Deborah A. Parks

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L‐kynurenine (KYN) and by determining the irreversible conversion of [3H] KYN to [3H] KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2‐oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT‐rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate‐specific. Thus, of the amino acids tested, only α‐aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3‐hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 μM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 μMfor KYN with 1 mM 2‐oxoglutarate, but a much higher Km(910 μM) with 1 mM pyruvate. Km values for 2‐oxoglutarate and pyruvate were 150 and 160 μM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate. At that time of almost complete neuronal destruction and pronounced astrocytic proliferation, enzyme activity in the lesioned striatum was almost twice as high as in controls, suggesting a preferential astroglial localization of brain KAT. The characteristics of KAT described here are compatible with a central role of the enzyme in brain KYNA function in vivo. Abnormal KAT activity, therefore, should be considered as an etiological factor in pathological phenomena related to dysfunction of excitatory amino acid receptors in the brain.

α-Amino-ω-phosphono carboxylates block ibotenate but not kainate neurotoxicity in rat hippocampus

Abstract (−) 2-Amino-7-phosphonoheptanoate and (−) 2-amino-5-phosphonovalerate were shown to possess selective and powerful antagonistic activity vis-a-vis the neurotoxic effects of ibotenic acid in the rat hippocampal formation. The neurotoxicity of kainic acid was not blocked by either drug.

Tetanus toxin fragment C as a vector to enhance delivery of proteins to the CNS

The non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C, TTC) has been used as a vector to enhance delivery of potentially therapeutic proteins to motor neurons from the periphery following an intramuscular injection. The unique binding and transport properties of this 50-kDa polypeptide suggest that it might also enhance delivery of proteins to neurons after direct injection into the CNS. Using quantitative fluorimetry, we found that labeled TTC showed vastly superior retention within brain tissue after intracerebral injection compared to a control protein (bovine serum album). Fluorescence microscopy revealed that injected TTC was not retained solely in a restricted deposit along the needle track, but was distributed through gray matter in a pattern not previously described. The distribution of injected protein within the extracellular space of the gray matter and neuropil was also seen after injection of a recombinant fusion protein comprised of TTC linked to the enzyme superoxide dismutase (TTC-SOD-1). Injections of native SOD-1 in contrast showed only minimal retention of protein along the injection track. Immunohistochemistry demonstrated that both TTC and TTC-SOD-1 were distributed in a punctate perineuronal and intraneuronal pattern similar to that seen after their retrograde transport, suggesting localization primarily in synaptic boutons. This synaptic distribution was confirmed using HRP-labeled TTC with electron microscopy along with localization within neuronal endosomes. We conclude that TTC may be a useful vector to enhance neuronal delivery of potentially therapeutic enzymes or trophic factors following direct injection into the brain.

A multi-domain protein system based on the HC fragment of tetanus toxin for targeting DNA to neuronal cells.

One goal of gene therapy is the targeted delivery of therapeutic genes to defined tissues. One attractive target is the central nervous system as there are several neuronal degenerative diseases which may be amenable to gene therapy. At present there is a lack of delivery systems that are able to target genes specifically to neuronal cells. Multi-domain proteins were designed and constructed to facilitate the delivery of exogenous genes to neuronal cells. Neuronal targeting activity of the proteins was achieved by inclusion of the HC fragment of tetanus toxin (TeNT), a protein with well-characterised tropism for the central nervous system. The yeast Gal4 DNA-binding domain enabled specific binding of DNA while the translocation domain from diphtheria toxin (DT) was included to facilitate crossing of the endosomal vesicle. One multi-domain protein, containing all three of these domains, was found to transfect up to 8% of neuroblastoma N18-RE105 cells with marker genes. Monitoring the transfection by confocal microscopy indicated that this protein-DNA transfection complex is to some extent localised at the cell surface, suggesting that further improvements to translocating this membrane barrier may yield higher transfection levels. The demonstration that this multi-domain protein can target genes specifically to neuronal cells is a first step in the development of novel vectors for the delivery of genes with therapeutic potential to diseased neuronal tissues.

Effect of proximal axotomy on GAP-43 expression in cortical neurons in the mouse

As an approach to understanding why central neurons fail to regenerate, we have studied the response to proximal axotomy of transcallosal neurons of the cerebral cortex of the mouse. Anatomical studies have indicated only very slight regenerative responses by this population of cortical neurons. To further examine the regenerative response of these cells, we have looked by in situ hybridization at the expression of GAP-43 mRNA following axotomy caused by a stab wound delivered within about 200 microm to 1.25 mm of the cell body. Axotomized transcallosal neurons were compared with near-by unaxotomized transcallosal neurons, as well as with distant unaxotomized cortical neurons in the contralateral hemisphere. All three populations of neurons had been pre-labeled with Fluoro-Gold to allow identification. No up-regulation of GAP-43 mRNA above background levels was detected for axotomized cortical neurons at 1, 3 or 7 days after injury. In contrast, increases in mean silver grain density of up to 8-fold were measured in axotomized spinal cord motor neurons used as positive controls. Thus, as a population, the transcallosal cortical pyramidal neurons did not show a significant regenerative response, as monitored by GAP-43 upregulation, even with very close axotomy. These results identify this population of neurons as among the least regenerative studied, and suggest that, on a molecular level, inherent neuronal properties play a role in the limited regenerative response to brain injury.

Neuronal binding of tetanus toxin compared to its ganglioside binding fragment (Hc)

The non-toxin 50 kD C-terminus peptide of the heavy chain of tetanus H(c) contains the ganglioside binding domain of tetanus toxin (TTX). H(c) retains much of the capacity of tetanus toxin for binding internalization and transport by neurons. For this reason tetanus H(c) has been studied as a vector for delivery of therapeutic proteins to neurons. We directly compared H(c) and TTX in the capacity to bind and be internalized by neurons by ELISA. Primary cultures of dissociated fetal cortical neurons were incubated with equimolar amounts of TTX or H(c). Neuronal associated tetanus protein was 4-8 fold greater on a molar basis with tetanus toxin compared to H(c) (1 h incubation). This increase in neuronal tetanus protein was evident with incubation in concentrations from 0.1 microM to 2 microM. There were greater amounts of TTX delivered to the cultured cells at both 0 degrees C (representing membrane bound tetanus protein) and 37 degrees C (bound and internalized tetanus protein). Unlike H(c), TTX showed significant continued accumulation of protein with increasing incubation durations. Neuronal associated TTX increased 2-3 fold over incubation times ranging from 1 to 8 h. Tetanus toxin appears to be clearly superior to the ganglioside binding fragment (H(c)) in the capacity for neuronal binding and internalization. Atoxic tetanus proteins containing additional molecular domains as well as H(c) may be more suitable vectors for linkage with therapeutic proteins and delivery to neurons.

Death of transcallosal neurons after close axotomy

The extent of cell death after axotomy may limit potential recovery after brain injury. We wished to determine the effect of axotomizing lesions on survival of transcallosally projecting cortical neurons. Transcallosal neurons were prelabeled by retrograde transport of the fluorescent dyes Fluoro-Gold and True Blue. A transcortical stab wound divided the field of labeled cortical cells into axotomized and unaxotomized groups. Little difference in labeled cell density was seen over the first few days after injury. Animals surviving at least 2 weeks after injury had clear loss of axotomized neurons. By 1 month after injury, the vast majority of axotomized labeled cells appeared to have died. Quantitative evaluation of labeled cells showed that the region of cortex within 1 mm of the axotomizing injury had less than 10% of the expected neuronal density in animals surviving at least 4 weeks after injury. Close axotomy appears to cause dramatic loss of transcallosal neurons even in adult animals.

Immunization does not interfere with uptake and transport by motor neurons of the binding fragment of tetanus toxin

The nontoxic binding domain of tetanus toxin (fragment C or TTC) readily undergoes retrograde axonal transport from an intramuscular injection site. This property has led to investigation of TTC as a possible vector for delivering therapeutic proteins to motor neurons. However, the vast majority of individuals in the developed world have been vaccinated with tetanus toxoid and have circulating antitetanus antibodies that cross‐react with TTC and may block the delivery of a TTC‐linked therapeutic protein. However, it is uncertain whether the immune response is capable of completely neutralizing an intramuscular depot of protein prior to its internalization by presynaptic nerve terminals, where it is inaccessible to antibody. We have evaluated uptake of rhodamine‐labeled TTC following intramuscular injection in normal animals and animals vaccinated with tetanus toxoid prior to injection of fluorescently labeled TTC. All animals demonstrated uptake of TTC, with fluorescence appropriately localized to the hypoglossal nerve and nucleus. The distribution and intensity of fluorescence within neurons and processes were indistinguishable between the two groups and were characteristic of TTC. Vaccinated animals showed levels of uptake of TTC into the brain comparable to those of immunologically naïve animals as measured by quantitative fluorimetry. All vaccinated animals had protective levels of antitetanus antibodies as measured by ELISA. Uptake of TTC by nerve terminals from an intramuscular depot is an avid and rapid process and is not blocked by vaccination associated with protection from tetanus toxin.

Effects of intrahypothalamic injection of quinolinic acid on anterior pituitary hormone secretion in the unanesthetized rat.

Bilateral intrahypothalamic injections of the brain metabolite quinolinic acid (QUIN) were made in an attempt to examine its effects on the secretion of LH, PRL, GH and TSH. Quin, a neuroexcitatory amino acid with close structural similarities to glutamate, kainate and N-methylaspartate, was infused into unanesthetized male rats, the animals sacrificed 7.5 min later, and serum hormone concentrations determined by radioimmunoassay. QUIN caused surges in LH, PRL and GH release (316, 607 and 1,134% of control, respectively, at 50 micrograms QUIN) without affecting the serum concentrations of TSH. At lower doses, a preferential effect of QUIN on PRL release was observed. All QUIN-induced hormonal changes were inhibited by concomitant administration of the specific antagonist (-)-2-amino-7-phosphonoheptanoic acid, indicating the presence of QUIN-sensitive receptors on neurons which are intimately associated with endocrine regulation. Moreover, because QUIN-treated animals exhibited behavioral signs of seizure activity and neuroendocrine dysfunction has been reported to occur in human convulsive disorders, the data are also of interest in view of a possible mechanistic link between epileptic phenomena and hormone secretion.

Localized tetanus in immunized mice

The capacity of tetanus toxin to enhance motor neuron excitability has suggested its potential use as a therapeutic. Widespread active vaccination against tetanus in all developed countries is considered the major obstacle to clinical use of the toxin. We wished to determine the response to localized intramuscular injection of tetanus toxin in both passively and actively immunized animals as an initial exploration into the possible use of tetanus toxin as a clinical therapeutic. Unvaccinated mice (n=18) underwent intramuscular injection of tetanus toxin into the gastrocnemius muscle (0.2 ng, 1 ng, 5 ng). All animals in the lowest dose group developed only local tetanus of the injected limb of at least 2 weeks duration, while all animals in the higher dose groups also rapidly developed generalized tetanus and were euthanized. Another group of mice (n=20) received anti-tetanus immunoglobulin (20-40 IU) at the time of toxin injection. These animals although dramatically resistant to the toxin developed predominantly local tetanus for over one month at doses of 2.5 microg and 5.0 microg. A third group of mice (n=30) underwent active vaccination with tetanus toxoid to induce protective anti-tetanus immunity and then was challenged with high dose toxin injection (5 ng, 50 ng, 0.5 microg, 1.25 microg, 2.5 microg, or 5 microg). All animals developed local tetanus in the injected limb at a dose of at least 0.5 microg. The severity and duration of local tetanus was generally related to dose, but was more variable in the actively vaccinated group than in the naive or passively immunized animals. Response to the toxin over the first few days was predictive of both the duration and maximal severity of the motor response. Although vaccination dramatically increases resistance to tetanus toxin, by virtue of its extremely high potency, the toxin can produce prolonged localized tetanus even in vaccinated animals with relatively small amounts of protein. These results suggest the possible use of tetanus toxin to enhance local motor activity in a variety of neurologic conditions even in immunized humans. This study in uniformly vaccinated animals also illustrates the potential difficulties in determining an appropriate dose of toxin in a human population with variable degrees of immunity.