Antisense suppression of TSC1 gene product, hamartin, enhances neurite outgrowth in NGF-treated PC12h cells.

Tuberous sclerosis complex (TSC) is an autosomal dominant inherited disorder characterized by benign tumors (hamartomas) in various organs. The brain is one of the most severely affected organs with neuropsychiatric disorders including epilepsy, mental retardation and autism. The identification of TSC genes (TSC1 and TSC2) and their gene products (hamartin and tuberin, respectively), revealed that they function together as a complex. However, mutations in TSC2 are often accompanied by more severe neurologic deficits. Here, we show that hamartin and tuberin play different roles in NGF-treated cultured neuronal cells PC12h. The level of hamartin in PC12h cells was slightly and gradually increased, while those of tuberin rapidly increased upon NGF-induced neuronal differentiation in PC12h cells. Antisense for TSC1 (TSC1-AS) or TSC2-AS reduced expression of hamartin or tuberin, respectively, and enhanced S-phase of cell cycle in PC12h cells. Suppression of hamartin significantly enhanced neurite outgrowth after NGF-treatment in PC12h cells, while suppression of tuberin inhibited neurite outgrowth. Expression of activated V14RhoA reverted TSC1-AS induced abnormal neurite development. These results suggest that loss of hamartin results in abnormal neurite elongation through Rho inactivation in NGF-treated PC12h cells, which may be associated with the neurological manifestations of TSC.

Dense distribution of macrophages in flexor aspects of the hand and foot of mid-term human fetuses.

In the developing human musculoskeletal system, cell death with macrophage accumulation occurs in the thigh muscle and interdigital area. To comprehensively clarify the distribution of macrophages, we immunohistochemically examined 16 pairs of upper and lower extremities without the hip joint (left and right sides) obtained from 8 human fetuses at approximately 10-15 weeks of gestation. Rather than in muscles, CD68-positive macrophages were densely distributed in loose connective tissues of the flexor aspects of the extremities, especially in the wrist, hand and foot. In contrast, no or fewer macrophages were evident in the shoulder and the extensor aspects of the extremities. The macrophages were not concentrated at the enthesis of the tendon and ligament, but tended to be arranged along other connective tissue fibers. Deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling revealed apoptosis in the hand lumbricalis muscles, but not in the area of macrophage accumulation. Likewise, podoplanin-positive lymphatic vessels were not localized to areas of macrophage accumulation. Re-organization of the connective tissue along and around the flexor tendons of the hand and foot, such as development of the bursa or tendon sheath at 10-15 weeks, might require the phagocytotic function of macrophages, although details of the mechanism remain unknown.

Monocular deprivation enhances the nuclear signalling of extracellular signal-regulated kinase in the developing visual cortex.

Monocular deprivation (MD) of vision leads to a loss of cortical response to the deprived eye in the early postnatal period (ocular dominance plasticity). The activity of several signal molecules, including extracellular signal-regulated kinase (ERK), has been reported as playing a crucial role in the ocular dominance plasticity. Although pharmacological inhibition of ERK disturbed the ocular dominance plasticity, it remains to be elucidated how the ERK activity is modulated by MD. We herein report the effects of MD on ERK activation in the visual cortex of young and adult rats. Phosphorylated ERK (pERK)-immunopositive cells are mainly distributed in layers II/III of the visual cortex. Following MD, we found a significant decrease in the density of pERK-immunopositive cells in the cortex receiving deprived-eye inputs in both young and adult animals. The amount of pERK protein also decreased in the input-deprived cortex as revealed by Western blotting. Regarding the subcellular localization of pERK, we found a significant increase in the pERK-immunopositive nucleus following MD in young animals. In these animals, the amount of pERK protein in the nuclear fraction of cortical tissue was significantly increased. No up-regulation of the nuclear pERK was observed in adults or following binocular deprivation. These findings suggest that ERK activation may therefore be regulated by different mechanisms between young and adult animals, and MD during the developing period may thus specifically up-regulate the nuclear signalling of ERK.

The TSC1 gene product hamartin interacts with NADE.

Hamartomatous brain lesions are a hallmark of brain pathology of tuberous sclerosis complex (TSC). To elucidate the mechanism of tumor development in the brain of TSC, we identified NADE (p75NTR-associated cell death executor) as an interactor for TSC1 gene product hamartin using a yeast two-hybrid system. In a pull-down assay, endogenous NADE was purified with the immobilized coiled-coil domain (CCD) of hamartin from the PC12h cell lysate. Immunofluorescence and immunoprecipitation confirmed the interaction of hamartin and NADE in cultured neurons and mouse brain lysate. Hamartin constitutively associated with NADE to prevent its proteasomal degradation. Suppression of hamartin with TSC1 small interfering RNA (siRNA) caused reduction of NADE and failed to lead to NGF-induced apoptosis in PC12h cells. These results indicate that hamartin binds to NADE to regulate neuronal cell function and loss of this association is likely to contribute to the brain pathology in TSC.