Donna M. Romano

Candidate gene for the chromosome 1 familial Alzheimer's disease locus

A candidate gene for the chromosome 1 Alzheimers disease (AD) locus was identified (STM2). The predicted amino acid sequence for STM2 is homologous to that of the recently cloned chromosome 14 AD gene (S182). A point mutation in STM2, resulting in the substitution of an isoleucine for an asparagine (N141l), was identified in affected people from Volga German AD kindreds. This N141l mutation occurs at an amino acid residue that is conserved in human S182 and in the mouse S182 homolog. The presence of missense mutations in AD subjects in two highly similar genes strongly supports the hypothesis that mutations in both are pathogenic.

DRAMATIC AGGREGATION OF ALZHEIMER ABETA BY CU(II) IS INDUCED BY CONDITIONS REPRESENTING PHYSIOLOGICAL ACIDOSIS

The cortical deposition of Aβ is an event that occurs in Alzheimer’s disease, Down’s syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Aβ, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Aβ at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Aβ1–40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Aβ1–40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Aβ1–40 and Aβ1–42 with submicromolar concentrations of Cu2+. Unlike Aβ1–40, Aβ1–42was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Aβ1–40 and histidine-modified human Aβ1–40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Aβ assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Aβ that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Aβ aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Aβ explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer’s disease.

Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate α-secretase activity

ADAM10, a member of a disintegrin and metalloprotease family, is an alpha-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimers disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated alpha-secretase activity of ADAM10 (>70% decrease), and elevated Abeta levels (1.5-3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.

ADAM10 Missense Mutations Potentiate β-Amyloid Accumulation by Impairing Prodomain Chaperone Function

The generation of Aβ, the main component of senile plaques in Alzheimers disease (AD), is precluded by α-secretase cleavage within the Aβ domain of the amyloid precursor protein (APP). We identified two rare mutations (Q170H and R181G) in the prodomain of the metalloprotease, ADAM10, that cosegregate with late-onset AD (LOAD). Here, we addressed the pathogenicity of these mutations in transgenic mice expressing human ADAM10 in brain. In Tg2576 AD mice, both mutations attenuated α-secretase activity of ADAM10 and shifted APP processing toward β-secretase-mediated cleavage, while enhancing Aβ plaque load and reactive gliosis. We also demonstrated ADAM10 expression potentiates adult hippocampal neurogenesis, which is reduced by the LOAD mutations. Mechanistically, both LOAD mutations impaired the molecular chaperone activity of ADAM10 prodomain. Collectively, these findings suggest that diminished α-secretase activity, owing to LOAD ADAM10 prodomain mutations, leads to AD-related pathology, strongly supporting ADAM10 as a promising therapeutic target for this devastating disease.

Ubiquilin 1 Modulates Amyloid Precursor Protein Trafficking and Aβ Secretion

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with α-, β-, or γ-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (α- and β-forms). Moreover, UBQLN1 knockdown increased levels of secreted Aβ40 and Aβ42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic “gatekeeper” that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Aβ.

Alzheimer's disease-associated ubiquilin-1 regulates presenilin-1 accumulation and aggresome formation

The Alzheimers disease (AD)‐associated ubiquilin‐1 regulates proteasomal degradation of proteins, including presenilin (PS). PS‐dependent γ‐secretase generates β‐amyloid (Aβ) peptides, which excessively accumulate in AD brain. Here, we have characterized the effects of naturally occurring ubiquilin‐1 transcript variants (TVs) on the levels and subcellular localization of PS1 and other γ‐secretase complex components and subsequent γ‐secretase function in human embryonic kidney 293, human neuroblastoma SH‐SY5Y and mouse primary cortical cells. Full‐length ubiquilin‐1 TV1 and TV3 that lacks the proteasome‐interaction domain increased full‐length PS1 levels as well as induced accumulation of high‐molecular‐weight PS1 and aggresome formation. Accumulated PS1 colocalized with TV1 or TV3 in the aggresomes. Electron microscopy indicated that aggresomes containing TV1 or TV3 were targeted to autophagosomes. TV1‐ and TV3‐expressing cells did not accumulate other unrelated proteasome substrates, suggesting that the increase in PS1 levels was not because of a general impairment of the ubiquitin‐proteasome system. Furthermore, PS1 accumulation and aggresome formation coincided with alterations in Aβ levels, particularly in cells overexpressing TV3. These effects were not related to altered γ‐secretase activity or PS1 binding to TV3. Collectively, our results indicate that specific ubiquilin‐1 TVs can cause PS1 accumulation and aggresome formation, which may impact AD pathogenesis or susceptibility.

RNA Interference-mediated Silencing of X11α and X11β Attenuates Amyloid β-Protein Levels via Differential Effects on β-Amyloid Precursor Protein Processing

Processing of the β-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by β- and α-secretase to produce APP-C99 and APP-C83, which are further cleaved by γ-secretase to produce amyloid β-protein (Aβ) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11α (MINT1, encoded by gene APBA1) and X11β (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11α and X11β alters APP processing and Aβ production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11α and X11β expression on APP processing and Aβ production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Aβ levels, but apparently not via attenuation of γ-secretase cleavage of APP. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor protein X11α is particularly attractive with regard to therapeutic potential given that side effects of γ-secretase inhibition due to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.

Increased recombination adjacent to the Huntington disease-linked D4S10 marker

Huntington disease (HD) is caused by a genetic defect distal to the anonymous DNA marker D4S10 in the terminal cytogenetic subband of the short arm of chromosome 4 (4p16.3). The effort to identify new markers linked to HD has concentrated on the use of somatic cell hybrid panels that split 4p16.3 into proximal and distal portions. Here we report two new polymorphic markers in the proximal portion of 4p16.3, distal to D4S10. Both loci, D4S126 and D4S127, are defined by cosmids isolated from a library enriched for sequences in the 4pter-4p15.1 region. Physical mapping by pulsed-field gel electrophoresis places D4S126 200 kb telomeric to D4S10, while D4S127 is located near the more distal marker D4S95. Typing of a reference pedigree for D4S126 and D4S127 and for the recently described VNTR marker D4S125 has firmly placed these loci on the existing linkage map of 4p16.3. This genetic analysis has revealed that the region immediately distal to D4S10 shows a dramatically higher rate of recombination than would be expected based on its physical size. D4S10-D4S126-D4S125 span 3.5 cM, but only 300-400 kb of DNA. Consequently, this small region accounts for most of the reported genetic distance between D4S10 and HD. By contrast, it was not possible to connect D4S127 to D4S125 by physical mapping, although they are only 0.3 cM apart. A more detailed analysis of recombination sites within the immediate vicinity of D4S10 could potentially reveal the molecular basis for this phenomenon; however, it is clear that the rate of recombination is not continuously increased with progress toward the telomere of 4p.

Clustering of multiallele DNA markers near the Huntington's disease gene.

Five highly informative multiallele restriction fragment length polymorphisms (RFLPs) of value for preclinical diagnosis of Huntingtons disease (HD) have been genetically characterized. One RFLP was uncovered by expansion of the D4S43 locus while three others are at D4S111 and D4S115, loci defined by NotI-linking clones. The final marker, D4S125, represents a recently discovered VNTR locus. All four loci map closer to the HD gene and to the telomere than D4S10, the original linked marker for HD. In combination with two multiallele RFLPs previously identified for D4S43 and another linked locus, D4S95, these five new multiallele markers will dramatically improve the speed and accuracy of predictive testing in HD, and increase its applicability by maximizing the chances of an informative test for anyone with appropriate family structure.

Mapping of cosmid clones in Huntington's disease region of chromosome 4

Huntingtons disease (HD) is tightly linked to genetic markers in 4p16.3. We have used a regional somatic cell hybrid mapping panel to isolate and map 25 cosmids to the proximal portion of 4p16.3 and 17 cosmids to the distal portion. The latter were positioned by long-range restriction mapping relative to previously mapped markers. One cosmid, L6 (D4S166), spans the critical breakpoint in the mapping panel that distinguishes proximal and distal 4p16.3. Four of the cosmids mapped distal toD4S90, the previous terminal marker on 4p, and stretched to within 75 kb of the telomere. Several of the cosmids that mapped between L6 andD4S90 were clustered near a number of previously isolated clones in a region with many NotI sites. Cosmid E4 (D4S168) was localized immediately proximal to the one remaining gap in the long-range restriction map of distal 4p16.3. Although pulsed field gel mapping with E4 failed to link the two segments of the map, the intervening gap was excluded as a potential site for theHD gene by genetic analysis.