Howard T.J. Mount

Aβ peptide immunization reduces behavioural impairment and plaquesin a model of Alzheimer's disease

Much evidence indicates that abnormal processing and extracellular deposition of amyloid-β peptide (Aβ), a proteolytic derivative of the β-amyloid precursor protein (βAPP), is central to the pathogenesis of Alzheimers disease (reviewed in ref. 1). In the PDAPP transgenic mouse model of Alzheimers disease, immunization with Aβ causes a marked reduction in burden of the brain amyloid. Evidence that Aβ immunization also reduces cognitive dysfunction in murine models of Alzheimers disease would support the hypothesis that abnormal Aβ processing is essential to the pathogenesis of Alzheimers disease, and would encourage the development of other strategies directed at the ‘amyloid cascade’. Here we show that Aβ immunization reduces both deposition of cerebral fibrillar Aβ and cognitive dysfunction in the TgCRND8 murine model of Alzheimers disease without, however, altering total levels of Aβ in the brain. This implies that either a ∼50% reduction in dense-cored Aβ plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic Aβ species.

Therapeutically effective antibodies against amyloid-β peptide target amyloid-β residues 4−10 and inhibit cytotoxicity and fibrillogenesis

Immunization of transgenic mouse models of Alzheimer disease using amyloid-β peptide (Aβ) reduces both the Alzheimer disease–like neuropathology and the spatial memory impairments of these mice. However, a therapeutic trial of immunization with Aβ42 in humans was discontinued because a few patients developed significant meningo-encephalitic cellular inflammatory reactions. Here we show that beneficial effects in mice arise from antibodies selectively directed against residues 4–10 of Aβ42, and that these antibodies inhibit both Aβ fibrillogenesis and cytotoxicity without eliciting an inflammatory response. These findings provide the basis for improved immunization antigens as well as attempts to design small-molecule mimics as alternative therapies.

Cyclohexanehexol inhibitors of Aβ aggregation prevent and reverse Alzheimer phenotype in a mouse model

When given orally to a transgenic mouse model of Alzheimer disease, cyclohexanehexol stereoisomers inhibit aggregation of amyloid β peptide (Aβ) into high-molecular-weight oligomers in the brain and ameliorate several Alzheimer disease–like phenotypes in these mice, including impaired cognition, altered synaptic physiology, cerebral Aβ pathology and accelerated mortality. These therapeutic effects, which occur regardless of whether the compounds are given before or well after the onset of the Alzheimer disease–like phenotype, support the idea that the accumulation of Aβ oligomers has a central role in the pathogenesis of Alzheimer disease.

BDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities.

While neurotrophins are critical for neuronal survival and differentiation, recent work suggests that they acutely regulate synaptic transmission as well. Brain-derived neurotrophic factor (BDNF) enhances excitatory postsynaptic currents in cultured dissociated hippocampal neurons within 2-3 min through postsynaptic, phosphorylation-dependent mechanisms. Moreover, BDNF modulates hippocampal long-term potentiation, in which postsynaptic NMDA (N-methyl-D-aspartate) receptors (NRs) play a key role. We now report that BDNF acutely increases tyrosine phosphorylation of the specific NMDA receptor subunit NR2B, which has recently been shown to play a role in long-term potentiation. Incubation of BDNF with cortical or hippocampal postsynaptic densities for 5 min increased tyrosine phosphorylation of the NR2B subunits in a dose-dependent manner. A maximal increase to 165% of control phosphorylation occurred at a BDNF concentration of 2 ng/ml. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, had no effect on NR2B phosphorylation. Further, BDNF action was selective, since it did not alter tyrosine phosphorylation of NR2A subunits. Our results suggest that tyrosine phosphorylation of NR2B subunits of the NMDA receptor may contribute to neurotrophin modulation of postsynaptic responsiveness and long-term potentiation.

α5GABAA Receptors Mediate the Amnestic But Not Sedative-Hypnotic Effects of the General Anesthetic Etomidate

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the α5 subunit (α5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by α5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not α5 null mutant (α5−/−) mice. In addition, etomidate impaired memory performance of WT but not α5−/− mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The α5−/− mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and α5−/− mice. Deletion of the α5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.

APH-1 Interacts with Mature and Immature Forms of Presenilins and Nicastrin and May Play a Role in Maturation of Presenilin·Nicastrin Complexes

APH-1 and PEN-2 genes modulate the function of nicastrin and the presenilins in Caenorhabditis elegans. Preliminary studies in transfected mammalian cells overexpressing tagged APH-1 proteins suggest that this genetic interaction is mediated by a direct physical interaction. Using the APH-1 protein encoded on human chromosome 1 (APH-11L; also known as APH-1a) as an archetype, we report here that endogenous forms of APH-1 are predominantly expressed in intracellular membrane compartments, including the endoplasmic reticulum andcis-Golgi. APH-1 proteins directly interact with immature and mature forms of the presenilins and nicastrin within high molecular weight complexes that display γ- and ε-secretase activity. Indeed APH-1 proteins can bind to the nicastrin Δ312–369 loss of function mutant, which does not undergo glycosylation maturation and is not trafficking beyond the endoplasmic reticulum. The levels of expression of endogenous APH-11L can be suppressed by overexpression of any other members of the APH-1 family, suggesting that their abundance is coordinately regulated. Finally, although the absence of APH-1 destabilizes the presenilins, in contrast to nicastrin and PEN-2, APH-1 itself is only modestly destabilized in cells lacking functional expression of presenilin 1 or presenilin 2. Taken together, our data suggest that APH-1 proteins, and APH-11 in particular, may have a role in the initial assembly and maturation of presenilin·nicastrin complexes.

Mature Glycosylation and Trafficking of Nicastrin Modulate Its Binding to Presenilins

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-β-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute γ-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.

Decreased brain-derived neurotrophic factor depends on amyloid aggregation state in transgenic mouse models of Alzheimer's disease.

Downregulation of brain-derived neurotrophic factor (BDNF) in the cortex occurs early in the progression of Alzheimers disease (AD). Since BDNF plays a critical role in neuronal survival, synaptic plasticity, and memory, BDNF reduction may contribute to synaptic and cellular loss and memory deficits characteristic of AD. In vitro evidence suggests that amyloid-β (Aβ) contributes to BDNF downregulation in AD, but the specific Aβ aggregation state responsible for this downregulation in vivo is unknown. In the present study, we examined cortical levels of BDNF mRNA in three different transgenic AD mouse models harboring mutations in APP resulting in Aβ overproduction, and in a genetic mouse model of Down syndrome. Two of the three Aβ transgenic strains (APPNLh and TgCRND8) exhibited significantly decreased cortical BDNF mRNA levels compared with wild-type mice, whereas neither the other strain (APPswe/PS-1) nor the Down syndrome mouse model (Ts65Dn) was affected. Only APPNLh and TgCRND8 mice expressed high Aβ42/Aβ40 ratios and larger SDS-stable Aβ oligomers (∼115 kDa). TgCRND8 mice exhibited downregulation of BDNF transcripts III and IV; transcript IV is also downregulated in AD. Furthermore, in all transgenic mouse strains, there was a correlation between levels of large oligomers, Aβ42/Aβ40, and severity of BDNF decrease. These data show that the amount and species of Aβ vary among transgenic mouse models of AD and are negatively correlated with BDNF levels. These findings also suggest that the effect of Aβ on decreased BDNF expression is specific to the aggregation state of Aβ and is dependent on large oligomers.

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS‐expressed Doppel or internally deleted PrP (‘ΔPrP’). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC‐like protein, ‘π’. Shadoo (Sho) is a hypothetical GPI‐anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N‐terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC‐deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC‐like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion‐infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC‐like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

Functional trkB neurotrophin receptors are intrinsic components of the adult brain postsynaptic density.

Neurotrophins have long been thought to act as target-derived factors that regulate the survival and differentiation of afferent neurons. Recently, brain-derived neurotrophic factor (BDNF) was shown to elicit rapid increases in synaptic activity of cultured hippocampal neurons by enhancing responsiveness to excitatory input. These findings suggest a postsynaptic localization of neurotrophin receptors. In this study, we examined the expression of trkB, a high-affinity receptor for BDNF, in the postsynaptic density (PSD), a proteinaceous specialization of the postsynaptic membrane. Western blot analyses with antibodies to trkB revealed localization to the PSD in adult rat cerebral cortex and hippocampus. Only the full-length, active form of trkB was detected in PSD samples. BDNF treatment of the adult cortical PSD resulted in a 5-fold increase in trkB autophosphorylation, supporting the contention that the PSD contains functional trkB. Truncated trkB, which does not contain the tyrosine kinase signaling domain, though present in membrane fractions, was undetectable in the PSD. The presence of trkB in the PSD is consistent with a role for neurotrophins in the regulation of synaptic activity via direct postsynaptic mechanisms.