SENS Foundation - Advancing Rejuvenation Biotechnologies

Announcing New Summer Internships

Daniel Kimbel — Tue, 31 Jan 2012 22:10:05 +0000

In the summer of 2012, the Academic Initiative will bring as many as three interns to the SENS Foundation Research Center in Mountain View, California to participate in SENS research for three months. These interns will receive monthly stipends and, if they are not local to the San Francisco Bay Area, a credit towards airfare.

Undergraduate, graduate, and medical students are all encouraged to apply. Applications from people who are not presently working towards university degrees will also be considered. After an initial selection process, the most promising candidates will be interviewed over the phone by the SENS researchers they would work with. Each major research program at the Research Center will limit itself to one intern, such that each intern will be working on a different project and will be selected by different researchers. It will be important for applicants to have prior lab experience, and more experienced applicants are more likely to be accepted than relatively inexperienced ones.

The application is available online here. Applications are due by March 31, 2012. The most promising applicants will be interviewed in April. As always, if you have any questions, you can contact us.

Welcome to the Academic Initiative's New Site

Daniel Kimbel — Tue, 03 Jan 2012 23:49:43 +0000

Welcome to the SENS Foundation Academic Initiative’s new website. In addition to containing more comprehensive information about the Initiative and what it does, this site offers a number of new features, including a listing of outreach projects and a searchable database of member profiles. The site leaves plenty of room for the Initiative to grow into, thanks to its new committee pages, its new media section, and its more streamlined navigation.

The single greatest feature of this new website -- an extension and enhancement of an overlooked, underused feature of our previous site -- is its ability to offer our members their own spaces. Each research project, outreach project, and volunteer committee has its own updatable page, so that people who are part of any given project or committee can edit and care for their own corner of our site.

With the launch of this new website and our greatly increased levels of student funding in 2012, now is the best time in the Initiative's history to become involved with the program. You can apply for membership here or apply to volunteer here.

NFT-Specific Tau Vaccine Arrests Tangle Progress

Michael Rae — Mon, 02 Jan 2012 02:37:46 +0000

Immunotherapy targeting the age-related accumulation of extracellular aggregates, in the form of ß-amyloid, is the first rejuvenation biotechnology to reach Phase III human clinical trials. The promise of this therapy for the treatment and prevention of Alzheimer's disease (and ultimately, of so-called "normal" brain aging) has sparked an interest in utilizing the same approach for other forms of aging damage, including the clearance of aggregated intracellular proteinaceous aging damage. Notably, as we have reviewed in a series of four previous posts, recent years have seen the appearance of a rising number preclinical studies of therapeutic vaccines targeting pathological tau species accumulating in the brains and spinal cords of transgenic rodent models of tauopathic neurodegeneration. These studies have reported -- somewhat surprisingly -- the antibody-mediated clearance of these primarily intracellular aging lesions, accompanied by functional improvements in treated animals.(4-8) These two forms of structural damage are major contributors to the age-related degeneration of the brain, whether it leads to frank dementia or to the diagnostic euphemism of "normal" age-related cognitive decline, and novel therapeutics to effect the removal of both from aging neurons will be key elements of a comprehensive panel of rejuvenation biotechnologies.

One limitation to the existing studies of vaccines targeting pathological tau has been that they have tended to initiate therapy before, at, or only very shortly after the appearance of tau pathology in the murine rodent system, and of the onset of cognitive or motor deficits -- a luxury which we cannot expect to be afforded in treating cases of frank dementia related to these inclusions, and that may not reflect the condition of the brain even prior to the appearance of gross cognitive and functional deficits in aging humans. Further complicating these matters, the combination of early intervention and the very rapid progression of tau pathology in these models has meant that these studies have given only limited information on the effects of the onset or later clearance of neurofibrillary tangles (NFT), which are the most prominent and mature form of tau pathology to appear in the brain during aging, in Alzheimer's disease, and with the genetic tauopathies. A group headed by Dr. Lars Ittner of the Laboratory for Translational Neurodegeneration at the University of Sydney, Camperdown has now provided the first evidence on these unresolved questions, furthering the case for human therapies targeting pathological tau.

Ittner's group performed their studies in pR5 mice, which express the mutant human tau species P301L, which is responsible for the human dementing tauopathy frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). These mice begin to accumulate tau that has been hyperphosphorylated at different epitopes early in life, but phosphorylation of the NFT-associated Ser422 and Ser396/S404 (the paired helical fragment-1 (PHF-1) epitope) first begins to appear at the 6 mo mark, accompanied by NFT pathology, which progresses thereafter throughout their shortened lifespans. The investigators accordingly engineered a vaccine targeting these NFT-associated species by linking a human tau peptide comprising phosphorylated PHF-1 Ser396/Ser404 to the immunogenic carrier protein KLH. This vaccine was then administered to animals before (4 mo), shortly after (8 mo), and long after (18 mo) the 6-month age of NFT onset, accompanied by complete Freund's adjuvant, and later followed by further adjuvant booster.(1)

By 17 mo, hippocampal and amygdalic neurons of untreated mice exhibited high levels of antibody staining for phorphoylated NFT-associated tau epitopes; even more notably, staingin was also high in the dystrophic neurites in subregions of these areas. Immunization targeting NFT tau epitopes had no effect on expression or distribution of either human mutant or total (transgenic plus native murine native) tau, but did result in robust (≥1:1000) antigen titers within four months of vaccination. Notably, even untreated pR5 mice exhibited low levels of such titers, while no such antibodies were evident in isochronic wild-type mice; this suggests a (weak) intrinsic immune response against the presence of aberrant tau species in mice bearing them.(1) The existence of such a response is consistent with previous findings in aging humans,(3) Alzheimer's disease patients,(3) and transgenic murine tauopathy.(2)

Tau-targeting vaccination substantially reduced the subsequent burden of pathological tau in neurons and dystrophic neurites, no matter when treatment was initiated:

Figure 1: Vaccination targeting pathological tau species reduces the further spread of antibody-labeled PHF-1 (left) and tau phosphorylated at Ser422 (center) when initiated shortly after NFT appearance; when administered before (Group I), shortly after (Group II), or long after (Group III) their appearance, it similarly impeded the appearance of Gallyas silver-stained NFTs per se (right),. Reproduced from (1) under the terms of Creative Commons License.

Limitations, and their Supercedance
One key limitation to this study is that no cognitive or motor function data are presented. In prior studies, however, preventive and therapeutic vaccination in similar murine models of tau neuropathy has led to relative improvements in such functions, proportional to the reduction in neuropathology.

Another limitation is of interpretation of the results, and possibly of the actual effects of the vaccine therapy itself. The authors note that the burden of NFT in treated animals from Group III -- aged 22 mo at autopsy, but whose immunization was delayed until 18 mo of age (ie, a full year after the onset of neuropathology) -- was similar to that in untreated mice shortly before (Group I) or shortly after (Group II) that onset at autopsy. Because autopsied Group II animals were of similar age to the the age at which vaccination was initiated in Group III, and unvaccinated Group II animals' neuropathology burden was similar to that present months after treatment in animals that were initially treated at a similar age, the authors suggest that the apparently similar NFT burdens after several months of vaccination in the latter group suggest that the treatment had arrested the further accumulation of tau pathology following vaccination, rather than having actively cleared it. However, as they also note, it is also possible that their tau vaccine did remove existing tau inclusions, but that the rate of active clearance of tau pathology was equal to the rate of new NFT accumulation.(1) This is quite plausible, granted the very rapid progression of tau pathology in these animals when left untreated, and even more so granted their relatively brief period of treatment: Group III was sufficiently far into their shortened life expectancy as to necessitate abbreviated (6 mo) therapy, relative to the 9-10 mo afforded to Groups I and II. Thus, a longer period of antibody-mediated clearance might have afforded a more thorough therapeutic effect.

At a minimum, even a vaccine that only arrested the burden of tau pathology could contribute to the prevention of significant age-related cognitive decline if administered in middle age. And in aging humans, the development of neurofibrillary tangles occurs over the course of decades, rather than months as in these animals; thus, even a moderate rate of true clearance could in principle be sufficient to lead to a net reduction in NFT burden in humans, and thus to the reversal of one component of brain aging.

In either case, several strategies could be explored to enhance the efficacy of such a vaccine. The most obvious is the administration of a series of booster shots, to increase therapeutic antibody titers or to restore putatively flagging numbers in the months (or in humans, perhaps years) after initial vaccination. Note that the investigators only collected a single data point on antibodies targeting pathological tau, taken relatively soon (4 mo) after a single vaccination at a single antigen dose, following which the same, single round of vaccination was left to work for a further 6 (Groups 1 and II) or 2 (Group III) months in younger and older animals, respectively; this clearly leaves open the potential that a series of rounds of treatment could have been more effective.

Another potential way to enhance the efficacy of a similar vaccine in humans would be to fortify the lysosome with novel hydrolytic enzymes, to enhance its capacity to clear out aggregates which might be targeted to the lysosome by anti-phospho-tau antibodies. While the involvement of lysosomal degradation of aberrant tau species in the therapeutic effects of immunotherapy was not evaluated in this study,(1) such an effect is consistent with the clearance of intraneuronal phospho-tau in these vaccination studies, and with much other data. Vaccination of a Parkinson's disease transgenic mouse model with α-synuclein, similarly, leads to a reduction of intracellular α-synuclein aggregates;(11) several studies have found that immunotherapy targeting β-amyloid leads to a reduction in intraneuronal forms of the aggregate, and work in Gunnar Gouras' laboratory, reviewed in an earlier post, finds that antibodies against Aβ can be internalized in AD neuronal culture models of Aβ accumulation and clear intraneuronal Aβ aggregates via the endosomal–lysosomal pathway.Additionally, the authors of the current study(1) also note that immunized K257T/P301S tauopathy mice exhibit changes in cathepsin levels, consistent with lysosomal degradation of pathological tau;(6) in this context, it is notable that the lysosomal-enhancing drug PADK (see a previous post) removes PHF-1 from hippocampal slices, in association with a normalization of impaired synaptic integrity.(10,11) I will note that the authors of the current study(1) do argue against such a mechanism, on the basis that "total tau levels remained unchanged upon immunization of JNPL3 mice [(5) below], and staining with total tau antibodies was comparable in immunized and control pR5 mice (this study), arguing against overtly increased degradation of tau" -- but of course, this does not argue against specific degradation of pathological tau species targeted by immunotherapy in their study.(1)

An additional possibility is that the clearance of other intraneuronal aggregates from aging neurons may itself facilitate the additional proteolytic or lysosomal removal of phospho-tau species. The relationship between the accumulation and neurotoxicity of Aβ and aberrant tau is complex,(13) but reducing one will almost certainly reduce the deleterious effects of the other; and, in particular, immunization against Aβ has been reported to lead to the clearance of early phospho-tau species.(14 (and see (15)) This adds a further argument in favor of a comprehensive approach to brain aging and Alzheimer's disease, suggesting synergistic rather than merely additive effects of combining therapies targeting Aβ in addition to malformed tau.

Finally, it is also possible that future vaccines might be more specifically targeted to especially pathologically important tau species, or be designed to elicit antibodies with higher avidity for their target. All of these avenues deserve exploration in future studies of this promising therapeutic approach.

Breaking from a March to a Run
This latest report(1) adds to a series of previous successful preclinical studies(4-8) of immunotherapies targeting pathological tau species for therapeutic clearance. This new study is notable for marking the entry of a new group of investigators into the tau vaccine space, suggesting that this area of rejuvenation biotechnology is attracting an increasing number of independent research teams. While limited in scope and in design, the new study adds to prior knowledge not only by testing a new vaccine, but importantly by evaluating its effects in animals in whom damage from tau inclusions was already extensive. While cognitive and motor functions were not evaluated, the clear ability of this vaccine to arrest -- and perhaps to reverse -- the burden of a life-long, accelerated deposition of aggressive pathological tau species offers promise for human translation into a treatment for genetic tauopathies, Alzheimer's disease, and the "normal" age-related decay of cogntitive, emotional, and neurological function that is only now finally being recognized as a disorder meriting therapy -- and ultimately, cure. The gaps in this study are opportunities for further experimentation, further refinement, further learning, and further therapeutic development; it marks progress, bringing us closer to the goal of a comprehensive panel of rejuvenation biotechnologies and the rescue of aging bodies and minds.

References

1: Bi M, Ittner A, Ke YD, Götz J, Ittner LM. Tau-Targeted Immunization Impedes Progression of Neurofibrillary Histopathology in Aged P301L Tau Transgenic Mice. PLoS One. 2011;6(12):e26860. Epub 2011 Dec 8. PubMed PMID: 22174735; PubMed Central PMCID: PMC3234245.

2: Rosenmann H, Grigoriadis N, Karussis D, Boimel M, Touloumi O, Ovadia H, Abramsky O. Tauopathy-like abnormalities and neurologic deficits in mice immunized with neuronal tau protein. Arch Neurol. 2006 Oct;63(10):1459-67. PubMed PMID: 17030663.

3: Rosenmann H, Meiner Z, Geylis V, Abramsky O, Steinitz M. Detection of circulating antibodies against tau protein in its unphosphorylated and in its neurofibrillary tangles-related phosphorylated state in Alzheimer's disease and healthy subjects. Neurosci Lett. 2006 Dec 20;410(2):90-3. PubMed PMID: 17095156.

4: Rosenmann H, Grigoriadis N, Karussis D, Boimel M, Touloumi O, Ovadia H, Abramsky O. Tauopathy-like abnormalities and neurologic deficits in mice immunized with neuronal tau protein. Arch Neurol. 2006 Oct;63(10):1459-67. PubMed PMID: 17030663.

5: Asuni AA, Boutajangout A, Quartermain D, Sigurdsson EM. Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements. J Neurosci. 2007 Aug 22;27(34):9115-29. PMID: 17715348 [PubMed - in process]

6: Boimel M, Grigoriadis N, Lourbopoulos A, Haber E, Abramsky O, Rosenmann H. Efficacy and safety of immunization with phosphorylated tau against neurofibrillary tangles in mice. Exp Neurol. 2010 Aug;224(2):472-85. Epub 2010 May 28. PubMed PMID: 20546729.

7: Boutajangout A, Ingadottir J, Davies P, Sigurdsson EM. Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain. J Neurochem. 2011 Aug;118(4):658-67. doi: 10.1111/j.1471-4159.2011.07337.x. Epub 2011 Jul 1. PubMed PMID: 21644996.

8: Boutajangout A, Quartermain D, Sigurdsson EM. Immunotherapy targeting pathological tau prevents cognitive decline in a new tangle mouse model. J Neurosci. 2010 Dec 8;30(49):16559-66. PubMed PMID: 21147995.

9: Wisniewski T, Sigurdsson EM. Murine models of Alzheimer's disease and their use in developing immunotherapies. Biochim Biophys Acta. 2010 Oct;1802(10):847-59. Epub 2010 May 13. PubMed PMID: 20471477; PubMed Central PMCID: PMC2930136.

10: 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.

11: 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.

12: Masliah E, Rockenstein E, Adame A, Alford M, Crews L, Hashimoto M, Seubert P, Lee M, Goldstein J, Chilcote T, Games D, Schenk D. Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease. Neuron. 2005 Jun 16;46(6):857-68. PMID: 15953415 [PubMed - indexed for MEDLINE]

13: Ittner LM, Götz J. Amyloid-β and tau--a toxic pas de deux in Alzheimer's disease. Nat Rev Neurosci. 2011 Feb;12(2):65-72. Epub 2010 Dec 31. Review. PubMed PMID: 21193853.

14: Oddo S, Billings L, Kesslak JP, Cribbs DH, LaFerla FM. Abeta immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome. Neuron. 2004 Aug 5;43(3):321-32. PMID: 15294141 [PubMed - indexed for MEDLINE]

15: Oddo S, Caccamo A, Tran L, Lambert MP, Glabe CG, Klein WL, LaFerla FM. Temporal profile of amyloid-beta (Abeta) oligomerization in an in vivo model of Alzheimer disease. A link between Abeta and tau pathology. J Biol Chem. 2006 Jan 20;281(3):1599-604. Epub 2005 Nov 10. PMID: 16282321 [PubMed - indexed for MEDLINE]

Holiday Appeal

Mike Kope — Sat, 24 Dec 2011 09:18:59 +0000

December 2011

Dear Friends,

The diseases of aging -- heart disease, Alzheimer's, macular degeneration -- are not only terrible, tragic, and debilitating, but potentially preventable. SENS Foundation is working to use regenerative medicine to repair the cellular and molecular damage that accumulates in all of our bodies over time. With the right application of such treatments, we could slow, or even reverse, the pathology of aging.

We call these innovative, damage-repairing treatments rejuvenation biotechnologies. In human and economic terms, their successful development would represent an incredible victory.

And yet, research into rejuvenation biotechnologies has received virtually no public funding. SENS Foundation has yet to receive monetary support from any government body. Other organizations in the field are in a similar position -- the Mayo Clinic, for instance, was recently denied funding by the NIH for follow-up work on its landmark senescent cell study published in Nature.

If rejuvenation biotechnologies are to be developed, private donors will need to step forward. SENS Foundation has led, and continues to lead, the charge towards robust therapies that address the diseases of aging. We are working ceaselessly to advance rejuvenation biotechnologies in the following ways:

We fund scientific work at universities and research institutes across the world -- including, next year, a new project and lab at Cambridge University.
We conduct our own in-house research at the rapidly-expanding SENS Foundation Research Center in Mountain View, California, which has nearly doubled its staff in 2011.
We develop the next generation of researchers focused on the development of rejuvenation biotechnologies through our Academic Initiative, which continues to award research grants to talented and enthusiastic university students.
We conduct outreach and forge new connections between individuals and organizations to grow the rejuvenation biotechnology field.
We hold conferences, most recently SENS5 in September of this year, to bring some of the world's most capable scientists together in one room to discuss the future of medicine.

SENS Foundation is a pioneer in the fledgling industry of rejuvenation biotechnology. We have a highly talented and ever-growing team of researchers, expanding facilities, and considerable experience in the field. Out work has the potential to make modern treatments for age-related diseases markedly more effective, but is gravely underfunded. For this reason, your contribution could have a huge impact on our organization and, as a result, on medicine itself.

We deeply appreciate the donations we have received thus far, and urge you to consider contributing to our cause. We know of none greater: that is why we have focused our careers on overcoming the diseases of aging. Either way -- whether or not you choose to give -- please accept our whole team's warmest holiday wishes.

Yours,

Mike and Aubrey

Click below to contribute

$30,000 in Academic Initiative Grants

Daniel Kimbel — Thu, 08 Dec 2011 01:22:01 +0000

The SENS Foundation Academic Initiative is pleased to announce that it will be awarding up to $30,000 in materials grants in 2012. These grants are available to undergraduate, graduate, and medical students, and may be used to cover the cost of laboratory materials for aging- and rejuvenation-related research projects. A typical grant will range from $500-$2000, but grants of up to $5000 may be awarded for group projects. These grants are meant to provide students with valuable experience in research and leadership, and to help set recipients on the course to a career in SENS-related research. As such, simple and straightforward "introductory-level" projects will receive full consideration.

The grant application can be found here. You can apply at any time. There may be a high level of competition, so students are encouraged to apply soon.

How to Disable a Cellular Bomb: Findings and Tools on the Machinery of ALT

Michael Rae — Sat, 03 Dec 2011 02:04:09 +0000

As discussed previously,

To develop an unbreachable defense against cancer, SENS Foundation is pursuing the WILT (Wholebody Interdiction of Lengthening of Telomeres) strategy (otherwise OncoSENS) of systematically deleting genes essential to the cellular telomere-maintenance mechanisms (TMM) from all somatic cells, while ensuring ongoing tissue repair and maintenance through periodic re-seeding of somatic stem-cell pools with autologous TMM-deficient cells whose telomeres have been lengthened ex vivo. In addition to the deletion of one or more genes coding for essential element(s) of the telomerase holoenzyme, success will also require the deletion of some essential element of the machinery [responsible] for the Alternative Lengthening of Telomeres (ALT) phenomenon, observed in a minority of cancer cells.

One of the characteristic phenotypic features of cells utilizing the ALT mechanism is the presence of telomeric DNA and the telomere binding proteins TERF1 and TERF2 in close association with a subset of the cell's promyelocytic leukaemia protein (PML) nuclear bodies. PML nuclear bodies are spherical nuclear structures which, in normal cells, are involved in DNA repair, senescence, apoptosis, and other functions. But their association with telomeric chromatin is rare, except in ALT cells, where they are instead hallmarks of the phenomenon; this has led to such assemblies being designated "ALT-associated PML bodies" (APBs). APBs not only contain recombination proteins demonstrated to be essential to telomere maintenance in ALT cells, but appear themselves to be involved in telomere recombination(1) and to coincide spatially and temporally with telomeric DNA synthesis.(2,3) Therefore, some have hypothesized that APBs may be the nexus of the “Alternative Lengthening of Telomeres” in such cells.

To test whether APBs might indeed be causally involved in the TMM of ALT cells, a team of German researchers devised the novel approach of generating APBs artificially, by recruiting APB subcomponents to telomeric or pericentric DNA, and observing the effects on assembly of APB-like structures and on telomere elongation.(4) To do this, the investigators generated fusion proteins of several APB constituents with the bacterial LacI repressor protein and fluorescent binding proteins as markers, and then exploited LacI's high-affinity binding with the operator region of lac operons to tether these proteins to the repeats of lac operator sequences that are stably integrated into the genome of U2OS osteosarcoma cell lines. To reveal the effects of chromosomal localization on such proteins' ability to form APBs and on the abilities of such experimentlaly-induced entities, the researchers tested the effects of APB constituent protein tethering in two different U2OS cell lines: one (F6B2) with such operator sequences integrated adjacent to telomeric DNA, and another (F42B8) whose sequences were localized pericentrically. Thus, transfection of one or the other cell line with LacI-APB component fusion constructs resulted in the tethering of the various APB component proteins at locations proximate to, or remote from, telomeric DNA.(4)

Bring a Friend
When tethered to telomeric DNA, LacI fusion constructs containing either of the two proteins (PML protein or Sp100) present in PML nuclear bodies, induced recruited the complementary protein in turn; tethering of "empty" fluorescent-labeled LacI constructs yielded no such results.(4) The bringing-together of these proteins at telomeric DNA induced the formation of apparent APBs, as suggested by the observations that (a) the PML protein assumed the same cap-like structures surrounding telomere repeats observed in APBs; (b) the structures appeared almost entirely localized with single telomere structures; and (c) the location of the recruited endogenous proteins appeared enriched with several SUMO isoforms, consistent with the presence of SUMO modifications of these proteins in endogenous PML nuclear bodies.(5)

Sussing Out SUMO
Indeed, modification of at least some APB constituents by SUMO proteins is known to be essential to the formation of APBs: PML protein contains a SUMO-interacting motif (SIM) that is not involved in SUMOylation but that that is required for PML nuclear body formation.(5) Again mirroring observations in native APBs, telomeric recruitment of LacI constructs with several SUMO isoforms led to colocalization of native PML, Sp100, and Rad17 (all present in endogenous APBs), again consistent with APB formation. A series of tests using constructs containing SUMO1 mutants either incapable of the noncovalent interactions with SIMs observed in the APBs in endogenous ALT cells, or incapable of covalent attachments, demonstrated that the role of this SUMO protein in the formation of the de novo APBs involves the former kind of interactions,(4) consistent with the need for noncovalent SIM binding in the formation of native PML nuclear bodies.

On the other hand, several telomere-associated proteins present in APBs in ALT (including TRF1, TRF2, and Rap1) do require SUMOylation, by the SUMO E3 ligase MMS21, and here again the investigators were able to use their tethering system to recapitulate observations of native APBs. Tethering of MMS21 to telomereic or pericentric DNA was "highly efficient in promoting APB assembly," recruiting substantial increases in colocalized PML protein. But importantly, only those MMS21-tethered telomeres with colocalized PML also colocalized with the APB component Rad9. This was hightly similar to what they observed in native APBs: "The vast majority (98%) of endogenous APBs (defined as colocalization of PML and TRF2) contained Rad9, and only 0.9% of the telomeres marked by TRF2 had Rad9 but no PML."(4) This is especially noteworthy, since there is evidence(5) that MMS21 plays a role in DNA repair, and the ALT TMM itself exploits (telomeric) DNA repair.

Back of the Line, Please
Inducing TRF1, TRF2, Rad9, NBS1, and MMS21 recruitment to telomeric DNA also increased formation of APBs.(4) On the other hand, tethering of two other APB constituents (Rad51 or Rad17) to telomeric DNA led to little (Rad51) or no (Rad17) recruitment of PML.(4) Despite this, endogenous Rad17 -- like NBS1 and Rad9 -- was enriched in the de novo APBs formed following PML tethering, consistent with native APBs. The authors note that a previous report (5) had found that knockdown or Rad51 with siRNA in U2OS cells had no effect on APB formation; combined, these results suggest that these repair factors may only be recruited to APBs after the initial assembly of other constituent proteins.

The Road to Perdition -- and a Bridge to Nowhere
In addition to observations at telomeric DNA, APB component proteins were also recruited when the researchers tethered PML to the pericentric lac operator sequences in the F442B8 U2OS line; in fact, these components were recruited "to a similar or even higher degree than at the telomeric sites." Again in general agreement with observations using telomerically-tethered proteins, recruiting MMS21 to pericentric DNA also led to elevated colocalization of APB proteins, whereas tethering of Rad51 at this locus did not.(4)

But the purpose of the investigation was not to determine if APBs could induced to form at these loci, but to determine the function of these structures: whether APBs play a causal role in telomere maintenance in ALT cells, or are merely phenotypic hallmarks of the ALT phenomenon. The investigators therefore tested the ability of de novo APBs to lengthen telomeres in a manner consistent with the ALT TMM.

When PML was tethered to telomeric chromosomal loci, the resulting de novo APBs were associated with elevated phosphorylated γ-H2AX histone. Phosphorylated γ-H2AX is a component of APBs, and indicator of double-strand break (DSB) repair, which is involved in the ALT TMM.(4) Similarly, the generation of de novo APBs at telomeric chromosomes was associated with increased non-replicative DNA synthesis, as detected by elevated pulsed 5-bromo-2′-deoxyuridine (BrdU) incorporation at the limited number of foci where it occurs during non-replicative phases of the cell cycle. Such increases in DNA synthesis, arising in association with the formation of APBs at telomeric DNA, are consistent with telomere lengthening. Again, a fluorescence in situ hybridization (FISH) probe was used to detect the percentage chromosomal ends too short to register on the probe, and thus primed for telomere extension by the ALT TMM. Tethering PML to telomeric DNA reduced the fraction of chromosomes with indetectably-short telomeres from ~43% to ~19%, suggesting the extension of critically-short telomeres; by contrast, tethering "empty" fluorescent-labeled LacI constructs to telomeric DNA led to no such increase.(4)

And importantly, none of these phenomena -- neither increased phosphorylated γ-H2AX, nor increased non-replicative BrdU incorporation, nor a reduction in indetectably-short telomeres -- appeared subsequent to APB formation at pericentric lac operator sequences. Thus, these phenomena -- each consistent with the lengthening of telomeres by an ALT-like mechanism -- were specific to the presence of APBs at telomeric DNA.(4)

Pause del Silenzio
It is worth here quoting the authors' own conclusions at length:

[Our findings] establish APBs as functional intermediates of the ALT pathway [my emphasis]. ... Accordingly, we conclude that the formation of bona fide APBs promotes the extension of the telomere repeat sequence by a DNA- repair-coupled synthesis process. Our study does not provide information on the nature of the telomere repeat template used for synthesis ... Furthermore, given the large number of partially contradicting results in the literature, it is well conceivable that different telomerase-independent mechanisms for telomere repeat extension exist. ... Thus, it will be important to further dissect the exact combination of protein factors that are sufficient to trigger telomere extension in APBs and to investigate the effects of their presence or absence. We anticipate that the experimental approach introduced here ... will allow us to precisely identify all protein components that are sufficient to form a telomeric PML-NB subcompartment structure, as well as the additional factors needed to induce telomere extension at these sites. This will serve to select protein targets for inhibiting telomere extension, and thus cell proliferation, in tumors that make use of the ALT pathway.(4)

All of the known constituents of APBs play a role in normal cellular metabolism. But full implementation of the WILT strategy will require not only that we "select [APB] protein targets for inhibiting telomere extension," but that one or more element of the ALT TMM be irreversibly disabled, in all of the cells of the body (and as a major intermediate goal, in those tissues in which ALT cancers most commonly arise). Thus, success with WILT will require a detailed elaboration of all the structures responsible for the lengthening of telomeres in ALT cells -- those discovered and undiscovered, constitutive of APBs and not -- and an understanding of their role in telomere lengthening in disease, as well as physiological functions in health. By providing significant support for the role of APBs in telomere extension, the beginnings of further understanding of the process of APB assembly and the roles of some constituent proteins in the APB TMM, and new tools for dissecting APB structure and function, Chung et al have taken us a significant step toward this benchmark, and thus toward a decisive cure for malignant disease.

References

1: Draskovic I, Arnoult N, Steiner V, Bacchetti S, Lomonte P, Londoño-Vallejo A. Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination. Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15726-31. Epub 2009 Aug 26. PubMed PMID: 19717459; PubMed Central PMCID: PMC2747187.

2: Nabetani A, Yokoyama O, Ishikawa F. Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body. J Biol Chem. 2004 Jun 11;279(24):25849-57. Epub 2004 Apr 9. PubMed PMID: 15075340.

3:Wu G, Lee WH, Chen PL. NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres. J Biol Chem. 2000 Sep 29;275(39):30618-22. PubMed PMID: 10913111.

4: Chung I, Leonhardt H, Rippe K. De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation. J Cell Sci. 2011 Nov 1;124(Pt 21):3603-18. Epub 2011 Nov 1. PubMed PMID: 22045732.

5: Shen TH, Lin HK, Scaglioni PP, Yung TM, Pandolfi PP. The mechanisms of PML-nuclear body formation. Mol Cell. 2006 Nov 3;24(3):331-9. PubMed PMID: 17081985; PubMed Central PMCID: PMC1978182

6: Potts PR, Yu H. Human MMS21/NSE2 is a SUMO ligase required for DNA repair. Mol Cell Biol. 2005 Aug;25(16):7021-32. PubMed PMID: 16055714; PubMed Central PMCID: PMC1190242.

With True Cells Come True Benefits: the Potential of Human Pluripotent Stem Cells Released in a Model of Parkinson's Disease

Michael Rae — Fri, 25 Nov 2011 22:36:31 +0000

Rejuvenation biotechnology encompasses a suite of advanced medical therapies, each of which removes, repairs, replaces, or renders harmless one of the forms of cellular or molecular damage that accumulates in an aging tissue over time and impairs its function. Through the comprehensive abatement of all such aging damage to levels approximating those of younger adults, tissue structure and function can be made more youthful, restoring the health and vigor of aging persons to that of persons years or decades younger. This approach is most prominently under pursuit in the development of cell therapy and tissue engineering, of which the most striking success to date has been the use of fetal and embryonic mesencephalic tissue grafts to replace dopaminergic (DA) neurons lost to the age-related neurodegenerative processes driving Parkinson's disease (PD).(0)

The most immediate cause of the loss of fine motor control that leads to the most prominent motion disorders characteristic of the disease is the local loss of DA release by substantia nigra pars compacta (SNc) DA neurons into their striatal targets. In studies to date, DA cells have been transplanted directly into the striatum, where they reinnervate and partly functionally integrate the tissue, locally restoring the needed release of DA that is essential to fine motor control.

While such grafts have led to striking temporary improvements in the major motion disorder symptoms in many patients, results have been limited, and significant improvements in the protocol are clearly required to realize the potential of cell therapy to help these patients. The grafts are significantly immunogenic, leading to rejection in some cases and necessitating immunosuppression in most patients. The nature of the cell source means that very few patients could ever hope to benefit from treatment, since the supply of fetal midbrain tissue is both inherently limited and subject to politically-imposed restrictions. And the actual benefits to patients have not been as robust as might have been hoped: the magnitude of clinical response has been highly variable, gains have proven impermanent, and ~15% of transplanted patients have developed substantial dyskinesias during the "off" phase of levodopa treatment.(0)

Many of these limitations are attributable to the crude cell sources used in these trials. An important step forward will be to move beyond the use of fetal mesencephalic tissue, and instead derive graft populations of pure DA neurons from pluripotent stem cells, such as embryonic stem cells (ESC), induced pluripotent stem cells (iPS), or somatic cell nuclear transfer (SCNT — "therapeutic cloning"). Any of these sources should substantially alleviate the problem of immunogenicity of fetal tissue: ESC-derived differentiated cells and progenitors appear to enjoy some degree of immunological privilege,(1) and iPS- or SCNT-derived cells would be immunologically native and free of rejection risk, with the possible exception of mitochondrially-derived immunogenicity in SCNT. As well, the graft-induced dyskinesias now appear to be largely attributable to the presence of serotonergic neurons in the mixed cell population present in the graft tissue,(2) which would not be present with DA cells specifically derived from pluripotent stem cells. And the nearly-unlimited replicative capacity of pluripotent cells, combined with an efficient and stable means of differentiation, would provide such cells in the quantities needed for widespread clinical use in frank PD and in "normative" brain aging.

The promise of this approach has been foreshadowed in murine models of PD, in which DA neurons derived from mouse ESC have been found highly effective in reversing motor symptoms. But the performance of ostensibly DA neurons derived from human pluripotent stem cells in the same systems has so far been poor, due to uncertain and unstable differentiation of the cells. In a new study,(3) a team of researchers led by Dr. Lorenz Studer of the Sloan-Kettering Institute’s Center for Cell Engineering have used their novel DA neuron differentiation strategy to resolve these difficulties, leading to robust and stable engraftment of human pluripotent stem cell-derived DA neurons into the striatum and substantial evidence of efficacy in two rodent models of the disease, and provided preliminary data on the viability of their approach in nonhuman primates.

A Superior Protocol
The authors had previously reported(4) a protocol for deriving mesencephalic DA neurons by nudging them through an intermediary stage as midbrain floor plate precursor cells. The floor plate is the developmental topos through which cells are thought to acquire DA neural progenitor characteristics. This entailed a dual inhibition of SMAD signalling in the cells through the simultaneous inhibition of BMP4 using Noggin (an inhibitor of BMP4), and activation of the Lefty/Activin/TGFβ pathway with the drug SB431542, and the approach has subsequently been independently validated by investigators at the MRC Centre for Regenerative Medicine.(5) As part of this new report,(3) two lines each of human ESC and iPS cells were used to derive DA neurons using either a modified version of this new strategy, or the currently-standard protocol(6) of moving such cells through a neural rosette intermediate for DA neuron derivation. The dual SMAD inhibition, floor-plate-intermediate protocol proved superior, generating a higher percentage of tyrosine hydroxylase-expressing (TH+) neurons, which unlike rosette-derived cells expressed markers of the developing mesencephalon and (importantly) led to far lower adventitious generation of serotonergic neurons.(3)

Dopaminergic Phenotype
One weakness common to many studies of derivation of differentiated cells from pluripotent precursors has been the almost exclusive reliance on cell-surface markers as indicators of ultimate fate, casting uncertainty over the nature of such cells and the correct interpretation of their observed therapeutic benefits, limitations, and adverse reactions in models of injury and disease. The authors loaned substantial credibility to their results by showing that their human ESC- and iPS-derived DA cells recapitulate multiple phenotypic characteristics of native SNc DA neurons and of SNc neurons grown from early postnatal mice, including not only high expression of their mature neuronal markers, but their characteristic electrophysiological phenotype and extensive fiber outgrowth.(3)

Success and Safety In Vivo
The investigators next performed in vivo tests of cells derived either from their own modified dual SMAD-inhibition protocol, or using the rosette method. Based on previous work, the investigators performed all transplantation when the therapeutic cells were in cell cycle exit; such cells proved to survive effectively in intact rodents, and were next tested by striatal injection into brains of mice whose SNc DA neurons had been unilaterally destroyed using 6-hydroxy-dopamine (6-OHDA), a standard model of PD.(3) Because it has proven challenging to develop differentiated cells that exhibit robust growth and strong engraftment in local neuronal tissue without leading to either "off-target" differentiation into undesired cell types, or tumorigenic overgrowth, the researchers opted to perform these tests using the radically immunodeficient NOD-SCID IL2Rgc null mouse, which "efficiently supports xenograft survival with particular sensitivity for exposing rare tumorigenic cells."(3) Moreover, neuronal cultures were grafted "as is" rather than following additional cell purification, to give maximum opportunity for any insufficiently- or improperly-differentiated cells to reveal themselves in overgrowth.

At four and a half months post-transplant, the fates of cells derived from the two protocols were dramatically different in vivo. In animals engrafted with DA neurons derived using the standard rosette method, administration of amphetamine led to stereotypic circling motions, caused by the drug's stimulation of the surviving DA neurons remaining on the unlesioned side of the brain. By contrast, engraftment with cells derived using the floor plate method rescued this behavior. Similarly, in animals treated with rosette-derived DA neurons, there were few human-derived DA neurons present at the graft site, which was however riddled with proliferating Ki-671+ cells, and exhibited "massive neuronal overgrowth."(3) The equivalent site in animals receiving floor plate-derived cells formed a distinctive hub, well-populated with human-derived DA neurons, and with <1% of total cells actively proliferating.(3)

To rule out any artifacts of their reliance on the immunodeficient mouse model, these experiments were repeated in rats immunosuppressed using cyclosporin A, this time comparing neural grafts using cells derived with their new protocol to sham surgery. Again, high numbers of transplanted DA neurons engrafted, survived, and branched out to integrate with surviving tissue without overgrowth; gene expression profiles suggested the presence of both nigral and ventral tegmental-area DA neurons. Treatment again eliminated rotational behavior following amphetamine administration, and also alleviated deficits in two tests of motor control that do not rely on drug-induced overactivation of surviving DA neurons: the stepping test of forelimb akinesia, and the specific use of the forelimb using the cylinder test.(3) In this model too, <1% of total cells had improperly differentiated into serotonergic neurons, and the few GFAP1 glial cells were host-derived; moreover, transplantation of their human pluripotent cell-derived DA neurons eliminated or improved each of the model motor disorders tested (see Figure 1).(3)

Figure 1. Floor-Plate (FP)-Derived Dopaminergic Neuron Grafts from Human ESC Rescue Motion Disorders in 6-OHDA-Lesioned Rats. Reproduced from (3).

Preliminary Promise in Primates
Finally, the investigators performed a minimal test the viability of their approach in a nonhuman primate model. In addition to being more closely related to our species, these animals' lesioned brains can acommodate the far larger numbers of cells than those of the much smaller rodent models, better reflecting what would be needed in actual human cell therapy. Their system allowed for ready derivation of 5 x 10⁷ DA neurons for testing, and following MPTP lesioning of the substantia nigra, each of two adult Rhesus monkeys received a total of six transplants of 1.25 x 10⁶ cells, injected into each of three sites spanning the posterior caudate and pre-commissural putamen on each side of the brain. One month later, GFP expression revealed high numbers of surviving mesencephalic DA neurons at each site, which coexpressed the human-specific cytoplasmic marker SC-121; these cells' fibers branched out of the graft cores for up to 3mm into surrounding host brain tissue. The sole note of caution was the suggestion of incomplete immunosuppression, based on the presence of Iba1+ host microglial cells in the grafts.(3)

Continuing, Perfecting, and Integrating a Rejuvenation Biotechnology
As the researchers conclude, their

novel [floor plate]-based [pluripotent stem cell] differentiation protocol faithfully recapitulates midbrain DA neuron development. ... Importantly, our study establishes a means of obtaining a scalable source of ... neurons for neural transplantation — a major step on the road towards considering a cell based therapy for Parkinson’s disease. Excellent DA neuron survival, function, and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.(3)

The most immediate next step in development of this potential therapy is to test the ability of these grafts to rescue the motion disorders of MPTP-lesioned nonhuman primates. Were such cells to prove themselves in this model, clinical trials in humans suffering with the crippling motion disorders of PD would be fully justified in light of the limited positive results already achieved in trials of cell therapy for this disorder. Engraftment with pure populations of pristine DA neurons should deliver far greater benefits than those previously observed using mixed fetal/embryonic tissue, reducing or eliminating the need for immunosuppressive thearapy and also minimizing or abrogating the dyskinesic side-effects that were apparently the result of contamination with serotonergic cells.(2) And, once proven safe and effective, the replicative capacity of pluripotent stem cells should allow such benefits to be delivered to far more patients.

Once these cells have proven their potential in humans, yet further refinement of the protocol can be expected to more fully alleviate the symptoms of PD. In this study,(3) as in the previous human(0) and preclinical work, transplanted cells have been injected directly into the striatum, where the loss of DA is acute. But native SNc DA neurons release DA not only at their striatal target sites, but throughout their soma and dendrites within the SNc itself; to fully restore the intact circuitry of the youthful, fully-functioning dopaminergic system will require protocols for the orthotopic transplantation of such cells into the neurodegenerative substantia nigra, recapitulating the physiological dopaminergic innervation of the striatum from this graft core. Fortunately, nonhuman primate work toward this goal is already underway.(10) The perfection of such protocols will enable us to move beyond even treatment of clinical PD, and into repair of the SNc and other DA neuronal structures lost during prodromal stages of the disease, and ultimately toward alleviating even the more subtle motion dysfunction caused by SNc DA neuronal losses suffered during "normal" brain aging.

Yet further gains will depend on combining this single rejuvenation biotechnology into a wider range of therapies to repair the agign brain. In the earlier trials, transplanted cells subsequently developed intraneuronal aggregates composed of α-synuclein, characteristic of the aging and particularly the PD brain, at a rate that appears to be modest as relates to graft survival times but perhaps faster than that in native, undiseased neurons.(7) The appearance of such damage in relatively recently-engrafted neurons contrasts with the temporal neuropathological staging of such pathology in the aging hindbrain, where it first appears in the lower brainstem, remote from the SNc and striatum, and gradually spreads forward toward it over time. There is strong evidence that the accumulation of such aggregates in extranigral sites plays a key role in PD, although its relationship with DA neuronal death is unclear and may well be independent; it is more likely that this more widespread damage underlies many of the nondopaminergic, levodopa-refractory symptoms of PD as a multisystem disorder.(8) Full brain rejuvenation will require the use of novel xenohydrolases(9) to clear aging neurons of these aggregates, both to maintain the benefits of transplanted cells, and to eliminate the troublesome and disabling symptoms that arise from aging damage to structures beyond the SNc.

A key barrier to the use of human pluripotent stem cells to rejuvenate the aging and neurodegenerative brain appears to have been broken. It is now our task to press on, treating Parkinson's disease and ultimately ending the age-related degeneration of the human brain.

References

0: Lindvall O, Björklund A. Cell therapy in Parkinson's disease. NeuroRx. 2004 Oct;1(4):382-93. Review. PubMed PMID: 15717042; PubMed Central PMCID: PMC534947.

1: English K, Wood KJ. Immunogenicity of embryonic stem cell-derived progenitors after transplantation. Curr Opin Organ Transplant. 2010 Dec 9. [Epub ahead of print] PubMed PMID: 21150615.

2: Politis M, Wu K, Loane C, Quinn NP, Brooks DJ, Rehncrona S, Bjorklund A, Lindvall O, Piccini P. Serotonergic neurons mediate dyskinesia side effects in Parkinson's patients with neural transplants. Sci Transl Med. 2010 Jun 30;2(38):38ra46. PubMed PMID: 20592420.

3: Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, Carrillo-Reid L, Auyeung G, Antonacci C, Buch A, Yang L, Beal MF, Surmeier DJ, Kordower JH, Tabar V, Studer L. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature. 2011 Nov 6. doi: 10.1038/nature10648. [Epub ahead of print] PubMed PMID: 22056989.

4: Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009 Mar;27(3):275-80. Epub 2009 Mar 1. Erratum in: Nat Biotechnol. 2009 May;27(5):485. PubMed PMID: 19252484; PubMed Central PMCID: PMC2756723.

5: Devine MJ, Ryten M, Vodicka P, Thomson AJ, Burdon T, Houlden H, Cavaleri F, Nagano M, Drummond NJ, Taanman JW, Schapira AH, Gwinn K, Hardy J, Lewis PA, Kunath T. Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus. Nat Commun. 2011 Aug 23;2:440. doi: 10.1038/ncomms1453. PubMed PMID: 21863007.

6: Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 2008 Jan 15;22(2):152-65. Erratum in: Genes Dev. 2008 May 1;22(9):1257. PubMed PMID: 18198334; PubMed Central PMCID: PMC2192751.

7: Braak H, Del Tredici K. Assessing fetal nerve cell grafts in Parkinson's disease. Nat Med. 2008 May;14(5):483-5. PubMed PMID: 18463652.

8: Lang AE, Obeso JA. Challenges in Parkinson's disease: restoration of the nigrostriatal dopamine system is not enough. Lancet Neurol. 2004 May;3(5):309-16. Review. PubMed PMID: 15099546.

9: Mathieu JM, Schloendorn J, Rittmann BE, Alvarez PJ. Medical bioremediation of age-related diseases. Microb Cell Fact. 2009 Apr 9;8:21. PubMed PMID: 19358742; PubMed Central PMCID: PMC2674406.

10: Redmond DE Jr, Weiss S, Elsworth JD, Roth RH, Wakeman DR, Bjugstad KB, Collier TJ, Blanchard BC, Teng YD, Synder EY, Sladek JR Jr. Cellular repair in the parkinsonian nonhuman primate brain. Rejuvenation Res. 2010 Apr-Jun;13(2-3):188-94. Review. PubMed PMID: 20370501; PubMed Central PMCID: PMC2946058.

Nothin' Gonna Hold Me Back: Clearance of Senescent Cells for Tissue Rejuvenation

Michael Rae — Mon, 07 Nov 2011 02:02:15 +0000

"Senescent" cells progressively restrict the body's capacity for tissue renewal and secrete factors that disrupt local tissue homeostasis. A new study provides proof-of-concept that ablation of these cells can delay -- and potentially contribute to the reversal of -- age-related tissue dysfunction and disease.

Aging bodies become increasingly burdened over time with dysfunctional cells resistant to apoptotic or other clearance. The most well-known of these are so-called "senescent" cells, originally characterized by Leonard Hayflick as mitotic cells that reached growth arrest after a limited replicative lifespan (later associated with telomere attrition) under unphysiological conditions in culture. Later research has revealed that few cells reach a "senescent" state through sheer replicative exhaustion: instead, senescence has emerged as a programmed response to DNA damage or oncogenic stress, and as part of the resolution of wound healing.(1) Unfortunately, the near-term benefits of these functions -- in preventing damaged cells from progressing to cancer, and in preventing fibrosis --are coupled to deleterious long-term consequences, whose effects worsen as the burden of such cells rises with aging. First, the loss of mitotic competence of stem cells denies proliferative tissues of the capacity for renewal. Secondly, the secretory and other phenotypes of such cells progressively derange local and systemic metabolism and tissue function, rendering tissues more vulnerable to metastasis, promoting systemic inflammation, and otherwise impairing tissue function.(1-4)

To bypass the disruptive effects of the age-related accumulation of senescent cells, some investigators are working on possible ways to manipulate the signaling pathways involved in enforcing the senescent phenotype. This approach bears with it great risks, however, because of the very purposes of senescence to which allusion was made above: returning senescent cells to their normal differentiated function and replicative capacity could lead cells bearing oncogenic mutations to progress into metastatic disease, and aberrant resumption of the wound-healing response leading to fibrosis.(1,4) The regenerative engineering solution to this dilemma is therefore the ablation of such cells, to eliminate their contribution to age-related loss of homeostasis without reactivating the more acute risks against which the senescence machinery was activated in the first place.(5)

As widely covered in the mainstream press, a successful proof-of-principle study for this rejuvenation biotechnology has now been performed.(6)

The study was performed using several founder lines, bred onto a background strain of mice hypomorphic for BubR1 (BubR1^H/H), a key component of the mitotic checkpoint machinery. Principal investigator Jan van Deursen, Professor of Biochem/Molecular Biology and of Pediatrics at the Mayo Clinic location in Minnesota, had already discovered(7) that BubR1^H/H mice "have a markedly shortened lifespan and exhibit a variety of age-related phenotypes, including infertility, lordokyphosis, sarcopenia, cataracts, [subcutaneous] fat loss, cardiac arrhythmias, arterial wall stiffening, impaired wound healing and dermal thinning."(6) Some, but not all, of these phenotypes were associated with a high age-related incidence in senescent (p16^Ink4a-positive) cells.(6-8) and van Deursen and colleagues had already demonstrated that breeding BubR1^H/H mice onto a p16^Ink4a homozygous-null genetic background attenuated their development of p16^Ink4a-senescent cell-associated aging phenotypes and modestly increased their very low survivorship.(8) Imputation of these results specifically to the animals' age-related, low-BubR1-driven rise in p16^Ink4a-expressing senescent cells was, however, limited: limited by the very nature of so-called "accelerated aging" models such as BubR1^H/H,(9) and limited by the lifelong, global absence of p16^Ink4a expression in the backcrossed mice.

Seeds of Destruction and Renewal

To impute aging phenotypes directly to p16^Ink4a-expressing senescent cells, van Deursen and colleagues with expertise in the aging and senescence of the relevant tissues developed and tested the effects of a pharmacologically-inducible system for the ablation of p16^Ink4a-expressing cells. To create this system, investigators modified an approach used in earlier research, in which mice were bred with a variant on the Gene-Directed Enzyme Prodrug Therapy (or "suicide gene") paradigm,(11) using a drug (AP20187) that activated fusion protein apoptosis machinery in cells in which the macrophage- and adipocyte-specific minimal Fabp4 promoter was transcriptionally active.(10) To generate mice in which p16^Ink4a-expressing cells could be similarly selectively ablated, van Deursen's team substituted a fragment of the p16^Ink4a gene promoter for the Fabp4 promoter, thereby generating BubR1^H/H;INK-ATTAC mice.(6) In such mice, then, p16^Ink4a would still be under normal physiological regulation, and still be induced in an abnormally high number of cells due to the mitotic checkpoint dysfunction caused by BubR1 hypomorphism, leading to the same abnormally-rapid accumulation of high burdens of p16^Ink4a-expressing senescent cells -- but administration of AP20187 would induce apoptosis selectively in such cells, purging the animals' tissues of senescent cells while leaving non-senescent cells unscathed.

Testing the System

A range of in vitro and in vivo tests was used to rigorously confirm the selectivity and sensitivity of the system's activation in, and ablation of, p16^Ink4a-positive senescent cells.(6) In early-aging (2-mo old) BubR1^H/H;INK-ATTAC mice, but not young (3-wk-old) mice, transcripts of the system and of reporter green fluorescent protein "were significantly elevated in [subcutaneous] adipose tissue, skeletal muscle and eye, but not in tissues in which endogenous p16^Ink4a is not induced, including liver and heart."(6) Moreover, subcutaneous adipose of prematurely-aged transgenic mice exhibited high levels of staining for the senescence marker senescence-associated-β-galactosidase (SAβ-gal) and expressed high levels of several established markers of senescence, including p21, p19, interleukin-6, (insulin-like growth factor binding protein-2 (Igfbp2), and Pai-1; primary BubR1^H/H;INK-ATTAC mouse embryonic fibroblasts forced artificially into senescence by oncogenic Ras or serial passage exhibited a subpopulation that was both GFP+ and stained positively SAβ-gal. BubR1^H/H;INK-ATTAC muscle cells and lens did not stain for SAβ-gal, but did exhibit selective induction of the "senescence genes."(6)

When BubR1^H/H;INK-ATTAC mouse bone marrow cells were pushed into senescence in vitro by the PPAR-γ-activating drug rosiglitazone, a subpopulation of the cells exhibited high levels of INK-ATTAC expression and GFP, coupled with SAβ-gal staining; subsequent to treatment with the INK-ATTAC activating drug, these cells rapidly entered into apoptosis, and within 48 h were either destroyed or in the cell death process.(6)

Ablation of Senescent Cells Retards Age-Related Tissue Degeneration
As a first approach, the investigators abrogated the premature age-related rise of p16^Ink4a-senescent cell burden in the tissues of BubR1^H/H;INK-ATTAC mice by initiating a lifelong course of senescent-cell-ablating AP20187 treatment at weaning. At 9-10 mo of age, such mice were then compared a to age-matched cohorts of untreated BubR1^H/H;INK-ATTAC mice, and to BubR1^H/H mice lacking the INK-ATTAC system for selective ablation of p16^Ink4a-expressing cells. Relative to both control cohorts of the same age, 9-10 mo old treated mice exhibited dramatically more youthful tissues. Consistent with earlier results, their burden of p16^Ink4a-positive senescent cells in muscle, eye, and adipose tissues were far lower. Their muscle fibers had larger diameters, their treadmill endurance was greater and they covered more distance on them. Treated mice had fewer cataracts, and less lordykyphosis. And they suffered less lipoatrophy, with larger fat deposits in multiple depots, higher individual adipocyte volumes, and more proliferating cells marked with BrDU (see Figure 1, below).(6) No treatment-related adverse events presented themselves.(6)

Figure 1: Amelioration of "Premature Aging" Phenotypes in Treated and Untreated BubR1^H/H;INK-ATTAC Mice. AP=AP20187 treatment. Reproduced from (6). © Nature Publication Group.

Importantly, "premature aging" phenotypes observed in BubR1^H/H mice over time, but that are in tissues where p16^Ink4a-positive senescent cells do not accumulate with aging, were not alleviated by drug treatment. Thus, these animals exhibit premature cardiac arrhythmias and stiffening of the arterial wall, and cardiac failure appears to be the main cause of death; yet these tissues are not burdened with an abnormally-high burden of p16^Ink4a-senescent cells, and accordingly, ablation p16^Ink4a-positive senescent cells in these animals had little tissue-specific or survivorship phenotypic impact.(6)

Following this initial test of abrogating the early, age-related rise in p16^Ink4a-expressing cell burden, the investigators probed the effects of leaving BubR1^H/H;INK-ATTAC to undergo 5 months of rapid "premature aging" (and thus, to the attendant accumulation of high levels of p16^Ink4a-positive cells and onset of "early-aging" phenotypes), and only then inducing ablation of senescent cells with the INK-ATTAC drug-activated system (see Figure 2 (g) below).(6) At that point, the animals' cataracts had already reached peak age-related severity, and remained stable after 5 months of further aging irrespective of treatment. But muscle fibers that continued to atrophy over the ensuing 5 months in control animals remained at their more youthful diameters in animals whose p16^Ink4a-positive senescent cells had been ablated, and treadmill times, distance traveled, and work outputs were maintained at substantially more youthful levels (Figure 2, (a) and (b) below). Similarly, the degeneration of adipose tissue cells and depots that occurred over the course of the next 5 months in control animals was virtually abrogated, leaving 10 mo-old animals with substantially the same subcutaneous and other fat tissue (in depth and in cell volume) as they had enjoyed in their relative youth, when treatment was first initiated.(6)

Figure 2: Ablation of Senescent p16^Ink4a-Expressing Cells Maintains Youthful Muscle and Adipose Tissues. Reproduced from (6). © Nature Publication Group.

Rejuvenation Implications

As noted above, studies involving the use of putative "premature aging" models must be interpreted with caution, as the designation inevitably involves an element of petitio principii: from a subset of similar phenotypes are drawn conclusions of similar aetiology, and from this, further conclusions about the "normal" degenerative aging process (and its biomedical amelioration) are too-readily drawn before the thesis itself has first been established.(9) Indeed, the degenerative aging process is by definition one in which the organism progressively accumulates damage to its cellular and molecular components over time, so any genetic or environmental factor that leads to a greater burden of such damage will bear some resemblance to the aging phenotype, irrespective of the causal origin of the defect or its relationship to "normal" aging.

In the case of this new report,(6) however, while caution is still merited, the nature of the intervention used makes the study relatively free of such complications. The investigators did not simply modulate or normalize the very thing that the mutation (in this case, to the mitotic checkpoint component BubR1) itself disrupts, as in other widely-publicized studies involving putative "accelerated aging" (eg. (12,13)). Rather, the defective checkpoint system was left to proceed, and one of its downstream consequences, which was still under normal regulation -- and one known to be directly induced by the normal degenerative aging process -- was reversed at the structural level, by clearing out the p16^Ink4a-positive senescent cells that had accumulated to an abnormal degree in their tissues. This left some aspects of the abnormal "progeroid" phenotype in these organisms (the cardiovascular defects) intact, but illustrated the dysfunctional consequences of having tissues riddles with such cells. While still of abnormal origin, there is no strong reason to think that the ongoing effects of a rising burden of such cells would not be similar -- and thus, that the effects of ablating such cells are uninformative about the effects of a similar intervention in "normally" aging bodies.^*

The links to aging phenotypes, and their near-arrest by ablation of p16^Ink4a-expressing senescent cells, appear to be dramatic illustrations of the deleterious effects of the age-related rise in the burden of senescent cells in genetically-intact mammals. The fact that it was the removal of such cells from aging tissues that arrested multiple aging phenotypes is of special importance to the rejuvenation biotechnology approach to preventing and reversing age-related disease and disability: it clearly identifies the damage itself, rather than the abnormal function of either p16^Ink4a (which was under normal, physiological regulation, rather than being pharmacologically modulated, or knocked out as in their previous report(8)) or BubR1 (mutation of which, and its direct metabolic sequelae, was not affected by the intervention).

And there are reasons to believe that the resulting arrest of multiple aspects of tissue aging by removal of p16^Ink4a-expressing senescent cells would indeed translate into the tissues of genetically-intact mice -- or humans.

Sarcopenia

The fact -- and disabling and fatal consequences -- of age-related decline in muscle quality and quantity is widely known, but the contribution of "senescent" cells to this degenerative process is not. While some studies have reported no decline in satellite cells (muscle progenitor cells) with aging, others (eg. (14,15)) have found age-related satellite cell attrition consistent with the senescence of a subset thereof; moreover, one such study (15) reported that decreases in the number and quality of satellite cells with aging are reliably associated with elevated expression of p16^Ink4a (contrary to (14)), and with secretory and proteomic abnormalities consistent with a rising burden of senescent cells. Consistent with a causal relationship, (16) reports that the prevalence of limited physical functioning in aging varies depending on p16^Ink4a allelic variation, consistent with variations in rate of stem cell attrition with senescence.

There is therefore good reason to expect that the profound arrest of sarcopenic phenotypes observed in p16^Ink4a-senescent cell ablated BubR1^H/H "premature aging" mice would translate into the human case.

Lipoatrophy

While less well-known (masked as it is and placed out of focus by the overall age-related body composition shift from lean mass to adiposity), there is none the less significant age-related subcutaneous lipoatrophy in aging, most visibly in the sunken appearance of the face. Part of this is a pathological redistribution of adipose from the subcutaneous to the visceral depot, but it now emerges that the subcutaneous depot becomes qualitative as well as quantitatively abnormal in the degenerative aging process also suffers genuine age-related lipoatrophy and lipodystrophy -- and that p16^Ink4a-driven cellular senescence is at the heart of it.

Subcutaneous adipose tissue contributes to maintenance of insulin sensitivity and other aspects of metabolic homeostasis, through the production of adipose-specific endocrine factors such as adiponectin. Surgical removal of subcutaneous fat reduces adiponectin levels and insulin sensitivity, and transplantation of subcutaneous fat increases both.(17) Slow-aging growth hormone receptor knockout (GHRKO) mice are obese, but highly insulin sensitive: in such animals, surgical removal of visceral adipose tissue impairs insulin secretion and peripheral insulin action, in part by reducing adiponectin production. (21) Moreover, while the link between excessive visceral adipose tissue and age-independent diabetes and metabolic syndrome widely known, recent studies suggest instead that it is the accumulation of senescent subcutaneous adipocyte progenitors -- and their abnormal metabolic function -- that drives similar diabetes-like phenotypes during the "normal" aging process.(3,18; cf. 19,20) Even in visceral fat, it has recently emerged that the obesity-driven rise in inflammation and insulin resistance is associated with an abnormal accumulation of senescent cells, albeit senescent endothelial cells rather than adipocytes.(20) It was this emerging line of research that van Deursen's collaborator Dr. James Kirkland presented at the fifth annual Strategies for Engineered Negligible Senescence Conference (SENS5) in September of this year,(18) and it was his expertise in the senescence of adipose tissue that he contributed to the new report on the effects of ablating such cells.(6)

Again, then, there is significant evidence consistent with a role of cellular senescence in age-related lipodystrophy and lipoatrophy, and for the benefits observed in treated mice in these studies to translate into aging humans. It is unfortunate that the investigators did not assess insulin secretion, insulin action, or systemic inflammation in early-aging BubR1^H/H;INK-ATTAC mice, with and without ablation of senescent adipose cells, but reasonable to be optimistic that doing so would yield some normalization of age-related metabolic abnormalities.

Cataract

There is only the most tentative of evidence suggesting a link between cellular senescence and cataract in "normal" aging.(22) Absence of evidence is, however, not evidence of absence, and certainly the inflammatory secretory profile of senescent cells would, if present, likely accelerate the degenerative course of the disease.

Cancer

A great deal of evidence has now been amassed that stromal cell senescence plays an important role in laying the groundwork for tumor metastasis, promoting cell proliferation with inflammatory cytokines, encouraging angiogenesis, and degrading the tumor-suppressive action of an intact extracellular matrix.(1) One clear disadvantage of using these "early-aging" mice is that they die too early to develop cancer -- too early for ablation of p16^Ink4a-positive senescent cells to impact the course of the disease. It would indeed be of great interest to see whether ablation of stromal p16^Ink4a-expressing senescent cells, in otherwise genetically intact INK-ATTAC animals without existing tumors, would lower the animals' risk for cancer, and put any tumors they might develop on a less malevolent trajectory, than untreated mice. It should be noted, however, that while a study on senescent cell ablation in genetically normal mice would provide at least some evidence on the effect of senescent cells (and their ablation) on promoting cancer, even such a study would likely show less effect than could be anticipated in a large mammal model, since even normally-aging mice rarely suffer metastatic disease to the extent of aging humans, as sheer primary tumor volume is generally sufficient to be fatal to mice.

Other Tissues

As the investigators note, the rapid age-related arterial stiffening and cardiac arrhythmias that appear to be at cause for the majority of deaths in BubR1^H/H mice were not attenuated by ablating p16^Ink4a-expressing senescent cells -- but these tissues had little burden of such cells, so this finding reinforces the conclusion that the multiple aging phenotypes arrested in these mice when senescent cells were ablated is attributable specifically to the removal of their baleful influence on local tissues. On the other hand, there are many other tissues -- notably, the kidney and articular cartilage -- where p16^Ink4a-expressing senescent cells appear to be a contributing factor to human and murine degenerative aging, but which were not evaluated in treated or control mice in this study, and it would be of interest to see the effects of ablation of p16^Ink4a-positive senescent cells.

Moreover, there are yet other cell types -- such as visceral adipose tissue macrophages and cytotoxic CD8+ T-cells -- in which the age-related supernumerary accumulation of dysfunctional and apoptosis-resistant cells appears to play a highly deleterious role on tissue function, but where the cells are not "senescent" cells in the classical sense of p16^Ink4a expression and the senescence-associated secretory profile observed in senescent fibroblasts. This study (6) cannot provide evidence directly on the effects of ablating such cells, but it does provide an analogous proof-of-concept for the approach. SENS Foundation is funding ongoing work in the lab of Dr. Janko Nikolich-Zugich to investigate the effects of clearance of anergic, "senescent" cytotoxic CD8+ T-cells on immunosenescence,(22) and is interested in the targeting of other such cells.(2)

Arrest vs Reversal

In the new study, p16^Ink4a-expressing senescent cells were ablated either at weaning or some months later, and assessed several months after the initial intervention. Remarkably enough, the removal of such cells arrested tissue degeneration, holding the muscles and adipose tissue (and, when administered before cataract was mature, lens opacification) at approximately the same relatively youthful condition prevalent when the inducing drug was first administered (see eg. Fig 2(a) above).(6) This is consistent with the deleterious effect of such cells on tissue function, and with the researchers' conclusion that “the observed improvements in skeletal muscle and fat of late-life treated 10-month-old BubR1^H/H;INK-ATTAC-5 mice reflect attenuated progression of age-related declines rather than a reversal of ageing”.(6) However, it would be useful to see a more thorough analysis of the effect of ablating p16^Ink4a-expressing senescent cells, and whether there may instead be evidence of a short-term rejuvenation of tissue function that is slowly lost over time to rising levels of other kinds of aging damage that INK-ATTAC activation does not address. Indeed, as illustrated by the lack of effect of p16^Ink4a-expressing cell ablation on lifespan, and by the ongoing degeneration of tissues (such as the heart) in which p16^Ink4a-postive senescent cells are not a driver of "early aging," true rejuvenation requires a comprehensive suite of rejuvenation biotechnologies to remove all forms of aging damage from the aging body.

Translation for Human Rejuvenation Biotechnologies

The investigators boldly, but rightly, conclude that

Our proof-of-principle experiments demonstrate that therapeutic interventions to clear senescent cells or block their effects may represent an avenue for treating or delaying age-related diseases and improving healthy human lifespan.(6)

How might the results of this intervention be translated for human rejuvenation therapies?

There is already evidence that senescent cells are targeted by the innate immune system.(24-28) Dr. Judith Campisi, in fact, has found that activating NKG2D receptors on natural killer (NK) cells engage MHC class I chain-related protein A and B (MICA/B) ligands on senescent cells, leading to their NK-induced apopotsis and subsequent clearance.(29) MICA/B ligands are also used to activate tumor cell destruction by NK cells via NKG2G binding, and tumors evolve resistance by several mechanisms to reduce cell-surface MICA abundance;(30) however, the natural selection mechanisms that drive the evolution of such defenses do not apply to growth-arrested cells. Dr. Campisi has found instead that a minority of senescent cells evade destruction by secreting high levels of matrix metalloproteinases (MMPs), which cleave MICA/B ligands and thereby prevent NKG2D binding.(29) This has led to the hypothesis that the great majority of such cells are destroyed over the lifetime by innate immunity, and that the specific senescent cells that do accumulate with aging are precisely those who had variants that allow MMP overexpression, in a kind of "one-off," very temporally-extended kind of selection process. Potentially, a kind of intervention that could overcome this resistance to endogenous clearance mechanisms would allow for the purgation of senescent cells from aging tissues.

SENS Foundation is currently funding work by PhD Candidate Kevin Perrott in Campisi's laboratory, screening compounds for their effectiveness in mitigating the negative impact of cells exhibiting the classical senescence-associated secretory phenotype (SASP)(1) following senescence induced by treatment with 10 gray of ionizing radiation by selective induction of apoptosis or modulating their secretions. To date, a screen of the collection of FDA-approved drugs in the Prestwick Library has identified a handful of potential candidates which have demonstrated effectiveness at lowering secretion of IL-6, a component of SASP whose concentration tends to rise systemically with aging and here used as a preliminary marker of SASP as a phenotype. In particular, Perrott has recently identified some members of a class of compounds that lower the SASP in irradiated-senescent cells, without reversing growth arrest, and he is currently investigating the mechanisms of this phenomenon.

It is clear that there is substantial distance yet to be traveled. Multiple cell types acquire distinctive "senescent" phenotypes on a cell-type-specific basis, and will require ablation to achieve comprehensive rejuvenation. However, this important proof-of-principle from Dr. van Deursen's laboratory, and the key validation of the scope of the effects of ablation of these particular senescent cells facilitated by his collaboration with the LeBrasseur and Kirkland labs at Mayo, stands as a key landmark in moving toward the removal of their baleful influence on aging tissues. As ever, SENS Foundation is committed to making investments in critical-path research to advance this key but heretofore-neglected line of biomedical research out of the laboratory, into the clinic, and to uniting the multiple strands of rejuvenation biotechnologies into a comprehensive panel for the restoration of the health, vigor, and open futures of aging humanity.

^*The principle caveat would be that the interaction of senescence with defective mitotic checkpoint function within such cells, and the effects upon their neighbors of their state and of their senescence-associated secretory phenotype, would very likely cause some phenomena that would not be observed in p16^Ink4a-senescent cells or in their effects on neighbors.

References

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18: Kirkland JL. Aging, Adipose Tissue, and Cellular Senescence. Abstracts of Strategies for Engineered Negligible Senescence (SENS) Fifth Conference. August 31-September 4, 2011. Cambridge, United Kingdom. Rejuvenation Res. 2011 Aug;14 Suppl 1:S11-45. PubMed PMID: 21847798.

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20: Villaret A, Galitzky J, Decaunes P, Estève D, Marques MA, Sengenès C, Chiotasso P, Tchkonia T, Lafontan M, Kirkland JL, Bouloumié A. Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence. Diabetes. 2010 Nov;59(11):2755-63. Epub 2010 Aug 16. PubMed PMID: 20713685; PubMed Central PMCID: PMC2963533.

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23: Smithey MJ, Renkema KR, Rudd BD, Nikolich-Žugich J. Increased apoptosis, curtailed expansion and incomplete differentiation of CD8+ T cells combine to decrease clearance of L. monocytogenes in old mice. Eur J Immunol. 2011 May;41(5):1352-64. doi: 10.1002/eji.201041141. Epub 2011 Apr 14. PubMed PMID: 21469120.

24: Chien Y, Scuoppo C, Wang X, Fang X, Balgley B, Bolden JE, Premsrirut P, Luo W, Chicas A, Lee CS, Kogan SC, Lowe SW. Control of the senescence-associated secretory phenotype by NF-{kappa}B promotes senescence and enhances chemosensitivity. Genes Dev. 2011 Oct 15;25(20):2125-36. Epub 2011 Oct 6. PubMed PMID: 21979375.v

25: Radaeva S, Sun R, Jaruga B, Nguyen VT, Tian Z, Gao B. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners. Gastroenterology. 2006 Feb;130(2):435-52. PubMed PMID: 16472598.

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28: Soriani A, Zingoni A, Cerboni C, Iannitto ML, Ricciardi MR, Di Gialleonardo V, Cippitelli M, Fionda C, Petrucci MT, Guarini A, Foà R, Santoni A. ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype. Blood. 2009 Apr 9;113(15):3503-11. Epub 2008 Dec 19. PubMed PMID: 19098271.

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SENS Foundation and Age-Related Macular Degeneration, Part 1

Tanya Jones — Thu, 03 Nov 2011 22:19:55 +0000

In this three part series, SENS Foundation researcher Max Peto describes the pathophysiology of age-related macular degeneration (ARMD), and how we direct our internal research efforts to reverse the pathology of this terrible, debilitating disease.

The first part of this series will discuss the macro-physiology of the eye, describing the major structural features of the eye. The second part will “zoom-in” to the biochemistry of the visual cycle and the pathophysiology of ARMD will be described. Finally the third part will include a discussion on how we are approaching the development of potential therapeutics internally at the SENS Foundation Research Center, and how these strategies relate to the pathophysiology of ARMD.

Part I – Macro-anatomy of the human eye

While first reading material associated with macular degeneration, I discovered that there are many parts of the eye, and these are necessarily small. An understanding of how these many small structures interact with one another and work together to enable vision is critical in understanding the pathophysiology of ARMD. However, there is an even greater level of detail involved in understanding the structure and function of the eye than one might first think. First, there are larger macrostructures, such as the macula, retina, choroid, and others. But then there are smaller microstructures, such as rod, cone, and RPE cells, and relationships among these are also critical to understanding ARMD. In this post, I’ll first describe the macrostructures (including pictures), and discuss how they are related to one another. In the next post (Part II), I’ll zoom into the microstructures and discuss the biochemical details of the pathophysiology of ARMD.

Macro-anatomy of the human eye

First, I’ll start off with a good picture:

I won’t focus on many of these parts of the eye, as they’re not particularly relevant to ARMD. But I now bring your attention to those which do. These are the:

Choroid (3),
Retina (7), and
Macula and fovea (8)

I’ll start by describing the macula (this is macular degeneration we’re talking about).

The macula is the darker-colored part of the above picture, labeled “8.”. The following is a useful picture of an actual macula of a right eye:

Note again how the macula (also called the “macula lutea” or the “macula of the retina”) is the dark spot in at the center of the retina in the back of the eye, and has a high density of photoreceptor cells, particularly in the “fovea” or “foveal region” of the macula. Note that the macula sits at the back of the eye, directly inward from the lens of the eye, enabling highly-focused central vision by specially-processing light that enters straight into the eye and travels directly to the back of the eye. From this information, one can see that the above picture is not quite back-to-front of the eye, but was taken obliquely.

The fovea is a notable part of the macula, which has the highest concentration and density of cone cells in the eye. The macula, and more specifically the fovea, enables high-acuity central vision.

The retina is large, thin structure, and is the shape of ~70% of a sphere that sits in the back of the eye on top of the choroid layer. I think of the retina as a spherical bowl or round-bottomed cylinder of nerve cells into which light is funneled. This can be better visualized by thinking about the first picture above, which is a cross-section of an illustrated eye. I’m referring to the place where the two layers are pulled away from the back of the eye, illustrating the retina resting on the choroid. The retinal nerve cells transmit light signals to the brain via the optic nerve.

The retina sits on the choroid layer, which is the vascularized layer providing a blood (and thus nutrient) supply to the eye. I will mention this again later, but keep in mind that the retinal pigment epithelial (RPE) cells sit almost directly on top of the choroid layer. This interplay between RPE cells, photoreceptor cells, and the choroid is going to be relevant in a later discussion on photoreceptor cell metabolism (particularly waste metabolism).

To review, from the back (deepest part) of the eye to the front:

The retina sits on the choroid layer, a layer consisting of blood vessels and capillaries which provide the eye with nourishment.
The retina is a spherical light-funnel which sits in the back of the eye on the choroid, and is composed of ten layers, including nerve tissue which transmits signals to the optic nerve which are later interpreted as vision in the brain.
The macular region is situated on the center of the retina in the back of the eye, directly inward from the lens where light is allowed into the eye.
The fovea is a special, central part of the macular region, and has a very high concentration of cone-type photoreceptor cells, which are critical in enabling high-acuity central vision (which is lost in ARMD).

At the RC, we frequently talk about retinal pigmented epithelial cells, also known as RPE cells. RPE cells are actually one layer of ten which make up the retina, so keep in mind that RPE cells are actually a small part of the retina (which is why they’re called retinal PE cells).

In the next post, I’ll elaborate on RPE cells, photoreceptor cell metabolism, the visual cycle, and how these interact to cause the pathophysiology of age-related macular degeneration.

Novel Abeta Vaccine Reports First Human Data

Michael Rae — Mon, 24 Oct 2011 01:27:14 +0000

Accumulation of soluble and insoluble aggregates of beta-amyloid protein (Aß) and other malformed proteins accumulate in brain aging and neurodegenerative disease, leading progressively to neuronal dysfunction and/or loss. These have long been widely accepted to be drivers of Alzheimer's disease (AD) and other age-related dementias and neurological disorders such as Parkinson's disease, and it has recently become increasingly clear that neuronal protein aggregates are the main driver of "normal" cognitive aging. To prevent and reverse the course of neurodegenerative disease and age-related cognitive dysfunction, the regenerative engineering solution is therapeutic clearance of extracellular aggregates (such as Aß plaques) and intracellular aggregates (such as soluble, oligomeric Aß).

Immunotherapeutic Aß clearance from the brain is a very active field of Alzheimer's research, with at least seven passive, and several second-generation active, Aß vaccines currently in human clinical trials.(1) Of all Aß immunotherapies, he furthest advanced along the clinical pipeline are the passive monoclonal antibody vaccines bapineuzumab/AAB-001 (Janssen/Elan/Pfizer) and solanezumab/LY2062430 (Eli Lilly), both of which are currently in Phase III clinical trials. Other approaches, still in preclinical development, include the use of beta-amyloid-targeting affibodies, DNA and peptide vaccination targeting beta-amyloid epitopes, and catalytic cleavage of the beta-amyloid peptide itself. We now have a published report of preliminary findings from the first Phase I trial in an Aß-targeting vaccine with novel properties, and with the benefit of preliminary findings of outcomes that have only emerged with the experience of its forerunners in previous clinical trials.

Novel Target Antigen

The new contender -- Hoffmann-La Roche/Morphosys candidate gantenerumab (R1450 or RO4909832) -- is the first fully human anti-Aβ monoclonal antibody to enter clinical development: previous candidates have been humanized versions of murine antibodies, or derived from antibody fragments, or antibodies already present in pooled human immunoglobulin for injection (IVIgG). Gantenerumab was selected from a human phage display library and "optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-β fibrils ... In peptide maps, both N-terminal and central portions of Aβ were recognized by gantenerumab."(2) This, too, is a novel charcteristic of the new passive vaccine: the investigators claim it is unique amongst therapeutic antibodies, and certainly most anti-Aβ antibodies in clinical development recognize B-cell epitopes located either at the N-terminus of the protein (as does bapineuzumab) or a central span (eg. solanezumab); those that exploit T-cell targeted epitopes (including those elicited through active vaccination) map primarily to the central span and C-terminus of the peptide.(1)

Novel Clearance Mechanism

Gantenerumab may also be distinct from its passive vaccine competitors in using a cell-mediated mechanism of action for the removal of Aβ from the brain. AN1792, the first Aβ-targeting vaccine to enter clinical trials, was an active vaccine, and were mediated through T-cell responses specific to the carboxy terminal of the peptide; to date, passive antibodies have appeared to work by inducing efflux of Aβ from the brain, either through passage of the antibody through the blood-brain barrier (BBB) followed by sequestering of brain Aβ and then cotransportation of IgG-Aβ immune complexes back through the BBB (eg bapineuzumab), or through the "peripheral sink" mechanism of binding systemic Aβ and eliciting drawdown of soluble Aβ from behind it (eg. solanezumab). While still in preclinical development, the approach that SENS Foundation finds most exciting (on first principles, and based on results to date) is catalytic clearance of the beta-amyloid peptide itself.*

In contrast to all of these, gantenerumab, appears to be the first passive vaccine to stimulate microglial phagocytosis of Aβ. "In functional assays gantenerumab induced cellular phagocytosis of human amyloid-β deposits in AD brain slices when co-cultured with primary human macrophages";(2) "In ex vivo studies of human brain slices from an independent sample of patients who had AD ... Gantenerumab induced phagocytosis of human amyloid in a dose-dependent manner ex vivo."(3) Promisingly, and consistent with such a mechanism, when tested model AD mice bearing transgenic familial AD mutations in amyloid precursor protein and presenillin-2, "gantenerumab showed sustained binding to cerebral amyloid-β and, upon chronic [5 mo] treatment, significantly reduced small amyloid-β plaques by recruiting microglia and prevented new plaque formation. Unlike other Aβ antibodies, gantenerumab did not alter plasma Aβ, suggesting undisturbed systemic clearance of soluble Aβ."(2)

Those findings have now been supplemented with the first data derived from human clinical trials.

Phase I Clinical Trial Data

In a Phase I clinical trial,(3) 18 mild to moderate AD patients, aged 50-90 y, recruited from 3 university medical centers, were randomized to receive 7 monthly intravenous infusions of placebo or gantenerumab (60 or 200 mg). These doses were derived from previous, unpublished, single- and multiple-dose Phase I studies. In practice, however, while all subjects receiving the lower dose of antibody received the full course of therapy, few patients in the higher-dose group received all 7 infusions, due to early termination (see below): "One patient received 2 infusions, 2 patients received 3 infusions, 2 patients received 4 infusions, and 1 patient received 5 infusions."(3) Sixteen of these patients underwent carbon 11–labeled positron emission tomographic imaging with the Aβ-binding imaging agent, Pittsburgh Compound B (PiB-PET).

The results: based on PiB-PET, "The mean (95% CI) percent change from baseline difference relative to placebo (n = 4) in cortical brain amyloid level was -15.6% (95% CI, -42.7 to 11.6) for the 60-mg group (n = 6) and -35.7% (95% CI, -63.5 to -7.9) for the 200-mg group (n = 6)."(3) (See Figures 3 and 4, reproduced from original report, below). The approximate doubling of brain Aβ clearance in the higher-dose group is all the more remarkable considering the truncated course of therapy experienced by most (see below), and appears on a nominal basis to represent a faster rate of clearance than that previously reported for bapineuzumab over the course of 18 months (and that, without clear evidence of a dose-proportionate response).(4)

Reproduced from (4). Copyright American Academy of Neurology and the Authors.

The reason for the early termination of the trial was -- unfortunately but not unexpectedly -- the appearance of an adverse reaction in the high-dose gantenerumab group: MRI revealed that 2 subjects underwent transient periods of focal cerebrovascular inflammation or vasogenic oedema, coinciding with sites with the greatest local clearance of Aβ deposits.(3) Their appearance, while not welcome, would have been anticipated, based on their earlier occurrence in Phase II trials with bapineuzumab(4) and possibly solanezumab,(5) but ongoing study of these "amyloid-related imaging abnomralities" (ARIA)(11) now suggests that they are less concerning than they had initially appeared. Data from cerebral imaging studies of the general, non-AD population of Rotterdam show that haemosiderin abnormalities (ARIA-H) consistent with cerebral microbleeds occur in 3-15% of older adults,(6) and several studies presented at the 2011 Alzheimer’s Association International Conference (AAIC, formerly ICAD) suggest that vasogenic oedema (ARIA-E) may also be common in untreated subjects with the disease. In fact, reports there and elsewhere (including preclinical studies on the mechanism of such abnormalities, and a central blinded review of sequential brain images from the Phase II bapineuzumab trial) suggest that their increased occurrence in patients treated with Aβ-binding therapeutic antibodies is not only relatively mild and transient, but a potentially positive rough indicator of successful mobilization of brain Aβ.*

The combination of initial, dose-dependent reduction of brain Aβ as detected on PiB-PET, plus (ironically) the occurrence of vasogenic oedema at locations adjacent to the sites where clearance is greatest, is tantalizing evidence that gantenerumab is efficacious in removing these malformed proteins from the brain. The study was too small and early-phase to permit assessment of cognitive outcomes, but with the success of the Phase I trial, a Phase IIb clinical trial is now underway, which will test the effects of subcutaneous gantenerumab (225 or 105 mg every 4 weeks for 104 weeks) vs. placebo on cognitive and functional outcomes in subjects with prodromal AD (identified based on partner-observed, gradual reductions in memory and cerebrospinal fluid biomarkers, but without dementia (Mini-Mental State Exam score ≥24).

The Race is On

With promising preliminary human data and what appears to be a mechanism of Aβ clearance, the advancement of gantenerumab into preliminary efficacy testing places one more damage-removal therapeutic into the race for disease-modifying therapies for AD. Should it ultimately be successful and its migroglial cell-mediated mechanism of action be validated, it would have the advantage of being potentially in synergy with lysosomal fortification with novel hydrolases to enhance microglial lysosomal hydrolysis of engulfed Aβ.

Rejuvenation biotechnology is the application of the principles of regenerative medicine to the damage to cellular and molecular structures that accumulate in aging tissues -- the structural damage that disables those structures' function and leads to loss of homeostasis and the progressive rise in frailty, disease, disability, and death that people now suffer with age. Because the damage is multifarious, a platform of rejuvenation biotechnologies, rather than a single, all-encompassing "youth pill," will be required to achieve the robust rejuvenation of aging humans, restoring youthful health and vitality.

SENS is a strategy for engineering negligible senescence, based on this heuristic -- not a prescriptive list of therapies in development whereby it shall be executed. The rapidly-expanding group of agents, each with a meaningfully-distinct mechanism of Aβ clearance, entering into the clinical pipeline and in increasingly advanced stages of human clinical testing for the arrest and reversal of Alzheimer's disease, bodes well for the early achievement of the first rejuvenation biotechnology. SENS Foundation is proud to be engaged in its mission of catalyzing the progress toward a mature rejuvenation biotechnology industry; with that proof of concept, the wider biomedical field should become more alive to the application of the damage-removal heuristic to the many different kinds of aging damage underlying age-related disease. The engagement of a wide range of scientists in academia and industry, and aggressive funding of rejuvenation research, will accelerate progress toward a comprehensive panel of rejuvenation biotechnologies, and the achievement of thoroughgoing biomedical restoration of youthful health, vigor, and longevity.

* The theoretical advantages of catalytic antibodies as a mechanism for the therapeutic clearance of malformed protein deposits, combined with promising results to date in preclinical studies using such antibodies to clear brain Aβ, were key considerations leading to SENS Foundation's funding of research into catalytic antibodies for the removal of senile cardiac amyloidosis.

References

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