Anne E. Luebke

Systemic 5-fluorouracil treatment causes a syndrome of delayed myelin destruction in the central nervous system

Background Cancer treatment with a variety of chemotherapeutic agents often is associated with delayed adverse neurological consequences. Despite their clinical importance, almost nothing is known about the basis for such effects. It is not even known whether the occurrence of delayed adverse effects requires exposure to multiple chemotherapeutic agents, the presence of both chemotherapeutic agents and the bodys own response to cancer, prolonged damage to the blood-brain barrier, inflammation or other such changes. Nor are there any animal models that could enable the study of this important problem. Results We found that clinically relevant concentrations of 5-fluorouracil (5-FU; a widely used chemotherapeutic agent) were toxic for both central nervous system (CNS) progenitor cells and non-dividing oligodendrocytes in vitro and in vivo. Short-term systemic administration of 5-FU caused both acute CNS damage and a syndrome of progressively worsening delayed damage to myelinated tracts of the CNS associated with altered transcriptional regulation in oligodendrocytes and extensive myelin pathology. Functional analysis also provided the first demonstration of delayed effects of chemotherapy on the latency of impulse conduction in the auditory system, offering the possibility of non-invasive analysis of myelin damage associated with cancer treatment. Conclusions Our studies demonstrate that systemic treatment with a single chemotherapeutic agent, 5-FU, is sufficient to cause a syndrome of delayed CNS damage and provide the first animal model of delayed damage to white-matter tracts of individuals treated with systemic chemotherapy. Unlike that caused by local irradiation, the degeneration caused by 5-FU treatment did not correlate with either chronic inflammation or extensive vascular damage and appears to represent a new class of delayed degenerative damage in the CNS.

Identification of a pore lining segment in gap junction hemichannels

The ability of certain connexins to form open hemichannels has been exploited to study the pore structure of gap junction (hemi)channels. Cysteine scanning mutagenesis was applied to cx46 and to a chimeric connexin, cx32E(1)43, which both form patent hemichannels when expressed in Xenopus oocytes. The thiol reagent maleimido-butyryl-biocytin was used to probe 12 cysteine replacement mutants in the first transmembrane segment and two in the amino-terminal segment. Maleimido-butyryl-biocytin was found to inhibit channel activity with cysteines in two equivalent positions in both connexins: I33C and M34C in cx32E(1)43 and I34C and L35C in cx46. These two positions in the first transmembrane segment are thus accessible from the extracellular space and consequently appear to contribute to the pore lining. The data also suggest that the pore structure is complex and may involve more than one transmembrane segment.

Efferent Protection from Acoustic Injury Is Mediated via α9 Nicotinic Acetylcholine Receptors on Outer Hair Cells

Exposure to intense sound can damage the mechanosensors of the inner ear and their afferent innervation. These neurosensory elements are innervated by a sound-activated feedback pathway, the olivocochlear efferent system. One major component of this system is cholinergic, and known cholinergic effects are mediated by the α9/α10 nicotinic acetylcholine receptor (nAChR) complex. Here, we show that overexpression of α9 nAChR in the outer hair cells of bacterial artificial chromosome transgenic mice significantly reduces acoustic injury from exposures causing either temporary or permanent damage, without changing pre-exposure cochlear sensitivity to low- or moderate-level sound. These data demonstrate that efferent protection is mediated via the α9 nAChR in the outer hair cells and provide direct evidence for a protective role, in vivo, of a member of the nAChR family.

Cochlear Function and Transgene Expression in the Guinea Pig Cochlea, Using Adenovirus- and Adeno-Associated Virus-Directed Gene Transfer

Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10(8) IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10(8) PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1(-), E3(-)) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1(-), E3(-)) adenovirus vectors.

SMOOTH PURSUIT EYE MOVEMENTS IN SCHIZOPHRENICS: QUANTITATIVE MEASUREMENTS WITH THE SEARCH-COIL TECHNIQUE

Eye movements of five schizophrenic and five normal subjects were measured with the magnetic-field search-coil technique. Subjects followed targets moving smoothly at various speeds, either unpredictably in a step-ramp fashion or predictably in a triangular wave. The tracking stimulus was either a small dot or a large, richly-textured image that occupied a large portion of the visual field. Tracking by schizophrenics was abnormal; it was punctuated by catch-up saccades that corrected for smooth following movements of inadequate velocity. We did not, however, find saccadic intrusions, such as square wave jerks. Under all tracking conditions steady-state gains (eye velocity/target velocity) and, in the case of step-ramps, average acceleration in the first 120 ms were lower in patients than in normal subjects. The differences were most pronounced for tracking of the small target, moving at the highest speed tested (30 degree/s), in the nonpredictable, step-ramp waveform. With this stimulus mean steady-state gain was 0.36 (SD +/- 0.12) for the schizophrenic patients and 0.73 (SD +/- 0.11) for the normal subjects. When the target was changed to the large-field stimulus or moved in a predictable (triangular-wave) fashion, tracking improved in both patients and normal subjects, and even more so when these features were combined.

A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function

The loss of cochlear hair cells, or the loss of their capacity to transduce acoustic signals, is believed to be the underlying mechanism in many forms of hearing loss. To develop viral vectors that allow for the introduction of genes directly into the cochleae of adult animals, replication-deficient (E1−, E3−) and replication-defective (E1−, E3−, pol−) adenovirus vectors were used to transduce the bacterial β-galactosidase gene into the hair cells of the guinea pig cochlea in vivo. Distortion product otoacoustic emissions, which monitor the functional status of outer hair cells, were measured throughout the viral infection periods to identify hair cell ototoxicity. The results demonstrated that the use of the (E1−, E3−) adenovirus vectors containing CMV-driven LacZ, compromised cochlear function when gradually introduced into scala tympani via an osmotic pump. However, when (E1−, E3−, pol−) adenoviral vectors containing CMV-driven LacZ were used to transduce cochlear hair cells, there was no loss of cochlear function over the frequency regions tested, and β-galactosidase (β-gal) was detected in over 80% of all hair cells. Development of a viral vector that infects cochlear hair cells without virus-induced ototoxic effects is crucial for gene replacement strategies to treat certain forms of inherited deafness and for otoprotective strategies to prevent hair cell losses to treat progressive hearing disorders. Moreover, in vivo (E1−, E3−, pol−) adenovirus mediated gene-transfer techniques applied to adult guinea pig cochleae may be useful in testing several hypotheses concerning what roles specific genes play in normal cochlear function.

Transition dynamics between pursuit and fixation suggest different systems

The offset of smooth pursuit eye movements is very different from the onset. The onset eye velocity is characterized by overshoot and ringing before settling to steady-state velocity. Yet, at pursuit offset, the eye velocity returns smoothly to zero. One reason for this difference may be that the pursuit system is nonlinear and behaves differently during acceleration and deceleration. After testing four subjects, we found no difference between acceleration and deceleration ringing dynamics, except for decelerations to 0 +/- 2 deg/sec, suggesting that around zero velocity, the central nervous system switches off pursuit and adopts fixation. This supports the idea that fixation and pursuit represent different neurological systems.

Gain changes of the cat's vestibulo-ocular reflex after flocculus deactivation

Motor learning can be demonstrated in the vestibulo-ocular reflex (VOR) by changing its gain (eye velocity/head velocity) with goggles and optokinetic (OK) drums. It is known that the flocculus is essential for this plasticity but there is controversy about whether the modifiable synapses mainly responsible are in the flocculus. To investigate this further we utilized the known reciprocal relationship between complex spikes and simple spikes in Purkinje cell discharges. By stimulating climbing fibers from the olive to the flocculus at 7 Hz, the simple spike rate of almost all recorded floccular cells could be driven to zero. This was termed floccular shutdown and is felt to effect a functional, reversible flocculectomy. Sixty single units in the flocculi of four cats were recorded. Stimulation of the climbing fibers at 7 Hz caused the discharge rate to decrease to zero in 95% of these cells. The gain of the horizontal VOR in three cats was driven repeatedly to twice or half its normal value by rotation within a moving OK drum and also by wearing magnifying or fixed-field goggles; this process required 3 days. If, on the 4th day, the cat was exposed to an OK drum rotating in the opposite direction, the gain was driven back to normal in 30 min. If, however, the climbing fibers were stimulated at 7 Hz during these 30 min, the gain did not return — learning was blocked. This verified that loss of floccular activity by this method abolishes VOR gain plasticity. Moreover, when 7 Hz stimulation first began, after 3 days of adaptation, the adapted gain remained at its adapted value, either half or twice normal, even in the face of floccular shutdown. This result appears incompatible with the hypothesis that the modifiable synapses are in the flocculus.

Lead exposure during development results in increased neurofilament phosphorylation, neuritic beading, and temporal processing deficits within the murine auditory brainstem

Low‐level lead (Pb) exposure is a risk factor for learning disabilities, attention deficit hyperactivity disorder (ADHD), and other neurological dysfunction. It is not known how Pb produces these behavioral deficits, but low‐level exposure during development is associated with auditory temporal processing deficits in an avian model, while hearing thresholds remain normal. Similar auditory processing deficits are found in children with learning disabilities and ADHD. To identify cellular changes underlying this functional deficit, Pb‐induced alterations of neurons and glia within the mammalian auditory brainstem nuclei were quantified in control and Pb‐exposed mice at postnatal day 21 by using immunohistochemistry, Western blotting, and 2D gel electrophoresis. Pb‐treated mice were exposed to either 0.1 mM (low) or 2 mM (high) Pb acetate throughout gestation and through 21 days postnatally. Pb exposure results in little change in glial proteins such as glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), or F4/80 as determined by Western blot analysis and immunohistochemistry. In contrast, Pb exposure alters neuronal structural proteins by inducing increased phosphorylation of both the medium (NFM) and high‐weight (NFH) forms of neurofilament within auditory brainstem nuclei. Axons immunolabeled for neurofilament protein show neuritic beading following Pb exposure both in vivo and in vitro, suggesting that Pb exposure also impairs axonal transport. Functional assessment shows no significant loss of peripheral function, but does reveal impairments in brainstem conduction time and temporal processing within the brainstem. These results provide evidence that Pb exposure during development alters axonal structure and function within brainstem auditory nuclei. J. Comp. Neurol. 506:1003–1017, 2008.

Identifying a Window of Vulnerability during Fetal Development in a Maternal Iron Restriction Model

It is well acknowledged from observations in humans that iron deficiency during pregnancy can be associated with a number of developmental problems in the newborn and developing child. Due to the obvious limitations of human studies, the stage during gestation at which maternal iron deficiency causes an apparent impairment in the offspring remains elusive. In order to begin to understand the time window(s) during pregnancy that is/are especially susceptible to suboptimal iron levels, which may result in negative effects on the development of the fetus, we developed a rat model in which we were able to manipulate and monitor the dietary iron intake during specific stages of pregnancy and analyzed the developing fetuses. We established four different dietary-feeding protocols that were designed to render the fetuses iron deficient at different gestational stages. Based on a functional analysis that employed Auditory Brainstem Response measurements, we found that maternal iron restriction initiated prior to conception and during the first trimester were associated with profound changes in the developing fetus compared to iron restriction initiated later in pregnancy. We also showed that the presence of iron deficiency anemia, low body weight, and changes in core body temperature were not defining factors in the establishment of neural impairment in the rodent offspring. Our data may have significant relevance for understanding the impact of suboptimal iron levels during pregnancy not only on the mother but also on the developing fetus and hence might lead to a more informed timing of iron supplementation during pregnancy.