathy (HBPN), an autosomal dominant disorder characterised by attacks of pain and weakness, atrophy, and sensory alterations of the shoulder girdle and upper limb muscles. METHODS: Four patients from separate kindreds with HBPN were evaluated. Upper extremity nerve biopsies were obtained during attacks from a person of each kindred. In situ hybridisation for common viruses in nerve tissue and genetic testing for a hereditary tendency to pressure palsies (HNPP; tomaculous neuropathy) were undertaken. Two patients treated with intravenous methyl prednisolone had serial clinical and electrophysiological examinations. One patient was followed prospectively through pregnancy and during the development of a stereotypic attack after elective caesarean delivery. RESULTS: Upper extremity nerve biopsies in two patients showed prominent perivascular inflammatory infiltrates with vessel wall disruption. Nerve in situ hybridisation for viruses was negative. There were no tomaculous nerve changes. In two patients intravenous methyl prednisolone ameliorated symptoms (largely pain), but with tapering of steroid dose, signs and symptoms worsened. Elective caesarean delivery did not prevent a typical postpartum attack. CONCLUSIONS: Inflammation, probably immune, appears pathogenic for some if not all attacks of HBPN. Immune modulation may be useful in preventing or reducing the neuropathic attacks, although controlled trials are needed to establish efficacy, as correction of the mutant gene is still not possible. The genes involved in immune regulation may be candidates for causing HBPN disorders.

Alterations in cell cycle regulation underlie cisplatin induced apoptosis of dorsal root ganglion neurons in vivo.

Cisplatin is used in the treatment of ovarian and testicular cancer. Twenty percent of patients cannot be optimally treated because of sensory neurotoxicity. Human and animal studies demonstrate that the dorsal root ganglion neuron is the primary target of drug injury. We have previously demonstrated that cisplatin causes neuronal apoptosis in vitro. We now report a reproducible animal model of cell death induced by cisplatin. Drug was administered for 1 or 2 cycles of 5 days separated by 5 days. Total dose administered was 0, 5, 7.5, 10, or 15 mg/kg. Ganglia from 34 animals were processed and examined using in situ hybridization for cyclin D1 messenger RNA and digoxigenin coupled TUNEL staining. Overall, 2.9 +/- 3.9% of neurons were TUNEL positive in treated rats compared with 0.2 +/- 0.3% in controls (P <.005). There was a strong positive correlation (r2 = 0.88; P = 0.018) between percentage of TUNEL stained DRG and cumulative dose of cisplatin. Two independent approaches to quantitation of in situ cyclin D1 hybridization were used; blinded grading by an observer and measurement of color density using digital image analysis. Both demonstrated dramatic upregulation of expression of cyclin D1 mRNA in treated compared with control rats. This demonstrates that apoptosis of neurons is preceded by aberrant reentry into G1 phase of the cell cycle in an animal model.

Suramin-induced neuropathy in an animal model.

Suramin is being used either alone, or in combination with other chemotherapeutic agents, in the treatment of hormone-refractory or metastatic prostate cancer. Use of this potentially valuable chemotherapy is limited by a dose-dependent polyneuropathy. It has been difficult in human studies to characterize peripheral suramin toxicity separately from cancer-related neuropathy. To characterize suramin-induced neuropathy in a rat model, adult rats were given either a single dose of 500 mg/kg (high dose) or 50 mg/kg (low dose) weekly suramin for 2 months. Electrophysiology and peroneal/sural nerve morphometry were performed. In high dose animals, neuropathy developed within 2 weeks, most severe in the digital sensory responses (DSR) (p<0.05) and tail and hind limb compound muscle action potential (p<0.001). Histologically, there was evidence of axonal degeneration and axon atrophy. With low dose suramin, the DSR (p<0.05) and tail distal sensory and motor responses (p<0.01) were most severely affected at 2 months. Axonal degeneration was seen in teased fibers from most animals. With TEM, there were abundant characteristic lysosomal inclusion bodies in DRG and Schwann cells. Electrophysiological and histological evidence of peripheral demyelination was rare, being observed in only one animal. Suramin induced a length, dose and time-dependent axonal sensorimotor polyneuropathy associated with axonal degeneration, atrophy, and accumulation of glycolipid lysosomal inclusions.

Nerve growth factor rescue of cisplatin neurotoxicity is mediated through the high affinity receptor: studies in PC12 cells and p75 null mouse dorsal root ganglia.

Nerve growth factor (NGF) rescues dorsal root ganglion neurons and PC12 cells from cisplatin-induced cell death. Two model systems were used to demonstrate that rescue is mediated through the high affinity NGF receptor. In dorsal root ganglion (DRG) neurons isolated from p75(-/-) and control mice, 20 ng/ml NGF completely prevented cisplatin-induced death. In PC12 cells, we overexpressed receptor chimeras between the tumor necrosis factor and NGF receptors. We demonstrated that activation of the intracellular domain of Trk A is responsible for the NGF rescue effect.

PMP22 transgenic dorsal root ganglia cultures show myelin abnormalities similar to those of human CMT1A.

Charcot-Marie-Tooth 1A (CMT1A) neuropathy is caused by duplication of the peripheral myelin protein 22 (PMP22) gene, leading to protein overexpression. Although this protein has a role in regulating Schwann cell growth and peripheral myelin compaction, how altered concentrations of PMP22 impair myelination is unknown. We established dorsal root ganglia (DRG) cultures from a transgenic rat overexpressing PMP22 (PMP22tg) to study the behavior of PMP22tg Schwann cells in early stages of development and myelination. We used reverse transcriptase-polymerase chain reaction and light and electron microscopy to study PMP22 expression and myelin formation. Myelin ultrastructure was evaluated in sural nerves from CMT1A patients to compare experimental and human findings. PMP22tg DRG cultures contained a greater number of internodes devoid of myelin, in the absence of remyelination, and increased periodicity of myelin lamellae compared with normal cultures. Widening of myelin lamellae was also observed in CMT1A biopsy specimens. Our results suggest that both functions of PMP22, in regulating Schwann cell differentiation and contributing to peripheral myelin compaction, are affected by its overexpression. The presence of similar myelin abnormalities in PMP22tg cultures and human nerves emphasizes the importance of developing in vitro models of hereditary neuropathies to study their underlying pathomechanisms.

Role of the extracellular matrix in myelination of peripheral nerve.

Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21-24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myelination occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co-cultured without ascorbic acid in the presence or absence of a function blocking anti-beta(1) integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination.

Plasma membrane calcium ATPase plays a role in reducing Ca(2+)-mediated cytotoxicity in PC12 cells.

In many cell types, cell death induced by a variety of insults is accompanied by an increase in intracellular calcium. The Ca(2+) homeostatic mechanisms affected by such insults, however, have not been fully determined. Recent evidence indicates that kainic acid-induced seizures alter plasma membrane calcium ATPase mRNA expression within vulnerable hippocampal cell populations before the onset of cell death. We examined the effects of altering plasma membrane calcium ATPase expression on cell vulnerability in rat pheochromocytoma 12 cells. Pheochromocytoma 12 cells are vulnerable to Ca(2+) overload induced by the Ca(2+) ionophore A23187. Reverse transcriptase-PCR and Western blot data indicated that plasma membrane calcium ATPase isoform 4b constitutes a major calcium pump isoform in the pheochromocytoma 12 cells. Therefore, permanently transfected pheochromocytoma 12-derived cell lines were established that either over-expressed plasma membrane calcium ATPase isoform 4b, or suppressed the expression of the endogenous plasma membrane calcium ATPase isoform 4. Over-expressing clones were less vulnerable to Ca(2+)-mediated cell death induced by A23187 whereas "antisense" clones were considerably more susceptible. These data indicate that regulation of plasma membrane calcium ATPase expression may be critical to cellular survival when cells are exposed to pathological increases in intracellular calcium.

Evidence for genetic heterogeneity in hereditary neuralgic amyotrophy.

Hereditary neuralgic amyotrophy (HNA) is a rare autosomal dominant disorder characterized by recurrent episodes of severe arm and shoulder pain with weakness, atrophy, and sensory impairment in a brachial plexus distribution. Recent studies mapped the HNA locus to chromosome 17q25. Two pedigrees with clinically typical HNA in which markers from chromosome 17q25 do not cosegregate with the disease and in which lod scores do not support linkage to chromosome 17q25 were identified.

MSP, a trypsin-like serine protease, is abundantly expressed in the human nervous system.

The goal of the present investigation was to determine the regional and cellular specific expression patterns of the newly identified serine protease, myelencephalon-specific protease (MSP), in the adult human brain (Scarisbrick et al. [1997b] J. Neurosci. 17:8156-8168). To assess the potential scope of MSP activity, Northern blot techniques were used to determine the relative abundance of MSP mRNA in 16 different adult human brain regions, and in the brain and peripheral tissues of the midgestation human fetus. The regional and temporal specific expression patterns of MSP mRNA were directly compared with those of tissue plasminogen activator (tPA), a serine protease strongly implicated in the development, ongoing plasticity, and response of the nervous system to injury and disease. mRNA encoding each protease was distributed widely throughout the normal adult human central nervous system (CNS), but the expression of each was only partially overlapping. Additionally, compared with tPA, MSP exhibited a more restricted distribution and delayed developmental onset. By immunohistochemical localization, MSP was present at moderate to high levels in neurons and oligodendroglia of the adult human brain, at a level closely resembling the relative abundance indicated by Northern blot. MSP was most abundantly expressed in the spinal cord, hippocampus, substantia nigra, and basal ganglia. The robust expression of MSP in clinically significant regions of the adult human CNS indicates that further study of this protease in terms of both normal brain physiology and neurodegenerative disorders is warranted.

IV immunoglobulin does not reverse established weakness in MS.

BACKGROUND: Immunoglobulin (Ig) administration induces remyelination in the Theiler's virus model of MS. METHODS: A randomized, double-blinded, placebo-controlled trial of IV immunoglobulin (IVIg) was performed in patients with MS who had persistent muscle weakness that had been stable for between 4 and 18 months to determine whether this would improve muscle strength (primary outcome: isometric muscle strength). Patients received either IVIg (0.4 g/kg) or placebo daily for 5 days, then single infusions every 2 weeks for 3 months (total, 11 infusions). Muscle groups identified by clinical measures to have unchanging significant weakness were the major targets for therapeutic response (targeted neurologic deficit [TND]). RESULTS: IVIg was well tolerated. An interim analysis after 67 patients were enrolled indicated no difference in the degree of change in strength between treatment groups in either the TND or non-TND muscle groups at 6 months, and the trial was terminated. There was no apparent benefit in relapse behavior or impairment measures during the 6-month observation period. Nor was there apparent benefit in either patients who remained clinically stable or in those with evidence of disease activity. Patients with active MS during the trial worsened in both TND and non-TND muscle groups. This worsening was seen regardless of whether the clinical manifestations of disease activity involved the TND muscle groups. CONCLUSIONS: IVIg does not reverse established weakness in MS. Measurements of isometric muscle strength were reliable (reproducible) indices of strength and may be sensitive, objective methods to document functional changes in impairment in future MS trials.

Ceramide initiates NFkappaB-mediated caspase activation in neuronal apoptosis.

The objective of the present study was to evaluate the role of ceramide in mediating apoptosis of dorsal root ganglion neurons induced by either nerve growth factor withdrawal or treatment with the chemotherapeutic agents suramin and cisplatin. Measurement of ceramide accumulation by mass spectrometry and the diacylglycerol kinase assay revealed elevation of intracellular ceramide only in suramin treated cultures. Ceramide-mediated neuronal cell death was inhibited by the caspase inhibitor zVAD.fmk. In these experimental models, ceramide accumulation mediated activation and nuclear translocation of the transcription factor NFkappaB and cyclin D1 protein expression. Specific inhibition of NFkappaB using a molecular decoy strategy resulted in increased cell viability accompanied by diminished caspase activity and cyclin D1 expression. Inhibition of NFkappaB did not alter intracellular ceramide levels. Our study suggests that ceramide generation occurs upstream of NFkappaB activation, cell cycle reentry, and caspase activation in the neuronal death pathway.

Mechanisms of neurotoxic injury and cell death.

Neurotoxic injury to the nervous system produces neuronal death or distal axonal degeneration. Neurotoxin-induced demyelination is relatively rare in the peripheral and central nervous systems. Major advances have occurred in our understanding of the mechanisms of apoptotic cell death. The pathways leading to apoptosis offer many new approaches to neuroprotection.

Preferential expression of myelencephalon-specific protease by oligodendrocytes of the adult rat spinal cord white matter.

Myelencephalon-specific protease (MSP) is a novel serine protease that is expressed predominantly in the nervous system. In the adult rat spinal cord, MSP mRNA expression was dramatically upregulated, in both the white and gray matter, after systemic exposure to the glutamate receptor agonist, kainic acid (KA) (Scarisbrick et al. J Neurosci 17: 8156-8168, 1997b). To determine the cell-specific expression patterns of MSP, we generated MSP-specific monoclonal antibodies. These have been used in immunohistochemical and in situ hybridization colocalization studies, to demonstrate that MSP mRNA and protein are produced predominantly by CNP-immunoreactive oligodendroglia, but not by GFAP-immunoreactive astrocytes, in the white matter of the normal adult cord. In vitro, the soma of oligodendrocytes were also densely MSP immunoreactive, as were their growth tips, while astrocytes were associated with lower levels. These findings suggest that the enzymatic activity of MSP is likely to be important in the biology of oligodendrocytes and/or in the maintenance of the nerve fiber tracts of the adult spinal cord.

Cloning and expression of glycolipid transfer protein from bovine and porcine brain.

Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.

Chemotherapeutic neuropathy.

Peripheral neurotoxicity is a dose-limiting side-effect for a number of effective chemotherapeutic agents, including platinum compounds, taxanes, and vinca alkaloids. New experimental chemotherapy drugs that cause neuropathy include suramin and Dolostatin-10. A better understanding of cellular mechanisms will lead to novel treatment strategies that will protect neurons without decreasing therapeutic efficacy.