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A comprehensive suite of rejuvenation biotechnologies must include the removal of extracellular aggregates from aging cells and tissues, particularly the brain. Recent work indicates that up-regulation of the activity of the native lysosomal pathway for clearance of beta-amyloid (Abeta) by the small molecule PADK can reverse existing Alzheimer-like pathology in mouse models, although caution is required in interpreting these results in the context of human disease.

As we have previously reviewed, a comprehensive suite of rejuvenation biotechnologies must include the removal of extracellular aggregates from aging cells and tissues. The most clinically-advanced such biotechnology is immunotherapy against aggregated beta-amyloid protein (Aβ), a characteristic neuropathological lesion that accumulates in the brain with age and contributes to  cognitive decline during “normal” brain aging and dementia in Alzheimer’s disease (AD).(0)

While  Aβ was originally identified neuropathologically in the form of the characteristic plaques that accumulate in the AD brain,  the view that soluble, intracellular oligomeric Aβ species are of equal or greater pathological significance has emerged and become dominant over the course of the last decade.(1-4) These Aβ species may be generated intraneuronally, or may be internalized from extracellular Aβ; irrespective of origin, evidence exists that the degradation of Aβ of either type may rely upon lysosomal hydrolysis, in both the neuron(2) and microglia,(6) and more surprisingly, that Aβ-targeting antibodies act via cellular uptake, followed by trafficking bound Aβ into the lysosome for disposal.

Dr. Ben Bahr and his colleagues with the Neurosciences Program and Division of Pharmaceutical Sciences at the University of Connecticut have for some time now been investigating the effects of elevating lysosomal activity using the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK). PADK  had previously been shown to reverse the AD-like abnormalities of the  induced in hippocampal slices treated with the lysosomal inhibitor chloroquine(7):

In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%.  … [C]hloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species , [restoration of synaptic composition(8)], and restored microtubule integrity.(7)

Similar lysosomal-endosomal, axonal, and synaptic pathology was later reported in the same model using  Aβ1–42,(8) and in vivo, intraperitoneal injections of PADK in rats were also shown to dose-dependently elevate forebrain levels of the lysosomal proteases cathepsin D by 50–100%, and cathepsin B by 40–80%.(8)

In a new study, Dr. Bahr’s group has extended this work into a transgenic mouse model of AD, testing PADK’s ability to retard, and to reverse, AD neuropathology and cognitive dysfunction in two models of transgenic AD mice: 10-11-month old APP(SwInd) mice, which  express a relatively low level of the parent strain transgene copy number and accumulate  relatively low levels of Aβ neuropathology, and  20–22 month old APPswe/PS1ΔE9 mice, with greater and longer-established burdens of Aβ. (6)

As in the hippocampal slice model, cathepsins B and D are upregulated in AD and aging brain, evidently to counteract rising levels of interneuronal Aβ; however, older APP(SwInd) mice fail to exhibit such an upregulation, suggesting that homeostatic responsiveness can be overcome .at high levels of proteotoxicity and/or that such a failure contributes to further degeneration in the older animals. Systemic PADK  injection of PADK in both models caused 3- to 8-fold increases in cathepsin B levels and similar elevations in the enzyme’s activity in lysosomal cell fractions, but did not affect other  Aβ-degrading enzymes (neprilysin and insulin-degrading enzyme). Vessicle-trafficking Rab protein levels were altered in vitro, without an effect on the lysosomal membrane protein LAMP-1, an essential receptor for chaperone-mediated autophagy.(6)

Accordingly PADK-induced lysosomal modulations cleared a significant amount of the intra- and extraneuronal burden of Aβ from treated mice, reducing intraneuronal Aβ regions of the hippocampus and piriform cortex by 63–73% in younger mice and by ~50% in older ones; in the latter animals, extracellular Aβ was concommitantly reduced by an impressive 76–85% (see Figure 1, below). Contrariwise, PADK had no  effect on recombinant human BACE1 activity, and cleavage products of α- and β-secretase were unchanged, weighing against modulation of Aβ production. Even as PADK lowered levels of the highly neurotoxic Aβ(1-42) species, it elevated levels of the  less pathogenic Aβ(1-38), suggesting that PADK-induced elevation of cathepsin B truncated Aβ(1-42) levels into a safer cleavage product.(6) These results are consistent with an earlier study,(9) not cited by the authors, which reported that virally-mediated overexpression of cathepsin B in aged model AD mice caused significant recession of existing plaque, associated with the production  of less-amyloidogenic   truncated Aβ peptides.

Figure 1. PADK decreases intra- and extraneuronal targeting antibody staining in aged APPswe/PS1ΔE9 mice. Reproduced from (6).

Model AD animals exhibited evidence of synaptic degeneration, in the form of 23–31% reductions in the synaptic markers synapsin II, synaptophysin, and GluA1 relative to wild type; PADK completely restored normal levels of GluA1, in both young and old AD mouse cohorts, and had similar effects on  synapsin II and synaptophysin. “The integrity of hippocampal dendritic fields was also found preserved in immunostained tissue sections, and the level of GluA1 immunoreactivity within each transgenic mouse correlated with the respective extent of cathepsin B enhancement in the brain.”(6)

As expected, model AD mice also exhibited substantial impairment of performance on cognitive-behavioral tests including the suspended rod, exploratory habituation, and spontaneous alternation behavior in a T-maze tests. PADK-induced Aβ clearance resulted in the complete restoration of normal function  in both young and old animals (Figure 2).

Figure 2. Rejuvenation of cognitive function in AD model mice. Reproduced from (6).

The results are surprisingly robust. Through a simple additive stimulation of the existing compensatory upregulation of the native lysosomal hydrolytic machinery,  intra- and even extraneuronal Aβ burden was substantially cleared, even in old animals with well-established disease. Moreover, the clearance was accompanied by the repair of disease-associated synaptic pathology, and a thorough rejuvenation of cognitive function. As a tribute to the power of the cellular waste-disposal machinery, these results are impressive.

Equally, they are not a solution to human brain aging. These animals, like most transgenic models of AD, exhibit no tau pathology nor significant neuronal loss — problems that will also have to be addressed in order to achieve the full prevention of AD and rejuvenation of aging brains. Constant stimulation of autophagy would be expected to lead to impaired protein synthesis, possibly contributing to sarcopenia, thrombocytopenia, or impaired wound healing or recovery from exercise, injury, or illness. Moreover, overexpression cathepsin B “has been associated with esophageal adenocarcinoma and other tumors.” And most profoundly, the very mechanism by which PADK lowers the Aβ burden in these animals — truncating the highly-amyloidogenic Aβ1–42 peptide into the less-aggregative Aβ1–38 — implies the limits of this therapy to maintain brain Aβ burden beneath the “threshold of pathology:” as with other such approaches involving the modulation of metabolic pathways so as to reduce their production of reactive metabolites, a chronic reduction in the rate of accumulation of damaging metabolic byproducts can only retard the course of the pathology. To permanently forestall, or even reverse, the course of the degenerative aging process in the brain as elsewhere requires therapies that directly remove, repair, replace, or render harmless such damage, maintaining or restoring the cellular and molecular structural integrity of the body and thus youthful and healthy functionality.

But these results still deserve to be highlighted for illustrating the potential of a true damage-removal strategy, such as  bolstering the inadequate native complement’s ability to degrade Aβ in neurons and microglia with novel lysosomal hydrolases that fully degrade Aβ peptides  without altering the activity of the endosomal-lysosomal system itself. And because  the effectiveness of Aβ-targeting immunotherapy ultimately rests on the ability of the lysosome to degrade antibody-bound Aβ, the results also emphasize the potential of a fortified lysosome to enhance the effectiveness of a true damage-removal approach to rejuvenate the aging brain.


0: Lemere CA, Masliah E. Can Alzheimer disease be prevented by amyloid-beta immunotherapy? Nat Rev Neurol. 2010 Feb;6(2):108-19.  PubMed PMID: 20140000; PubMed Central PMCID: PMC2864089.

1: Ono K, Yamada M. Low-n oligomers as therapeutic targets of Alzheimer’s disease. J Neurochem. 2011 Apr;117(1):19-28. doi: 10.1111/j.1471-4159.2011.07187.x. Epub 2011 Feb 9. Review. PubMed PMID: 21244429.

2: Tampellini D, Gouras GK. Synapses, synaptic activity and intraneuronal abeta in Alzheimer’s disease. Front Aging Neurosci. 2010 May 21;2. pii: 13. PubMed PMID: 20725518; PubMed Central PMCID: PMC2912028.

3: Klyubin I, Betts V, Welzel AT, Blennow K, Zetterberg H, Wallin A, Lemere CA, Cullen WK, Peng Y, Wisniewski T, Selkoe DJ, Anwyl R, Walsh DM, Rowan MJ. Amyloid beta protein dimer-containing human CSF disrupts synaptic plasticity: prevention by systemic passive immunization. J Neurosci. 2008 Apr 16;28(16):4231-7. PubMed PMID: 18417702; PubMed Central PMCID: PMC2685151.

4: Walsh DM, Selkoe DJ. A beta oligomers – a decade of discovery. J Neurochem. 2007 Jun;101(5):1172-84. Epub 2007 Feb 5. Review. PubMed PMID: 17286590.

5: Butler D, Hwang J, Estick C, Nishiyama A, Kumar SS, Baveghems C, Young-Oxendine HB, Wisniewski ML, Charalambides A, Bahr BA. Protective effects of positive lysosomal modulation in Alzheimer’s disease transgenic mouse models. PLoS One. 2011;6(6):e20501. Epub 2011 Jun 10. PubMed PMID: 21695208; PubMed Central PMCID: PMC3112200.

6: Yang CN, Shiao YJ, Shie FS, Guo BS, Chen PH, Cho CY, Chen YJ, Huang FL, Tsay HJ. Mechanism mediating oligomeric Aβ clearance by naïve primary microglia. Neurobiol Dis. 2011 Jun;42(3):221-30. Epub 2011 Jan 8. PubMed PMID: 21220023.

7: Bendiske J, Bahr BA. Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis–an approach for slowing Alzheimer disease? J Neuropathol Exp Neurol. 2003 May;62(5):451-63. PubMed PMID: 12769185.

8: Butler D, Brown QB, Chin DJ, Batey L, Karim S, Mutneja MS, Karanian DA, Bahr BA. Cellular responses to protein accumulation involve autophagy and lysosomal enzyme activation. Rejuvenation Res. 2005 Winter;8(4):227-37. PubMed PMID: 16313222.

9: Mueller-Steiner S, Zhou Y, Arai H, Roberson ED, Sun B, Chen J, Wang X, Yu G, Esposito L, Mucke L, Gan L. Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer’s disease. Neuron. 2006 Sep 21;51(6):703-14. PubMed PMID: 16982417.

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