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diseases, as endolysosomal and autophagosomal dysfunction has been demonstrated in multiple neurodegenerative diseases (Tresse et al., 2010; Urwin et al., 2010; Ginsberg et al., 2010; Harris and Rubinsztein, 2011), including Alzheimer’s, Parkinson’s, Huntington’s, and FTLD.

In Parkinson’s disease, mutations in genes encoding important endolysosomal/autophagosomal proteins have been shown to result in disease, and mutations in PD-associated genes with unclear functions have also been shown to result in endolysosomal/autophagosomal aberrations. For example, mutations in ATPase type 13A2 (ATP13A2), a component of the lysosomal acidification machinery, have been found in patients with hereditary parkinsonism (Williams et al., 2005). Fibroblasts derived from these patients exhibit defective clearance of autophagosomes and accumulation of α-synuclein (Usenovic et al.,

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2012; Ramirez et al., 2006); additionally, these mutations result in decreased activity of lysosomal enzymes and reduced degradation of substrates (Dehay et al., 2012). Mutations in PINK (PTEN- induced putative kinase) and PARK2 (which encodes a component of the ubiquitin ligase complex) are also known Mendelian causes of Parkinson’s disease and are associated with defective mitophagy (Geisler et al., 2010; Narendra et al., 2008; Valente et al., 2004). Additionally, mutations in VPS35, which encodes an endosomal protein involved in retrograde transport between endosomes and the trans-Golgi, have been shown to result in Parkinson’s

disease and result in decreased autophagosome biogenesis (Chartier-Harlin et al., 2011; Zimprich et al., 2011). Finally, missense mutations in leucine-rich repeat kinase 2 (LRRK2), which codes for a large protein called dardarin of uncertain function with functional GTPase and kinase domains, are the most common known genetic cause of Parkinson’s disease (Hedrich et al., 2006). Disease-associated mutations have been shown to affect vesicular trafficking, autophagy, cytoskeletal function, and protein synthesis in both cell and animal models of Parkinson’s (Martin et al., 2016; MacLeod et al., 2013).

In Alzheimer’s disease (AD), most data suggesting a link to endolysosomal/autophagosomal dysfunction is more correlative. For example, affected brains from Alzheimer’s patients as well as from animal models exhibit dystrophic neurites which contain accumulations of autophagosomes and autolysosomes (Lee et al., 2010; Nixon and Yang, 2011). Beclin-1, the mammalian ortholog of yeast Atg6 (Liang et al., 1998), has been shown to be decreased in AD patients and may be one of the initial signs of autophagic dysfunction in AD (Pickford et al., 2008). Abnormal upregulation of Rab GTPases, including Rab5 and Rab7, has been shown in cholinergic basal forebrain neurons microdissected from postmortem brains (Ginsberg et al., 2010) and, additionally, a genome-wide analysis shows transcriptional upregulation of autophagy-related genes in affected Alzheimer’s brain (Lipinski et al., 2010), possibly indicating a compensatory mechanism in response to autophagic dysfunction. However, perhaps the strongest link between AD and lysosomal dysfunction comes from work with presenilin-1 (PS1). Mutations in PSEN1 (presenilin 1) are one of the most common causes of

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familial AD. It has been reported that loss of PS1 or AD-associated mutations in PSEN1 result in impaired lysosomal acidification and degradative ability (Lee et al., 2010; Avrahami et al., 2013; Dobrowolski et al., 2012). In presenilin-1 knockout mouse blastocysts, Lee et al. reported defective clearance of autophagic vacuoles as well as defective lysosome acidification as measured by LysoTracker fluorescence (a pH sensitive dye whose intensity correlates with acidity). Lee et al. further provide evidence that loss of PS1 results in a failure of the V0a1 subunit of the vATPase to be properly N-glycosylated in the ER, resulting in impaired delivery of PS1 to autolysosomes and lysosomes (Lee et al., 2010).

In FTLD specifically, dysregulation of the endolysosomal/autophagosomal network has also been linked to disease. For example, mutations in the charged multivesicular body protein 2B (CHMP2B) gene are a known cause of disease. CHMP2B is a subunit of the ESCRT-III complex and plays a role in sorting ubiquitinated proteins into the intraluminal vesicles of late endosomes for ultimate degradation upon lysosomal fusion (Urwin et al., 2010; Filimonenko et al., 2007; Rusten and Stenmark, 2009; Katzmann et al., 2002). Disease-associated mutations in

CHMP2B result in the deletion of the C-terminus of the protein (Urwin et al., 2010) and expression of these C-terminal deletion mutant CHMP2Bs in human neuroblastoma cells results in the formation of enlarged late endosomes (van der Zee et al., 2008). Furthermore, patient fibroblasts as well as cortical neurons also exhibit enlarged endosomes (Urwin et al., 2010). This defect was attributed to impairments of endosome-lysosome fusion, as late endosomes in CHMP2B mutant cells were defective in their ability to recruit the Rab7 GTPase, a key player in the fusion of endosomes-lysosomes and lysosomes-autophagosomes (Bucci et al., 2000; Hyttinen et al., 2013; Jager et al., 2004). Mutant CHMP2B thus disturbs endosomal and autophagic trafficking (Urwin et al., 2010; Filimonenko et al., 2007; Lee et al., 2007), intimating at a general role for endolysosomal/autophagosomal disturbances in the development of FTLD.

In addition, mutations in VCP, which encodes valosin-containing protein (VCP), also cause a familial form of FTLD. Specifically, these VCP mutations cause a dominantly inherited, multisystem degenerative disease that affects muscle, bone, and brain. VCP has been shown to

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have a multitude of activities within the cell including cell-cycle regulation, DNA repair, organelle biogenesis, protein quality control, endolysosomal sorting, and autophagosome biogenesis and maturation (Ritz et al., 2011; Tresse et al., 2010; Ju et al., 2009; Braun et al., 2002). VCP is thought to be able to execute this variety of functions due to its N-terminal domain, which can interact with many different adaptor proteins. The conformation of this N-terminal domain is determined by the presence of ATP or ADP in the nucleotide binding pocket (Abramzon et al., 2012; Tang et al., 2010). FTLD-associated mutations alter the shape of the binding pocket, and this is thought to increase the interactions of VCP with some adaptors and decrease its interactions with others (Fernandez-Saiz and Buchberger, 2010).

Among its many functions, VCP has been shown to be essential for maturation of autophagosomes, as knockdown of VCP (or expression of dominant-negative VCP) results in the accumulation of immature autophagic vesicles (Tresse et al., 2010). FTLD-associated mutations in VCP have similarly been shown to interfere with and alter autophagosomal pathways, blocking transport of ubiquitinated cargo to lysosomes and also impairing the maturation of autophagosomes (Ju et al., 2009; Ritz et al., 2011). In addition, large LAMP1+/LAMP2+ vacuoles with accumulated LC3 and TDP-43 are seen in myoblasts of patients with VCP mutations (Ju et al., 2009; Tresse et al., 2010).

Mutations in CHMP2B and VCP thus intimate that dysregulation of endolysosomal/autophagosomal pathways may contribute to disease pathogenesis in FTLD.

1.2.5 Summary

Multiple lines of evidence are converging on dysregulation of the endolysosomal/autophagosomal network as a central common theme in neurodegenerative processes. Many neurodegenerative diseases exhibit impaired neuronal autophagy and accumulation of toxic proteins, with detrimental effects for cells. Proper lysosomal and autophagosomal function is vital to clear aggregates and maintain cellular homeostasis. This is of even more importance in the post-mitotic neuron, which cannot reduce the effects of damaged organelles or protein aggregates through cell division and must maintain cellular clearance and

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recycling of proteins over long distances between the cell body and distal processes (Lee et al., 2011b).

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