The microtubule-associated protein tau, and the cytoplasmic protein ubiquitin, are constituents of pathological neurofibrillary tangles found in Alzheimer's disease. In order to see if there is any physiological relationship between these proteins in a functioning human system, human neuroblastoma (LAN-5) cells were grown in vitro and differentiated to a neuronal phenotype. Cell extracts were analyzed by SDS-PAGE, immunoblot, and immunoprecipitation techniques. The colocalization of ubiquitin and tau immunoreactivity was noted in 12- and 35-kDa bands, predominantly located in a cell membrane fraction. The bands were also isolated by immunoprecipitation with the Alz-50 antibody and then identified with a ubiquitin antiserum. These findings show a relationship between tau and ubiquitin in a human neural cell line. This interaction suggests that tau may normally be degraded by an ubiquitin-dependent mechanism and alterations in it may contribute to the formation of neuro-fibrillary pathology.

Regulation by hormones of nervous system development is well recognized in humans, laboratory animals and cultured nerve cells. Thyroxine (T4) and triiodothyronine (T3), the major thyroid hormones, act on brain development and maturation by binding to T3 nuclear receptors. T3 responsive genes have been identified with the T3 receptor as a superfamily of genes including cortisol and estrogens, necessary for adaptation and survival. Less defined are T3 and T4 actions on the peripheral nervous system. In chromaffin cells explanted from the adrenal of immature rats, T3 induces the enzyme tyrosine hydroxylase, involved in catecholamine synthesis. This action is similar but, so far, apparently independent from Nerve Growth Factor promotion of sympathetic and chromaffin cell growth. Mechanisms based on nuclear binding depend on multiple receptors functionally diversified; their selectivity of action over a wide range of early and late developmental patterns is an attractive hypothesis to be further explored.

The structural basis of the cellular disturbance in Alzheimer’s disease (AD) may involve the cytoskeleton. One of the major constituents of the cytoskeleton is the microtubule network. This is composed primarily of tubulin which has a molecular weight of 55 kd and assembles, under certain conditions, to form the microtubules (Cleveland et al., 1977). Other microtubular components include the microtubule associated proteins (MAPS), MAP I and MAP II, and Tau proteins, which co-purify with MAPs. “Tau” represents a class of several proteins which will be referred to collectively as Tau protein.

1. Specific binding of [3H]dexamethasone to cytosol and the activation of bound hormone-receptor complexes were studied in the kidney of immature (3-week) and mature (26-week) Long-Evans male rats. 2. The concentration of specific binding sites was significantly higher (25%) in the kidney of immature rats as compared with mature, while dissociation constants (Kd) remain unaltered at both ages. 3. Heat activation (25 degrees C for 45 min) significantly enhanced the binding of [3H]dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both ages to the same extent. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice versa) gave similar results to the non-mixed groups. 4. Ca2+ activation (0 degree C for 45 min with 20 mM Ca2+) also enhanced the nuclear and DNA-cellulose binding at both ages but to a greater magnitude in immature rats. 5. Differences in the number of specific binding sites and some of the physicochemical properties of kidney glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.

Tau protein and Chromobindin A have several features in common but are not identical. Both consist of a small group of closely related proteins which can form aggregates. Both have a similar range of molecular weights (53–62 kDa) and isoelectric points (6.0–7.5). While Chromobindin A is known to be membrane associated, there is evidence that Tau protein also interacts with phospholipids. Both, not present in all tissues, can be found in the adrenal medulla. Despite these similarities both classes of proteins are unique and immunologically distinct. A rabbit antisera to Tau does not cross react with Chromobindin A. In addition, while protein kinase C and Ca/Calmodulin-dependent protein kinase II phosphorylate Tau protein, they do not phosphorylate Chromobindin A, demonstrating the specificity of these kinases for Tau protein phosphorylation.

Postnatal growth of skeletal muscle (m. gastrocnemius) was compared in rats under euthyroid, hypothyroid and hypothyroid-rehabilitated conditions. In normal (euthyroid) animals, gastrocnemius muscle grows significantly in terms of weight (150 x) from birth to the young adult and, in terms of total contractile myofibril protein (15 x) and myosin ATPase activity (10 x) between days 25 and 90. Rats made hypothyroid (with 0.1% w/v propylthiouracil, PTU) from birth show reduced growth. At 25 days (weaning), compared with euthyroid, muscle weight is only 25% of normal, and a similar reduction is found in total DNA, RNA, protein, myofibril protein, and myosin ATPase activity. These deficits, already significant by day 10, are more marked by day 50 due to the near arrest of growth. Hypothyroid rats allowed to recover by PTU withdrawal after day 25 (rehabilitated) undergo marked compensatory muscle growth. By day 90, muscle weight and protein content increase 50 x, DNA 7 x and RNA 17 x. Over this period, total myofibrillar protein and myosin ATPase increase 20-40 x, but are still below those of 90-day controls, suggesting that the severe growth retardation had not yet been fully compensated. Early thyroid deficiency drastically reduces the normal age-related growth of skeletal muscle and severely retards the development of contractile elements, affecting muscle hypertrophy (protein content) more than cell proliferation (DNA content). Rehabilitation compensates to a major degree for this growth retardation. These results underline the key role of thyroid hormones in regulating development and maturation of skeletal muscle throughout the preweaning and postweaning phases of growth.

Pineals from male Long‐Evans rats (60–65 days old; adapted to a 0700–1900 photoperiod) were cultured for 6 h either in light (1200–1800) or in dark (1800–2400). The objective was to ascertain the effects of tryptophan (trp) phenylalanine (phe) levels ratios in the culture medium on melatonin levels in the pineals their respective media. Total culture (pineal + medium) melatonin levels, determined by RIA, were similar under all conditions. However, in cultures during the early dark phase (1800–2400) lower trp:phe ratios in the medium led to lower pineal:medium ratios of melatonin content. In cultures during the late light phase (1200–1800) the trp:phe ratio had little impact on the pineal:medium distribution of melatonin. Trp:phe ratio rather than absolute level of either amino acid appeared responsible for this effect. Functionally this means that during early dark phase, but not late light phase, movement of melatonin from cultured pineal to medium is progressively facilitated by lower trp:phe ratios. It remains to be determined to what extent darkness per se and/or endogenous pineal rhythmic mechanisms have a permissive role in the action of trp:phe ratio on pineal melatonin release. A melatonin compartmentalization/release effect of these or other amino acids, or their ratios, has not been reported previously may possibly contribute to mechanisms for melatonin's transport or release at night.

Hypothalamic melatonin levels of Long-Evans male rats were studied at three ages (25, 55-60 and 90 days), at four times of the day in the autumn (6:00, 12:00, 18:00 and 24:00), and at two times (12:00 and 24:00) in the spring using radiommunoassay. Melatonin levels increased markedly at noon at 55-60 days of age, compared with the levels at the same time of the day at 25 and 90 days. This increase persisted in autumn and spring. The 24-hr pattern in hypothalamic melatonin was the inverse of that in the pineal, with the levels at noon higher than those at midnight. This pattern was detectable at 25 days of age although the difference in melatonin between 12:00 and 24:00 hr was not great. The day/night difference was prominent by 55-60 days of age and disappeared by adulthood (90 days). This 24-hr pattern was similar in spring and autumn in the three ages studied. Although in the 55-60-day-old group the melatonin ratio (noon/midnight) was the same in autumn and spring, the absolute levels of melatonin in spring were significantly lower. The findings are consistent with the general concept of a modulatory role of melatonin in control of hypothalamo-hypophyseal GnRH and gonadotropin function, and the timing of the developmental maturation of this neuroendocrine axis. Demonstration of the mechanism of melatonin's action at the hypothalamic level will be facilitated by further definition of quantitative developmental changes.

The ubiquitin-dependent protein degradation system plays a major role in the removal of abnormal and denatured proteins which may form insoluble aggregates in pathological conditions or during other cellular stress. Neuritic plaques and neurofibrillary tangles in sections of Alzheimer's cortex contain insoluble aggregates of proteins and are shown here to specifically immunostain with an antiserum to ubiquitin-protein conjugates. Plaque core amyloid and normal neurons do not immunostain and sodium dodecyl sulphate (SDS)-insoluble tangle preparations are not ubiquitin-positive on slot blots. The possible role and consequences of ubiquitination in tangle and plaque production in Alzheimer's disease are discussed.

We have assessed the activity (nmol/mg protein/h) of glutamic acid decarboxylase (GAD) in discrete hypothalamic nuclei before and after sexual maturation in the developing female rat. Activity in other brain regions including the cortex, septum and caudate-putamen was also assessed. While there appears to be a general rise (approximately 30%), with age, in GAD activity, the rise is most marked, and highly significant (P less than 0.001), in the anterior portion of the hypothalamus (56%). In contrast, no significant increase of GAD activity was found in the medical basal hypothalamus.

The binding of [3H]dexamethasone-receptor complexes to purified nuclei was studied in the cerebral hemispheres of immature (3-week-old) and mature (26-week-old) Long-Evans male rats to determine the age-related changes, if any, in the physicochemical properties of glucocorticoid receptors. Our data show that heat activation (for 45 min at 25 degrees C) significantly enhances the nuclear binding of [3H]dexamethasone-receptor complexes in rats of both ages, with a greater magnitude in immature rats. Ca2+ activation (20 mM Ca2+ for 45 min at 0 degree C) also enhances the nuclear binding of bound receptor complexes but to a similar extent at both ages. These findings indicate that some of the physicochemical properties (e.g. heat activation) of glucocorticoid receptor change, while others (e.g. Ca2+ activation) remain unchanged at different phases of the lifespan.

Specific binding of [3H]dexamethasone to cytosol and translocation of bound receptor complexes to purified nuclei were studied in the skeletal muscle of immature (3-week) and mature (26-week) Long-Evans male rats. A marked decrease (57%) in the specific binding sites with no apparent change in dissociation constant (Kd) was observed in the skeletal muscle of mature rats compared to immature. Heat activation (25 degrees C for 45 min) significantly enhances the nuclear binding of steroid-receptor complexes in the skeletal muscle of rats of both the ages at almost similar level. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice-versa) gave similar values. Interestingly, Ca2+-activated (0 degree C for 45 min with 20 mM Ca2+) nuclear translocation was significantly higher (27%) in the skeletal muscle of immature rats compared to mature. Our results indicate that glucocorticoid receptor level and some physicochemical properties change with age in the skeletal muscle of rats.

Long-Evans male rats were made hypothyroid from birth by the addition of 6-N-propylthiouracil (PTU) to their drinking water (0.1%). A group of animals was rehabilitated beginning at postnatal day 25 by withdrawal of the PTU from the drinking water. Subsequently, the rats were tested for a variety of behavioral tasks. Serum concentrations of thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) were determined by radioimmunoassay. At 50 days of age, PTU-treated rats had non-detectable levels of T4 but an eight-fold increase of TSH. In 50-day-old, neonatally hypothyroid but rehabilitated rats, serum TSH and T3 were normal, although T4 was still significantly lower. At 90 days of age, basal levels of TSH and thyroid hormones were normal in the rehabilitated rats, but thyroid hormone secretion in response to various types of neural stress was markedly altered. Comparison of passive avoidance learning revealed no significant alteration in the memory retention of either PTU-treated or rehabilitated animals. The 50-day-old, rehabilitated rats showed increased locomotor activity both in running-wheel and in hole-board tests; this hyperactivity, though markedly reduced, still persisted at day 90. In the early phase of rehabilitation (50 days of age), decreases in exploratory activity and lack of habituation occurred with the hole-board test; by the late phase of rehabilitation (90 days of age) these behavioral parameters had become normal. These results suggest generally longer periods of plasticity of the brain and better prospects for rehabilitation from neonatal cretinoid retardation than commonly believed. Specifically, the pituitary - thyroid system and neural mechanisms integrating adaptive behavior possess considerable capacity for spontaneous recovery from hypothyroidism; certain types of altered neuroendocrine and behavioral responses appear to be less amenable to rehabilitation or require longer periods for complete rehabilitation.