CLOCK Genes and Circadian Rhythmicity in Alzheimer Disease.

Disturbed circadian rhythms with sleep problems and disrupted diurnal activity are often seen in patients suffering from Alzheimer disease (AD). Both endogenous CLOCK genes and external Zeitgeber are responsible for the maintenance of circadian rhythmicity in humans. Therefore, modifications of the internal CLOCK system and its interactions with exogenous factors might constitute the neurobiological basis for clinically observed disruptions in rhythmicity, which often have grave consequences for the quality of life of patients and their caregivers. Presently, more and more data are emerging demonstrating how alterations of the CLOCK gene system might contribute to the pathophysiology of AD and other forms of dementia. At the same time, the impact of neuropsychiatric medication on CLOCK gene expression is under investigation.

Stereological analysis of the reorganization of the dentate gyrus following entorhinal cortex lesion in mice.

Denervation of the dentate gyrus by entorhinal cortex lesion has been widely used to study the reorganization of neuronal circuits following central nervous system lesion. Expansion of the non-denervated inner molecular layer (commissural/associational zone) of the dentate gyrus and increased acetylcholinesterase-positive fibre density in the denervated outer molecular layer have commonly been regarded as markers for sprouting following entorhinal cortex lesion. However, because this lesion extensively denervates the outer molecular layer and causes tissue shrinkage, stereological analysis is required for an accurate evaluation of sprouting. To this end we have performed unilateral entorhinal cortex lesions in adult C57BL/6J mice and have assessed atrophy and sprouting in the dentate gyrus using modern unbiased stereological techniques. Results revealed the expected increases in commissural/associational zone width and density of acetylcholinesterase-positive fibres on single brain sections. Yet, stereological analysis failed to demonstrate concomitant increases in layer volume or total acetylcholinesterase-positive fibre length. Interestingly, calretinin-positive fibres did grow beyond the border of the commissural/associational zone into the denervated layer and were regarded as sprouting axons. Thus, our data suggest that in C57BL/6J mice shrinkage of the hippocampus rather than growth of fibres underlies the two morphological phenomena most often cited as evidence of regenerative sprouting following entorhinal cortex lesion. Moreover, our data suggest that regenerative axonal sprouting in the mouse dentate gyrus following entorhinal cortex lesion may be best assessed at the single-fibre level.

Dexamethasone selectively suppresses microglial trophic responses to hippocampal deafferentation.

Hippocampal deafferentation increases the expression of insulin-like growth factor-1 by microglia, and of ciliary neurotrophic factor and basic fibroblast growth factor by astroglia in fields and periods of reactive axonal growth. Glucocorticoids attenuate lesion-induced hippocampal sprouting, possibly by reducing trophic signals that stimulate growth. With an interest in this hypothesis, the present studies evaluated the influence of systemic treatment with the synthetic glucocorticoid dexamethasone on entorhinal lesion-induced increases in neurotrophic factor expression in young adult rat hippocampus. Daily dexamethasone injections almost completely blocked increases in insulin-like growth factor-1 messenger RNA content, but did not perturb increases in ciliary neurotrophic factor or basic fibroblast growth factor messenger RNA content, in the deafferented dentate gyrus molecular layer. To determine if the suppression of insulin-like growth factor-1 expression was secondary to a general inhibition of microglial responses, and to identify the time period of glucocorticoid sensitivity, additional rats were prepared to evaluate the effects of semi-chronic (i.e. daily) and single dexamethasone injections on microglial proliferation, ED-1 immunoreactivity (a marker of microglial reactivity) and insulin-like growth factor-1 messenger RNA expression. Semi-chronic dexamethasone treatment attenuated all three measures of deafferentation-induced microglial reactivity. However, a single dexamethasone injection given two (but not one or three) days postlesion inhibited deafferentation-induced increases in insulin-like growth factor-1 messenger RNA content, without having significant effects on other measures. These results demonstrate that dexamethasone treatment preferentially suppresses microglial, as opposed to astroglial, trophic responses to deafferentation, and suggest that glucocorticoids attenuate reactive axonal sprouting by inhibiting the microglial production of insulin-like growth factor-1.

Deafferentation-induced increases in hippocampal insulin-like growth factor-1 messenger RNA expression are severely attenuated in middle aged and aged rats.

Deafferentation of the adult rat dentate gyrus induces reactive axonal growth by surviving afferent systems. In middle aged and aged rats, axonal sprouting is delayed and reduced relative to young adults. The cause for this age-related decline is not known, but it may reflect a decrement in trophic signals which initiate sprouting. Insulin-like growth factor-1 may play a role in sprouting because it (i) promotes axonal growth, (ii) is expressed at elevated levels by microglia just prior to sprouting onset, and (iii) is expressed with better spatiotemporal correspondence to hippocampal sprouting than other trophic factors examined. The present study used in situ hybridization to evaluate the influence of age on deafferentation-induced insulin-like growth factor-1 messenger RNA expression in the dentate gyrus. Messenger RNA levels were markedly elevated 4 days after an entorhinal cortex lesion at 3 months of age. Comparable lesions at 12 months did not significantly increase labeling whereas lesions at 18-26 months caused only a modest increase at 8 days postlesion. These data demonstrate that deafferentation induces more modest and delayed increases in insulin-like growth factor-1 expression in middle aged and aged rats than in young adults. The loss of reactive insulin-like growth factor-1 expression at ages exhibiting an attenuated sprouting response supports (i) an association between insulin-like growth factor-1 and sprouting and (ii) the possibility that impairments in the expression of this factor contributes to reduced axonal plasticity with age.

Astroglial ciliary neurotrophic factor mRNA expression is increased in fields of axonal sprouting in deafferented hippocampus.

Evidence that ciliary neurotrophic factor promotes axonal sprouting and regeneration in the periphery raises the possibility that this factor is involved in reactive axonal growth in the brain. In situ hybridization was used in the present study to determine whether ciliary neurotrophic factor mRNA expression is increased in association with axonal sprouting in deafferented adult rat hippocampus. In untreated rats, ciliary neurotrophic factor cRNA labeling density was high in the olfactory nerve, pia mater, and aspects of the ventricular ependyma and was relatively low within areas of white matter (fimbria, internal capsule) and select neuronal fields (hippocampal cell layers, habenula). After an entorhinal cortex lesion, hybridization was markedly increased in fields of anterograde degeneration, including most prominently the ipsilateral dentate gyrus outer molecular layer and hippocampal stratum lacunosum moleculare. Labeling in these fields was increased by 3 days postlesion, was maximal at 5 days, and returned to normal levels by 14 days. Double labeling demonstrated that, in both control and experimental tissue, ciliary neurotrophic factor mRNA was colocalized with glial fibrillary acidic protein immunoreactivity in astroglia, but it was not colocalized with markers for oligodendrocytes or microglia. These results demonstrate that astroglial ciliary neurotrophic factor expression is increased in fields of axonal and terminal degeneration and that increased expression is coincident with 1) increased insulin-like growth factor-1 and basic fibroblast growth factor expression and 2) the onset of reactive axonal growth. The synchronous expression of these glial factors in fields of deafferentation suggests the possibility of additive or synergistic interactions in the coordination of central axonal growth.

Heparan sulfate and chondroitin sulfate glycosaminoglycan attenuate beta-amyloid(25-35) induced neurodegeneration in cultured hippocampal neurons.

beta-Amyloid peptide has been reported to be toxic to neurons in vitro and in vivo. The fragment of the beta 1-42 peptide believed to be responsible for this toxicity consists of amino acids 25 to 35. beta-amyloid protein, heparan sulfate (HS) glycosaminoglycan (GAG), and proteoglycan (PG) are all localized throughout the senile plaques found in Alzheimer's disease. Chondroitin sulfate (CS) and dermatan sulfate have also been found at the periphery of senile plaques. We have found that both HS and CS prevented neurite fragmentation and toxicity normally induced by beta 25-35. HS and CS by themselves did not have a significant influence on cell viability, indicating that their protective actions were not due to a general trophic effect. In contrast, cultures treated with HS and beta 1-42 did not show significantly reduced toxicity compared to cultures treated with beta 1-42 alone despite specific binding interactions. These data indicate that one function of GAGs in the brain may be to protect neurons from select toxic insults and injury, and additionally suggest that HS interacts differently with different beta-amyloid fragments. These data further suggest that different beta-amyloid fragments may induce distinct mechanisms of toxicity in vitro.