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.

Cisplatin-induced apoptosis in rat dorsal root ganglion neurons is associated with attempted entry into the cell cycle.

Platinum compounds induce apoptosis in malignant cells and are used extensively in the treatment of cancer. Total dose is limited by development of a sensory neuropathy. We now demonstrate that when rats are administered cisplatin (2 mg/kg i.p. for 5 d), primary sensory neurons in the dorsal root ganglion die by apoptosis. This was reproduced by exposure of dorsal root ganglion neurons and PC12 cells to cisplatin (3 microg/ml) in vitro. Apoptosis was confirmed by electron microscopy, DNA laddering, and inhibition by the caspase inhibitor z-VAD.fmk (100 microM). Cell death in vitro was preceded by upregulation of cyclin D1, cdk4, and increased phosphorylation of retinoblastoma protein; all are indicators of cell cycle advancement. The level of p16(INK4a), an endogenous inhibitor of the cyclin D1/cdk4 complex decreased. Exposure of PC12 cells and dorsal root ganglion neurons to increased levels of nerve growth factor (100 ng/ ml) prevented both apoptosis and upregulation of the cell cycle markers. Cancer cells without nerve growth factor receptors (gp140TrkA) were not protected by the neurotrophin. This indicated that cisplatin may kill cancer cells and neurons by a similar mechanism. In postmitotic neurons, this involves an attempt to re-enter the cell cycle resulting in apoptosis which is specifically prevented by nerve growth factor.

Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the adult rat spinal cord: regulation by the glutamate receptor agonist kainic acid.

Previous in vitro studies indicate that select members of the neurotrophin gene family, namely brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), contribute to survival and differentiation of spinal cord motoneurons. To investigate the potential roles of these factors in the adult spinal cord, we examined their cellular localization and regulation after systemic exposure to an excitotoxic stimulus, kainic acid (KA). Of the neurotrophins examined, NT-4/5 mRNA was most robustly expressed in the lumbosacral spinal cord of the normal adult rat, including expression by neurons throughout the gray matter, and in a subpopulation of white and gray matter glia. Both BDNF and NT-3 mRNAs were also densely expressed by alpha motoneurons of lamina IX, but were detected at lower levels elsewhere in the gray matter. NT-3 mRNA was additionally expressed by spinal cord glia, but was less widespread compared to NT-4/5. In response to systemic administration of KA, NT-4/5 and BDNF mRNAs were dramatically upregulated in a spatially and temporally restricted fashion, whereas levels of NT-3 mRNA were unchanged. These results provide strong in vivo evidence to support the idea that BDNF, NT-3, and in particular, NT-4/5, play a role in the normal function of the adult spinal cord. Furthermore, our results indicate that the actions of BDNF and NT-4/5 participate in the response of the cord to excitotoxic stimuli, and that those of NT-4/5 and NT-3 include both neurons and glia.

Suramin induced ceramide accumulation leads to apoptotic cell death in dorsal root ganglion neurons.

Suramin is an experimental antineoplastic agent that is currently being tested in clinical trials for a number of human cancers. In previous clinical trials, it has been noted that a significant percentage of patients treated with suramin develop a peripheral neuropathy. Both the cytotoxic (chemotherapeutic) and neurotoxic mechanisms of action of this compound are unknown. Evidence presented in this study suggests that both effects may be due to extensive disruption in glycolipid transport and/or metabolism. Suramin treated dorsal root ganglion cultures revealed an accumulation of the GM1 ganglioside and ceramide. Exposure of cultures to suramin, a cell permeable ceramide analog, or sphingomyelinase lead to apoptotic cell death demonstrated by electron microscopy, bis-benzimide staining and DNA laddering on gel electrophoresis. Furthermore, a significant increase in intracellular ceramide preceded cell death in suramin treated neurons. We propose that suramin induced ceramide accumulation within neurons leads to apoptotic cell death.

Phase I trial of dolastatin-10 (NSC 376128) in patients with advanced solid tumors.

Dolastatin-10 (dola-10) is a potent antimitotic peptide, isolated from the marine mollusk Dolabela auricularia, that inhibits tubulin polymerization. Preclinical studies of dola-10 have demonstrated activity against a variety of murine and human tumors in cell cultures and mice models. The purpose of this Phase I clinical trial was to characterize the maximum tolerated dose, pharmacokinetics, and biological effects of dola-10 in patients with advanced solid tumors. Escalating doses of dola-10 were administered as an i.v. bolus every 21 days, using a modified Fibonacci dose escalation schema. Pharmacokinetic studies were performed with the first treatment cycle. Neurological testing was performed on each patient prior to treatment with dola-10, at 6 weeks and at study termination. Thirty eligible patients received a total of 94 cycles (median, 2 cycles; maximum, 14 cycles) of dola-10 at doses ranging from 65 to 455 microg/m2. Dose-limiting toxicity of granulocytopenia was seen at 455 microg/m2 for minimally pretreated patients (two or fewer prior chemotherapy regimens) and 325 microg/m2 for heavily pretreated patients (more than two prior chemotherapy regimens). Nonhematological toxicity was generally mild. Local irritation at the drug injection site was mild and not dose dependent. Nine patients developed new or increased symptoms of mild peripheral sensory neuropathy that was not dose limiting. This toxicity was more frequent in patients with preexisting peripheral neuropathies. Pharmacokinetic studies demonstrated a rapid drug distribution with a prolonged plasma elimination phase (t 1/2z = 320 min). The area under the concentration-time curve increased in proportion to administered dose, whereas the clearance remained constant over the doses studied. Correlation analysis demonstrated a strong relationship between dola-10 area under the concentration-time curve values and decrease from baseline for leukocyte counts. In conclusion, dola-10 administered every 3 weeks as a peripheral i.v. bolus is well tolerated with dose-limiting toxicity of granulocytopenia. The maximum tolerated dose (and recommended Phase II starting dose) is 400 microg/m2 for patients with minimal prior treatment (two or fewer prior chemotherapy regimens) and 325 microg/m2 for patients who are heavily pretreated (more than two prior chemotherapy regimens).

Relative importance of basement membrane and soluble growth factors in delayed and immediate regeneration of rat sciatic nerve.

Sciatic nerve regeneration was studied in two groups of rats. In group I, nerves were transected and transplanted immediately to the contralateral side. In group II, nerves were transected and transplanted 30 days later to the contralateral side. At 4 weeks, group II had an average nerve action potential amplitude of 784 +/- 292 microV and 43.2% +/- 6.7% of myelinated fibers were > 4 microns in diameter. In comparison, the respective measurements were 94 +/- 35.6 microV (p = 0.05) and 29.5% +/- 1.9% (p = 0.04) in group I. At 8 weeks, there were no significant differences in these measurements between groups. These data suggest that the environment in the distal stump improves early regeneration of nerve fibers when that stump was transected 30 days earlier. These and previous findings suggest that soluble trophic factors may be important in initiation of axonal regeneration.

Characteristics of neurite outgrowth from rat spinal ganglia: effects of serum and segmental level.

The outgrowth of neurites from spinal ganglia in tissue culture may be affected by many variables including medium constituents and the age of ganglia. If E15 rats were used, the rate of outgrowth from ganglion explants in response to nerve growth factor was independent of the cranial-caudal position from which the ganglia were derived. This contrasts with the cranial-to-caudal gradient present during embryonic development. The effect of serum on this outgrowth was also studied and ten of 17 lots of human placental serum, two of three lots of pooled human placental serum, and one of two lots of fetal calf serum significantly inhibited the outgrowth of neurites. This has relevance to the design of tissue culture studies and may be important in the interpretation of in vitro studies of disease mechanisms.

Localization and expression of ciliary neurotrophic factor (CNTF) in postmortem sciatic nerve from patients with motor neuron disease and diabetic neuropathy.

Ciliary neurotrophic factor (CNTF) is thought to play an important role in the maintenance of the mature motor system. The factor is found most abundantly in myelinating Schwann cells in the adult sciatic nerve. Lack of neuronal growth factors has been proposed as one possible etiology of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Growth factor replacement therapies are currently being evaluated as a treatment for motor neuron disease. In this report we determined whether the expression of CNTF in sciatic nerve differed in patients with motor neuron disease compared to controls or patients with another form of axonopathy. We identified 8 patients (7 with ALS and 1 with SMA) with motor neuron disease and 6 patients with diabetic motor neuropathy who had autopsy material available. Immunoperoxidase staining showed reduced CNTF expression in nerves of patients with motor neuron disease but not in patients with diabetic motor neuropathy. Decreased CNTF appears be associated with primary motor neuron disease rather than a generalized process of axon loss. This result supports suggestions that CNTF deficiency may be an important factor in the development of motor neuron disease.

Ultrastructural changes produced in dorsal root ganglia in vitro by exposure to ethylene oxide from hemodialyzers.

Ethylene oxide (EO) gas, used to sterilize hemodialysis equipment, is a known neurotoxin and residual gas remains in dialyzers after airing. If tissue culture medium is incubated in dialyzers and then rat dorsal root ganglion (DRG) cultures are exposed to this medium, characteristic changes occur. These include axonal swellings and the death of neurons after five days. Electron microscope findings were compared between DRG grown in control medium containing 10% calf serum, DRG incubated in the same medium exposed to the blood compartment of capillary flow or plate dialyzers and DRG grown in medium supplemented with 10% dialysis patient serum. Electron microscope findings included frequent axonal swellings with the accumulations of membrane bound vesicles and the apparent disruption of microtubules. There were also conspicuous accumulations of membrane bound vesicles, dense bodies, myeloid figures, and areas of vesicular degeneration in Schwann cells. These changes were seen with dialyzer exposed medium or with medium containing patient serum but not with controls or with medium incubated in plate dialyzers. The changes were similar to those reported in nerve biopsy specimens from patients with known EO neuropathy.

Interstitial hyperosmolarity may cause axis cylinder shrinkage in streptozotocin diabetic nerve.

Maximal conduction velocity values of nerves of diabetic rats 20 weeks after streptozotocin intoxication were found to be intermediate between those of onset-control and those of end-control groups. The abnormality of conduction velocity of the streptozotocin group might therefore be attributed to a failure of maturation. Detailed electron microscopic morphometry of myelinated fibers (MFs) indicates that more than lack of maturation is involved. Whereas the number of lamellae and the perimeter of axis cylinders of myelinated fibers of the three study groups suggested that growth continues, cross-sectional area of the axis cylinders of the streptozotocin group was smaller than those of either control group. Scored evaluation of fiber shape and the measured index of circularity, which related perimeter and transverse axis cylinder area, also indicated that a selective shrinkage of axis cylinders had occurred. This selective alteration in size and shape of axis cylinders is identical to that described after hyperosmolar fixation. Compared with that of controls, the serum of streptozotocin rats is hyperosmolar. It would seem reasonable to attribute the axis cylinder changes to shrinkage. Whether an additional maturational effect is operative as well cannot be resolved from our data.

The endoneurial content of lead related to the onset and severity of segmental demyelination.

The endoneurial lead and water content was serially evaluated in the nerves of rats fed lead carbonate and related to the onset and severity of segmental demyelination and remyelination. Lead began to accumulate significantly in the endoneurium by 5 days, reached a maximum level (71 microgram/g dry weight) by 34 days, and then fell to the perineurial level (28 microgram/g dry weight) by 3 months. The water content of endoneurium did not become significantly increased until the 50th day. Extensive teased fiber grading of pathologic abnormalities carried out on the same animals showed that segmental demyelination began between the 20th and 35th days and worsened progressively. This provides the first evidence that high endoneurial lead concentration precedes segmental demyelination and nerve edema. It suggests that the random Schwann cell damage is more likely to be due to a direct toxic effect of lead rather than to a factor associated with edema or increased endoneurial pressure. Contrary to our expectations, lead content does not parallel water content, as would be expected if lead entry into the endoneurium were associated with an abrupt breakdown of the blood-nerve barrier. A further new finding is the decrease in endoneurial lead content at a time when edema and the pathologic lesions are progressing. This may suggest the development of lead removal mechanisms.

Blood nerve barrier in rat and cellular mechanisms of lead-induced segmental demyelination.

Feeding of lead carbonate to rats causes widespread and reproducible segmental de- and remyelination of myelinated fibers (MFs) of peripheral nerve. Such segmental demyelination might be explained by increased permeability of endoneurial capillaries to serum containing protein-bound lead. The perineurium of control and lead nerves was impermeable to fluorescein-labeled bovine albumin (FBA) and to horseradish peroxidase (HRP). Epineurial capillaries in both conditions allowed HRP to pass freely between and, to a lesser extent, through endothelial cells. Confirming earlier work, endoneurial capillaries of control rats did not appear to allow HRP to pass between endothelial cells, but allowed some to pass by pinocytosis through endothelial cells where it was taken up by macrophages. Contrary to expectation, flooding of the endoneurium with HRP was seen in only 1 of 36 tissue blocks of lead nerves from rats fed 4% lead carbonate for 7 1/2 and 12 weeks. Abundant HRP reaction product was seen in the epineurium in more than half of these tissue blocks. HRP was not generally found in endoneurial fluid, even in lead nerves with marked edema and widespread segmental de- and remyelination. These findings are against a massive breakdown of the blood nerve barrier, so that HRP passes freely into the endoneurium between endoneurial endothelial cells. It was our impression that HRP reaction product was slightly increased in endoneurial endothelial cells and macrophages of lead nerves as compared to control nerves. These studies suggest that there may be an increased transfer of HRP through endoneurial cells in lead neuropathy. The studies do not provide additional evidence that an altered blood nerve barrier is involved in the development of segmental demyelination in lead neuropathy.

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.

Neuropathy associated with hyperlipidemia.

We describe six patients with painful polyneuropathy associated with hyperlipidemia. Each had mild, slowly progressive neuropathy characterized by pain in feet, without proximal extension or involvement of hands. Weakness and autonomic symptoms and signs were absent. Three patients had normal tendon reflexes; three others had decreased ankle reflexes. Serum cholesterol levels were moderately increased; serum triglyceride levels were exceedingly high. In one patient, symptoms resolved with correction of hypertriglyceridemia. No other cause of peripheral neuropathy was found. Marked increases in serum triglycerides may cause painful small-fiber neuropathy.

The sensitivity and specificity of anti-GM1 antibody testing.

Elevated titers of antibodies directed at ganglioside epitopes have been associated with multifocal motor neuropathy (MMN), motor variant of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), amyolrophic lateral sclerosis (ALS), and other motor neuropathies. Anti-GM1 antibodies were measured in 675 patients: 180 age- and sex-stratified healthy blood bank controls, 132 normal controls who had full neurologic assessment including electromyography, 121 patients with definite ALS, 19 patients with pure sensory neuropathy, and 173 consecutive patient serum samples submitted for GM1 antibody testing. Antibodies to three ganglioside epitopes were determined by ELISA: IgM and IgG anti-monosialo GM1, asialo GM1, and disialo GD1b. Antibody titers for normal subjects and patients with ALS were used to determine normal values and borderline levels below which 99% of normal and 99% of ALS patient titers were found. Clinical evaluation of the next 173 consecutive patients referred for anti-GM1 antibody testing revealed 36 patients with motor neuropathies. Sera from 18 of these patients had titers above the 99% normal threshold and 14 had titers above the ALS and normal borderline threshold. All 14 with elevated sera titers were from patients with motor neuropathy or neuronopathy. Sixteen patients met the clinical and electrophysiologic criteria for MMN; 10 had elevated titers. Ten patients had the motor variant of CIDP without conduction block and three had elevated titers. Anti-IgM asialo GM1 antibodies had the highest sensitivity and specificity. High-titer IgM antibodies against monosialo GM1 occurred only in patients with various forms of pure motor neuropathy (100% specificity). The sensitivity was 50% for this referral-based population.

How frequently does classic amyotrophic lateral sclerosis develop in survivors of poliomyelitis?

There is a paucity of reports of classic amyotrophic lateral sclerosis (ALS) developing in survivors of paralytic poliomyelitis. We describe a patient with classic ALS and an antecedent paralytic disease thought to have been poliomyelitis from which she recovered completely. If the paucity of ALS preceded by true poliomyelitis is not merely a matter of underreporting, antecedent paralytic poliomyelitis may have a protective role against the development of ALS. This has implications relevant to pathogenesis and to projected secular trends of ALS incidence since the introduction of poliomyelitis vaccines. There is a need to establish the incidence of cases of classic ALS in patients with antecedent poliomyelitis.

The effect of nerve growth factor, ciliary neurotrophic factor, and ACTH analogs on cisplatin neurotoxicity in vitro.

Cisplatin, used to treat ovarian, bladder, and testicular cancers, causes a sensory dose-limiting neuropathy. Preliminary observations in humans and animals suggest that nerve damage may be prevented by ACTH analogs, particularly those belonging to the melanocortin class, and by nerve growth factor (NGF). We established a rat embryo dorsal root ganglion model to study cisplatin neurotoxicity. The drug reproducibly inhibited axonal growth at concentrations similar to that known to produce toxicity in neurons. The inhibition was prevented in a dose-dependent fashion by simultaneous exposure to alpha-melanocyte stimulating hormone (alpha-MSH) or ACTH but not by excess NGF or ciliary neurotrophic factor (CNTF). The ACTH peptides were not effective in preventing suramin-induced neurotoxicity in the same model. Drug interaction and dose-response studies showed that ACTH and alpha-MSH do not act by potentiation of NGF action. ACTH analogs appear to protect against cisplatin-induced neurotoxicity directly at the cellular level.

Seronegativity for type 1 antineuronal nuclear antibodies ('anti-Hu') in subacute sensory neuronopathy patients without cancer.

We followed 21 patients with sensory neuronopathy without evidence of cancer for up to 23 years. All were seronegative for type 1 antineuronal nuclear antibodies (ANNA-1, also called "anti-Hu"). We additionally studied 67 seropositive patients with sensory neuropathy or a related neurologic syndrome. Ninety-one percent of the seropositive patients had a small-cell lung carcinoma. One, with a normal chest x-ray, had been followed for 7 years for sensory neuronopathy of indeterminate cause before serologic testing for ANNA-1 led to the discovery of the tumor by CT. We conclude that ANNA-1 seropositivity in a patient with sensory neuronopathy is strong evidence for an underlying small-cell lung cancer.

Role of the blink reflex in the evaluation of sensory neuronopathy.

Because of an incidental observation that the blink reflex was normal in paraneoplastic sensory neuronopathy (SN) and frequently abnormal in nonparaneoplastic SN, the authors reviewed the electromyographic records of patients with SN in whom blink reflex studies were performed. The blink reflex was normal in all 17 patients with paraneoplastic SN and abnormal in 20 of 43 patients with nonparaneoplastic SN. Although it does not exclude paraneoplastic SN, an abnormal blink reflex favors a nonparaneoplastic etiology.

Lack of progression of neurologic deficit in survivors of paralytic polio: a 5-year prospective population-based study.

We completed a prospective, population-based cohort study of polio survivors in Olmsted County, Minnesota, between 1986 and 1993. We identified 50 individuals who had had paralytic polio between 1935 and 1960, as representative of all 300 cases of paralytic polio in the county. We completed detailed quantitative clinical and electrophysiologic studies at entry and after 5 years. These studies demonstrated stable neuromuscular function within the cohort, although 60% of the individuals were symptomatic. In two-thirds of the symptomatic patients, the causes of their symptoms were unrelated to earlier polio. For the 20% of patients who had unexplained muscle pain, perception of weakness, and fatigue, a mechanical disorder most likely underlies their symptoms.