Lie PP, Yang DS, Stavrides P, Goulbourne CN, Zheng P, Mohan PS, Cataldo AM, Nixon RA. Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons. Cell Rep. 2021 Apr 27;35(4):109034. PubMed.
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Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP)
Lysosomes are degradative organelles that serve to coordinate the turnover of macromolecules with metabolic flux. In non-neuronal cells, lysosomes and lysosome-related organelles, defined by their acidic lumen and complement of lysosomal membrane proteins, have been shown to be heterogenous with respect to size, ultrastructure, dynamics, and content of hydrolytic enzymes. Defects in lysosome function have particularly prominent effects in the brain because neurons are long-lived cells that are subject to special proteotoxic challenges. This is evident by the occurrence of neurodegenerative disorders that arise from the age-related accumulation of toxic protein aggregates such as extracellular Aβ deposits and intracellular neurofibrillary tangles found in Alzheimer's disease (AD).
Given that many of the aggregation-prone proteins that accumulate in neurodegenerative diseases affect neuronal axons and/or synapses, a crucial question is whether mature degradative lysosomes enter distal axons to locally degrade substrate proteins or if their distribution and dynamics are restricted to the somatodendritic compartment and possibly proximal axons.
In this new study in Cell Reports, Nixon and colleagues have tackled this question by characterizing the distribution, functional and biochemical features, and axonal trafficking of degradative organelles in the mouse brain in vivo using a plethora of markers and criteria. They provide strong evidence that mature acidic lysosomes containing active cathepsins are absent from axons and, instead, undergo selective retrograde transport to neuronal somata. In contrast, non-acidic (or moderately acidified) LAMP1 carriers arising from the trans-Golgi network are seen to enter axons and be transported anterogradely to synapses, consistent with their proposed role in presynaptic biogenesis (Vukoja et al., 2018). Acidified late endosomes also are transported exclusively retrogradely in axons. These data also call for a more cautionary use of the term lysosome to mark acidic degradative organelles rich in hydrolytic enzymes.
These results thus suggest that the degradative capacity of distal axons is limited. As a consequence, it appears that dystrophic axonal varicosities observed in AD models and in AD brain are caused by their failure to be degraded locally. Although the exact reasons for this failure require further studies, this new work by Lie et al. argues for an important role in retrograde delivery of aggregation-prone cargoes, e.g. in AD, via autophagic vesicles or late endosomes to neuronal somata to achieve proper clearance. Boosting retrograde transport of autophagic vesicles and/or late endosomes may thus provide a viable therapeutic option to combat AD in humans.
References:
Vukoja A, Rey U, Petzoldt AG, Ott C, Vollweiter D, Quentin C, Puchkov D, Reynolds E, Lehmann M, Hohensee S, Rosa S, Lipowsky R, Sigrist SJ, Haucke V. Presynaptic Biogenesis Requires Axonal Transport of Lysosome-Related Vesicles. Neuron. 2018 Sep 19;99(6):1216-1232.e7. Epub 2018 Aug 30 PubMed.
Johannes Gutenberg-University Mainz, Medical School
In the 1950s, Christian De Duve identified a new cellular organelle, the lysosome, or “digestive body,” which proved to be packed with degradative activity and to be the end of the line in essential cellular pathways such as autophagy or the endo-lysosomal system. Dysfunction of the organelle was connected to multiple disorders, including neurodegeneration. However, besides its important role in destroying cellular components, the lysosome turned out to be a real scientific “troublemaker”: Due to its complex maturation and the existence of several precursors, the identification of a fully digestive (mature) lysosome proved tricky, resulting in several misinterpretations. Moreover, in neurons vesicular processes are compartmentalized and active long-distance trafficking and shuttling processes occur.
From the 1990s on, the Nixon lab has proposed that disturbances in the endosomal-lysosomal pathway occur early in Alzheimer’s disease pathogenesis (Cataldo et al., 1990, 1991; Pensalfini et al., 2020) and they have demonstrated the key importance of autophagy in AD (Bordi et al., 2016). This recent study from their lab by Lie et al. now focuses on the identification of mature (!) lysosomes and their differentiation from lysosomal precursors in neurons. In fact, Lie et al. demonstrate that fully active lysosomes are restricted to the neuronal soma and are not found in axons. Non-degradative axonal organelles, previously misidentified as lysosomes, are acidified and provided with lysosomal components during their retrograde transport to the soma. Investigating this process in AD models, the authors show that the pre-degradative components accumulate in dystrophic axons without achieving full lysosomal identity.
This study has clear implications for the overall degradative potential of axonal structures, but even more, 70 years after the initial characterization of lysosomes, it illustrates the precautions necessary in accurately identifying them in neurons and in drawing any conclusions about their functional implications. Further, this elegant and detailed study underlines the importance of the degradative autophagy-lysosomal pathway for the maintenance of neuronal protein homeostasis and, therefore, for preventing AD, and it certainly calls for special care when addressing organelle function and trafficking, in particular when it comes to lysosomes.
Being aware that endosomal dysfunction in neurons is basically the earliest known pathobiology specific to AD, the stringent analysis of the endosomal-lysosomal and autophagy-lysosomal pathways/systems will promote our understanding of AD pathogenesis.
References:
Cataldo AM, Thayer CY, Bird ED, Wheelock TR, Nixon RA. Lysosomal proteinase antigens are prominently localized within senile plaques of Alzheimer's disease: evidence for a neuronal origin. Brain Res. 1990 Apr 16;513(2):181-92. PubMed.
Cataldo AM, Paskevich PA, Kominami E, Nixon RA. Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):10998-1002. PubMed.
Bordi M, Berg MJ, Mohan PS, Peterhoff CM, Alldred MJ, Che S, Ginsberg SD, Nixon RA. Autophagy flux in CA1 neurons of Alzheimer hippocampus: Increased induction overburdens failing lysosomes to propel neuritic dystrophy. Autophagy. 2016 Dec;12(12):2467-2483. Epub 2016 Nov 4 PubMed.
Pensalfini A, Kim S, Subbanna S, Bleiwas C, Goulbourne CN, Stavrides PH, Jiang Y, Lee JH, Darji S, Pawlik M, Huo C, Peddy J, Berg MJ, Smiley JF, Basavarajappa BS, Nixon RA. Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease. Cell Rep. 2020 Nov 24;33(8):108420. PubMed.
Tufts University School of Medicine
In this paper, Lie et al. rigorously show that mature lysosomes are not present in axons. The generation of a new transgenic mouse model expressing human cathepsin D under the control of Thy1 promoter allowed the investigators to clearly demonstrate the absence of lysosomes in axons in vivo.
By employing pH-sensitive fluorescent reporters, immunostaining, and electron microscopy, Lie et al. identified vesicles generated at the trans-Golgi network (TGN) that carry lysosomal hydrolases into the axon and which can be mistaken for lysosomes if not rigorously characterized. These findings are particularly important because they clarify a controversial topic in the field of neurobiology.
The localization of mature lysosomes to the soma of the neuron requires that axonal proteins, e.g. BACE1, be retrogradely transported to be degraded in lysosomes. These findings further support our recent observation that BACE1 accumulates in axons in vitro and in vivo in the absence of the clathrin adaptor GGA3 (Lomoio et al., 2020). Our studies demonstrated that BACE1 is degraded in the lysosomes, and that GGA3 is a key regulator of BACE1 trafficking and degradation (Koh et al., 2005; Tesco et al., 2007). Our most recent data elucidate a specific role for GGA3 in coordinating BACE1 axonal trafficking. Our findings indicate that GGA3 loss of function, due to genetic deletion or to an AD-linked GGA3 variant, induces an impairment of BACE1 retrograde trafficking, most likely by affecting the subset of vesicles that co-transport both proteins and are mainly retrograde-directed. As a consequence, BACE1 cannot be trafficked back to the soma, where it is normally degraded in mature lysosomes, and instead starts accumulating in the axon, triggering axonal dystrophies in vitro and in vivo.
Given that GGAs sort mannose 6-phosphate receptors from the TGN to the endosomal-lysosomal system, it would be also interesting to study the impact of GGA3 loss of function on the axonal delivery of TGN-derived transport carriers to retrogradely transported pre-degradative vesicles (Puertollano et al., 2001).
References:
Lomoio S, Willen R, Kim W, Ho KZ, Robinson EK, Prokopenko D, Kennedy ME, Tanzi RE, Tesco G. Gga3 deletion and a GGA3 rare variant associated with late onset Alzheimer's disease trigger BACE1 accumulation in axonal swellings. Sci Transl Med. 2020 Nov 18;12(570) PubMed.
Koh YH, von Arnim CA, Hyman BT, Tanzi RE, Tesco G. BACE is degraded via the lysosomal pathway. J Biol Chem. 2005 Sep 16;280(37):32499-504. PubMed.
Tesco G, Koh YH, Kang EL, Cameron AN, Das S, Sena-Esteves M, Hiltunen M, Yang SH, Zhong Z, Shen Y, Simpkins JW, Tanzi RE. Depletion of GGA3 stabilizes BACE and enhances beta-secretase activity. Neuron. 2007 Jun 7;54(5):721-37. PubMed.
Puertollano R, Aguilar RC, Gorshkova I, Crouch RJ, Bonifacino JS. Sorting of mannose 6-phosphate receptors mediated by the GGAs. Science. 2001 Jun 1;292(5522):1712-6. PubMed.
Juntendo University Graduate School of Medicine
Lysosome-related organelles in the axons of intact and dystrophic brains
Lysosomes are multifunctional, membrane-bound, acidic organelles that contain various acid hydrolases that degrade excess, old, and unneeded intracellular and extracellular materials, including cytoplasmic organelles, into biologically active monomers. Acidity of mature lysosomes can range from pH 4.5 to 5.0, and they lack mannose 6-phosphate receptors. The intracellular processes of mature lysosomes in neurons are basically similar to those in mammalian cells under normal conditions.
The structural and functional properties of axons differ from the dendrites and soma of neurons. Axons have long and thin extending compartments that include synaptic regions, and they are specialized at delivering information to other neurons. The maintenance of homeostasis requires the inevitable removal of excess, old, and unneeded substances from the limited compartments of axons. Recent studies have clarified the ability of autophagic responses to eliminate metabolites and unneeded structures in axons, particularly in the synaptic region (Yue et al., 2007; Koike et al., 2017). There is a question of whether degradation in neurons occurs within the axonal compartment in mature lysosomes or in other types of acidic compartments. Thus far, numerous in vivo and in vitro studies have attempted to answer this basic question.
Recently, Lie et al. reported a study that addressed this problem. For this, they first engineered transgenic mice expressing a neuron-specific lysosomal aspartic proteinase, cathepsin D (CtsD), and then examined vesicular CTSD acquisition, acidification, and trafficking within the autophagic-lysosomal pathway in vivo. They showed a very clear-cut result wherein mature lysosomes are restricted from axons. Previously, work using living cells expressing the mannose 6-phosphate receptor fused with green fluorescent protein (GFP-CI-MPR) had shown that at steady state, the bulk of GFP-CI-MPR localized to the trans-Golgi network (TGN), but significant amounts were also detected in peripheral, tubulo-vesicular structures as well as in early endosomes and at the plasma membrane (Waguri et al., 2003). Similar to those results, Lie et al. noted the presence of various types of tubulo-vesicular structures with TGN-derived transport carriers (TCs) that supply lysosomal components to axonal organelles, such as autophagic vacuoles (AV). They used electron microscopy to describe how distinctive TCs containing TGN and lysosomal markers enter axons and engage AVs and late endosomes (LEs). Such events are observed at the proximal portion of an axon but not in the distal portion. Lie et al. did not examine whether TCs and AVs fused with TCs (amphisomes or LEs) appear within the axon initial segment (AIS), which is long enough to be identified as the proximal axonal region and may have circumstances that differ from those of a proper axon. Moreover, to examine these events under disease conditions, they used a mouse model of amyloidosis and found that this process was markedly upregulated in the dystrophic axons of the PS/APP mice. To further confirm results from in vivo studies, Lie et al. performed in vitro studies using cultured neurons and concluded that the lack of mature lysosomes in axons is further strengthened by the observation that most axonal LAMP1 vesicles are labeled by a TGN38 homolog, while carrying lysosomal proteins such as TRPML1 and ACP2, which indicates their identity as either TCs or LEs/amphisomes but not lysosomes.
Lie et al. used recent genetic, molecular, and cell biology techniques to draw a clear-cut conclusion concerning the characteristic features of lysosomes in the axons of intact and dystrophic brains. That is, restricted entry of lysosomes into axons explains the unique lysosome distribution in neurons as well as their vulnerability toward neuritic dystrophy in a disease state.
References:
Yue Z. Regulation of neuronal autophagy in axon: implication of autophagy in axonal function and dysfunction/degeneration. Autophagy. 2007 Mar-Apr;3(2):139-41. PubMed.
Koike M, Shibata M, Sunabori T, Yamaguchi J, Sakimura K, Komatsu M, Tanaka K, Uchiyama Y. Purkinje Cells Are More Vulnerable to the Specific Depletion of Cathepsin D Than to That of Atg7. Am J Pathol. 2017 Jul;187(7):1586-1600. Epub 2017 May 11 PubMed.
Waguri S, Dewitte F, Le Borgne R, Rouillé Y, Uchiyama Y, Dubremetz JF, Hoflack B. Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells. Mol Biol Cell. 2003 Jan;14(1):142-55. PubMed.
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