Jorge Busciglio Scott Brady

This live discussion is an update of last year's initial Live Discussion of axonal transport as an underlying factor in neurodegeneration. Jorge Busciglio and Scott Brady led this live discussion on 17 June 2003. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

View Transcript of Live Discussion — Posted 26 August 2006

Background Text
By Jorge Busciglio and Scott Brady

Neuronal cells are highly sensitive to transport defects because of their highly polarized morphology and large number of specialized microdomains. For their survival and proper function, neurons depend on the efficient delivery of proteins from the cell body to neuritic processes. Axons in particular are highly susceptible to transport deficiencies because they lack the elements necessary for protein synthesis. In this context, we have suggested that defects in protein transport play a critical role in Alzheimer's disease and other neurodegenerative conditions (Morfini et al., 2002a). Experimental evidence indicates that kinase and phosphatase activities are key regulators of fast axonal transport. Two major serine-threonine protein kinases, glycogen synthase kinase 3b (GSK3b) and cyclin-dependent kinase 5 (CDK5), have been implicated as major kinases responsible for both normal and pathological phosphorylation of tau protein in AD. Moreover, GSK3a, which is highly homologous to GSK3b, has been recently implicated in the modulation of Ab production (see ARF related news story). Both CDK5 and GSK3b have also been shown to regulate kinesin-driven motility. Specifically, GSK3b phosphorylates kinesin light chains in vivo and causes the release of kinesin from membrane-bound organelles (MBOs), leading to a reduction in kinesin-I-driven motility (Morfini et al., 2002b). Given the essential role of axonal transport in neuronal function, a misregulation of transport induced by an imbalance in specific kinase/phosphatase activities within neurons may represent an early and critical step of neuronal pathology.

Presenilin Mutations: They Do More Than Increase Ab

Significant evidence indicates that presenilin-1 (PS1) is essential for g-secretase activity. At the same time, there is also considerable evidence to suggest that PS1 has additional physiological functions, including control of calcium homeostasis, cell-cycle regulation, neurite outgrowth, apoptosis, membrane trafficking, and synaptic plasticity. In particular, PS1 has been implicated in regulating intracellular trafficking, maturation, and delivery to the cell surface of selected transmembrane proteins. Such effects of PS1 have been shown for the membrane proteins AbPP, TrkB and ICAM-5/telencephalin (Cai et al., 2002; Naruse et al., 1998; ARF related news story). One way in which PS1 might modulate intracellular protein trafficking is by regulating kinesin-based motility.

To determine whether PS1 alters kinesin-based protein transport, we utilized presenilin-1 knockout (PS1-/-) and mutant human PS1 knock-in M146V (KIM146V) mice and cultured their cells. We show that PS1 and GSK3b coimmunoprecipitate and colocalize in specific neuronal compartments, particularly growth cones (Pigino et al., 2003). Both FAD mutations in PS1, or the absence of PS1, increased relative levels of GSK3b activity. One possibility is that both PS1 and GSK3ß may be components of a trafficking regulatory complex at specific subcellular locations that is misregulated by the absence of PS1 or by the presence of PS1 mutations. Concomitant with increased GSK3b activity, PS1 deletion or PS1 mutations increased relative levels of kinesin light chain phosphorylation, and markedly reduced the amount of kinesin bound to MBOs. Consistent with a deficit in kinesin-mediated fast axonal transport, densities of synaptophysin and syntaxin-I containing vesicles and mitochondria were reduced in neuritic processes, but not cell bodies, of KIM146V hippocampal neurons. Similarly, we found reduced levels of PS1, AbPP, and synaptophysin in sciatic nerves of KIM146V mice. These results suggest that PS1 modulates GSK3b activity and normally affects the release of kinesin from MBOs at sites of vesicle delivery and membrane insertion.

In summary, we propose a model whereby mutations in PS1 compromise neuronal function by misregulating GSK3 activity, which would cause premature release of kinesin cargoes and impaired delivery of MBOs such as mitochondria to appropriate neuronal compartments. This ARF discussion will focus on the molecular mechanisms that may lead to disturbances of axonal transport and neurodegeneration in AD, including PS1 mutations, altered GSK3 kinase activity, tau hyperphosphorylation, and Ab production.

I suggest we discuss these questions during the chat:

1. The amyloid cascade hypothesis still draws majority support in the field as the predominant, if incomplete, explanation for AD. Can we integrate the axonal transport and the amyloid hypotheses? If yes, how do they fit together?

2. What is/are the earliest event(s) that disrupt axonal transport? Elevated GSK/CDK5 activity? Do we know it is occurring in AD? How can we find out?

3. In FAD, PS mutations would not only increase Ab production, but also increase GSK activity and disrupt transport. How about LOAD?

4. How could the relative roles of Ab generation and transport disruption by FAD PS1 be assessed? Video microscopy of FAD transgenic mouse neurons? What other methods are suitable?

5. How does PS1 modulate GSK3b? Most FAD PS1 mutations cause a gain of its AbPP-proteolytic function. If a different mechanism is at play, what is it and how do we find out?

6. How about GSK3a?

7. Are there therapeutic implications to this further development of the axonal transport hypothesis of AD?

References:
Cai D, Leem JY, Greenfield JP, Wang P, Kim BS, Wang R, Lopes KO, Kim SH, Zheng H, Greengard P, Sisodia SS, Thinakaran G, Xu H. Presenilin-1 regulates intracellular trafficking and cell surface delivery of beta-amyloid precursor protein. J Biol Chem. 2003 Jan 31;278(5):3446-54. Epub 2002 Nov 14. Abstract

Morfini G, Pigino G, Beffert U, Busciglio J, Brady ST. Fast axonal transport misregulation and Alzheimer's disease. Neuromolecular Med. 2002a;2(2):89-99. Review. Abstract

Morfini G, Szebenyi G, Elluru R, Ratner N, Brady ST. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. EMBO J. 2002b Feb 1;21(3):281-93. Abstract

Naruse S, Thinakaran G, Luo JJ, Kusiak JW, Tomita T, Iwatsubo T, Qian X, Ginty DD, Price DL, Borchelt DR, Wong PC, Sisodia SS. Effects of PS1 deficiency on membrane protein trafficking in neurons. Neuron. 1998 Nov;21(5):1213-21. Abstract

Pigino G, Morfini G, Pelsman A, Mattson, MM, Brady ST and Busciglio J. Alzheimer's presenilin-1 mutations impair kinesin-based axonal transport. J Neurosci, 1 June 2003. 23(11). Abstract

	Comments on Live Discussion
	Comment by:  Nikolaos Tezapsidis (Disclosure)
	Submitted 26 August 2006  |  Permalink	Posted 26 August 2006
	I would like to invite the audience to broaden their consideration of the topic of presenilin and axonal transport by including our current findings (Tezapsidis et al. 2003). Full text of the electronic version is publicly available. In our previous study (Johnsingh et al. 2000), we had demonstrated that presenilin 1 (PS1) interacts with the cytoplasmic linker protein 170/ Restin (CLIP-170). CLIP-170 is a microtubule plus-associated protein that directs minus-end movement of cargo-loaded vesicles. In our current paper, we disrupted this interaction in the neuronal cell lines SY5Y and N2a by transfecting vectors that drive the expression of peptide fragments corresponding to their binding domains (BDPs). Interestingly, disrupting the PS1/CLIP-170 complex is associated with both decreased secretion of endogenous A  Read more I would like to invite the audience to broaden their consideration of the topic of presenilin and axonal transport by including our current findings (Tezapsidis et al. 2003). Full text of the electronic version is publicly available. In our previous study (Johnsingh et al. 2000), we had demonstrated that presenilin 1 (PS1) interacts with the cytoplasmic linker protein 170/ Restin (CLIP-170). CLIP-170 is a microtubule plus-associated protein that directs minus-end movement of cargo-loaded vesicles. In our current paper, we disrupted this interaction in the neuronal cell lines SY5Y and N2a by transfecting vectors that drive the expression of peptide fragments corresponding to their binding domains (BDPs). Interestingly, disrupting the PS1/CLIP-170 complex is associated with both decreased secretion of endogenous Ab and decreased uptake of exogenous Ab from the medium. BDP-expressing cells were also more resistant to the surges of Ab secretion that are normally induced by thapsigargin and ionomycin, which elevate intracellular calcium concentrations, and by PS1 mutations linked to familial Alzheimer's disease. Uptake of Ab by SY5Y cells was amplified when preincubated with ApoE and was mediated through lipoprotein receptor-related protein (LRP). BDP-expressing cells or cells treated with PS1 antisense oligonucleotides took up less Ab from the medium compared to controls, indicating that the PS1/CLIP-170 interaction is involved and that PS1 cannot be substituted. In this study, we also mapped the minimum binding domains (mBDPs) of PS1 and CLIP-70 to regions corresponding to the N-terminal end of the large cytoplasmic loop of PS1 and to the C-terminal end of CLIP-170 that contains its metal-binding motif. The connection to axonal transport arises because our data from in-vitro taxol-polymerization of tubulin and confocal immmunofluorescence suggest that PS1, via CLIP-170, may serve as an anchor to microtubules for specific subcellular fractions containing amyloidogenic fragments. Interestingly, Notch is absent from this population of microtubule-binding subcellular fractions, and its cleavage was unaffected in cells transfected with the PS1-based BDP. This raises the possibility that the interaction of PS1 with CLIP-170 could provide a conceptual basis for antiamyloidogenic therapeutic strategies with improved specificity. However, this approach may be hampered by low efficiency, since it may also block Ab clearance from the interstitial space of the CNS. Our study is complementary to Jorge Busciglio's current paper. For example, CLIP-170 as a PS1 partner is subject to regulation by phosphorylation. GSK3b could well be among the various kinases/phosphatases capable of either promoting or inhibiting the binding of CLIP-170 to the microtubules. GSK3b inhibition by lithium is known to significantly increase the presence of CLIP-170-associated proteins (CLASP2) at distal MT ends, which should attract more CLIP-170, which should attract more PS1. This could be investigated more thoroughly. Minus-end directed motion within cells (i.e., retrograde transport) generally uses the motor protein dynein, while kinesin does the job in the other direction (anterograde transport). Even so, there is evidence suggesting that organelles of the same microtubule track can move in both directions (see Ma and Chisholm, 2002). At the molecular level, dynactin, previously thought to be exclusively part of the dynein/dynactin complex, can also bind kinesin II. Thus, there is room for both Busciglio's suggestion that PS1 regulates anterograde transport and our suggestion that PS1 regulates retrograde transport. We reported that mutated PS1 binds more tightly to CLIP-170 and this brings about more Ab. Busciglio and colleagues reported that mutated PS1 affects GSK3b activity, which slows down axonal transport. Perhaps these are linked; in fact, there is an abnormal cycle of increased retrograde vs. anterograde motion within AD neurons which results in making more Ab. Such a proposition would suggest that cytoskeletal abnormalities (i.e., tau phosphorylation) may actually precede amyloid build-up. In summary, these are the major points of our study: 1. Prior to its cleavage to become part of the g-secretase complex, PS1 interacts with CLIP-170. This interaction may be relevant to the docking step that Berezovska et al. describe in the same issue of J. Neurosci that features Jorge Busciglio's current paper. (See also schematic diagram in our paper.) 2. Neuronal cell lines take up Ab following its binding to ApoE through the LRP. This is confirmation of Kang et al., 2000, who worked with a different cell type. 3. Disruption of the PS1/CLIP-170 interaction led to decreased Ab production, decreased Ab uptake, but no effect on Notch cleavage. Thus, targeting this interaction can be more specific than targeting g-secretase activity and might avoid Notch-related side effects. Here is an interesting twist: How important is Ab uptake by neurons? Is it a clearance mechanism, a signaling event, or does it contribute to neurotoxic build-up inside? Before these questions are answered, it will not be known whether blocking the PS1/CLIP-170 interaction could be efficient as a therapeutic strategy. 4. PS1 may be involved in the docking step of intracellular vesicle transport toward the minus end of microtubules. A tighter binding of mutated PS1 to CLIP-170 at the tip of the microtubules may create conditions that favor the assembly of the g-secretase complex. View all comments by Nikolaos Tezapsidis

	Submit a Comment on this Live Discussion
	Cast your vote and/or make a comment on this live discussion.  If you already are a member, please login. Not sure if you are a member? Search our member database. *First Name   *Last Name   Country or Territory: USA Canada Afghanistan Albania Algeria American Samoa Andorra Angola Anguilla Antarctica Antigua and Barbuda Argentina Armenia Aruba Australia Austria Azerbaijan Bahamas Bahrain Bangladesh Barbados Belarus Belgium Belize Benin Bermuda Bhutan Bolivia Bosnia and Herzegovina Botswana Bouvet Island Brazil British Indian Ocean Territory British Virgin Islands Brunei Bulgaria Burkina Faso Burma Burundi Cambodia Cameroon Canada Cape Verde Cayman Islands Central African Republic Chad Chile China Christmas Island Cocos Islands Colombia Comoros Congo Cook Islands Corte d'lvoire (formerly Ivory Coast) Costa Rica Croatia Cuba Cyprus Czech Republic Denmark Djibouti Dominica Dominican Republic East Timor Ecuador Egypt El Salvador Equatorial Guinea Eritrea Estonia Ethiopia Falkland Islands Faroe Islands Fiji Finland France French Guiana French Polynesia French Southern Territories Gabon Gambia Georgia Germany Ghana Gibraltar Greece Greenland Grenada Guadeloupe Guam Guatemala Guinea Guinea-Bissau Guyana Haiti Heard and McDonald Islands Honduras Hong Kong Hungary Iceland India Indonesia Iran Iraq Ireland Israel Italy Ivory Coast (now known as Corte d'lvoire) Jamaica Japan Jordan Kazakhstan Kenya Kiribati Korea, North Korea, South Kuwait Kyrgyzstan Laos Latvia Lebanon Lesotho Liberia Libya Liechtenstein Lithuania Luxembourg Macau Macedonia ( Former Yugoslav Republic of) Madagascar Malawi Malaysia Maldives Mali Malta Marshall Islands Martinique Mauritania Mauritius Mayotte Mexico Micronesia ( Federated States of) Moldova Monaco Mongolia Montserrat Morocco Mozambique Myanmar Namibia Nauru Nepal Netherlands Netherlands Antilles New Caledonia New Zealand Nicaragua Niger Nigeria Niue Norfolk Island Northern Mariana Islands Norway Oman Pakistan Palau Panama Papua New Guinea Paraguay Peru Philippines Pitcairn Island Poland Portugal Puerto Rico Qatar Reunion Romania Russia Rwanda S. Georgia and S. Sandwich Isls. Saint Kitts & Nevis Saint Lucia Saint Vincent and The Grenadines Samoa San Marino Sao Tome and Principe Saudi Arabia Senegal Seychelles Sierra Leone Singapore Slovakia Slovenia Somalia South Africa Spain Sri Lanka St. Helena St. Pierre and Miquelon Sudan Suriname Svalbard and Jan Mayen Islands Swaziland Sweden Switzerland Syria Taiwan Tajikistan Tanzania Thailand Togo Tokelau Tonga Trinidad and Tobago Tunisia Turkey Turkmenistan Turks and Caicos Islands Tuvalu U.S. Minor Outlying Islands U.S.A. Uganda Ukraine United Arab Emirates United Kingdom Uruguay Uzbekistan Vanuatu Vatican City Venezuela Vietnam Virgin Islands Wallis and Futuna Islands Western Sahara Yemen Yugoslavia (Former) Zaire Zambia Zimbabwe *Login Email Address   *Password    Minimum of 8 characters *Confirm Password   Stay signed in?   Comment: (If coauthors exist for this comment, please enter their names and email addresses at the end of the comment.) References: *Enter the verification code you see in the picture below: This helps Alzforum prevent automated registrations. Terms and Conditions of Use: Printable Version By clicking on the 'I accept' below, you are agreeing to the Terms and Conditions of Use above.