Michael J. Kubek

Impaired growth and neurological abnormalities in branched-chain α-keto acid dehydrogenase kinase-deficient mice

The BCKDH (branched-chain alpha-keto acid dehydrogenase complex) catalyses the rate-limiting step in the oxidation of BCAAs (branched-chain amino acids). Activity of the complex is regulated by a specific kinase, BDK (BCKDH kinase), which causes inactivation, and a phosphatase, BDP (BCKDH phosphatase), which causes activation. In the present study, the effect of the disruption of the BDK gene on growth and development of mice was investigated. BCKDH activity was much greater in most tissues of BDK-/- mice. This occurred in part because the E1 component of the complex cannot be phosphorylated due to the absence of BDK and also because greater than normal amounts of the E1 component were present in tissues of BDK-/- mice. Lack of control of BCKDH activity resulted in markedly lower blood and tissue levels of the BCAAs in BDK-/- mice. At 12 weeks of age, BDK-/- mice were 15% smaller than wild-type mice and their fur lacked normal lustre. Brain, muscle and adipose tissue weights were reduced, whereas weights of the liver and kidney were greater. Neurological abnormalities were apparent by hind limb flexion throughout life and epileptic seizures after 6-7 months of age. Inhibition of protein synthesis in the brain due to hyperphosphorylation of eIF2alpha (eukaryotic translation initiation factor 2alpha) might contribute to the neurological abnormalities seen in BDK-/- mice. BDK-/- mice show significant improvement in growth and appearance when fed a high protein diet, suggesting that higher amounts of dietary BCAA can partially compensate for increased oxidation in BDK-/- mice. Disruption of the BDK gene establishes that regulation of BCKDH by phosphorylation is critically important for the regulation of oxidative disposal of BCAAs. The phenotype of the BDK-/- mice demonstrates the importance of tight regulation of oxidative disposal of BCAAs for normal growth and neurological function.

Effects of subconvulsive and repeated electroconvulsive shock on thyrotropin-releasing hormone in rat brain

Male Sprague-Dawley rats were given a single electroconvulsive shock (ECS) on alternate days and sacrificed 48 hrs after 1, 3, or 5 seizures. The content of TRH in hippocampus, pyriform cortex and amygdala was increased 2.5-fold, 5.4-fold and 4.3-fold respectively, 48 hrs. after 3 alternate-day electroconvulsive shocks (ECS) and remained unchanged after 2 additional shocks. Pyriform cortex exhibited a significant intermediate increase (1.7-fold) after only 1 ECS. In a second study, rats were sacrificed 48 hrs after a series of 5 alternate-day ECS vs. subconvulsive shocks (SCS). SCS had no significant effect in these same regions, but was seen to alter TRH in striatum. These results provide an interesting parallel to several aspects of clinical electroconvulsive treatment (ECT) of depression. Together with other findings, these data suggest also, that endogenous TRH may play a role in the modulation of convulsive seizures.

Effect of electroconvulsive shock on the content of thyrotropin-releasing hormone in rat brain.

Five grand-mal seizures were electrically induced in rats on alternate days. Forty-eight hours following the last seizure, TRH was quantitated in extracts of anterior cortex, hippocampus, striatum, thalamus plus midbrain, and hypothalamus. When compared to sham treated controls, TRH was found to be elevated 5-fold in the hippocampus and 2-fold in the striatum with no changes observed in the remaining regions. Since the time chosen for analysis excludes acute post-ictal effects, these results draw attention to a prolonged alteration of TRH levels in specific brain regions in an animal model of electroconvulsive treatment.

Attenuation of kindled seizures by intranasal delivery of neuropeptide-loaded nanoparticles.

SummaryThyrotropin-releasing hormone (TRH; Protirelin), an endogenous neuropeptide, is known to have anticonvulsant effects in animal seizure models and certain intractable epileptic patients. Its duration of action, however, is limited by rapid tissue metabolism and the blood—brain barrier. Direct nose-to-brain delivery of neuropeptides in sustained-release biodegradable nanoparticles (NPs) is a promising mode of therapy for enhancing CNS neuropeptide bioavailability. To provide proof of principle for this delivery approach, we used the kindling model of temporal lobe epilepsy to show that 1) TRH-loaded copolymer microdisks implanted in a seizure focus can attenuate kindling development in terms of behavioral stage, after-discharge duration (ADD), and clonus duration; 2) intranasal administration of an unprotected TRH analog can acutely suppress fully kindled seizures in a concentration-dependent manner in terms of ADD and seizure stage; and 3) intranasal administration of polylactide nanoparticles (PLA-NPs) containing TRH (TRH-NPs) can impede kindling development in terms of behavioral stage, ADD, and clonus duration. Additionally, we used intranasal delivery of fluorescent dye-loaded PLA-NPs in rats and application of dye-loaded or dye-attached NPs to cortical neurons in culture to demonstrate NP uptake and distribution over time in vivo and in vitro respectively. Also, a nanoparticle immunostaining method was developed as a procedure for directly visualizing the tissue level and distribution of neuropeptide-loaded nanoparticles. Collectively, the data provide proof of concept for intranasal delivery of TRH-NPs as a viable means to 1) suppress seizures and perhaps epileptogenesis and 2) become the lead compound for intranasal anticonvulsant nanoparticle therapeutics.

Thyrotropin-releasing hormone in the treatment of intractable epilepsy

Intractable seizures remain a significant therapeutic challenge despite current advances in the treatment of epilepsy. Thyrotropin-releasing hormone, the first neuroendocrine releasing factor to be isolated and fully characterized, was also the first releasing factor investigated as a possible neurotransmitter/neuromodulator outside the hypothalamus. Basic and clinical research has revealed a distinct neuroanatomic distribution and a neurochemical role for thyrotropin-releasing hormone in seizure modulation. Thyrotropin-releasing hormone and selected analogs were reported to have antiepileptic effects in several animal seizure paradigms, including kindling and electroconvulsive shock. Clinically, thyrotropin-releasing hormone treatment has been reported to be efficacious in such intractable epilepsies as infantile spasms, Lennox-Gastaut syndrome, myoclonic seizures, and other generalized and refractory partial seizures. Herein, we review evidence that suggests that thyrotropin-releasing hormone and selected thyrotropin-releasing hormone analogs may represent a new class of novel antiepileptic drugs, namely, antiepileptic neuropeptides and provide insights into potential new treatments for the intractable epilepsies.

Prolonged seizure suppression by a single implantable polymeric-TRH microdisk preparation

Thyrotropin-releasing hormone (TRH; Protirelin) is an endogenous neuropeptide known to have anticonvulsant effects in several seizure models and in intractable epileptic patients. Like most neuropeptides, its duration of action may be limited by a lack of sustained site-specific bioavailability. To attempt to provide long-term delivery, we attached TRH to a biodegradable polyanhydride copolymer as a sustained-release carrier. Utilizing the rat kindling model of temporal lobe epilepsy, a single TRH microdisk implanted stereotaxically into the seizure focus (amygdala) significantly suppressed kindling expression when assessed by the number of stimulations required to reach each behavioral stage and to become fully kindled (8.63 +/- 0.92 vs. 16.17 +/- 1.37; Mean +/- S.E.M.). Two indices of seizure severity, afterdischarge duration (Mean +/- S.E.M., sec.) (stimulated amygdala [87.40 +/- 5.47 vs. 51.80 +/- 15.65] and unstimulated amygdala [89.60 +/- 5.55 vs. 48.67 +/- 15.8] and clonus duration (71.2 +/- 5.94 vs. 29.40 +/- 8.87; Mean +/- S.E.M., sec.), were also significantly reduced by a single polymeric-TRH implant. Fifty days after initiation of the study a significant reduction in clonus duration (53.90 +/- 3.27 vs. 40.09 +/- 4.14) still remained in the TRH-implanted groups. This report is the first to provide evidence in support of in situ microdisk pharmacotherapy for potential neuropeptide delivery in intractable epilepsy and possibly other neurological disorders.

Regional distribution of leucine-enkephalin in hypothalamic and extrahypothalamic loci of the human nervous system

The recent discovery of the endogenous opioid peptide, leucine-enkephalin (L-E), throughout vertebrate species prompted investigation of its potential presence in the human CNS using a specific radioimmunoassay. Twenty CNS structures were dissected from 5 human brains obtained at autopsy and were extracted with 2 N and glacial acetic acid. L-E immunoreactivity, identical to synthetic standard, was found in all structures examined. Highest concentrations (range 76-650 pmol/g wet wt.) were found in the pituitary, infundibular stalk, globus pallidus, putamen, substantia nigra, amygdala, head of caudate, and hypothalamus. Lowest concentrations (range 23-49) were found in frontal cortex and cerebellum. L-E was also found in the spinal cord. It is concluded that L-E immunoreactivity is present in the human CNS. The correspondence between the distribution of L-E and the opiate receptor suggests that L-E represents a biologically significant endogenous opiate in man. The high concentrations of L-E in the human hypothalamus and pituitary raises the possibility of a neuroendocrine role for this peptide.

Elevated TRH levels in pyriform cortex after partial and fully generalized kindled seizures

In a previous study we reported significant elevations of TRH in neocortex, hippocampus and combined amygdala/pyriform cortex in rats 48 h after the last of a series of stage 5 kindled seizures. In the present study, to determine whether the increases in TRH were proportional to the intensity of the convulsions, and the degree of development of the kindling process, we compared the effects of partially kindled (stage 2) vs fully kindled (stage 5) seizures. As a further refinement, we examined separately the TRH responses in the pyriform, cingulate and frontal cortices. The responses were especially marked in the pyriform cortex, where TRH increased 7-fold after stage 5 kindled convulsions, compared with 2-fold increases after stage 2-3 seizures. Increases were seen in other cortical regions, as well, but only after stage 5 seizures. These findings are consistent with reports suggesting that the increases in brain TRH occurring after convulsions are aftereffects of the seizures, possibly representing homeostatic anticonvulsant responses, and that the pyriform cortex is a site that is uniquely activated by convulsions.

Distribution of thyrotropin-releasing hormone (TRH) in the hippocampal formation as determined by radioimmunoassay

The distribution of thyrotropin-releasing hormone (TRH) in the hippocampal formation was determined using a radioimmunoassay (RIA) specific for TRH. RIA of hippocampal subregions revealed that the CA3 region of the hippocampal formation contained the highest amount of TRH, followed by intermediate levels in region CA1 and the dentate gyrus. The hilus and subiculum contained the lowest levels. The issue of whether hippocampal TRH is derived from extrinsic and/or intrinsic sources was evaluated by making lesions of the major subcortical afferent to the hippocampus, the fornix pathway. Analysis of the hippocampal formation by RIA revealed that the ventral hippocampus contains higher levels of TRH than the dorsal hippocampus (6.01 +/- 0.62 pg/mg tissue weight vs 1.11 +/- 0.19 pg/mg tissue weight). Lesions of the fornix produced significant decreases in ventral TRH to 52.9% of its control level and in dorsal TRH to 28.8% of its control level. The results from these studies suggest that (1) there is a differential distribution of TRH in the hippocampal formation, (2) the hippocampal formation might be composed of extrinsic and intrinsic sources of TRH, and (3) extrinsic sources of TRH might enter the hippocampus via the fornix pathway. In addition (4) the greater post-lesion decrement in ventral vs dorsal hippocampal TRH suggests that TRH fibers traversing the fornix innervate the ventral hippocampal formation in preference to its dorsal counterpart.

Intranasal Delivery of a Thyrotropin‐Releasing Hormone Analog Attenuates Seizures in the Amygdala‐Kindled Rat

Purpose: Thyrotropin‐releasing hormone (TRH) is known to have anticonvulsant effects in several animal seizure models and is efficacious in treating patients with certain intractable epilepsies. However, the duration of TRHs action is limited due to low bioavailability and difficulty penetrating the blood–brain barrier (BBB). Since direct nose to brain delivery of therapeutic compounds may provide a means for overcoming these barriers, we utilized the kindling model of temporal lobe epilepsy to determine if intranasal administration of a TRH analog, 3‐methyl‐histidine TRH (3Me‐H TRH), could significantly inhibit various seizure parameters.