Citations

In prion-infected mice, both the Notch-1 intracellular domain transcription factor (NICD) and the disease-causing prion protein (PrP Sc ) increase in the brain preceding dendritic atrophy and loss. Because the drug LY411575 inhibits the γ-secretase-catalyzed cleavage of Notch-1 that produces NICD, we asked whether this γ-secretase inhibitor (GSI) might prevent dendritic degeneration in mice with scrapie. At 50 d postinoculation with Rocky Mountain Laboratory (RML) prions, mice were given GSI orally for 43–60 d. Because we did not expect GSI to produce a reduction of PrP Sc levels in brain, we added quinacrine (Qa) to the treatment regimen. Qa inhibits PrP Sc formation in cultured cells. The combination of GSI and Qa reduced PrP Sc by ≈95% in the neocortex and hippocampus but only ≈50% in the thalamus at the site of prion inoculation. The GSI plus Qa combination prevented dendritic atrophy and loss, but GSI alone did not. Even though GSI reduced NICD levels to a greater extent than GSI plus Qa, it was unable to prevent dendritic degeneration. Whether a balance between NICD and dendrite growth-stimulating factors was achieved with GSI plus Qa but not GSI alone remains to be determined. Although the combination of GSI and Qa diminished PrP Sc in the brains of RML-infected mice, GSI toxicity prevented us from being able to assess the effect the GSI plus Qa combination on incubation times. Whether less toxic GSIs can be used in place of LY411575 to prolong survival remains to be determined.

Abstract Notch and its ligands have been implicated in the regulation and differentiation of various CD4+ T-helper cells. Regulatory T cells (Tregs), which express the transcription factor Foxp3, suppress aberrant immune responses that are typically associated with autoimmunity or excessive inflammation. Previous studies have shown that transforming growth factor beta (TGFβ1) induces Foxp3 expression and a regulatory phenotype in peripheral T cells. Here, we show that pharmacologic inhibition of Notch signaling using γ-secretase inhibitor (GSI) treatment blocks (1) TGFβ1-induced Foxp3 expression, (2) the up-regulation of Foxp3-target genes, and (3) the ability to suppress naive T-cell proliferation. In addition, the binding of Notch1, CSL, and Smad to conserved binding sites in the foxp3 promoter can be inhibited by treatment with GSI. Finally, in vivo administration of GSI results in reduced Foxp3 expression and development of symptoms consistent with autoimmune hepatitis, a disease previously found to result from dysregulation of TGFβ signaling and regulatory T cells. Together, these findings indicate that the Notch and TGFβ signaling pathways cooperatively regulate Foxp3 expression and regulatory T-cell maintenance both in vitro and in vivo.

Cells that have evolved to produce large quantities of secreted proteins to serve the integrated functions of complex multicellular organisms are equipped to compensate for protein misfolding. Hepatocytes and plasma cells have well developed chaperone and proteasome systems to ensure that secreted proteins transit the cell efficiently. The number of neurodegenerative disorders associated with protein misfolding suggests that neurons are particularly sensitive to the pathogenic effects of aggregates of misfolded molecules because those systems are less well developed in this lineage. Aggregates of the amyloidogenic (Aβ 1–42 ) peptide play a major role in the pathogenesis of Alzheimer's disease (AD), although the precise mechanism is unclear. In genetic studies examining protein–protein interactions that could constitute native mechanisms of neuroprotection in vivo , overexpression of a WT human transthyretin (TTR) transgene was ameliorative in the APP23 transgenic murine model of human AD. Targeted silencing of the endogenous TTR gene accelerated the development of the neuropathologic phenotype. Intraneuronal TTR was seen in the brains of normal humans and mice and in AD patients and APP23 mice. The APP23 brains showed colocalization of extracellular TTR with Aβ in plaques. Using surface plasmon resonance we obtained in vitro evidence of direct protein–protein interaction between TTR and Aβ aggregates. These findings suggest that TTR is protective because of its capacity to bind toxic or pretoxic Aβ aggregates in both the intracellular and extracellular environment in a chaperone-like manner. The interaction may represent a unique normal host defense mechanism, enhancement of which could be therapeutically useful.

A number of hypotheses regarding how anti‐Aβ antibodies alter amyloid deposition have been postulated, yet there is no consensus as to how Aβ immunotherapy works. We have examined the in vivo binding properties, pharmacokinetics, brain penetrance, and alterations in Aβ levels after a single peripheral dose of anti‐Aβ antibodies to both wild‐type (WT) and young non‐Aβ depositing APP and BRI‐Aβ42 mice. The rapid rise in plasma Aβ observed after antibody (Ab) administration is attributable to prolongation of the half‐life of Aβ bound to the Ab. Only a miniscule fraction of Ab enters the brain, and despite dramatic increases in plasma Aβ, we find no evidence that total brain Aβ levels are significantly altered. Surprisingly, cerebral spinal fluid Aβ levels transiently rise, and when Ab:Aβ complex is directly injected into the lateral ventricles of mice, it is rapidly cleared from the brain into the plasma where it remains stable. When viewed in context of daily turnover of Aβ, these data provide a framework to evaluate proposed mechanisms of Aβ attenuation mediated by peripheral administration of an anti‐Aβ monoclonal antibody (mAb) effective in passive immunization paradigm. Such quantitative data suggest that the mAbs are either indirectly enhancing clearance of Aβ or targeting a low abundance aggregation intermediate.—Levites, Y., Smithson, L. A., Price, R. W., Dakin. R. S., Yuan, B., Sierks, M. R., Kim, J., McGowan, E., Reed, D. K., Rosenberry, T. L., Das, P., Golde, T. E. Insights into the mechanisms of action of anti‐Aβ antibodies in Alzheimer's disease mouse models. FASEB J. 20, E2002–E2014 (2006)

ABSTRACT γ‐Secretase cleavage is the final proteolytic step that releases the amyloid β‐peptide (Aβ) from the amyloid β‐protein precursor (APP). Significant evidence indicates that the presenilins (PS) are catalytic components of a high molecular weight γ‐secretase complex. The glycoprotein nicastrin was recently identified as a functional unit of this complex based on 1) binding to PS and 2) the ability to modulate Aβ production following mutation of a conserved DYIGS region. In contrast to the initial report, we find that overexpression of wild‐type (WT) nicastrin increases Aβ production, whereas DYIGS mutations (MT) have little or no effect. The increase in Aβ production is associated with an increase in γ‐secretase activity but not with a detectable increase in PS1 levels. Subcellular fractionation studies show that WT but not MT nicastrin matures into buoyant membrane fractions enriched in γ‐secretase activity. These data support the hypothesis that nicastrin is an essential component of the γ‐secretase complex. The finding that WT nicastrin overexpression can increase γ‐secretase activity without altering levels of the presumed catalytic component (PS) of the enzyme may point to a role for nicastrin in facilitating cleavage by regulating substrate interactions with the γ‐secretase complex.