In this elegant study, Iwata et al. injected a recombinant AAV vector (rAAV-NEP) into the hippocampus to achieve high-level neprilysin expression. Elevated intrahippocampal Aβ levels in neprilysin-deficient mice were almost completely reversed to wild-type levels by rAAV-NEP gene transfer. In young Tg2576 mutant APP mice, unilateral injection of rAAV-NEP resulted in a significant reduction in Aβ concentration in the ipsilateral hippocampus. Interestingly, the authors found that the transduced neprilysin was axonally transported from the ipsilateral to contralateral side, resulting in increased neprilysin activity and decreased Aβ levels in the contralateral hippocampus, as well. To determine whether rAAV-NEP gene transfer could reduce Aβ deposition in aged Tg2576 mice, 18-month-old mice were injected with the vector, and quantitative assessment of Aβ deposition was performed 12 weeks later. Aβ loads in rAAV-NEP-injected mice were reduced by 25 and 50 percent in the entire hippocampal formation and the ipsilateral stratum radiata, respectively, compared to mice injected with an...  Read more

In this elegant study, Iwata et al. injected a recombinant AAV vector (rAAV-NEP) into the hippocampus to achieve high-level neprilysin expression. Elevated intrahippocampal Aβ levels in neprilysin-deficient mice were almost completely reversed to wild-type levels by rAAV-NEP gene transfer. In young Tg2576 mutant APP mice, unilateral injection of rAAV-NEP resulted in a significant reduction in Aβ concentration in the ipsilateral hippocampus. Interestingly, the authors found that the transduced neprilysin was axonally transported from the ipsilateral to contralateral side, resulting in increased neprilysin activity and decreased Aβ levels in the contralateral hippocampus, as well. To determine whether rAAV-NEP gene transfer could reduce Aβ deposition in aged Tg2576 mice, 18-month-old mice were injected with the vector, and quantitative assessment of Aβ deposition was performed 12 weeks later. Aβ loads in rAAV-NEP-injected mice were reduced by 25 and 50 percent in the entire hippocampal formation and the ipsilateral stratum radiata, respectively, compared to mice injected with an inactivated form of neprilysin. These results, as well as those reported previously by Marr et al. and Leissring et al., demonstrate that enhanced neprilysin activity can decelerate Aβ deposition, providing strong rationale for a therapeutic approach for Alzheimer’s disease based on increasing the activity of Aβ-degrading proteases. Given that neprilysin is capable of cleaving a number of biologically relevant neuropeptides, the potential for mechanism-based toxicity associated with this approach clearly warrants further study. Animals transgenic for neprilysin are apparently fine, however, indicating that the approach may well be tolerated.

The fact that Aβ levels were reduced only partially in wild-type, neprilysin-deficient, or Tg2576 mice despite high levels of transduced neprilysin activity (>10-fold) suggests that Aβ is present in specific forms or specific cellular compartments that are not susceptible to cleavage by neprilysin. This is particularly evident in the studies of unilateral injection into young Tg2576 mice, which resulted in similar reductions in soluble Aβ in the ipsilateral and contralateral hippocampal formations, despite a much higher level of neprilysin activity on the ipsilateral side. AAV-transduced neprilysin was found to colocalize with synaptic and axonal markers, and likely degrades secreted Aβ, while other Aβ-degrading enzymes such as the ECEs (see Eckman et al., 2001, 2003) appear to degrade intracellular Aβ.

References:
Marr RA, Rockenstein E, Mukherjee A, Kindy MS, Hersh LB, Gage FH, Verma IM, Masliah E. Neprilysin gene transfer reduces human amyloid pathology in transgenic mice. J Neurosci. 2003 Mar 15;23(6):1992-6. Abstract

Leissring MA, Farris W, Chang AY, Walsh DM, Wu X, Sun X, Frosch MP, Selkoe DJ. Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron. 2003 Dec 18;40(6):1087-93. Abstract

Eckman EA, Watson M, Marlow L, Sambamurti K, Eckman CB. Alzheimer's disease beta-amyloid peptide is increased in mice deficient in endothelin-converting enzyme. J Biol Chem. 2003 Jan 24;278(4):2081-4. Epub 2002 Dec 02. Abstract

Eckman EA, Reed DK, Eckman CB. Degradation of the Alzheimer's amyloid beta peptide by endothelin-converting enzyme. J Biol Chem. 2001 Jul 6;276(27):24540-8. Epub 2001 May 03. Abstract

View all comments by Christopher Eckman
View all comments by Elizabeth Eckman

The manuscript by Iwata, Saido and colleagues provides the compelling demonstration that rAAV-mediated overexpression of neprilysin significantly reduces Aβ levels and decelerates amyloid deposition in brains of transgenic mice.

The most provocative finding of the study is that injection of rAAV encoding neprilysin into the entorhinal cortex leads to selective reductions in Aβ levels in the ipsilateral hippocampus, and selective diminution of amyloid deposition in the outer molecular layer of the dendate gyrus ipsilateral to the injection. In addition, there appeared to be a striking decrease in Aβ levels in the contralateral hippocampus, despite only a small increase in neprilysin activity in detergent extracts prepared from this tissue. Why are these findings of interest? The classical retrograde HRP labeling studies of Steward and Scoville, 1976 revealed that there are two major projection pathways for neurons in the entorhinal cortex. The first is an almost exclusively ipsilateral projection of layer II...  Read more

The manuscript by Iwata, Saido and colleagues provides the compelling demonstration that rAAV-mediated overexpression of neprilysin significantly reduces Aβ levels and decelerates amyloid deposition in brains of transgenic mice.

The most provocative finding of the study is that injection of rAAV encoding neprilysin into the entorhinal cortex leads to selective reductions in Aβ levels in the ipsilateral hippocampus, and selective diminution of amyloid deposition in the outer molecular layer of the dendate gyrus ipsilateral to the injection. In addition, there appeared to be a striking decrease in Aβ levels in the contralateral hippocampus, despite only a small increase in neprilysin activity in detergent extracts prepared from this tissue. Why are these findings of interest? The classical retrograde HRP labeling studies of Steward and Scoville, 1976 revealed that there are two major projection pathways for neurons in the entorhinal cortex. The first is an almost exclusively ipsilateral projection of layer II neurons, via the perforant pathway, to the outer molecular layer of the dentate gyrus; the second is a bilateral projection of layer III neurons to regio superior, which includes the distal dendrites of CA1 pyramidal cells and the subiculum. Thus, the data from Iwata et al. can be interpreted to suggest that neprilysin is axonally transported from the entorhinal cortex to presynaptic sites of established terminal fields where the protease encounters Aβ and degrades it. It is difficult to offer any other conclusion.

These observations offer support for our earlier studies (Buxbaum et al., 1998), in which we demonstrated that APP is axonally transported from the entorhinal cortex to the dentate gyrus where APP-CTFs accumulate, and more recently, for studies in which we observed a selective reduction of Aβ deposition in the outer molecular layers of the dentate gyrus following knife lesions of the perforant pathway (see Lazarov et al., 2002 in ARF related news story).

View all comments by Sangram Sisodia

Re: Causes of premature death in APP transgenic mice…and how to alleviate them.

The premature death of APP Tg mice is a practical problem—as anyone knows who has talked to biotech or pharma companies about licensing a strain of mice that lives “unpredictably.” But even more than that, it is a major scientific problem that was puzzling us in the mid 90’s, soon after entering this field.

Contrary to finding out what the underlying reasons were, the “executive summary” of the outcome reads rather simply: Premature death of APP Tg mice is caused by excitotoxicity, as shown by massive neuronal death in the hippocampus. I refer the interested readers to four of our publications (spear-headed by D. Moechars) listed below, directly or indirectly addressing the problem.

To a large extent, premature death was caused by environmental “stress” defined in its broadest sense, i.e., occasional or persistent infections, high background noise levels, poor training of animal caretakers and researchers entering the rooms, some room cohabitants (other strains of Tg mice). Premature...  Read more

Re: Causes of premature death in APP transgenic mice…and how to alleviate them.

The premature death of APP Tg mice is a practical problem—as anyone knows who has talked to biotech or pharma companies about licensing a strain of mice that lives “unpredictably.” But even more than that, it is a major scientific problem that was puzzling us in the mid 90’s, soon after entering this field.

Contrary to finding out what the underlying reasons were, the “executive summary” of the outcome reads rather simply: Premature death of APP Tg mice is caused by excitotoxicity, as shown by massive neuronal death in the hippocampus. I refer the interested readers to four of our publications (spear-headed by D. Moechars) listed below, directly or indirectly addressing the problem.

To a large extent, premature death was caused by environmental “stress” defined in its broadest sense, i.e., occasional or persistent infections, high background noise levels, poor training of animal caretakers and researchers entering the rooms, some room cohabitants (other strains of Tg mice). Premature death was and is “normalized” to that of other Tg (non-APP) strains and (nearly) to that of wild-type non-Tg strains by improving these conditions in our animal house, i.e., SPF or IVC cages, silence or soft background music, no “dilettante” but well-instructed animal caretakers and researchers handling the mice, strict application of sanitary rules, inverted day-night cycle, etc. This set of measures (not tested individually) appears to alleviate the environmental stress and thereby prevent premature death, but we occasionally see it reappear in the conventional animal rooms when the above conditions are not applied rigorously.

The data support a direct relation of APP-metabolites Aβ and β-CTF to augmented excitability of APP Tg mice and to excitotoxicity as the underlying cause for premature death. We have observed premature death only in APP Tg mice and not in the 50+ other Tg strains expressing different transgenes that we have generated and studied over the last 15 years. It is perfectly in line with the experimental demonstration that mutant presenilins increase Aβ levels and effectively decrease the threshold for excitotoxicity (Schneider et al., 2001).

That brings us full circle, since a decrease in Aβ-levels then must decrease premature death, as demonstrated by Leissring and coworkers. It is safe to predict that their double APP (x IDE or NEP) Tg mice also will have decreased excitotoxicity relative to the parent APP single Tg mice.

Intriguingly, neuronal deficiency of PS1 effectively decreases Aβ levels, but contributes negatively to neuronal excitability by increasing intracellular calcium store. This also implicates β-CTF, which are increased in PS1(n-/-) mice (Dewachter et al., 2002). One wonders what happens to the β-CTF in the double-Tg mice: Are they also chewed up by the overexpressed proteinases or not?

And I do want to quote from an old comment by Chris Exley (Submitted 16 November 2001) referred to in and below this story: “It would seem that the 'fog' has already begun to clear. It just went, apparently, unnoticed.” This notion pertains to many discoveries in the AD field these days!

References:
Moechars D, Lorent K, Dewachter I, Baekelandt V, De Strooper B, Van Leuven F. Transgenic mice expressing an alpha-secretion mutant of the amyloid precursor protein in the brain develop a progressive CNS disorder. Behav Brain Res 1998;95:55-64. Abstract

Moechars D, Dewachter I, Lorent K, Reverse D, Baekelandt V, Naidu A, Tesseur I, Spittaels K, Haute CV, Checler F, Godaux E, Cordell B, Van Leuven F. Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain. J Biol Chem 1999;274:6488-6492. Abstract

Moechars D, Lorent K, De Strooper B, Dewachter I, Van Leuven F. Expression in brain of amyloid precursor protein mutated in the alpha-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice. EMBO J 1996;15:1265-1274. Abstract

Moechars D, Lorent K, Van Leuven F. Premature death in transgenic mice that overexpress a mutant amyloid precursor protein is preceded by severe neurodegeneration and apoptosis. Neuroscience 1999;91:819-830. Abstract

Schneider I, Reverse D, Dewachter I, Ris L, Caluwaets N, Kuiperi C, Gilis M, Geerts H, Kretzschmar H, Godaux E, Moechars D, Van Leuven F, Herms J. Mutant presenilins disturb neuronal calcium homeostasis in the brain of transgenic mice, decreasing the threshold for excitotoxicity and facilitating long-term potentiation. J Biol Chem 2001;276:11539-11544. Abstract

Dewachter I, Reverse D, Caluwaerts N, Ris L, Kuiperi C, Van den Haute C, Spittaels K, Umans L, Serneels L, Thiry E, Moechars D, Mercken M, Godaux E, Van Leuven F. Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci 2002;22:3445-3453. Abstract

View all comments by Fred Van Leuven