Effects of apoE on neuronal signaling and APP processing in rodent brain.

We previously reported that primary neuronal cells treated with apolipoprotein E (apoE) or an apoE-derived peptide (EP) increased ERK activation and decreased JNK activation via apoE receptors. Here, we examined if the effects observed in vitro were observed in vivo. Similar to our observations in primary neurons, in vivo we found that injections of 2muM EP into the rat hippocampus increased the levels of ERK activation and decreased JNK activation. However, the time course of these effects was slower in vivo. Immunohistochemical analysis of the tissue showed prominently increased ERK phosphorylation and decreased JNK phosphorylation in neuronal cells throughout the hippocampus, particularly in the CA3 regions. To determine if apoE was endocytosed by neurons, we conjugated fluorescent microspheres with the EP and injected them into the rat hippocampus. After 7 days, the microspheres were present in neurons. We also examined the in vivo effects of apoE on ApoEr2 and APP processing. EP and full-length apoE3 and apoE4 increased C-terminal fragments of ApoEr2 and APP after a single injection, multiple injections, and chronic infusion paradigms. ApoE3 produced higher levels of ApoEr2 and APP C-terminal fragments than apoE4. These results demonstrate that apoE alters ApoEr2 and APP processing in vivo. The increase in ERK activation is consistent with a role for apoE in a neuronal response to stress, and the decrease in JNK activation suggests that apoE may have anti-apoptotic effects, over several days.

Multiple pathways of apolipoprotein E signaling in primary neurons.

Apolipoprotein E is a genetic risk factor for Alzheimer's disease, and the apoE protein is associated with beta-amyloid deposits in Alzheimer's disease brain. We examined signaling pathways stimulated by apoE in primary neurons in culture. ApoE and an apoE-derived peptide activated several intracellular kinases, including prominently extracellular signal-regulated kinase 1/2 (ERK1/2). ERK1/2 activation by apoE was blocked by an inhibitor of the low-density lipoprotein receptor family, the specific NMDA glutamate receptor antagonist MK 801 and other calcium channel blockers. Activation of apoE receptors also induced tyrosine phosphorylation of Dab1, an adaptor protein of apoE receptors, but experiments in Dab1 knockout neurons demonstrated that Dab1 was not necessary for ERK activation. In contrast, apoE treatment of primary neurons decreased activation of c-Jun N-terminal kinase, a kinase that interacts with another apoE receptor adaptor protein, c-Jun N-terminal kinase-interacting protein. This change also depended on interactions with the low-density lipoprotein receptor family but was independent of calcium channels. c-Jun N-terminal kinase deactivation by apoE was blocked by gamma-secretase inhibitors and pertussis toxin. These results demonstrate that apoE affects several signaling cascades in neurons: increased disabled phosphorylation, activation of the ERK1/2 pathway (dependent on calcium influx via the NMDA receptor) and inhibition of the c-Jun N-terminal kinase 1/2 pathway (dependent on gamma-secretase and G proteins).

Postnatal development of the cerebellum and the CNS adrenergic system is independent of norepinephrine and epinephrine.

A fundamental question in the formation of the nervous system is the extent to which a neurotransmitter contributes to the development of the neurons that synthesize and release it. A complementary question is whether neurotransmitter signaling contributes to the development of postsynaptic targets. Prior studies have suggested that adrenergic signaling may promote adrenergic neuronal proliferation or survival and may be critical for the postnatal development of the cerebellum. To test these possibilities genetically, we studied mice that are unable to synthesize norepinephrine and epinephrine (NE/E), the endogenous adrenergic receptor ligands, due to a disruption the gene for dopamine beta-hydroxylase. These mice develop postnatally in the absence of NE/E. Here we report that the adrenergic neurons of these mutant mice are present in normal numbers and locations and exhibit typical innervation patterns throughout the central nervous system (CNS), as assessed by immunostaining for tyrosine hydroxylase and the NE transporter. Furthermore, cerebellar cortical development (size, foliation, layering, cell number, and position), which proceeds to a large degree postnatally, is unaltered in the mutants. These results indicate that the fate and innervation pattern of the adrenergic neurons, as well as the development of the cerebellum, do not depend on postnatal signaling by NE/E. The results also suggest that when restoration of adrenergic signaling is performed in this mutant mouse model (by administering a synthetic precursor of NE), reversal of phenotypes is due to the synthesis and release of NE/E from adrenergic terminals that are distributed normally within the CNS.