WO2011066430A2 - Therapeutic agents for neurodegenerative diseases - Google Patents

Abstract

Methods of treating diseases involving protein aggregation are provided. Methods of identifying and selecting an autophagosome/lysosome fusion enhancer are also provided.

Description

THERAPEUTIC AGENTS FOR NEURODEGENERATIVE DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional patent application serial number 61/264,597, filed November 25, 2009, U.S. Provisional patent application serial number 61/287,112, filed December 16, 2009, and U.S. Provisional patent application serial number 61/284,752, filed December 23, 2009, each of which is herein incorporated by reference.

FIELD OF INVENTION

[0002] The disclosure relates to diagnostic and therapeutic methods, assays for screening for active agents, and to therapeutic agents all relating to conditions that involve the nervous system, including neurodegenerative diseases.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under Grant No. NS-054022, awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] Autophagy was initially characterized as a non-selective degradation process in response to starvation. Nutrient-independent autophagic functions have recently become evident. This so called "basal" autophagy, while not essential for survival, appears to function as intracellular quality control machinery crucial for protecting individuals from devastating disorders, such as neurodegenerative disease.

[0005] Autophagy is the primary degradation pathway responsible for the disposal of long-lived proteins, macromolecular complexes and organelles. Autophagy consists of two discrete but essential steps: the formation of autophagosomes that sequester cytosolic constituents, and the delivery of autophagic substrates to lysosomes where the contents are degraded. The ability of autophagosomes to sequester substrates of diverse sizes and origins provides a unique degradative capacity that complements the proteasome system. However, because autophagosomes lack intrinsic protease activities, productive autophagy involves the efficient fusion of autophagosomes to lysosomes. Although the characterization of the essential autophagy-related genes (ATG) has yielded insight into the mechanism of autophagy activation and autophagosome formation, the fusion processes of autophagosomes to lysosomes remain poorly understood. [0006] The second unresolved issue in autophagy lies with its unique substrate repertoire.

Autophagy has been predominantly characterized as a non-selective degradative pathway activated by starvation. In this context, autophagy degrades cytosolic contents and organelles non-discriminatively to supply cells with essential macromolecules and energy for survival. However, it has become apparent that autophagy is not solely dedicated to nutrient management. Although this nutrient-independent "basal" autophagy remains poorly defined, evidence suggests that its main function is to enforce intracellular quality control by selectively disposing of protein aggregates and damaged organelles, including mitochondria. Such an activity is best illustrated by the finding that neural-specific ablation of atg5 or atg7, two genes involved in autophagy, leads to accumulation of ubiquitin-positive protein aggregates and progressive loss of neurons in mice (Hara et al , Nature 441 : 885-889 (2006); Komatsu et al, Nature 441 : 880-884 (2006)). This form of autophagy is referred to herein as basal quality-control (QC) autophagy to better define its specific function. Accordingly, despite their shared dependence on a common ATG machinery and ability to degrade cytosolic constituents via lysosomes, basal QC autophagy and starvation-induced autophagy are distinct in their function, nature and substrate specificity. There is, however, little molecular understanding of what distinguishes these two fundamental autophagic modes. Nor is it known how basal QC autophagy achieves substrate specificity.

[0007] The protein deacetylase HDAC6 plays a role in the cellular management of protein aggregates. Unique among histone deacetylase (HDAC) family members, HDAC6 has an intrinsic ubiquitin-binding activity and associates with both microtubules and the F-actin cytoskeleton (Gao et al. , Mol. Cell Biol. 27: 8637-8647 (2007); Hubbert et al , Nature 417: 455-458 (2002); Kawaguchi et al, Cell 115: 727-738 (2003); Seigneurin-Berny et al. , Mol. Cell Biol. 21: 8035-8044 (2001); Zhang et al., Mol. Cell 27: 197-213 (2007)). While the HDAC6-actin interaction has been primarily linked to the regulation of cell motility, its association with the microtubule network and ubiquitinated proteins has led to the finding that HDAC6 is a regulatory component of the aggresome, the microtubule organizing center (MTOC)-localized inclusion body where excess protein aggregates are deposited. The formation of aggresomes, which are related to Lewy bodies found in many forms of neurodegenerative diseases, is proposed to protect cells by concentrating toxic protein aggregates to the MTOC where they are processed by autophagy (Kopito, Trends Cell Biol. 10: 524-530 (2000)). Through its ubiquitin-binding BUZ finger motif, HDAC6 binds and facilitates the transport of ubiquitinated misfolded proteins via the microtubule network to form the aggresome (Kawaguchi et al. , 2003). Evidence suggests that HDAC6 also plays a role in the eventual clearance of aggresomes, implying a functional connection between HDAC6 and autophagy (Iwata et al. , J. Biol. Chem. 280: 40282-40292 (2005); Pandey et al., Nature 447: 859-863 (2007)). [0008] Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder and is characterized by the selective degeneration of dopaminergic neurons in the substantia nigra. The neurological lesions are frequently accompanied by the appearance of prominent cytoplasmic inclusion bodies, the Lewy bodies, which contain ubiquitin-positive protein aggregates. The prevalence of the Lewy bodies in PD patients has led to a central proposal that aberrant accumulation of protein aggregate is a contributing factor to the development of Parkinsonism. Given the state of the art, additional methods for treating diseases relating to protein aggregation and methods for selecting autophagosome/lysosome fusion enhancers are needed.

SUMMARY

[0009] In an aspect, the disclosure provides a method of identifying an autophagosome/lysosome fusion enhancer. In embodiments, the method comprises contacting a host cell comprising a double tag system with an agent, where the double tag system comprises a first tag component, a second tag component, and a targeting component. The targeting component targets the double tag system to an autophagosome. Autophagolysosomes are detected in the contacted host cell and the number of autophagolysosomes in the contacted host cell is compared to the number of autophagolysosomes in a host cell that has not been contacted with the agent. An increase in the number of autophagolysosomes indicates that the agent is an autophagosome/lysosome fusion enhancer.

[0010] In an aspect, the disclosure provides a method of identifying an enhancer of HDAC6- dependent F-actin assembly at a site of protein aggregation under non-starvation conditions. In embodiments, the method comprises contacting a host cell comprising a double tag system with an agent, where the double tag system comprises a first tag component, a second tag component, and a targeting component. The targeting component targets the double tag system to autophagosomes.

Autophagolysosomes are detected in the contacted host cell and the number of autophagolysosomes in the contacted host cell is compared to the number of autophagolysosomes in a host cell that has not been contacted with the agent. An increase in the number of autophagolysosomes indicates that the agent is an enhancer of HDAC6-dependent F-actin assembly at a site of protein aggregation under non-starvation conditions.

[0011] In an aspect, the disclosure provides a method of HDAC6 recruitment of cortactin at a site of protein aggregation during autophagosome -lysosome fusion under non-starvation conditions. In embodiments, the method comprises contacting a host cell comprising a double tag system with an agent, where the double tag system comprises a first tag component, a second tag component, and a targeting component. The targeting component targets the double tag system to autophagosomes.

Autophagolysosomes are detected in the contacted host cell and the number of autophagolysosomes in the contacted host cell is compared to the number of autophagolysosomes in a host cell that has not been contacted with the agent. An increase in the number of autophagolysosomes indicates that the agent is an enhancer of HDAC6 recruitment of cortactin at a site of protein aggregation during autophagosome- lysosome fusion under non-starvation conditions.

[0012] In an aspect, the disclosure provides a method of treating a disease relating to protein aggregation in a subject in need of treatment. In embodiments, the method comprises administering to the subject at least one autophagosome/lysosome fusion enhancer in an amount effective to treat the disease.

[0013] In an aspect, the disclosure provides a method of treating a neurodegenerative disease in a subject in need of treatment. In embodiments, the method comprises administering to the subject at least one autophagosome/lysosome fusion enhancer in an amount effective to treat the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A shows a graph of ubiquitin immuno-blot signal intensity for wild type (WT), HDAD6 knockout (HDAC6 KO) and knockout (KO) mouse embryonic fibroblasts (MEFs) reconstituted with HDAC6 constructs. (IB) shows a Western blot of LC3, actin, and HDAC6 in WT and HDAC6 KO MEFs treated with MG132. (1C) shows images of cells treated with MG132 and immunostained with antibodies to ubiquitin and LC3. (ID) shows images of WT and ATG5 KO MEF cells treated with MG132 and immunostained with antibodies to ubiquitin and LC3.

[0015] Figure 2A shows images of wild type and HDAC6 KO MEF cells transfected with pcDNA, pcDNA-HDAC6WT, HDAC6CD or HDAC6ABuz, along with mCherry-GFP-LC3 as indicated. (2B) shows a graph of the total number of yellow vesicles quantified and presented as percentage ( ) of total mCherry-GFP-LC3 dots with standard deviation. (2C) shows the fusion detected for wild type and HDAC67- MEF cells. (2D) shows electron microscope images of wild type and HDAC6 KO MEFs in normal growth conditions, wherein yellow arrows are autophagosomes, red arrows are

autophagolysosomes, and green arrowheads are multilamellar bodies. (2E) shows the quantification of autophagosomes and autophagolysosomes.

[0016] Figure 3A shows autophagosome-Iysosome fusion in wild type and HDAC6 KO MEF cells with or without starvation using mCherry-GFP-LC3. (3B) shows Western blots for LC3, HDAC6 and GAPDH in wild type and HDAC6 KO MEF cells cultured in Hank's solution followed by

immunoblotting with an antibody for LC3, HDAC6, and GAPDH. (3C) shows degradation of [¹⁴C]- valine labeled long-lived protein in wild type and HDAC6 KO MEF cells.

[0017] Figure 4A shows images of wild type and HDAC6 KO MEF cells treated with MG132 and immunostained with antibodies to Lamp-1 (a lysosome marker, red) and ubiquitin (green), and F-actin was detected by phalloidin (blue). Arrows indicate ubiquitin-positive aggregates that are surrounded by F- actin and LAMP-I. (4B) shows images of wild type and HDAC6 KO MEF cells transfected with mCherry-GFP-LC3, followed by treatment with latrunculin A (LtA) or nocodazole (NOC). (4C) shows the percentage of yellow dots formed for the cells in Figure 4B. (4D) shows fusion for autophagosomes (APG) and lysosomes (Lys) isolated from fed mouse hepatocytes and treated with or without latrunculin (LatA) as indicated, labeled with the antibody, and subjected to in vitro fusion assay in the presence or absence of purified actin. (4E) shows fusion for autophagosomes (APG) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes and treated with or without latrunculin (LatA) as labeled. (4F) shows fusion for autophagosomes (APG) and lysosomes (Lys) isolated from HDAC6 KO MEF cells and treated with or without latrunculin (LatA) as indicated.

[0018] Figure 5A shows different subcellular fractions isolated from wild type cells and subjected to SDS-PAGE and immunoblot for the indicated proteins: Horn, homogenate; APG, autophagosomes; APL, autophagolysosomes; LYS, lysosomes. (5B) shows fusion for autophagosomes (APG) and lysosomes (Lys) isolated from fed cells and treated with or without lantrunculin (LatA) as labeled. (5C) shows wild type and ATG5 KO MEF cells treated with MG132 and immunostained with antibodies to ubiquitin (green) and treated with phalloidin (red) to detect F-actin, with arrows indicating ubiquitin-positive protein aggregates.

[0019] Figure 6A shows wild type and HDAC6 KO MEF cells treated with MG132 and immunostained with antibodies to cortactin (red), ubiquitin (green), and phalloidin for F-actin (blue), with arrows indicating ubiquitin-positive aggregates that were colocalized with F-actin and cortactin. (6B) shows wild type MEF cells transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for LAMP-1 (red, to label lysosome), or ubiquitin (green), and phalloidin for actin (blue). (6C) shows wild type MEF cells transfected with control or cortactin siRNA, treated with MG132, and subjected to filter trap assay using an ubiquitin antibody, with the knockdown level of endogenous cortactin confirmed by immunoblotting using an antibody to cortactin and GAPDH in the right panel. (6D) shows percentage of yellow dot formation for autophagosome-lysosome fusion in U20S cells transfected with control siRNA and cortactin siRNA, analyzed with or without starvation using the mCherry-GFP-LC3 reporter as described in Figure 3A. (6E) shows percentage of yellow dot formation for autophagosome-lysosome fusion for wild type MEF cells transfected with mCherry-GFP-LC3 plasmid and cotransfected with wild type, 9KQ (acetylation-mimic), or 9KR (deacetylation-mimic) cortactin expressing plasmids.

[0020] Figure 7A shows the hippocampus and cerebral cortex regions from 6-month-old wild type and HDAC6 KO littermates subjected to immunostaining with a ubiquitin antibody and counter-stained with hematoxylin, with red arrows indicating ubiquitin-positive neuritic aggregates and black arrows indicating cytoplasmic aggregates. (7B) shows apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining. (7C) shows immunostaining for ubiquitin (green) in frontal eye sections of I-day-old (dl) and 30-day-old (d30) fly eyes of control and HDAC6- depleted flies. (7D) shows light micrographs (left) and corresponding Richardson-stained frontal eye sections (right) of 1 -day-old and 30-day-old fly eyes of control and HDAC6-depleted flies.

[0021] Figures 8A-D show images of HeLa cells transfected with GFP-Cherry-LC3 reporter plasmid and treated with rapamycin and subsequently with (Figs. 8B-C) and without Hanks Solution (HBSS; Fig. 8A). (8D) shows a Western blot for LC3 I and LC3 II of HeLa cells without rapamycin treatment and with rapamycin treatment, in the presence or absence of HBSS.

[0022] Figure 9A shows HDAC6 KO MEF cells transfected with control and ATG5 siRNA, treated with MG132, and immunostained with antibodies to ubiquitin (green) and LC3 (Red). Arrows indicate ubiquitin positive protein aggregates. (9B) shows wild type and HDAC6 KO MEFs transfected with mCherry-GFP-LC3 and co-stained with monodansylcadaverine (MDC) to visualize autophagosomes.

[0023] Figure 10A shows lysotracker DND-99 staining of wild type (WT) and HDAC67- MEFs (HDAC6 KO). (10B) shows a graph of the number of lysotracker positive puncta in the cells shown in Figure 10A. (IOC) shows immunoblots for cathepsin D of the same cells maintained in the presence or absence of serum to determine lysosomal pH through the cleavage of pro-cathepsins into their mature forms.

[0024] Figure 11 shows representative images of in vitro autophagosome-lysosome fusion assay between autophagosomes (APG) and lysosomes (Lys) from wild type (WT) and HDAC67- MEFs (HDAC6 KO).

[0025] Figures 12A-K show electron microscope images of wild type MEFs in normal growth conditions. Figures 12A-B show autophagosomes and Figs. 12C-I show autophagolysosomes. Figs. 12J-K show low magnification images from which Figs. 12A-I were taken.

[0026] Figures 13A-K show electron microscope images for HDAC6 KO MEFs under normal growth conditions. Figs. 13A-F show autophagosomes and Figs. 13G-I show autophagolysosomes. Figs. 13J and K show low magnification images from which Figs. 13A-I were taken.

[0027] Figure 14 shows images of HDAC6 KO MEFs with immunogold labeling for LC3.

[0028] Figure 15A shows a graph of autophagy for wild type and HDAC6 KO MEFs as determined from increased LC3-II amount after lysosomal protease inhibition (Pepstatin A and E-64D each 10 μg/mL). (15B) shows Western blots for wild type and HDAC6 KO MEFs, incubated with normal media or HBSS with/without 20 niM NH₄C1 and 100 μg/mL leupeptin, using anti-LC3, anti-HDAC6 and anti- GAPDH antibodies. (15C) shows a Western blot of wild type and HDAC6 KO MEFs cell lysates using anti-p62, anti-LC3, anti-HDAC6, and anti-GAPDH antibodies.

[0029] Figure 16A shows wild type and HDAC6 KO MEFs transfected with Rotenone,

Deferoxamine, H₂0₂, and Hank's solution, with yellow signals indicating non-acidic autophagosomes and red signals indicating acidic autophagolysosomes. (16B) shows a graph of the total number of yellow vesicles quantified from 3 independent experiments and presented as percentage of total mCherry-GFP- LC3 dots (red plus yellow) with standard deviation.

[0030] Figure 17A shows a graph of the percentage of cells with actin-surrounded aggregates in each genotype as quantified from 3 independent experiments. (17B) shows images of HDAC6KO MEFs stably expressing human HDAC6WT, HDAC6CD, or HDAC6ABUZ mutant, treated with MG132, and stained with antibodies for ubiquitin (green), for human HDAC6 (red), and with phalloidin for actin (blue).

[0031] Figures 18A-D show graphs of percentage ( ) fusion observed for wild type (Figs. 18A, 18C) and HDAC6-/- MEFs (Figs. 18B, 18D) treated with or without latrunculin (LatA) as indicated and subjected to in vitro fusion assay in the presence or absence of purified actin. MEFs were cultured in serum (+) (Figs. 18A-B) or serum (-) (Figs. 18C-D) conditions.

[0032] Figure 19A shows images of control and cortactin-knockdown MEFs treated with 2.5 μΜ MG132 for 24 hrs, incubated in normal growth media without MG132 for 18 hrs, and subjected to immunocytochemistry using anti-ubiquitin antibody (green) and phalloidin (red). (19B) shows Western blots using anti-ubiquitin, anti-cortactin and anti-GAPDH antibodies of control and cortactin-knockdown MEFs treated with 2.5 μΜ MG132 for 18 hrs, washed with phosphate buffered saline (PBS), and incubated with full media.

[0033] Figure 20 shows a Western blot for ubiquitin of purified autophagosome and lysosome factions from wild type MEFs under normal growth media or serum starvation.

[0034] Figure 21 shows a model for HDAC6-dependent clearance of ubiquitinated protein aggregates by autophagy.

[0035] Figure 22 shows images of wild type MEFs treated with MG132 and immunostained with antibodies to LC3, ubiquitin, and F-actin.

[0036] Figure 23 shows images of wild type MEFs treated with rotenone or DMSO and immunostained for LC3, phalloidine, and DNAse I.

[0037] Figure 24A-D show graphs of fusion detected for wild type and HDAC6 KO MEFs in the presence or absence of cortactin and actin.

[0038] Figure 25A shows a Western blot for LAMP-1, cortactin, and actin in autophagosome and lysosome fractions from fed or starved mouse liver. (25B) shows a Western blot for cortactin, actin, and GADPH in autophagosome and lysosome fractions from wild type MEFs under normal growth media or serum starvation. DETAILED DESCRIPTION

[0039] Before any embodiments are described in detail, it is to be understood that the claims are not limited to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings.

[0040] As described herein, the inventors have identified that the ubiquitin-binding deacetylase HDAC6 and cortactin-dependent F-actin cytoskeleton are involved in basal quality control (QC) from nutrient-regulated autophagy. Without being limited by any particular mechanism it seems that HDAC6 promotes the fusion of autophagosomes and lysosomes associated with basal QC autophagy by recruiting a cortactin-dependent actin remodeling machinery to ubiquitinated protein aggregates, where the assembly of F-actin facilitates autophagosome/lysosome fusion and clearance of autophagic substrates. It further appears that ubiquitin-dependent, actin-remodeling machinery promotes QC autophagy by stimulating the fusion of autophagosomes and lysosomes, thus providing a regulatory mechanism of autophagy. Deficiency in HDAC6 -dependent fusion machinery causes a failure in autophagy designated for the clearance of protein aggregate or damaged mitochondria, two of the most prominent and common toxic entities found in patients afflicted with neurodegenerative disease. These findings provide a molecular framework to understand basal QC autophagy and non-selective nutrient-regulated autophagy, as well as new insights into the importance of autophagosome/lysosome fusion events in the development of neurodegenerative diseases.

[0041] In a broad sense, the disclosure provides a method for treating diseases relating to protein aggregation. A basal quality control (QC) autophagy mechanism is described herein which identifies the involvement of HDAC6 in the association of autophagosomes and lysosomes in non-starvation induced autophagy. Thus, the disclosure provides methods for identifying, screening, and using an agent that acts as an autophagosome/lysosome fusion enhancer. Further provided are methods for treating diseases, such as neurodegenerative diseases, relating to protein aggregation using an autophagosome/lysosome fusion enhancer. The disclosure also provides methods for identifying autophagosome/lysosome fusion enhancers using a double tag system for determining the number of autophagolysosomes in a cell.

[0042] In an aspect, the disclosure provides a method of treating a disease associated with protein aggregation wherein the method comprises contacting a patient in need of such treatment with an effective amount of an agent that enhances HDAC6 activity. In embodiments the agent enhances the fusion event between an autophagosome and a lysosome. In embodiments the agent enhances HDAC6 activity under non-starvation conditions.

[0043] As used herein, "protein aggregation" relates to the fibrilization or formation of insoluble structures from completely or partially unfolded peptides, where the peptide can bind to itself or to other proteins in the cell in an unnatural way. [0044] In an aspect, the disclosure provides a method of treating a disease involving protein aggregation in a subject. In embodiments, the disease involving protein aggregation is a

neurodegenerative disease. Non-limiting examples of a neurodegenerative disease include

Adrenoleukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease

(Spinocerebellar ataxia type 3), MELAS - Mitochondrial Encephalopathy, Lactic Acidosis and Stroke, Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease), Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, and Toxic

encephalopathy.

[0045] In some embodiments, the method of treatment can be used in combination with other known active agents and/or treatment regimes that are known in the art for any of the diseases disclosed herein. In such embodiments, one of skill in the art will be able to determine the proper course of treatment, including whether the treatments should be co-administered, administered in sequence or series; dosing, routes of administration, determining the endpoints in one or the other, or both, treatments, or whether the treatments are synergistic in effect, and the like.

[0046] As used herein, "autophagolysosome" refers to fusion product between the autophagosome and lysosome, which has also been termed "autolysosome" in the art. The fusion and association of these two cellular structures involves the outer membrane of the autophagosome fusing in the cytoplasmic space with a lysosome to form an autophagolysosome, where its contents are typically degraded via acidic lysosomal hydrolases.

[0047] In some embodiments, methods of treating neurodegenerative diseases comprise

administering to a subject in need of treatment an autophagosome/lysosome fusion enhancer in an amount effective to treat the disease. In certain embodiments, methods of treating neurodegenerative diseases comprise administering an autophagosome/lysosome fusion enhancer in combination with rapamycin and/or other autophagy enhancing agent(s), as are known in the art. Thus, in certain instances, the methods are effective in instances wherein the administration of rapamycin and/or other autophagy inducers alone may be ineffective in patients, such as in patients having a disease involving defects in autophagosome/lysosome fusion function. Further, in certain instances, rapamycin and/or other autophagy inducers can increase the formation of autophagosomes at a rate, or to an extent, that is greater than a cell's ability to fuse the autophagosomes with lysosomes. Thus, in certain embodiments, rapamycin and/or other autophagy inducers can be more effective for treating a neurodegenerative disease in the presence of an autophagosome/lysosome fusion enhancer.

[0048] As used herein, "autophagy inducer" or "autophagy enhancer" refers to any agent that can increase or stimulate autophagy in a cell. In some embodiments, an autophagy inducer can increase the formation of autophagosomes, and include the non-limiting examples of rapamycin and other inhibitors of the mammalian target of rapamycin (mTOR or FRAP1), including Temsirolimus (CCI-779), Deforolimus (Ridaforolimus, AP23573 and MK-8669), Everolimus (RAD001), AZD8055, OSI-027, BEZ235 (NVP- BEZ235), INK-128, XL388, P2281, P529, GSK2126458, KU-0063794, WAY-001, WAY-600, WYE- 687, Wyeth-BMCL-200910075-9b, Wyeth-BMCL-200910096-27, KU-BMCL-200908069-5, KU- BMCL-200908069-1, PI-103, WYE-354, Torinl, PP242, PP30, PP487, PP121, and XL765

(SAR245409) (reviewed in Liu et al, Drug Discov. Today Ther. Strateg. 6(2): 47-55 (2009)),

GSK1059615 (Carnero, Expert Opin. Investig. Drugs 18(9): 1265-1277 (2009)), deferoxamine and its derivatives, and other chelators that induce hypoxia such as desferoxamine and transition metals like cobalt chloride. In some embodiments, other autophagy enhancing agents include, but are not limited to, perhexiline, niclosamide, rottlerin, lithium, L-690,330, carbamazepine, sodium valproate, verpamil, loperamide, amiodarone, nimodipine, nitrendipine, niguldinpine, pimozide, calpastatin, calpeptin, clonidine, rilmenidene, 2' ,5'-Dideoxyadenosine, NF449, minoxidil, penitrem A, Fluspirilene, trifluoperazine, trehalose, SMER10, SMER18, SMER28, and SMER analogs (Renna et al. , JBC 285: 11061-11067 (2010)).

[0049] As used herein, "autophagosome/lysosome fusion enhancer" relates to an agent that increases the fusion of an autophagosome to a lysosome. In certain embodiments, an autophagosome/lysosome fusion enhancer is selected from HDAC6, active fragments of HDAC6, and polypeptides having about 90% identity to HDAC6 that retain autophagosome/lysosome fusion enhancing activity. In certain embodiments, an autophagosome/lysosome fusion enhancer is an agent that encodes and/or increases expression of a polypeptide selected from HDAC6, active fragments of HDAC6, and polypeptides having about 90% identity to HDAC6 that retain autophagosome/lysosome fusion enhancing activity. In certain embodiments, an autophagosome/lysosome fusion enhancer can directly induce expression of a polypeptide selected from HDAC6, active fragments of HDAC6, and polypeptides having about 90% identity to HDAC6. In embodiments, HDAC6 expression can be induced with MCI 575 (Duong et al., Mol. Cancer Res. 6(12): 1908-1919 (2008)). In certain embodiments, an autophagosome/lysosome fusion enhancer can indirectly induce expression of a polypeptide selected from HDAC6, active fragments of HDAC6, and polypeptides having about 90% identity to HDAC6. In certain embodiments, an autophagosome/lysosome fusion enhancer can enhance HDAC6 activity under typical nutrient conditions such as, for example, non-starvation conditions or non-nutrient depleted conditions. In certain embodiments, an autophagosome/lysosome fusion enhancer can directly interact with HDAC6 to enhance HDAC6 activity under typical nutrient conditions such as, for example, non-starvation conditions or non- nutrient depleted conditions. In certain embodiments, an autophagosome/lysosome fusion enhancer can indirectly interact with HDAC6 to enhance HDAC6 activity under typical nutrient conditions such as, for example, non-starvation conditions or non-nutrient depleted conditions. In certain embodiments, an autophagosome/lysosome fusion enhancer can directly enhance an activity other than HDAC6 activity, that subsequently enhances HDAC6 activity through downstream action within a biochemical cascade. Embodiments also provide an agent that can act as an autophagosome/lysosome fusion enhancer and can direct and induce the fusion of an autophagosome to a lysosome (i.e., HDAC6-like fusion activity) in the absence of HDAC6. Certain exemplary autophagosome/lysosome fusion enhancers include those identified using the methods and assays discussed herein. Thus, in certain embodiments, examples of non-limiting autophagosome/lysosome fusion enhancing agents include, but are not limited to, proteins, peptides, small molecules, polynucleotides, polysaccharides, antibodies and antibody fragments, etc. that can increase the activity or amount of HDAC6 in a cell.

[0050] As used herein, "HDAC6 activity" includes the ability of HDAC6 to bind to ubiquitin and ubiquitinated proteins, to associate with microtubles and the F-actin cytoskeleton, and to recruit cortactin to protein aggregates under non-starvation conditions. HDAC6 activity can also be taken to mean enabling fusion between an autophagosome and a lysosome. In embodiments, HDAC6 activity is modulated after an autophagosome/lysosome fusion enhancer is administered. In some embodiments, HDAC6 activity is increased after an autophagosome/lysosome fusion enhancer is administered. In some embodiments, HDAC6 activity is decreased after an autophagosome/lysosome fusion enhancer is administered.

[0051] As used herein, "expression levels" refers to a measure of the production of a biological product encoded by a nucleic acid sequence, such as gene sequence and typically refers to the relative or absolute amount or activity of the gene product. This biological product, referred to herein as a "gene product," may be a nucleic acid or a polypeptide. The nucleic acid is typically an RNA molecule which is produced as a transcript from the gene sequence. The RNA molecule can be any type of RNA molecule, whether either before (e.g., precursor RNA) or after (e.g., mRNA) post-transcriptional processing. cDNA prepared from the mRNA of a sample is also considered a gene product. The polypeptide gene product is a peptide or protein that is encoded by the coding region of the gene, and is produced during the process of translation of the mRNA. For example, "HDAC6 expression levels" can refer to the relative amount of an HDAC6-related biomolecule, such as mRNA encoding HDAC6 amino acid sequence or HDAC6 protein.

[0052] In certain embodiments, autophagosome/lysosome fusion enhancers are administered in a pharmaceutically acceptable composition, such as in or with a pharmaceutically acceptable carrier. In such embodiments, pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.

[0053] As used herein, a "subject in need of treatment" refers to a subject having been diagnosed with a disease, e.g., a disease involving protein aggregration, such as a neurodegenerative disease. A subject can also be one who has been determined as likely to develop a disease such as, for example, a subject having a form of a gene indicating susceptibility of developing the disease, or a subject in whose family the disease is more frequent than normal. Symptoms, diagnostic tests, and prognostic tests for each of the above-mentioned conditions are described in, e.g., the Diagnostic and Statistical Manual of Mental Disorders, 4 ' ed., 1994, Am. Psych. Assoc.; and Harrison's Principles of Internal Medicine©, " 16th ed., 2004, The McGraw-Hill Companies, Inc. The subject to be treated in the method provided herein can be any mammalian subject in need of treatment such as, for example, a mammalian subject diagnosed with, or exhibiting clinical indications of a neurodegenerative disease. In some embodiments the subject has been diagnosed with a neurodegenerative disease. In alternative embodiments, the subject comprises a non-human animal model (e.g., mouse, rat, rabbit, etc.) of a disease that is engineered to model a corresponding human disease in aspects of, for example, clinical symptoms, pathology, disease progression, etc. In other embodiments the mammalian subject is a patient, such as a human patient. In yet further embodiments, the subject is a human patient that has Parkinson's disease, has been diagnosed with Parkinson's disease, exhibits one or more genetic or clinical signs of Parkinson's disease, or has an increased likelihood of developing Parkinson's disease.

[0054] "Pharmaceutically acceptable" means suitable for use in a human or other mammal. The terms "pharmaceutically acceptable carriers" and "pharmaceutically acceptable excipients" are used interchangeably and refer to substances that are useful for the preparation of a pharmaceutically acceptable composition. In certain embodiments, pharmaceutically acceptable carriers are generally compatible with the other ingredients of the composition, not deleterious to the recipient, and/or neither biologically nor otherwise undesirable.

[0055] Certain exemplary pharmaceutically acceptable carriers include, but are not limited to, substances useful for topical, ocular, parenteral, intravenous, intraperitoneal intramuscular, sublingual, nasal and oral administration. "Pharmaceutically acceptable carrier" also includes agents for preparation of aqueous dispersions and sterile powders for injection or dispersions. Examples of pharmaceutically acceptable carriers and excipients are discussed, e.g., in Remington Pharmaceutical Science, 16th Ed. Certain exemplary techniques and compositions for making dosage forms are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0056] "Administering" refers to administration of agents as needed to achieve a desired effect. Exemplary routes of administration include, but are not limited to, oral, rectal, nasal, sublingual, buccal, intramuscular, subcutaneous, intravenous, transdermal, and parenteral administration. Such

administration can be, in certain embodiments, by injection, inhalation, or implant.

[0057] It is desirable that the route of administration and dosage form of the preparation be selected to maximize the effect of the treatment. Typical examples of the administration route include oral routes as well as parenteral routes, including intracerebral, intraperitoneal, intraoral, intrabronchial, intrarectal, subcutaneous, intramuscular and intravenous routes. However, the prophylactic/therapeutic agent for neurodegenerative diseases according to the present invention is preferably administered directly to a target site that requires the prevention/treatment of a neurodegenerative disease. Specifically, the prophylactic/therapeutic agent can be administered to the target site by injection, catheter, incision or other suitable means. Typical examples of the dosage form include sprays, capsules, liposomes, tablets, granules, syrups, emulsions, suppositories, injections, ointments and tapes.

[0058] One skilled in the art can select an appropriate dosage and route of administration depending on the patient, the particular neurodegenerative disease being treated, the duration of the treatment, concurrent therapies, etc. In certain embodiments, a dosage is selected that balances the effectiveness with the potential side effects, considering the severity of the neurodegenerative disease.

[0059] For oral therapeutic administration, the composition may be combined with one or more carriers and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, foods and the like. Such compositions and preparations should contain at least 0.1 % of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 0.1 to about 100% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0060] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. The above listing is merely representative and one skilled in the art could envision other binders, excipients, sweetening agents and the like. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.

[0061] The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0062] In general, the daily dose contains from about 0.1 mg to about 2000 mg. More preferably, each dose of a compound contains about 0.5 to about 60 mg of the active ingredient. This dosage form permits the full daily dosage to be administered in one or two oral doses. More than once daily or twice daily administrations, e.g., 3, 4, 5 or 6 administrations per day, are also contemplated herein.

[0063] As used herein, an "amount effective" refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, clinical indications or symptoms, or causes of a disease, or any other desired alteration of a biological system. Accordingly, methods of treatment as disclosed herein can slow or halt the progression of a disease, or reverse a disease, such as a neurodegenerative disease. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study.

Methods of identifying autophagosome/lyosome fusion enhancers

[0064] In an aspect, methods of identifying autophagosome/lysosome fusion enhancers are provided. In certain embodiments, the method comprises contacting a cell with an agent and using an

autophagosome/lysosome fusion reporter assay to detect autophagosome/lysosome fusion. In certain embodiments, when the agent increases the rate of autophagosome/lysosome fusion, the agent is considered to be an autophagosome/lysosome fusion enhancer. In certain embodiments, when the agent increases the amount of autophagosome/lysosome fusion detected, the agent is considered to be an autophagosome/lysosome fusion enhancer. In various aspects described herein, an increase in rate or amount in the above embodiments encompasses any detectable increase including, for example, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more. [0065] Any suitable assay can be used to detect autophagosome/lysosome fusion and determine whether a test compound is an autophagosome/lysosome enhancer. In various embodiments, an assay for identifying an autophagosome/lysosome fusion enhancers comprises a eukaryotic host cell comprising a reporter gene for autophagosome/lysosome fusion is used to test agents for the ability to enhance autophagosome/lysosome fusion. In some embodiments, the reporter gene comprises a double tag system that includes a first tag component, a second tag component, and a targeting component. In certain embodiments, the first tag component and the second tag component are distinguishable from each other, e.g., because they are based on different detection technology or emit signals of different wavelengths. The targeting component, in certain embodiments, targets the double tag system to autophagosomes. In certain embodiments, a first tag component is a moiety that is detectable in both autophagosomes and autophagolysosomes, i.e., it is detectable in an autophagosome environment (pH ~ 5.7 to 6.4) and it is detectable in an autophagolysosome environment (pH ~ 4.7 to 4.8). The first tag component is a moiety that is detectable in pH neutral to acidic environments. In certain embodiments, a second tag component is a moiety that is detectable in only one of autophagosomes and autophagolysosomes. The second tag is sensitive to pH and is inactivated at neutral or acidic pH. Thus, the second tag component can be detectable in an autophagosome environment but not in an autophagolysosome environment; or the second tag component can be detectable in an autophagolysosome environment but not in an

autophagosome environment. In certain embodiments, a second tag component emits a detectably different signal in an autophagosome environment versus an autophagolysosome environment. For example, the wavelength of the emitted light from a tag component changes based on the pH.

[0066] One skilled in the art can design an appropriate double tag system that comprises a first tag component, a second tag component, and a targeting component that are covalently connected to one another and/or will associate within a cell. Certain non-limiting exemplary double tag systems are described herein.

[0067] In certain embodiments, the first tag component, the second tag component, and the targeting component are expressed in the host cell as a single polypeptide. That is, in certain embodiments, the polynucleotide sequence encoding the first tag component, the polynucleotide sequence encoding the second tag component, and the polynucleotide coding sequence for the targeting component are operably linked such that the three components are expressed as a single polypeptide. Further, the three components can be in any order in the polypeptide. For illustratory purposes, an example includes the three components in the order: first tag component - second tag component - targeting component or the three components in the order: second tag component - targeting component - first tag component.

[0068] In certain embodiments, when the first tag component, the second tag component, and/or the targeting component comprises more than one polypeptide subunit, a first subunit can be expressed with at least one of the other components as a single polypeptide. In certain such embodiments, one or more of the other subunits can be expressed as separate polypeptides such that they will associate with the first subunit. In certain embodiments, a first subunit can be expressed with one of the other components as a single polypeptide and a second subunit can be expressed with the other component as a single polypeptide, such that association of the subunits results in formation of a complex comprising the first tag component, the second tag component, and the targeting component.

[0069] In certain embodiments, the double tag system is expressed in a host cell from a suitable expression vector. One skilled in the art can select an appropriate expression vector according to the particular application and host cell selected. In certain embodiments, the double tag system is expressed from a polynucleotide incorporated into the genome of the host cell. Methods of incorporating polynucleotides into host cell genomes are known in the art.

[0070] Nonlimiting exemplary targeting components include, but are not limited to, LC3A, LC3B, GABARAP, GABARAPLl, and GABARAPL2. The targeting component comprises a region that has an affinity for at least one region, molecule, or component that is associated with an autophagosome.

Nonlimiting examples of tag components include fluorescent proteins such as, for example, mCherry, mStrawberry, mRFPl, DsRed, tdTomato, TagRFP-T, UMFP-1, UMFP-2, UMFP-3, UMFP-4, Tl, dimer2, mHoneydew, dTomato, tdTomato, GFP, EGFP, and YFP. Nonlimiting examples of tag components also include creatine kinase, β-galactosidase, alkaline phosphatase, luciferase, lactamase, CAT, and aequorin.

[0071] Exemplary host cells include, but are not limited to, eukaryotic cells that are capable of forming autophagosomes and autophagolysosomes. In certain embodiments, a host cell is a mammalian cell. In certain embodiments, a host cell is selected from neural cells, fibroblast cells, HeLa cells, CHO cells, 293 cells, COS cells, and the like. In certain embodiments, a host cell is deficient in HDAC6. In certain embodiments, a host call can be made HDAC6 deficient by transfecting the cell with an inhibitory RNA to HDAC6. Certain exemplary inhibitory RNAs include siRNAs, microRNAs, and antisense RNAs. In certain embodiments, a host cell is HDAC6 deficient because of a genetic mutation that has been introduced into the cell, has arisen spontaneously, or has been caused by nonspecific mutagenesis of the cell, e.g., chemical mutagenesis. One skilled in the art can make a host cell HDAC6 deficient using any suitable method known in the art. In certain embodiments, a host cell comprises HDAC6.

[0072] One skilled in the art can select an appropriate double tag system according to the intended application, including a suitable first tag component, a suitable second tag component, and a suitable targeting component. One skilled in the art can also select a suitable host cell.

[0073] A non-limiting exemplary double tag system is described, e.g., in Pankiv et al, J. Biol. Chem. 282: 24131-24145 (2007), e.g., at pages 24138 to 24139, including Figures 5 and 6, which is incorporated by reference herein. In that double tag system, the first component is mCherry, the second component is GFP, and the targeting component is LC3B. The host cells exemplified in that reference for that system are HeLa cells.

[0074] In certain embodiments, an assay for selecting autophagosome/lysosome fusion enhancers comprises a host cell including a double tag system is provided. In certain embodiments, the number and/or proportion of autophagosomes and autophagolysosomes in the host cell is determined. In certain embodiments, the number and/or proportion of autophagosomes and autophagolysosomes in the host cell is determined in the presence of a test agent. In certain embodiments, the host cell is also contacted with an autophagy inducer, such as described above, both with and without the test agent. In certain such embodiments, the number and/or proportion of autophagosomes and autophagolysosomes in the host cell is determined in the presence of the autophagy inducer, with and without the test agent. Thus, in certain embodiments, a test agent can be selected that enhances the activity of an autophagy inducer.

[0075] For a double tag system in which the first tag component is detectable in both

autophagosomes and autophagolysosomes, and the second tag component is detectable in

autophagosomes but not autophagolysosomes, the number of autophagosomes can be determined by counting the number of spots in the host cell that comprise both tag components. In certain such embodiments, the number of autophagolysosomes can be determined by counting the number of spots in the host cell that comprise only the first tag component. The proportion of autophagosomes can be determined, in certain embodiments, by dividing the number of autophagosomes by the total number of autophagolysosomes and autophagosomes. The proportion of autophagolysosomes, in certain embodiments, can be determined by dividing the number of autophagolysosomes by the total number of autophagolysosomes and autophagosomes. Alternatively, the proportion of autophagosomes can be determined, in certain embodiments, by determining the ratio of the signal from the second tag component to the signal from the first tag component (i.e., proportion autophagosomes = (signal second tag component)/(signal first tag component)). Similarly, the proportion of autophagolysosomes can be determined, in certain embodiments, by determining the ratio of the signal from the first tag component less the signal from the second tag component to the total signal from the first tag component (i.e., proportion autophagolysosomes = (signal first tag component - signal second tag component)/(signal first tag component).

[0076] For a double tag system in which the first tag component is detectable in both

autophagosomes and autophagolysosomes, and the second tag component is detectable in

autophagolysosomes but not autophagosomes, the number of autophagosomes can be determined by counting the number of spots in the host cell that comprise only the first tag component. In certain such embodiments, the number of autophagolysosomes can be determined by counting the number of spots in the host cell that comprise both tag components. The proportion of autophagosomes can be determined, in certain embodiments, by dividing the number of autophagosomes by the total number of

autophagolysosomes and autophagosomes. The proportion of autophagolysosomes, in certain embodiments, can be determined by dividing the number of autophagolysosomes by the total number of autophagolysosomes and autophagosomes. Alternatively, the proportion of autophagosomes can be determined, in certain embodiments, by determining the ratio of the signal from the first tag component less the signal from the second tag component to the total signal from the first tag component (i.e., proportion autophagosomes = (signal first tag component - signal second tag component)/(signal first tag component). Similarly, the proportion of autophagolysosomes can be determined, in certain

embodiments, by determining the ratio of the signal from the second tag component to the signal from the first tag component (i.e., proportion autophagolysosomes = (signal second tag component)/(signal first tag component)).

[0077] For a double tag system in which the first tag component is detectable in both

autophagosomes and autophagolysosomes, and the second tag component emits a detectably different signal in autophagolysosomes versus autophagosomes, the number of autophagosomes can be determined by counting the number of spots in the host cell that comprise the first tag component and the second tag component emitting the signal expected in autophagosomes. In certain such embodiments, the number of autophagolysosomes can be determined by counting the number of spots in the host cell that comprise the first tag component and the second tag component emitting the signal expected in autophagolysosomes. The proportion of autophagosomes can be determined, in certain embodiments, by dividing the number of autophagosomes by the total number of autophagolysosomes and autophagosomes. The proportion of autophagolysosomes, in certain embodiments, can be determined by dividing the number of

autophagolysosomes by the total number of autophagolysosomes and autophagosomes. Alternatively, the proportion of autophagosomes can be determined, in certain embodiments, by determining the ratio of the signal emitted by the second tag component in autophagosomes to the total signal from the first tag component (i.e., proportion autophagosomes = (signal emitted from second tag component in

autophagosomes)/(signal first tag component). Similarly, the proportion of autophagolysosomes can be determined, in certain embodiments, by determining the ratio of the signal emitted by the second tag component in autophagolysosomes to the signal from the first tag component (i.e., proportion autophagolysosomes = (signal emitted by second tag component in autophagolysosomes)/(signal first tag component)).

[0078] One skilled in the art can determine additional suitable methods for determining the number and/or proportion of autophagosomes and autophagolysosomes. [0079] In various embodiments, autophagosome/lysosome fusion enhancers can include small molecules, peptides, proteins, antibodies and antibody fragments, polysaccharides, and polynucleotides including DNA and RNA, such as plasmids, vectors, cDNAs, siRNAs, microRNAs, antisense RNAs, etc.

[0080] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and

"containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to illustrate aspects and embodiments of the disclosure and does not limit the scope of the claims.

[0081] While a number of aspects and embodiments are detailed by the disclosure, variations of those aspects and embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description and fall within the scope of the disclosure and claims. The examples that follow are intended merely to be illustrative of certain aspect and embodiments of the disclosure, and should not be interpreted to be limiting to the claims.

EXAMPLES

Example 1: Materials and Methods

[0082] Cell lines and plasmids. Wild type and HDAC6 knock out (KO) mouse embryonic fibroblasts (MEFs) reconstituted with various HDAC6 constructs were prepared as described previously (Gao et al , 2007). mCherry-GFP-LC3 construct was a gift from Dr. Terje Johansen and is described in Pankiv et al , 2007. Lipofectamine LTX (Invitrogen) was used for transfection.

[0083] Antibodies and reagents. Anti-mouse HDAC6 antibody was generated against amino acids 991 to 1149 of HDAC6. The following antibodies/reagents were also used: anti-HDAC6 (H-300; Santa Cruz), anti-acetyl-a-tubulin (Sigma), anti-ubiquitin (Biomol and Calbiochem), anti-p62 (Santa Cruz), anti-LAMP-1 (Hybridoma Bank, Iowa), anti-LC3 (a generous gift from Dr. Ron R. Kopito), Latrunculin A (Sigma), Nocodazole (Sigma), Phalloidin-Alexa Fluor 647, and phalloidin-rhodamine (Molecular Probes).

[0084] Immunofluorescence microscopy. Immunostaining was performed as described previously (Hubbert et al, 2002; Lee et al, J. Biol. Chem. 279: 30265-30273 (2004)). Cells were cultured on glass coverslips, followed by treatment with MG132 at 2.5 μΜ for 24 hr. Cells were washed with phosphate buffered saline (PBS), incubated with full growth media for 18 hrs, and then processed for immunostaining. Images were acquired by a spinning-disk confocal microscope (Olympus ZX-70 or Leica DM16000C) equipped with an ORCA ER charge -coupled-device camera.

[0085] Filter- Trap assay. Filter-trap assay for aggregates was performed as described (Scherzinger et al , Cell 90: 549-558 (1997)). The total protein load was normalized to the volume of the soluble fraction.

[0086] Long-live protein degradation assay. Long-lived protein assay was done according to the published protocol (Pattingre et al , Methods Enzymol. 390: 17-31 (2004)). Briefly, cells were labeled for 18 hrs at 37°C with 0.2 mCi/mL of -[¹⁴C] valine in complete medium DMEM (GIBCO), 10% fetal bovine serum (Hyclone), and 1% penicillin-streptomycin. Unincorporated radioactivity was removed by three times rinse with PBS. Cells were then incubated in Hanks' balanced salt solution (HBSS, GIBCO), plus 0.1% of bovine serum albumin (BSA), and 10 mM valine (Sigma) to stimulate autophagy. When involved, 10 mM 3-MA (3-methyladenine; Sigma) was added throughout the chase period to inhibit the formation of autophagic vacuoles. After the first hour of incubation, at which time short-lived proteins were being degraded, the medium was replaced with the fresh medium, and incubation was continued for an additional 4 hrs period. Thereafter, the medium was precipitated with trichloroacetic acid (TCA) added to a final 10% concentration. After centrifugation for 10 min at 2000 rpm at 4°C, the acid-soluble radioactivity was measured by liquid scintillation counting. Cells were washed twice with cold 10% TCA and dissolved at 37°C in 0.2 N NaOH. Radioactivity was then measured by liquid scintillation counting. The rate of long-lived protein degradation was calculated from the ratio of the acid-soluble radioactivity in the medium to the cell acid precipitable fraction.

[0087] In vitro fusion assay. Autophagosomes, autophagolysosomes, and lysosomes were isolated from cultured cells using a protocol that was modified from previous reference (Marzella et al., J. Cell Biol. 93: 144-154 (1982)). Briefly, cells were lysed by nitrogen cavitation and a pellet enriched in autophagic and lysosomal components and in mitochondria was prepared by differential centrifugation. The different components of this pellet were separated by centrifugation in a discontinuous metrizamide gradient as described before (Yu et al , J. Cell Biol. 171: 87-98 (2005)). Fractions were collected from the interfaces of the gradient, washed by centrifugation in 0.25 M sucrose and resuspended in 10 mM MOPS pH 7.2/0.25 M sucrose. Autophagosomes were labeled by sequential 5 min incubations at room temperature with an antibody against LC3 and an FITC-conjugated secondary antibody. The lysosomes in the lysosomal-enriched fraction were labeled in a similar manner but with a primary antibody against LAMP-2 and a Cy5 -conjugated secondary antibody. Labeled fractions were washed and resuspended in fusion buffer (10 mM HEPES pH 7, 10 mM KC1, 1.5 mM MgCl₂, 1 mM DTT in 0.25 M Sucrose).

Labeled fractions were mixed and supplemented with 2 mM CaCl₂, 2 mM GTP, 3 mM ATP and an energy regenerating system (8 mM Phopshocreatine and 0.16 mg/mL creatine phosphokinase). After 30 min incubation at 37°C, the reaction was spotted in a coverslip and stopped by fixation with 8% formaldehyde in 0.25 M sucrose for 15 min on ice. After addition of mounting media images of the slides were acquired with an Axiovert 200 fluorescence microscope (Carl Zeiss Ltd., Thornwood, NY).

Quantification was performed using Image J software (NIH, MD) with the JACoP plug-in. At least 3 different experiments and 4-6 different fields per condition and experiment were imaged and quantified (average total number of particles was aproximately 800-1200 particles). Fluorescence structures larger than 4 diameters above the average particle diameter were eliminated from the counting as they were probably "vesicular" clumps impossible to completely eliminate from the preparation without affecting the stability of the other vesicle membranes. Fusion events were quantified as the percentage of vesicles positive for both fluorophores. Mean values of the 8 different fields were using to calculate the mean value of the different experiments.

[0088] The protocol for the isolation and labeling of autophagosomes and lysosomes from livers of fed or 6 hrs starved mice was identical but homogenization was used instead to disrupt the hepatocytes. In this case, lysosomes were isolated from wild type mice and labeled as above, whereas autophagosomes were isolated from GFP-LC3 mice to avoid need of further labeling of that fraction. Where indicated, autophagosomes and/or lysosomes were treated with 200 μΜ lantrunculin A for 20 min at 25 °C and recovered by centrifugation before adding them to the fusion reaction. Rabbit skeletal muscle actin (20 μg/mL) was added in some reactions during fusion.

[0089] Electron Microscope (EM) image analysis. The following criteria was used to assign autophagosome versus autophagolysosomes. APG (autophagosome): The presence of double membrane was the defining component. The density inside of autophagosomes was similar to that of the surrounding cytosol. APGL (Autophagolysosome): Vesicle with single membrane (sometimes there was residual double membrane but very little) and contained amorphous materials, and the lumen of the vesicle was usually of lighter density than the surrounding cytosol.

[0090] Immunogold labeling was performed in cells fixed in 4% paraformaldehyde/0.1%

gluteraldehyde in 0.1 M cacodylate buffer. Briefly, cellular monolayers were for 1 hr at room

temperature, dehydrated, embedded in Lowicryl and cut in ultrathin sections. Each grid was washed in 50 mM glycine in PBS, blocked and preincubated in the antibody incubation buffer for 1 hr. Blocked grids were incubated with the antibody against LC3 for 2 hr, extensively washed and incubated with the gold- conjugated secondary antibody (1 : 100) for another 2 hr. Control grids were incubated with either an irrelevant IgG and the secondary antibody under the same conditions, or only with the secondary antibody. After extensive washing, samples were fixed a second time for 5 min in 2% gluteraldehyde washed and negatively stained with 1% uranyl acetate for 15 min. All grids were viewed on a JEOL lOOCX II transmission electron microscope at 80 kV. [0091] Fly stocks. All Drosophila stocks were maintained at 25 °C on standard yeast agar media except where noted. UAS-dHDAC6 and UAS-dHDAC6RNAi strains were described previously (Pandey et al., 2007). The GMR-GAL4 strain was obtained from the Bloomington Stock Center (Bloomington, IN). For each genotype and condition, at least 100-300 flies were evaluated.

[0092] Histology and immunohistochemistry of Drosophila eye sections. Fly heads of the appropriate genotype were collected and fixed in 4% paraformaldehyde in PBS for 2 hrs at room temperature or overnight at 4°C. The samples were then dehydrated using a series of one hour ethanol incubations (50%, 70%, 80%, 90%, 95%, and twice at 100%). Following dehydration, samples were infiltrated by JB-4 infiltration solution (JB-4 plus solution A and benzoyle peroxide), plasticized for 2-3 days at 4°C, then processed to JB-4 embedding and sectioned with a microtome (LEICA model

RM2255). For histological analysis, sectioned tissues were desiccated overnight at room temperature and subjected to Richardson's staining. For ubiquitin detection, sectioned tissues were blocked at 1% BSA plus 1 % goat serum for 1 hr followed by incubation with mouse monoclonal antibody against Ubiquitin (Zymed) at 1 : 100 for 3-4 hrs. Samples were washed with PBS three times for 5 min each time, and stained with an anti-mouse secondary antibody conjugated to Alexafluor 488 and DAPI (Polysciences, Inc.), cover slipped and analyzed by fluorescence microscopy.

[0093] Immunohistochemical analysis for mouse sections. Mice were transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). Brains were post-fixed in the same fixative for 3 hrs in 4°C, incubated with 30% sucrose in 4°C for overnight and embedded in O.C.T. compound. For immunohistochemical analysis, frozen brain tissue sections (20 μηι) were subjected to immunostaining with anti-ubiquitin antibody followed by the Vectastain ABC kit (Vector Laboratory). After

immunostaining, sections were counter-stained using Meyer's hematoxylin. Apoptotic neurons were visualized by TUNEL assay using the In Situ Cell Death Detection Kit, TMR red (Roche).

[0094] Statistical analysis. Two-tailed Student's t-test was conducted for statistic analysis of quantitative data.

Example 2: Loss of HDAC6 results in defects in protein aggregate clearance but not autophagy activation

[0095] HDAC6 knockdown cells are deficient in aggresome formation (Kawaguchi et al , 2003). To determine if HDAC6 is also involved in protein aggregate degradation, we treated mouse embryonic fibroblasts (MEFs) derived from HDAC6 knockout (KO) and matching wild type mice with the proteasome inhibitor MG132. Levels of accumulated ubiquitinated aggregates were then analyzed by a filter trap assay. Figure 1A shows results of filter trap analysis of MG132-induced SDS-insoluble ubiquitinated aggregates generated in wild type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. Signal intensity from the ubiquitin immuno-blot (bottom panel) was quantified and presented as the average of means from three independent experiments with standard deviation (Top). Accumulation of ubiquitin-positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6-CD and ABUZ mutants were observed. Wild-type, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytic inactive mutant) or HDAC6 ABUZ (ubiquitin-binding deficient mutant) were analyzed for the level of HDAC6 using an anti- human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody. As shown in Figure 1 A, ubiquitinated protein aggregates accumulated by about 3 fold in HDAC6 KO MEFs compared to wild type MEFs, indicating that HDAC6 KO MEFs were deficient in protein aggregate degradation. The abnormal accumulation of protein aggregates was completely reversed in HDAC6 KO MEFs reconstituted with wild type HDAC6, but not catalytic- inactive or ubiquitin binding-deficient mutants (Figure 1A), indicating that enzymatic activity and ubiquitin-binding activity were both involved in HDAC6 to promote the clearance of ubiquitinated protein aggregates.

[0096] Autophagy is considered the dominant mechanism for protein aggregate clearance. To unravel the mechanistic link between HDAC6 and autophagy, we investigated whether autophagy was properly activated in HDAC6 knockout (KO) MEFs upon proteasome inhibition. We assessed autophagy activation by first monitoring the conversion of LC3 from the cytosolic form, LC3-I, to the

autophagosome-associated form, LC3-II. Figure IB shows results for wild type and HDAC6 KO MEFs treated with protease inhibitor MG132 and subjected to Western blot analysis for LC3, actin, and HDAC6. Unexpectedly, despite the defect in protein aggregate clearance, conversion to LC3-II was induced in HDAC6 KO MEFs upon MG132 treatment, suggesting that autophagosomes formed normally in the absence of HDAC6 (Figure IB).

[0097] To further evaluate autophagosomes involved in protein aggregate clearance, we examined the formation of LC3-positive autophagosomes in wild type and HDAC6 KO MEFs treated with MG132 and then chased in normal medium for 18 hrs. Figure 1C shows results for cells treated with MG132 and immunostained with antibodies to ubiquitin (green) and LC3 (Red). Arrows indicate ubiquitin-positive aggregates that co-localize with LC3-positive autophagosomes (scale bar = 10 μηι). The results show that prominent LC3-positive autophagic vesicles were induced and readily found surrounding ubiquitin- positive protein aggregates in both cell types.

[0098] This staining was not caused by non-specific incorporation of LC3 into protein aggregates (Kuma et al. , Autophagy 3: 323-328 (2007)), as no LC3-positive structures were observed in ATG5 deficient MEFs subjected to the same treatment (Figure ID and Figure 9A). Figure ID shows results for wild type and ATG5 KO MEFs treated with 2.5 μΜ MG132 for 1 day and incubated with normal growth media for 18 hrs, with MEFs immunostained with anti-LC3 (red) and anti-ubiquitin antibody (green). Shown in Figure 9A are HDAC6 KO MEF cells transfected with control and ATG5 siRNA, treated with MG132, and immunostained with antibodies to ubiquitin (green) and LC3 (red), wherein arrows indicate ubiquitin positive protein aggregates (scale bar = 25 μηι). HDAC6 KO MEFs were defective in aggregate clearance but not in autophagosome induction or targeting. These results indicated that autophagosome formation proceeded normally in the absence of HDAC6 but that autophagy-dependent protein aggregate degradation was defective.

Example 3: HDAC6 is involved in fusion of autophagosomes and Iysosomes associated with basal quality control (QC) autophagy

[0099] Autophagosomes fuse to Iysosomes to degrade their contents. The inability of HDAC6 KO MEFs to degrade protein aggregates despite the apparent induction of autophagosome formation prompted us to determine if HDAC6 is involved in autophagosomes-lysosomes fusion. Fusion efficiency was determined using an assay including a pH sensitive, double tagged mCherry-GFP-LC3 (dtLC3) reporter. GFP fluorescence is lost in acidic compartments while mCherry can fluoresce at neutral and acidic pH. Thus, mCherry-GFP-LC3 labels non-acidic autophagosomes as yellow fluorescence (positive for both green and red) and acidic autophagolysosomes as red mCherry (Pankiv et al , 2007). To test fusion efficiency, we expressed mCherry-GFP-LC3 in wild type and HDAC6 KO MEFs under normal nutrient conditions. Shown in Figure 2A are results for wild type and HDAC6 KO MEFs transfected with pcDNA, pcDNA-HDAC6WT, HDAC6CD, or HDAC6ABuz, along with mCherry-GFP-LC3 as indicated. Yellow signals indicated non-acidic autophagosomes and red signals indicated acidic

autophagolysosomes (scale bar, 10 μηι). We observed a prominent increase in the number of yellow fluorescence-vesicles in HDAC6 KO MEFs as compared to wild type control, indicating an accumulation of autophagosomes resulting from a defect in autophagosome-lysosome fusion (i.e., failure to form autophagosome -lysosome fusion).

[00100] Indeed, the double-positive mCherry-GFP-LC3 vesicles were also positive for an

autophagosome marker, MDC (mono-dansyl-cadaverine, Figure 9B). Shown in Figure 9B are wild type and HDAC6 KO MEF cells transfected with mCherry-GFP-LC3 and co-stained with

monodansylcadaverine (MDC) to visualize autophagosome, wherein yellow indicates non-acidic autophagosomes and red signals indicates acidic autophagosomes in the left panel. The middle panel shows MDC staining as blue color, and the right panel shows the merged image, with arrows indicating autophagosomes labeled with mCherry-GFP-LC3 as yellow and MDC as blue (scale bar = 10 μηι). The observed increase in yellow fluorescence -vesicles in HDAC6 KO MEFs could not be attributed to loss of lysosomal acidification as the lysosomal pH was in the normal range in both wild type and HDAC6 KO MEFs (Figure 10). Figure 10A shows lysotracker DND-99 staining of wild type and HDAC67- MEF cells (HDAC6 KO), Figure 10B shows the quantification of the number of lysotracker positive puncta in the cells shown in Figure 10A, and Figure IOC shows immunoblots for cathepsin D of the same cells maintained in the presence or absence of serum to determine lysosomal pH through the cleavage of pro- cathepsins into their mature forms, wherein arrows indicate the precursor (p) and mature (m) forms of cathepsin. The percentage of cathepsin processed into mature form in each condition was calculated by densitometry. LAMP-1 was also shown. The acidification of the lysosomal system was comparable in wild type and HDAC6 KO MEF cells. Lastly, Figure 2B shows the total number of yellow vesicles quantified from three independent experiments (greater than 12 cells each) and presented as a percentage ( ) of total mCherry-GFP-LC3 dots (red plus yellow) with standard deviation. These results strongly indicated that HDAC6 was involved in efficient autophagosome-Iysosome fusion under normal nutrient conditions. The accumulation of yellow vesicles (i.e. autophagosomes) in HDAC6 KO MEFs was efficiently reversed by the reintroduction of wild type, but not catalytic inactive (CD) or ubiquitin-binding deficient (ABUZ) mutant HDAC6 (Figure 2B).

[00101] As further evidence that HDAC6 directly regulates autophagosome-Iysosome fusion, we purified fractions enriched in autophagosomes or lysosomes from wild type and HDAC6 KO (HDAC67-) MEFs and analyzed their fusion efficiency in an in vitro assay. Figure 2C shows results from in vitro fusion assays wherein autophagosomes (APGs) and lysosomes (Lys) purified from wild type and HDAC67- MEFs were subjected to heterotypic and homotypic in vitro fusion assays (representative fields are shown in Figure 11), with values expressed as means with the standard error (S.E.) of the percentages of fusion from 3 independent experiments (more than 10 images per each experiment). Shown in Figure 11 are results from in vitro fusion between autophagosomes (APG) and lysosomes (Lys) from wild type and HDAC67- MEF cells (HDAC6 KO), with arrows indicating colocalizataion of green and red vesicles, inset panels showing higher magnification images of the events, and the quantification of these experiments shown in Figure 2C. As shown in Figure 2C and Figure 11, lysosomes (LYS) and autophagosomes (APG) from HDAC6 KO MEFs showed about a two fold reduction in fusion compared with those purified from wild type MEFs, while the homotypic fusion of autophagosomes or lysosomes was not significantly affected. Thus both in vivo and in vitro fusion assays showed that HDAC6 mediated efficient fusion of autophagosomes and lysosomes.

[00102] An autophagosome-Iysosome fusion deficiency would predict an accumulation of autophagosomes. We therefore analyzed autophagic structures in control and HDAC6 KO MEFs by transmission electron microscopy (EM). Shown in Figure 2D are electron microscope images of wild type and HDAC6 KO MEFs in normal growth conditions, with yellow arrows for autophagosomes, red arrows for autophagolysosomes, and green arrowheads for multilamellar bodies. Shown in Figure 12 are electron microscopy images of wild type MEF cells in normal growth conditions, wherein Figures 12A-B are autophagosomes, Figures 12C-I are autophagolysosomes, Figures 12J-K are low magnification images, and letters indicate the cellular location of the corresponding magnified image (APG = autophagosome; APGL = autophagolysosome). In wild type MEFs, autophagolysosomes were prominent and easily identified (Figure 2D, left panel, red arrows, and Figure 12).

[00103] In contrast, very few such structures were observed in HDAC6 KO MEFs (Figure 2D, right panel and Figure 13). Shown in Figure 13 are electron microscopy images of HDAC6 KO MEF cells in normal growth conditions, wherein Figures 13 A-F are autophagosomes, Figures 13G-I are

autophagolysosomes, Figures 13 J-K are low magnification images, and letters indicate the cellular location of the corresponding magnified image (APG = autophagosome; APGL = autophagolysosome). Instead, HDAC6 deficient cells accumulated large numbers of double-membrane autophagosome structures (Figure 2D, right panel, yellow arrows), many of which contained multilamellar bodies (MLB, green arrowheads) that were rarely found in wild type MEFs. The autophagic origin of the vesicular structures that accumulated in HDAC6 KO MEFs was confirmed by immunogold as all of them were positive for LC-3 labeling (Figure 14). For immunogold labeling for LC3 of HDAC67- MEFs shown in Figure 14, a general area (top left) and higher magnification pictures of individual autophagic vacuoles are shown, with arrows pointing to gold particles associated to the inner or outer membrane of the autophagic vesicles (av, autophagic vesicles; Id, lipid droplet; lm?, possible limiting membrane in formation). Figure 2E shows quantification of autophagosomes and autophagolysosomes, with error bar = standard error. Quantification of autophagic structures confirmed a marked increase (about 2 fold) in the ratio of autophagosome/autophagolysosome in HDAC6 KO MEFs (Figure 2E). Thus, fusion assays in vitro and in vivo and morphometric analysis all supported a role for HDAC6 in the fusion of autophagosomes to lysosomes under basal conditions. This was further confirmed through measurement of autophagic flux analysis and p62 accumulation (Figure 15). As shown in Figure 15 A, autophagy flux was determined as increased LC3-II amount after lysosomal protease inhibition (Pepstatin A and E-64D, each 10 μg/mL) and presented as mean + S.D. from 3 independent experiments. Measurement of the degradation of LC3-II under basal conditions revealed a decrease of almost 50% in the accumulation of LC3-II upon inhibition of lysosomal degradation with leupeptine, supporting reduced rates of delivery of this molecule to lysosomes through autophagosome/lysosome fusion. Shown in Figure 15B are representative Western blots for autophagy flux assay, wherein wild type and HDAC6 KO MEFs were incubated with normal media or HBSS with/without 20 niM NH₄C1 and 100 μg/mL leupeptin for 6 hrs, cell lysates were subjected to Western analysis using anti-LC3, anti-HDAC6, and anti-GAPDH antibodies, and all measurements were normalized by GAPDH. Shown in Figure 15C are Western blots of wild type and HDAC6 KO MEFs cell lysates using anti-p62, anti-LC3, anti-HDAC6, and anti-GAPDH antibodies.

Example 4: HDAC6 is dispensable for non-selective, starvation-induced autophagy

[00104] To determine whether HDAC6 plays a general role in autophagosome-lysosome fusion, we used the mCherry-GFP-LC3 fusion assay to determine whether HDAC6 mediates autophay induced by starvation. Although HDAC6 KO MEFs were defective in supporting autophagosome-lysosome fusion under normal nutrient conditions, the fusion efficiency was completely normalized and comparable to that of control wild type MEFs when subjected to starvation. Shown in Figure 3A are results from

autophagosome-lysosome fusion analyzed in wild type and HDAC6 KO MEFs with or without starvation (6 hrs) using mCherry-GFP-LC3 as described in Figure 2A. Supporting this observation, starvation- induced LC3-II conversion (Figure 3B) and long-lived protein degradation were normal and even more robust in HDAC6 KO than in wild type MEFs (Figure 3C). Shown in Figure 3B are results from wild type and HDAC6 KO MEFs cultured in Hank's solution for 3 hrs followed by immunoblotting with an antibody for LC3, HDAC6, and GAPDH. Figure 3C shows long-lived protein degradation in wild type and HDAC6 KO MEF cells, wherein the degradation of [¹⁴C] -valine labeled long-lived protein was measured in the presence or absence of 3-methyl adenine (3MA, inhibits the formation of autophagic vacuoles). The average of percentage degradation from 3 independent experiments was evaluated (error bar = standard deviation).

[00105] Measurement of LC3 autophagic flux further confirmed normal fusion of autophagosomes to lysosomes in HDAC6 KO MEFs upon starvation (Figures 15A-B, described above). Altogether, these results demonstrated that HDAC6 was not involved in starvation-induced autophagy.

[00106] Shown in Figure 16 are results from wild type and HDAC6 KO MEFs transfected with mCherry-GFP-LC3 and treated with Rotenone, Deferoxamine, H₂0₂, and Hank's solution for 5 hrs as indicated. In Figure 16 A, yellow signals indicate non-acidic autophagosomes and red signals indicate acidic autophagolysosomes (scale bar = 25 mm). The total number of yellow vesicles quantified from 3 independent experiments (greater than 10 cells each) as a percentage of total mCherry-GFP-LC3 dots (red plus yellow) with standard deviation is shown in Figure 16B.

Example 5: HDAC6 recruits an F-actin network to facilitate autophagosome-lysosome fusion

[00107] Our analysis showed that HDAC6-deficient autophagosomes and lysosomes remained defective in fusion even when they were mixed in vitro (Figure 2C). This finding suggested that the fusion defect likely involved mechanisms independent of long-range microtubule-dependent transport, a process known to be regulated by HDAC6. In addition to microtubules, HDAC6 associates with and regulates actin membrane ruffles, a specialized form of the F-actin cytoskeleton (Gao et al , 2007). F- actin remodeling has been reported to promote fusion of specific vesicular compartments, including those involving lysosomes (Eitzen et al , J. Cell Biol. 158: 669-679 (2002); Jahraus et al , Mol. Biol. Cell 12: 155-170 (2001); Kjeken et al. , Mol. Biol. Cell 15: 345-358 (2004)). We next determined if the actin cytoskeleton plays a role in QC autophagy associated with protein aggregates. Shown in Figure 4A are results from wild type and HDAC6 KO MEFs treated with MG132 and immunostained with antibodies to Lamp-1 (a lysosome marker, red) and ubiquitin (green) as indicated. F-actin was detected by phalloidin (blue). Arrows indicate ubiquitin-positive aggregates that were surrounded by F-actin and LAMP-I. Shown in Figure 17A is the percentage of cells with actin-surrounded aggregates in each genotype as quantified from 3 independent experiments (greater than 100 cells per experiment, representative images are shown in Figure 4A and Figure 9B). Shown in Figure 17B are HDAC6KO MEFs stably expressing human HDAC6WT, HDAC6CD, or HDAC6ABUZ mutant that were treated with MG132 and stained with antibodies for ubiquitin (green), for human HDAC6 (red) and with phalloidin for actin (blue), wherein arrows indicate aggregates positive for F-actin. We found that aggresome-like protein aggregates were frequently surrounded by or co-localized with prominent F-actin structures in wild type MEFs (about 30%, Figure 4A, top panel and Figure 17A). In stark contrast, little concentration of F-actin was found around protein aggregates in HDAC6 KO MEFs (Figure 4A, bottom panel). The formation of the F-actin structure was efficiently restored by the re-introduction of wild type HDAC6, but not catalytic inactive (CD), or ubiquitin-binding deficient (ABUZ) mutant HDAC6, which was not associated with ubiquitinated protein aggregates (Figure 17). These results suggested that HDAC6 promoted local assembly of an F-actin network at autophagic substrates.

[00108] We next determined if the formation of an F-actin network is involved in autophagosome- Iysosome fusion. As shown in Figures 4B-C, the mCherry-GFP-LC3 reporter assay showed that autophagosome-Iysosome fusion was effectively suppressed when cells were treated with latrunculin A (LatA), which sequestered free G-actin thereby inhibiting F-actin polymerization. For results shown in Figures 4B-C, wild type and HDAC6 KO MEFs were transfected with mCherry-GFP-LC3, followed by treatment with latrunculin A (LtA, 100 nM) or nocodazole (NOC, 250 nM) for 6 hrs and analyzed as described in Figure 3 A. Consistent with our analysis (Figure 2C), treatment with the microtubule destabilizing agent nocodazole (NOC) had only a mild effect in this assay. To gain further evidence that F-actin polymerization was involved in efficient autophagosome-Iysosome fusion, we treated the isolated autophagosome (APG) and lysosome (LYS) fractions with latrunculin and assessed the fusion efficiency in vitro. Shown in Figure 4D are results for autophagosomes (APG) and lysosomes (Lys) isolated from fed mouse hepatocytes treated with or without latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labeled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. Differences with untreated samples were significant for p<0.01. As shown in Figure 4D, latrunculin (LatA) treatment significantly reduced fusion of purified

autophagosomes and lysosomes in vitro. After pretreatment of APG and LYS with latrunculin, the fusion activity could be restored by the addition of purified actin (Figure 4D), demonstrating that the effect of latrunculin was actin dependent.

[00109] As HDAC6 was selectively involved in fusion associated with basal QC but not starvation- induced autophagy (Figure 3), we compared the effect of latrunculin on autophagosomes and lysosomes purified under fed or starved conditions. Remarkably, the autophagic compartments purified from cells or mice subject to starvation were much more resistant to latrunculin A than those purified from cells maintained in normal medium or from fed animals (Figure 4E, Figure 18 A, and Figure 18C). Results shown in Figure 4E are for autophagosomes (APG) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes treated with or without latrunculin (LatA) as labeled and subjected to in vitro fusion assay. Differences with untreated samples were significant for p<0.05. Shown in Figure 18 are autophagosomes (APG) and lysosomes (Lys) purified from wild type (Figures 18 A, 18C) and HDAC67- MEFs (Figures 18B, 18D) that were treated with or without with latrunculin (LatA) as indicated and subjected to in vitro fusion assay in the presence or absence of purified actin. MEFs were cultured in serum (+) (Figures 18A-B) or serum (-) (Figures 18C-D) conditions. Values shown are means with the standard error (S.E.) of the percentages of fusion from 3 independent experiments.

[00110] These results indicated that the assembly of F-actin was selectively involved in basal but not starvation-induced autophagy. Consistent with the proposal that HDAC6 regulated autophagy via promoting F-actin network formation, latrunculin A treatment did not further inhibit fusion of autophagosomes and lysosomes purified from HDAC6 KO MEFs (Figure 4F, Figure 18B, and Figure 18D). Results shown in Figure 4F are for autophagosomes (APG) and lysosomes (Lys) isolated from HDAC6 KO MEFs treated with or without latrunculin (LatA) as indicated and subjected to in vitro fusion assay. Altogether, these findings supported the conclusion that HDAC6 promotes autophagosome- Iysosome fusion associated with basal QC autophagy by recruiting an F-actin network to autophagic substrates.

[00111] The distinct involvement of HDAC6 and actin cytoskeleton may reflect the function unique to different autophagic modes. For example, as nutrient-regulated autophagy serves to replenish macromolecules to sustain cell survival under starvation, it follows that autophagosomes formed under starvation are not endowed with selectivity so that they can efficiently recycle macromolecules by non- discriminatively sequestering cytosolic contents and fusing to lysosomes with great efficiency. In contrast, quality control (QC) autophagy target aberrant protein aggregates and damaged organelles but spare their normal counterparts. Thus, this form of autophagy is equipped with built-in "selectivity". The involvement of an ubiquitin-binding deacetylase, HDAC6, for basal QC but not starvation-induced autophagy suggests that the substrate selectivity involves ubiquitin modification. Supporting this hypothesis, the ubiquitin-binding deficient HDAC6-ABUZ mutant does not associate with protein aggregates (Figure 17B) and cannot support autophagosome-Iysosome fusion (Figures 2A-B). Under basal conditions, autophagosomes contain ubiquitinated proteins (Pankiv et al, J. Biol. Chem. 282:

24131-24145 (2007)). We also found that purified autophagosome fractions are enriched for ubiquitin- positive protein aggregates (Figure 20). Figure 20 shows purified autophagosome and lysosome fractions from wild type MEFs under normal growth media or serum starvation that were subjected to immunoblot for ubiquitin. Ubiquitinated protein aggregates were detected in the base of the wells visible upon transfer of the stacking gel. These results support the idea that ubiquitinated proteins are a major class of substrate for basal QC autophagy.

[00112] Although HDAC6 regulates efficient QC autophagy, the recruitment of autophagosomes to protein aggregates is independent of HDAC6 (Figure 1C). This activity is likely mediated by another ubiquitin-binding protein, p62, which binds LC3 with high affinity and promotes protein aggregate clearance (Bjorkoy et al, J. Cell Biol. Ill: 603-614 (2005); Seibenhener et al, Mol. Cell Biol. 24: 8055- 8068 (2004)). Thus, HDAC6 and p62 are both involved in protein aggregate clearance but they regulate different components in autophagy. We propose that HDAC6 and p62 independently recognize and bind specific ubiquitin-moieties that mark protein aggregates or other QC autophagic substrates, where they recruit and assemble components for autophagy: autophagosomes, lysosomes and the actin network. By concentrating autophagic components to the substrates and stimulating the fusion of autophagosomes with lysosomes, such an arrangement would enable QC autophagy to achieve specific and efficient removal of protein aggregates or damaged organelles that arise sporadically in the cytoplasm (see Figure 21). This mechanism, however, would provide less advantage to the non-selective bulk degradation induced by starvation, whose substrates are abundant, less geographically constrained and readily accessible to lysosomes. The degradation of defective mitochondria, another substrate of QC autophagy (mitophagy), was recently shown to be mediated by parkin, an ubiquitin E3-ligase (Narendra et al, J. Cell Biol. 183: 795-803 (2008)). HDAC6 and cortactin are also involved in parkin-dependent clearance of damaged mitochondria (data not shown). Our results support a central role for ubiquitin modification and HDAC6-regulated F-actin remodeling in establishing QC autophagy.

Example 6: Autophagosomes are associated with actin and involved in F-actin network formation at ubiquitinated protein aggregates

[00113] Our data indicated that F-actin polymerization assists autophagosome-Iysosome fusion (Figure 4). This raises a question as to the source of actin that feeds into F-actin polymerization. Figure 5A shows results from biochemical characterization of autophagic compartments isolated from HDAC6 knockout cells. Different subcellular fractions (75 μg protein) isolated from wild type (WT) were subjected to SDS-PAGE and immunoblot for the indicated proteins (Horn: homogenate; APG:

autophagosomes; APL: autophagolysosomes; LYS: lysosomes). We found that actin is abundantly present in the autophagosomal but almost undetectable in lysosomal fractions (Figure 5A). This spatial distribution suggests a possibility that autophagosome-associated actin might become polymerized by an HDAC6 -dependent mechanism to stimulate fusion to lysosomes. Supporting this hypothesis, we found that latrunculin A treatment of purified autophagosomes significantly inhibited autophagosome-Iysosome fusion while the same treatment in lysosomes had little effect on fusion (Figure 5B). For results shown in Figure 5B, autophagosomes (APG) and lysosomes (Lys) isolated from fed cells were treated with or without lantrunculin (LatA) as labeled and subjected to in vitro fusion assay. Differences with untreated samples were significant for p<0.01. If autophagosomes were the main sources of actin, one would also predict that autophagosomes would be involved to establish the F-actin network at autophagic substrates. For results shown in Figure 5C, wild type and ATG5 KO MEFs were treated with MG132 and immunostained with antibodies to ubiquitin (green) as indicated. F-actin was detected by phalloidin (red). Arrows indicated ubiquitin-positive protein aggregates. Collectively, these data indicate that

autophagosomes are associated with actins, which can be assembled into an F-actin network at the autophagic substrates by an HDAC6 -dependent mechanism. Indeed, we found that ATG5 knockout MEFs cannot generate an F-actin network at protein aggregates (Figure 5C).

Example 7: HDAC6 recruits cortactin to assemble an F-actin network essential for autophagosome- Iysosome fusion and protein aggregate clearance.

[00114] Our results indicated that HDAC6 promotes the formation of an F-actin network at protein aggregates (Figure 4A). Cortactin, a component of the F-actin polymerization machinery is a substrate of HDAC6 (Zhang et al., Mol. Cell 27: 197-213 (2007)). We therefore determined if cortactin is involved in the HDAC6-dependent F-actin assembly at protein aggregates. For results in Figure 6A, wild type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to cortactin (red), ubiquitin (green), and phalloidin for F-actin (blue) as indicated. Arrows indicated ubiquitin-positive aggregates that were colocalized with F-actin and cortactin. We found that cortactin is concentrated at F- actin positive, aggresome-like protein aggregates in control MEFs (Figure 6A). In contrast, little concentration of cortactin was found in HDAC6 KO MEFs, indicating that HDAC6 recruits cortactin to protein aggregates (Figure 6A, Bottom Panels). Knockdown of cortactin by siRNA completely prevented the formation of the F-actin network at inclusion bodies but had little effect on lysosome distribution (Figure 6B, bottom Panels). For results in Figure 6B, wild type MEFs were transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for LAMP-1 (red, to label lysosome), or ubiquitin (green) and phalloidin for actin (blue). Note that F-actin staining at protein aggregates was lost but lysosomes remained concentrated in cortactin knockdown cells (arrow). This result demonstrated a role of cortactin in the assembly of the F-actin network at protein aggregates.

[00115] The failure to form the F-actin network was accompanied by a prominent defect in protein aggregate clearance (Figure 6C and Figure 19B) and the accumulation of abnormally large aggresomes in cortactin deficient cells (Figure 6B, bottom panels, and Figure 19A). Wild type MEFs were transfected with control or cortactin siRNA, treated MG132 2.5 μΜ for 18 hrs and subjected to filter trap assay using an ubiquitin antibody (Figure 6C). The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody to cortactin and GAPDH in the right panel. Figure 19A shows control and cortactin-knockdown MEFs treated with 2.5 μΜ MG132 for 24 hrs, incubated in normal growth media without MG132 for 18 hrs, and subjected to immunocytochemistry using anti-ubiquitin antibody (green) and phalloidin (red), with arrows indicating ubiquitin aggregates. Figure 19B shows control and cortactin-knockdown MEFs treated with 2.5 μΜ MG132 for 18 hrs. Cells were incubated with full media for the indicated times after washing 3 times with PBS, and cell lysates were subjected to Western analysis using anti-ubiquitin, anti-cortactin, and anti-GAPDH antibodies. To detect ubiquitinated protein aggregates, the stacking gel was transferred and blotted with anti-ubiquitin antibody.

[00116] These findings, for the first time, demonstrate that F-actin remodeling is involved in autophagy-dependent protein aggregate degradation. The mCherry-GFP-LC3 reporter assay showed that cortactin knockdown also led to significant inhibition of autophagosome-Iysosome fusion under normal, but not starvation conditions (Figure 6D). For results shown in Figure 6D, U20S cells were transfected with control siRNA and cortactin siRNA. Autophagosome-Iysosome fusion was analyzed with or without starvation (6 hrs) using the mCherry-GFP-LC3 reporter as described in Figure 3A.

[00117] Lastly, to investigate the potential importance of cortactin acetylation, we assessed the effects of wild type, acetylation-resistant (KR) or acetylation-mimicking (KQ) mutant cortactin on

autophagosome-Iysosome fusion. As shown in Figure 6E, the expression of the acetylation-mimicking mutant (KQ), but not wild type or acetylation-resistant mutant (KR), significantly inhibited

autophagosome-Iysosome fusion, indicating that acetylated cortactin cannot support fusion. For results shown in Figure 6E, mCherry-GFP-LC3 plasmid was cotransfected with wild type, 9KQ (acetylation- mimic), or 9KR (deacetylation-mimic) cortactin expressing plasmids into wild type MEFs.

Autophagosome-Iysosome fusion was analyzed as described in Figure 3A. Taken together, these results suggest that HDAC6 recruits and deacetylates cortactin, thereby promoting F-actin remodeling for autophagosome-Iysosome fusion and protein aggregate clearance. Example 8: HDAC6 deficiency causes ubiquitinated protein aggregate accumulation and neurodegeneration

[00118] Basal QC autophagy was proposed to remove toxic protein aggregates and protect neurons (Hara et al., 2006; Komatsu et al., 2006). We therefore investigated if there were any neurodegenerative phenotypes in HDAC6-deficient animals. While HDAC6 knockout mice were fertile and viable, they developed prominent ubiquitin-positive aggregates in the brain as early as six months of age whereas very few such structures were found in littermate controls (Figure 7A). The hippocampus and cerebral cortex regions from 6-month-old wild type and HDAC6 KO littermates were subjected to immunostaining with an ubiquitin antibody and counter-stained with hematoxylin. Red arrows indicate ubiquitin-positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin-positive structures were rarely observed in control littermates (scale bar = 50 μηι). TUNEL assay demonstrated the presence of apoptotic cell death in brains of HDAC6 KO mice, but not the wild type littermate controls (Figure 7B; scale bar = 100 μηι). Similar neurodegenerative phenotypes were observed in strains of transgenic Drosophila that express a HDAC6 siRNA in photoreceptor neurons (Figures 7C-D). Figure 7C shows HDAC6 depletion in the Drosophila eye lead to ubiquitin-positive pathology, as determined from immunostaining for ubiquitin (green) in frontal eye sections of I-day-old (dl) and 30-day-old (d30) fly eyes. The eyes of HDAC6-depleted flies (GMR:GAL4/UAS-HDAC6RNAi) developed ubiquitin- positive cytoplasmic inclusions that become more prominent at day 30 (d30) (blue = DAPI). Figure 7D shows depletion of HDAC6 in the Drosophila eye lead to age-dependent degeneration, with light micrographs (left) and corresponding Richardson-stained frontal eye sections (right) of 1 -day-old and 30- day-old fly eyes. The eyes of control flies (GMR:GAL4/+) and HDAC6-depleted flies

(GMR:GAL4/UAS-HDAC6RNAi) showed normal highly organized ommatidial array at day 1. 30-day- old control animals also showed no defects, but 30-day-old HDAC6-depleted flies showed degeneration with disorganization of the ommatidial array and loss of normal eye architecture (40X and 80X).

Together, these results demonstrated that HDAC6 deficiency leads to age-dependent neurodegeneration and ubiquitinated protein aggregate accumulation.

[00119] The role of autophagy in preventing toxic protein aggregate accumulation and

neurodegeneration has been established, e.g., in mice lacking ATG5 or ATG7, both of which are involved in autophagy activation (Hara et al., 2006; Komatsu et al., 2006). We found that HDAC6 knockout mice and knockdown Drosophila transgenic flies accumulated ubiquitin-positive aggregates and developed spontaneous neurodegeneration (Figure 7). However, HDAC6 deficiency did not affect autophagy activation (Figure IB); rather, it impaired autophagosome-Iysosome fusion (Figure 2). This data indicates that fusion defects are implicated in the development of neurodegenerative disease (Figure 7). [00120] In HDAC6 KO MEFs, defects in fusion to lysosomes led to a buildup of autophagosomes. EM analysis revealed that many of these autophagosomes showed abnormal morphology and contents (Figure 2D). Similar abnormal autophagic structures were also found prominently accumulated in dystrophic axons in Alzheimer's disease (AD) patients (Nixon et al., J. Neuropathol. Exp. Neurol. 64: 113-122 (2005)) and in a mouse model for frontotemporal dementia (FTD), the second most common form of presenile dementia (Leroy et al., Am. J. Pathol. 171 : 976-992 (2007)). These similarities suggest the possibility that neurons affected by AD or FTD and cells deficient in HDAC6 might share a common defect: autophagosome-Iysosome fusion. In fact, it has been speculated that abundant autophagic structures observed in AD patients could involve a failure to form functional autophagolysosomes (Nixon et al., 2005). Thus, a defect in lysosome fusion, rather than autophagy activation per se, might be a factor contributing to the pathogenesis of certain neurodegenerative diseases. It was recently reported that inactivation of components of the ESCRT complex, which regulates late endocytic multiple vesicular bodies (MVB), can lead to an autophagosome-Iysosome fusion defect, protein aggregate accumulation, and frontotemporal dementia (FTD) (Filimonenko et al., J. Cell Biol. 179: 485-500 (2007); Lee et al., Curr. Biol. 17: 1561-1567 (2007)). Together, these observations suggest a causative relationship between autophagosome-Iysosome fusion defects and the development of neurodegenerative disease. If this hypothesis is correct, simply activating formation of autophagosomes, the step targeted by most of the commonly used autophagy activating drugs, might not be the most effective therapeutic approach to neurodegenerative disease. Supporting this view, in the Drosophila spinal bulbar muscular atrophy (SBMA) model, the neuroprotective effect of rapamycin, which potently activates autophagy, was abrogated in the HDAC6 mutant background (Pandey et al., 2007). Instead, we speculate that agents that stimulate autophagosome-Iysosome fusion might provide an alternative and effective way to enhance the degradative capacity of autophagy, thereby protecting neurons. Thus, the autophagosome-Iysosome fusion machinery could be an attractive therapeutic target for developing novel therapies for

neurodegenerative diseases.

Example 9: Rapamycin induced autophagosomes robustly but did not promote autophagosome- Iysosome fusion

[00121] The pKa of GFP is about 6.0, making it difficult to use GFP fusions to follow

autophagosomes by fluorescence microscopy after they become acidified following fusion with endosomes or lysosomes to produce amphisomes or autophagolysosomes. However, the pKa of the monomeric red fluorescent protein mCherry is less than 4.5, making the protein acid-stable. A double-tag strategy can be used by expressing mCherry-GFP-LC3B in Hela cells, it is possible to visualize LC3B in acidic vesicles displaying red fluorescence only, and in neutral structures displaying both red and green (appears as yellow) fluorescence. It can be verified that acidic structures visualized with mCherry-GFP- LC3B are due to fusions between autophagosomes and endolysosomes/lysosomes by looking for colocalization with the endocytic pathway marker AlexFluor 647 dextran.

[00122] GFP-Cherry-LC3 reporter plasmid was transfected into HeLa cells. Cells were treated with rapamycin (0.25 μg/mL) for 4 hr (Figure 8A) alone or followed by incubation in HBSS (Hanks Solution) for 5 or 10 min (Figures 8B-C). Figure 8D shows a Western blot for LC3 I and LC3 II of HeLa cells without rapamycin treatment and with rapamycin treatment, in the presence or absence of HBSS. The GFP-Cherry-LC3 fusion reporter labeled autophagosomes as yellow dots (red+green) and

autophagolysosomes as red dots. Double -positive yellow vesicles with rapamycin rapidly changed to single positive red vesicles after Hank solution incubation. The accumulation of double positive (red+green) yellow vesicles indicated that rapamycin induced autophagosomes robustly but did not promote autophagosome-Iysosome fusion appreciably. However, the appearance of single positive red vesicles upon the inclusion of Hank Solution, which lacks amino acids and some other compounds, indicated that the rapamycin-induced autophagosomes underwent rapid fusion to lysosomes. The fusion to lysosomes was verified by the degradation of LC3 upon Hank Solution treatment. This result suggests that it is possible to stimulate autophagosome-Iysosome fusion in cells that are treated with rapamycin, and identify agents that can convert GFP-Cherry-LC3 fusion reporter vesicles from yellow to red dots. In principle, the combined effect of rapamycin or other autophagy-activating agents with

autophagosome/lysosome fusion enhancers can enhance the degradative capacity of autophagy.

Example 10

[00123] As shown in Figure 22, F-actin associated with ubiquitinated aggresomes but not with autophagosomes itself. Wild type MEFs were treated with MG132 as described above and

immunostained with antibodies to LC3 (green), ubiquitin (red), and F-actin-Blue). Arrows indicate LC3 positive autophagosomes that does not co-localize with F-actin, and arrowheads indicate ubiquitinated aggregates that associate with LC3 positive autophagosomes and F-actin (scale bar = 10 mm).

[00124] As shown in Figure 23, autophagosomes were positive for G-actin rather than F-actin. Wild type MEFs were treated with rotenone or DMSO for 18 hrs and subjected to immunostaining with anti- LC3 antibody (autophagosomes, red), phalloidine (F-actin, blue), and DNAse I (G-actin, green).

[00125] As shown in Figure 24, the effect of cortactin on autophagosome/lysosome fusion was examined. Autophagosomes (APG) and lysosomes (Lys) purified from wild type (Figures 24A and 24C) and HDAC67- MEFs (Figures 24B and 24D) were subjected to heterotypic in vitro fusion assays in the presence or absence of the indicated amounts of purified cortactin and 1 mg of purified actin. Values presented in Figure 24 are means with the standard error (S.E.) of the percentages of fusion from 3 independent experiments.

[00126] As shown in Figure 25A-B, cortactin and actin were enriched in basal autophagosomes which contained ubiquitinated protein. In Figure 25A, purified autophagosome (APG) and lysosome (Lys) fractions from fed or starved mouse liver were analyzed by Western blotting for LAMP-1, cortactin, and actin. In Figure 25B, purified autophagosomes and lysosome fractions from wild type MEFs under normal growth media or serum starvation were analyzed by Western blotting for cortactin, actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Ubiquitinated protein aggregates were detected in the base of the wells visible upon transferring of the stacking gel.

Claims

We Claim:

1) A method of identifying an autophagosome/lysosome fusion enhancer, comprising:

a) contacting a host cell comprising a double tag system with an agent, wherein the double tag system comprises a first tag component, a second tag component, and a targeting component, wherein the targeting component targets the double tag system to autophagosomes ;

b) detecting autophagolysosomes in the contacted host cell; and

c) comparing the number of autophagolysosomes in the contacted host cell to the number of autophagolysosomes in a host cell that has not been contacted with the agent;

wherein an increase in the number of autophagolysosomes indicates that the agent is an

autophagosome/lysosome fusion enhancer.

2) The method of claim 1 , wherein the enhancer induces HDAC6-dependent F-actin assembly at a site of protein aggregation under non-starvation conditions.

3) The method of claim 1 , wherein the enhancer induces HDAC6 recruitment of cortactin at a site of protein aggregation during autophagosome-lysosome fusion under non-starvation conditions.

4) A method of identifying an enhancer of HDAC6-dependent F-actin assembly at a site of protein aggregation under non-starvation conditions, comprising:

a) contacting a host cell comprising a double tag system with an agent, wherein the double tag system comprises a first tag component, a second tag component, and a targeting component, wherein the targeting component targets the double tag system to autophagosomes ;

b) detecting autophagolysosomes in the contacted host cell; and

c) comparing the number of autophagolysosomes in the contacted host cell to the number of autophagolysosomes in a host cell that has not been contacted with the agent;

wherein an increase in the number of autophagolysosomes indicates that the agent is an enhancer of HDAC6-dependent F-actin assembly at a site of protein aggregation under non-starvation conditions.

5) A method of identifying an enhancer of HDAC6 recruitment of cortactin at a site of protein aggregation during autophagosome-lysosome fusion under non-starvation conditions, comprising:

a) contacting a host cell comprising a double tag system with an agent, wherein the double tag system comprises a first tag component, a second tag component, and a targeting component, wherein the targeting component targets the double tag system to autophagosomes ; b) detecting autophagolysosomes in the contacted host cell; and

c) comparing the number of autophagolysosomes in the contacted host cell to the number of autophagolysosomes in a host cell that has not been contacted with the agent;

wherein an increase in the number of autophagolysosomes indicates that the agent is an enhancer of HDAC6 recruitment of cortactin at a site of protein aggregation during autophagosome-lysosome fusion under non-starvation conditions.

6) The method of any one of claims 1-5, further comprising contacting the host cell with at least one autophagy inducer in an amount to induce autophagy.

7) The method of claim 6, wherein the autophagy inducer is rapamycin.

8) The method of any one of claims 1-7, wherein the first tag component is detectable in autophagosomes and autophagolysosomes and the second tag component is detectable in autophagosomes but not in autophagolysosomes.

9) The method of claim 1-7, wherein the first tag component is detectable in autophagosomes and autophagolysosomes and the second tag component is detectable in autophagolysosomes but not in autophagosomes.

10) The method of claim 1-7, wherein the first tag component is detectable in autophagosomes and autophagolysosomes and the second tag component emits a detectably different signal in autophagosomes versus autophagolysosomes.

11) The method of claim 1-7, wherein the first and/or second tag component is pH sensitive.

12) The method of claim 1-7, wherein the host cell is deficient in HDAC6.

13) A method of treating a disease relating to protein aggregation in a subject in need of treatment, the method comprising administering to the subject at least one autophagosome/lysosome fusion enhancer in an amount effective to treat the disease.

14) A method of treating a neurodegenerative disease in a subject in need of treatment, the method comprising administering to the subject at least one autophagosome/lysosome fusion enhancer in an amount effective to treat the disease.

15) The method of claim 13 or 14, further comprising administering to the subject at least one autophagy inducer.

16) The method of claim 15, wherein at least one autophagy inducer is rapamycin.

17) The method of claim 13 or 14, wherein the autophagosome/lysosome fusion enhancer alters HDAC6 activity relative to the HDAC6 activity before the autophagosome/lysosome fusion enhancer is administered. 18) The method of claim 17, wherein the autophagosome/lysosome fusion enhancer increases HDAC6 activity relative to the HDAC6 activity before the autophagosome/lysosome fusion enhancer is administered.

19) The method of claim 13 or 14, wherein the autophagosome/lysosome fusion enhancer alters HDAC6 expression levels relative to the HDAC6 expression levels before the autophagosome/lysosome fusion enhancer is administered.

20) The method of claim 19, wherein the autophagosome/lysosome fusion enhancer increases HDAC6 expression levels relative to the HDAC6 expression levels before the autophagosome/lysosome fusion enhancer is administered.

21) The method of claim 13 or 14, wherein the autophagosome/lysosome fusion enhancer modulates autophagosome and lysosome fusion relative to autophagosome and lysosome fusion before the autophagosome/lysosome fusion enhancer is administered.

22) The method of claim 21, wherein the autophagosome/lysosome fusion enhancer increases autophagosome and lysosome fusion relative to autophagosome and lysosome fusion before the autophagosome/lysosome fusion enhancer is administered.

23) The method of claim 13, wherein the disease relating to protein aggregation comprises a neurodegenerative disease.

24) The method of claim 23, wherein the neurodegenerative disease is Parkinson's disease.