Immunohistochemistry (IHC) experiments with the antibody mEM48, which preferentially recognizes aggregated mHTT (Gutekunst et al., 1999), revealed age-dependent development of mHTT-positive profiles in the striatum of Q175KI mice. Brain sections from 2.5-mo-old Q175 (not shown) only exhibited faint and diffuse nuclear mHTT staining without identifiable aggresomes/inclusion bodies (IBs), while at 6 and 10 mo of age, mHTT-positive IBs were readily detected progressively with age (Figure 1A2–A3). There were no similar mHTT immunoreactive puncta in WT striatum (Figure 1A1). These observations are consistent with previous findings in Q175 and other HD mouse models (Carty et al., 2015; Li et al., 1999; Menalled et al., 2003). To further determine the locations of mHTT IBs in Q175, mEM48-immunostained sections were counterstained with cresyl violet (Figure 1A4) to distinguish nuclear IBs (Figure 1A4, arrowheads) from extranuclear IBs, which were localized predominantly in the neuropil (Figure 1A4, arrows) and detected, but rarely, in the cytoplasmic portion of the perikaryon. Thus, our results demonstrate an age-dependent increase in mHTT aggresomes in the Q175 model.

To reveal the ultrastructural locations and features of the mHTT aggregates, immunogold EM (IEM) with antibody mEM48 was performed. EM images (Figure 1B) demonstrated that the IEM with this antibody was highly specific in detecting mHTT IBs which were localized in the neuronal nuclei, dendrites, and axons. Ultrastructurally, most IBs were cotton-ball shaped and composed of fine fibrous or granular elements, somewhat similar to the unbundled short fibrils/protofibrils found in vitro with recombinant mHTT protein fragments (Ko et al., 2018; Kolla et al., 2021; Mario Isas et al., 2021), and similar to the structure of NII type inclusions we found in the human HD brain (Berg et al., 2024). Notably, however, human neuritic IBs often displayed a more heterogeneous composition, such as fine fibrils mixed with AVs or bundles composed of microtubule-like filaments, which were not found in the mouse neuritic IBs. Thus, the result suggests a homogeneous aggresome pathology in the mouse model, possibly reflecting a more rapid formation rate than in humans.

Previous immunoblotting studies have observed fragmentation of mHTT molecules in the human brain (Kim et al., 2001; Mende-Mueller et al., 2001), including our own study which detects mHTT fragments of 45–48 kDa, which predominantly exist in HD striatum (Berg et al., 2024). To assess HTT molecular species in the Q175 mouse brain, we employed multiple antibodies that preferentially detected mHTT over wild-type HTT, including MAB1574 (Clone 1C2) (Figure 1C1 and C2), its epitope containing a 38-glns stretch (Trottier et al., 1995), and mAb PHP2 (Figure 1C1), reacting with the peptide sequence QAQPLLPQP within the proline-rich domain of HTT (Ko et al., 2018). Both detected full-length mHTT and a~120 kDa fragment in the Q175 model (Figure 1C1; 1C2 left and 1C3 top two graphs). By contrast, MAB5490 (Figure 1C2 right), reacting with aa115-129 of HTT (C-terminal to the region of polyQ stretch-containing exon 1), detected both wild type and mutant forms of HTT (Figure 1C3, bottom right graph). A~48 kDa HTT fragment may correspond to the 45–48 kDa fragment seen in human brain (Berg et al., 2024), and was detected by both MAB1574 and MAB5490 antibodies in some samples (Figure 1C2) but its levels in the Q175 models and the control TRGL were not statistically significantly different (Figure 1C3, bottom left graph). Thus, the result suggests that HTT fragmentation is not obvious in the Q175 brains, unlike the far more prevalent occurrence of this phenomenon in human HD striatum.

Our earlier study in human HD brains Berg et al., 2024 found a high degree of colocalization of mHTT/Ub or Ub/p62 colocalized signals in IBs, suggesting a relationship between mHTT and the autophagy machinery since p62 and Ub are adaptor proteins mediating autophagic cargo sequestration. Similarly, in Q175 mice, mHTT/Ub or Ub/p62 signals were highly colocalized in IBs, particularly in NIIs, in striatal neurons of Q175 mice (Figure 2—figure supplement 1). Additional triple IF labeling experiments (mHTT/Ub/p62) with brain sections from 6- and 10-mo-old Q175 mice identified IBs positive for mHTT, p62, and Ub within or outside the nuclei (Figure 2A, arrows and arrowheads, respectively). The 10-mo-old Q175 mice exhibited more and larger mHTT-positive IBs than 6-mo mutants (Figure 2A, first column), consistent with Figure 1A and the literature (Carty et al., 2015; Deng et al., 2021). Thus, our data demonstrate a close spatial relationship among mHTT and autophagy receptor protein p62 and Ub in the Q175 model, which is consistent with our observations in human HD brain (Berg et al., 2024).

Double IF labeling of mHTT with a LY marker CTSD (Figure 2B1) detected small punctate mHTT signal in CTSD-positive vesicles, suggesting a pool of mHTT within the ALP. Consistent with this LM finding, IEM with antibody MAB1574 directed against mHTT clearly demonstrated that the AVs in either cell bodies, dendrites, and axons were labeled with concentrated silver-enhanced gold particles (Figure 2B2), and the specificity of the IEM labeling was very high, as reflected by a much higher number of silver-enhanced gold particles associated with AVs versus the minimal number of silver-enhanced gold particles with mitochondria that represent the background labeling (Figure 2B3). Together, the LM and IEM findings suggest that mHTT molecules exist within ALP vesicles in the absence of definable aggresome/IB structures, similar to the IEM finding from a recent report (Zhou et al., 2021).

We crossed TRGL mice (Lee et al., 2019) with Q175 to generate TRGL/Q175 and assessed AV/LY subtypes in striatal neurons with a hue angle-based analysis method (Lee et al., 2019; Lee et al., 2022). Our pilot studies in young TRGL/Q175 mice (2.5–6-mo of age) hardly detected autophagy alterations, compared to the control TRGL mice (not shown). Previous studies have reported that autophagy impairment was hardly detected in 6-mo Q175/GFP-LC3 mice (Wold et al., 2016), but alterations in a limited number of autophagy markers (e.g. protein levels of p62, LC3, p-Beclin-1) were found in brain homogenates of 12–15-mo Q175 mice (Abd-Elrahman et al., 2017; Heikkinen et al., 2021; Wold et al., 2016). Therefore, to further validate the status of the ALP and to discover potential additional autophagy alterations, we expanded our study to old mice at 17-mo of age, where we found the following ALP alterations.

To investigate potential neurodegeneration in our model, brain sections from 17-mo-old mice were double-labeled with the medium spiny neuron marker DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa)(Ouimet et al., 1998) and a general neuronal marker NeuN. We found that the intensity of DARPP-32-IR significantly diminished in TRGL/Q175 compared to TRGL (Figure 4A and B). However, there were no alterations in NeuN-IR, including Area Covered and the Number of NeuN-positive neurons (Figure 4A and B), and there were clear examples of neurons showing strong NeuN signal but faint or no DARPP-32 signal (Figure 4A, arrows). Such a finding is consistent with other studies in Q175 and Q140 mice where neuronal loss was not found even though a reduction in DARPP-32 was seen in the same study, and that neuronal loss only occurred quite late, e.g., around 2 y of age (Deng et al., 2021; Hickey et al., 2008; Peng et al., 2016; Rothe et al., 2015). Thus, our data support the notion that changes in DARPP-32 may indicate alterations in its protein level and a loss of phenotype pattern, which is a potentially common prelude to neuronal loss in other neuronal cell types, e.g., basal forebrain cholinergic neurons (Jiang et al., 2022).

As a prelude to investigate effects of pharmacological autophagy stimulation with a mTORi, INK, on TRGL/Q175 mice, we first performed a safety, pharmacokinetic, and pharmacodynamic evaluation of INK in 6-mo-old WT mice, administered via oral gavage. The WB results (Figure 5—figure supplement 1A) indicated that all doses tested, from 2.5 mg/kg (mpk) to 10-mpk, exhibited target engagement as revealed by the reductions in the protein levels of mTOR targets, i.e., p-ULK1 (S757, phosphorylated by mTOR), p-p70S6K (not shown), or phosphorylated S6 ribosomal protein (p-S6, at S240/244), implying high BBB permeability of this compound and target engagement. Consistently, measurements for the levels of INK in cerebellar homogenates of WT mice treated with 1, 3, and 10-mpk INK revealed dose-dependent brain levels of INK (Figure 5—figure supplement 1B).

We then decided on an oral gavage treatment regimen for INK as 4-mpk, daily, for 3 wk, to be administered in 6-mo-old TRGL/Q175. The brains from the mice (7-mo of age after the 3 wk treatment duration) were then analyzed by multiple experimental approaches. By immunoblotting, target engagement was verified, as indicated by decreased levels of p-mTOR (S2481, autophosphorylation site), p-ULK1 (S757) and p-S6 (S240) in brain homogenates from INK-treated TRGL/Q175 compared to Veh-treated TRGL/Q175 (Figure 5A and B). Additionally, there were also INK-induced changes in marker proteins located downstream of autophagy induction, including, for example, increased levels of p-ATG14 in INK-treated samples (Figure 5A and B). It is interesting that although INK did not induce any alterations in the transgene product tfLC3-I or -II, there was a trend of decreased endogenous LC3-I, leading to a statistically significant increase in the ratio of LC3-II/I, and such a result in LC3-I reduction was reproduced in a repeated experiment (Figure 5A and B). Thus, the pilot study in WT mice and the actual study in TRGL/Q175 mice together established mTORi INK’s BBB permeability, target engagement, and ability to modify molecular events in the ALP.

We took advantage of the tfLC3 construct reporting in vivo autophagy flux (Lee et al., 2019) to assess INK effects on AV/LY subtypes in TRGL/Q175. Confocal images from INK- or Veh-treated TRGL/Q175 and Veh-treated TRGL brain sections of 7-mo-old mice, immuno-stained with anti-CTSD antibody and detected with a third fluorophore, were collected (Figure 6A) and processed for hue-angle based quantitative analysis using the protocol described previously (Lee et al., 2019). The analysis in striatal neurons (Figure 6B) revealed reversal of the two main ALP alterations. First, the pre-existing abnormally lowered LY number in TRGL/Q175-Veh (Figure 6B, LY group, red vs green), was reversed by INK treatment (Figure 6B, LY group, red vs blue). Second, INK reduced the number of AL in TRGL/Q175-Veh compared to TRGL-Veh (Figure 6B, AL group, red vs blue), implying improved AL clearance and likely providing the basis for restoration of normal LY number. Together, the data suggest that autophagy phenotype in 7-mo Q175 is measurable but milder than that in the 17-mo-old mice shown in Figure 3A and that INK has the potential to modulate the autophagy pathway and restore normal function at this mild disease stage. mTOR inhibition mitigates mHTT-related aggresome pathology.

Importantly, we found that HD-like pathology such as mHTT IBs and associated protein aggregation of adaptor proteins, p62, and Ub, was ameliorated by INK treatment. Confocal images demonstrated that mHTT positive particles/inclusions were substantially reduced in INK-treated 7-mo-old TRGL/Q175, compared to Veh-treated TRGL/Q175 mice (Figure 7A). Moreover, p62-IR or Ub-IR also changed in a similar trend (Figure 7A), consistent with the high degree of colocalization of mHTT/p62/Ub signals shown in Figure 2A. The corresponding quantitative analysis, shown as Area Covered by either mHTT-, p62-, or Ub-IR on a per cell basis, confirmed statistically significant decreases in the Total Area Covered by mHTT- and p62-signals (Figure 7B1). With the assistance of the endogenous tfLC3 signal (particularly the mRFP signal) in the TRGL to identify the association status of the mHTT-, p62-, or Ub-IR with AVs, the images were additionally quantified to generate the separated ‘Area Covered by the AV-associated Form’ (Figure 7B2) and ‘Area Covered by the AV-unassociated Form’ (Figure 7B3) of mHTT-, p62-, or Ub- signals. The results collectively suggest decreased mHTT-, p62-, or Ub-IR in treated compared to untreated TRGL/Q175, implying that INK treatment was able to promote the clearance of mHTT and related receptor proteins p62 and/or Ub.

Finally, we evaluated neurodegenerative changes by employing DARPP-32 IHC on brain sections of Veh-treated or INK-treated TRGL/Q175. Similar to what was found in 17-mo-old mice (Figure 4), the group of TRGL/Q175-Veh, even at this young age (7-mo), already exhibited a diminished DARPP-32-IR signal compared to that in TRGL-Veh (Figure 8A and B). However, no differences between the untreated and INK-treated TRGL/Q175 groups were established by quantitative analysis (Figure 8A and B). Thus, the result suggests a lack of effectiveness of INK in reversing this pre-existing phenotype, implying that earlier intervention may be necessary (Marchionini et al., 2022).

In the current study, we assessed ALP alterations in the Q175 model by analyzing protein markers in all phases of the ALP including autophagy induction signaling, membrane nucleation/AP formation, autophagy adaptor proteins, and AL formation/substrate degradation. By immunoblot analysis, alterations in the tested marker proteins even at 17 mo of age in TRGL/Q175 mice were limited to a reduction in p-P70S6K (T389), similar to that reported previously (Ravikumar et al., 2004), and a reduced ratio of p-ULK1 (S757)/total ULK1 as a result of increased total ULK1. While these two alterations together would suggest a lower mTOR activity and autophagy activation, consequential alterations of downstream markers, particularly proteins in the ATG14-containing Beclin-1/VPS34 complex, were not detected. Although 4 out of the 10 tested TRGL/Q175 mice exhibited very low levels of p-ATG14, implying a deficit in this complex as previously reported (Wold et al., 2016), variation among animals was too great to draw statistical conclusions. In this regard, mice used in the current study were heterozygous, which results in less autophagy alteration than that reported in homozygous mice using certain autophagy markers (Abd-Elrahman et al., 2017). It may also be noted in our experience that immunoblot analysis of brain tissue averages contributions from varied cell types that may respond to disease states in opposite directions and mask changes in a given cell population. Thus, we also applied a neuron-specific analysis using our neuron-specific tfLC3 reporter and hue-angle-based image quantification approach, which yielded more sensitive detection and findings.

In brain sections from 17-mo-old TRGL/Q175 mice, we observed increased numbers of AL including the population of pa-AL and reduced numbers of LY. Such changes in AL/pa-AL were not readily observed in TRGL/Q175 mice at 2 mo (not shown) or 7 mo of age (e.g. Figure 6B, which only exhibited a reduction in LY number), indicating that even if these alterations signify an existence of impairments in the late phase of the ALP, they do not occur until a later age (e.g. 17 mo). Importantly, this pattern of late-onset ALP pathology progression, e.g., AL/pa-AL increases, is consistent with our findings from HD human brains (Berg et al., 2024) where enlarged CTSD-positive AL accumulate and cluster in affected neuronal populations at the late disease stages (HD3 and HD4). These findings suggest that the overall autophagy function in heterozygous Q175 is largely maintained until the relatively late disease stage and provide a rationale to stimulate autophagy as a therapeutic intervention when the autophagy machinery, especially those involved in clearance, is still functional, which would generate beneficial effects. We obtained support for this concept in the subsequent study with mTORi INK when administered to TRGL/Q175 at 6 mo of age for 3 wk.

One of the key findings from this study is the reduction of mHTT-IR in neurons detected by immunolabeling after the 3 wk oral treatment with mTORi INK (Figure 7). This beneficial outcome from the drug treatment is interpreted as a primary result of the manipulation/stimulation of autophagy by the compound, leading to enhanced autophagy clearance of mHTT species including smaller, presumably soluble, species and larger aggregates such as the IBs. This interpretation is supported by the following considerations and evidence. First, in theory, the compound used for the treatment is a mTORi, so it is expected to enhance autophagy (Bensalem et al., 2021; Kim and Guan, 2015). Second, target engagement is achieved as clearly reflected by the diminished levels of mTOR-mediated phosphorylation on mTOR itself (S2481), ULK1 (S757), and p-S6 (S240/S244), along with changes in proteins downstream of mTOR, such as p-ATG14 (S29) and LC3. Third, hue-angle based confocal image analysis of AL/LY subtypes reveals a reduced AL number and an increased LY number in INK-treated TRGL/Q175 compared to Veh-treated TRGL/Q175. The findings from this analysis of AL/LY subtypes highlight the advantages of crossing TRGL with Q175 mice to evaluate autophagy modulators on the ALP, consistent with a notion of developing better tools capable of investigating autophagy flux in vivo (Nixon, 2013; Vodicka et al., 2014).

Our confocal microscopy analyses revealed mHTT signal in CTSD-positive vesicles, which can be classified as AL based on their LC3 fluorescence, and IEM detected mHTT-silver-enhanced gold particles in AP and AL including lipofuscin granules. Both findings indicate the existence of mHTT species inside the ALP as autophagy substrates. The presence of low levels of mHTT in CTSD-positive AL also implies a constitutive functional degradation event of mHTT in AL (Figure 2B1, 10-mo-old mouse). This is in contrast to the case in young AD mouse models where early-onset autophagy-stress exhibited severe substrate accumulation (e.g. reflected by strong LC3 signal) due to deficits of degradative functions within AL. (Lee et al., 2022). A reduced level in the portion of AV-associated mHTT signals (Figure 7B2) by mTORi INK further suggests that mHTT degradation has been enhanced after manipulating the ALP. This AV-associated mHTT pool is considered to be corresponding to the mHTT-silver-enhanced gold signal under IEM (Figure 2B2), i.e., amorphous and existing within the lumen of AV. Such mHTT localization and ultrastructural features are consistent with recent findings that mHTT proteins exogenously added to Q175 brain sections for incubation are recruited to multivesicular bodies/amphisomes, AL, and residual bodies (lipofuscin granules), and ultrastructurally localized to non-fibrillar, electron-lucent regions within the lumen of these organelles (Zhou et al., 2021).

It is notable that the AV-non-associated form of mHTT (i.e. did not show colocalization with the tfLC3 signal implying that they might not have contact with AVs) decrease more obviously after drug treatment, along with the reductions in p62 and Ub signals (Figure 7B3). These mHTT species are mainly larger aggresomes/IBs, including those within the nuclei. Based on our mHTT-IEM observation (Figure 1B), the IBs in Q175 brain are fibrillar aggregates in the nucleus and cytoplasm, exhibiting a homogeneous feature in shape (i.e. round/oval cotton-ball) and in content (i.e. short fine fibrils), similar to the nuclear IBs we found in human HD brains (Berg et al., 2024), whose cytoplasmic and neuritic IBs, however, also exhibit more heterogeneous compositions such as a mixture of fine fibrils with AVs, fingerprint-like structures or bundles of microtubules. In the current study, we did not observe that a whole IB is contained within an AP or AL in our EM examination, raising a question of how these larger aggregates could be degraded by the autophagy machinery after an autophagy activation by mTOR inhibition. This issue is informed by findings that the mHTT species are dynamic and the aggregates can form as different phases (liquid-like or solid-like), making it possible for mHTT species to move out or into IBs (Aktar et al., 2019; Liu et al., 2015; Peskett et al., 2018; Riguet et al., 2021) and to shuttle between the nucleus and the cytoplasm (Atwal et al., 2007; Xia et al., 2003). Thus, it is speculated that under the autophagy-activated condition induced by mTOR inhibition, the enhanced clearance of mHTT in the ALP pool would promote movements of mHTT species from the IB forms, leading to a reduction on the overall mHTT load.

Even if INK is a mTORi mainly targeting autophagy, we cannot completely exclude additional contributions from the UPS for the observed reduction of mHTT species given the crosstalk between the two proteolytic systems. It is well accepted that autophagy plays a more important role in the clearance of larger protein aggregates such as aggresomes/IBs (Sarkar and Rubinsztein, 2008a). For example, autophagy is involved in the degradation of p62 bodies (Bjørkøy et al., 2005) and autophagy markers are colocalized with cytoplasmic mHTT IBs (Iwata et al., 2005). However, there is also evidence for degradation of polyQ aggregates in the nuclei (Iwata et al., 2009), which contain p62 condensates as a hub for UPS-mediated nuclear protein turnover (Fu et al., 2021). We observed a high level of colocalization of mHTT with p62 and Ub in aggregates at both intranuclear and extranuclear locations (Figure 2A), which is presumably more related to the UPS hub and autophagy, respectively. It is speculated that the reduction of mHTT in the cytoplasm due to the stimulation of autophagy may partially relieve the burden of the proteasome in both the cytoplasm and the nucleus so that the nuclear proteasome operates more effectively. One of the observations reported here which may support the above speculation is the reductions of AV-non-associated forms of mHTT/p62/Ub (Figure 7B3), given that some of these aggregates should exist within the nucleus and, therefore, their reduced levels (in the nucleus) may reflect increased intranuclear UPS activity, besides the other possibility that they may travel from the nucleus to the cytosol for clearance (by autophagy) as discussed above.

Lowering mHTT via interfering protein production (e.g. through RNAi, antisense oligonucleotides) has been an attractive strategy in HD therapeutic development (Kordasiewicz et al., 2012; Tabrizi et al., 2019). Given that mTOR regulates multiple cellular pathways including protein synthesis (Magnuson et al., 2012; Wang and Proud, 2006), the inhibition of mTOR as was done in the present study would potentially affect protein synthesis as well. Thus, while our results of decreases in mHTT signals (Figure 7) can be interpreted as a result of autophagy-mediated clearance of mHTT, a possibility cannot be excluded that mTOR inhibition may result in a reduction in HTT production which may also contribute to the observed results – future studies should determine how significant such a contribution is.

In summary, Q175 models, as detected in the current study, develop ALP alterations late in the disease progression. Although milder than what is seen in late-stage HD human brain (Berg et al., 2024), there are similarities in ALP pathobiology between human HD brains and Q175 brains. These include the late-onset nature, mHTT as an ALP substrate, increased AL/pa-AL, mouse IB ultrastructural feature similar to the common fine fibril type of human IBs. The late-onset mild ALP pathology in Q175 is different from the severe AV accumulation in mouse models of AD as mentioned above (Lee et al., 2022). Together, the mild and late-onset nature of ALP alterations in Q175 provides a basis for manipulating autophagy to be a promising therapeutic strategy if the manipulations (e.g. stimulating autophagy with mTORi as in this study) are applied when the autophagy machinery is largely undamaged. This has been validated to be successful in this study as demonstrated by the INK-induced reduction of mHTT species along with other beneficial effects, consistent with findings from similar studies and supporting the general notion of lowering mHTT as a therapeutic strategy for HD (Barker et al., 2020; Cortes and La Spada, 2014; Ravikumar et al., 2004). However, it should be noted that the current study is an experimental therapeutic attempt in a mouse model which, consistent with previous reports (Ravikumar et al., 2004), is just a proof of concept for manipulating autophagy (e.g. via inhibiting mTOR in the current setting) as a potential therapeutic. The clinical implications from such studies require further rigorous verifications where early diagnosis and earlier interventions would be critical factors to be considered.

Q175 mice (B6J.zQ175 Knock-In mice, CHDI-81003003) (Menalled et al., 2012; Menalled et al., 2003) were obtained from the Jackson Lab (Stock No. 027410) on a C57BL/6 J background. The zQ175 KI allele has the mouse Htt exon 1 replaced by the human HTT exon 1 sequence containing a~190 CAG repeat tract, and the average size for the mice used in this study was 200 CAG. Q175 genotyping and CAG sizing were conducted by Laragen, Inc (Culver City, CA). The TRGL (Thy-1 mRFP-eGFP-LC3) mouse model, expressing tandem fluorescence-tagged LC3 (tfLC3 or mRFP-eGFP-LC3) in neurons, was generated, genotyped by PCR and maintained in a C57BL/6 J background as described previously (Lee et al., 2019). The TRGL was crossed with Q175 to generate the new TRGL/Q175 model featuring the tfLC3 probe as an in vivo autophagy reporter.

The mice were maintained in the Nathan Kline Institute for Psychiatric Research (NKI) animal facility and housed in a 12 hr light/dark cycle. All animal procedures were performed following the National Institutes of Health Guidelines for the Humane Treatment of Animals, with approval from the Institutional Animal Care and Use Committee at the NKI (AP2018-624). Animals of both sexes were used in this study. Details for mouse ages are in the Figure Legend and/or Results. All efforts were made to minimize animal suffering and the number of animals used. Mouse sample sizes were estimated based on similar experimental procedures in our prior studies (Lee et al., 2019; Lee et al., 2022; Yang et al., 2017; Yang et al., 2011; Yang et al., 2014), where a minimum of 5 mice per each mouse genotype per experimental condition would yield statistically significant differences in revealing alterations of autophagy in AD transgenic animal models vs wild-type mice.

INK-128 (ChemScene) was formulated in the sterile filtered vehicle (Veh) – 0.5% carboxymethyl cellulose (Sigma, Cat #5678) and 0.05% Tween 80 in water – at different concentrations to achieve the various desired dosages. The mixture was homogenized with a tissue homogenizer, stored in a sterile container at room temperature for up to 2 wk, and stirred prior to each dosing. It was administered to mice via oral gavage at a dose volume of 5 ml/kg, which was a relatively smaller volume of drug solution (generally 100–150 µl depending on the mouse body weight) aimed at avoiding the suppression of appetite. Mice receiving the Veh (also at a solution volume of 5 ml/kg) served as the control group. Randomization of grouping: mice from each pool of sex and genotype of a particular age were pulled at random and put into either vehicle or drug treatment groups to make sure of even distribution of sexes and genotypes in the vehicle and drug treatments. Oral gavage was conducted with 20 G-38mm Cadence Science Malleable Stainless-Steel Animal Feeding Needles #9921 (Fisher Sci Cat #.14-825-275). Each of the INK-128 dosing tests (ranging from 2.5 to 10 mg/kg [mpk] with various durations) and the 3 wk daily 4 mpk treatment procedures were performed once.

INK was formulated into 0.5% carboxymethyl cellulose and 0.05% Tween-80 in water to be administered orally. 6 mo old C57B6/J male mice were treated with compound at 1, 3, or 10 mpk or vehicle. 6, 12, or 24 hr after administration, mice were terminally anesthetized with pentobarbital, and cerebellum was dissected and flash frozen on liquid nitrogen and stored at –80 °C until analysis. Tissue was homogenized and extracted with acetonitrile (containing 0.1% formic acid). Extract was analyzed using a LC-MS/MS (Waters Xevo TQ MS) method with a lower limit of quantitation of 13 nM.

To obtain tissues for experiments, the animals were anesthetized with a mixture of ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW). Mice for light microscopic (LM) analyses were usually fixed by cardiac perfusion using 4% paraformaldehyde (PFA) in 0.1 M sodium cacodylate buffer [pH 7.4, Electron Microscopy Sciences (EMS), Hatfield, PA]. Following perfusion fixation, the brains were immersion-fixed in the same fixative overnight at 4 °C. For transmission electron microscopic (EM) study, 4% PFA was supplemented with 2% glutaraldehyde (EMS). For biochemical analyses such as immunoblotting, the brains were flash frozen on dry ice and stored at –70 °C. When both morphological and biochemical analyses were to be performed on the same brain, the brain was removed after brief perfusion with saline. One hemisphere was frozen at –70 °C and the other half was immersion-fixed in 4% PFA for 3 d at 4 °C.

The following primary antibodies were used in this study. (1) from Cell Signaling Technology: tHTT rabbit mAb (total HTTs, #5656), MTOR rabbit mAb (#2983); p-MTOR (S2481 autophosphorylation) pAb (#2974), p-MTOR (S2448) pAb (#2971), p70S6K rabbit mAb (#2708), p-p70S6K (S371) pAb (#9208), p-p70S6K (T389) rabbit mAb (#9235), S6 ribosomal protein mAb (#2317), p-S6 ribosomal protein (S240/244) rabbit mAb (#5364), ULK1 pAb (#4773), ULK1 rabbit mAb (#6439), p-ULK1 (S757) pAb (#6888), p-ULK1 (S317) pAb (#37762), ATG5 rabbit mAb (#12994), ATG14 pAb (#5504), p-ATG14 (S29) rabbit mAb (#92340), p-Beclin 1 (S30) rabbit mAb (#35955), VPS34 rabbit mAb (#4263), p-VPS34 (S249) pAb (#13857), TRAF6 rabbit mAb (#8028). (2) from Millipore-Sigma: ntHTT mAb (mEM48, #MAB5374; N-Terminus-specific), HTT mAb (1C2, #MAB1574; epitope: N-terminal part of the human TATA Box Binding Protein (TBP) containing a 38-glns stretch), HTT mAb (#MAB5490; epitope: human HTT aa115-129), ATG5 pAb (#ABC14), LC3 pAb (#ABC929), NeuN (#MAB377), β−actin mAb (#A1978). (3) from other vendors: HTT mAb MW8 (epitope: HD exon 1 with 67Q; Develop Studies Hybridoma Bank, University of Iowa), HTT mAb PHP2 (CHDI-90001516–2, Coriell/CHDI), Beclin 1 mAb (BD Biosciences, #612113), LC3 pAb (Novus Biologics, #NB100-2220), p62/SQSTM1 mAb (BD Biosciences, #610832) or C-term-specific p62/SQSTM1 Guinea Pig pAb (Progen Biotechnik #C-1620); ubiquitin pAb (Dako Agilent, #Z0458), ubiquitin pAb (Abcam, #ab7780), DARPP32 (Abcam, #40801), LAMP1 rat mAb (Developmental Studies Hybridoma Bank, #H4A3), CTSB goat pAb (Neuromics, #GT15047). (4) In-house made: CTSD pAb (RU4). CTSD sheep pAb (D-2–3) (Cataldo et al., 1990).

The following secondary antibodies and reagents for immunoperoxidase labeling were purchased from Vector Laboratories (Burlingame, CA): biotinylated goat anti-rabbit or -mouse IgG/IgM, Vectastain ABC kit (PK-4000), and DAB Peroxidase Substrate Kit (SK-4100). Mouse on Mouse (M.O.M) detection kit (BMK-2201), normal-goat (S-100), and normal-donkey (S-2000–20) serum blocking solution were also from Vector Lab. The following secondary antibodies for immunofluorescence were purchased from Thermo Fisher Scientific (Waltham, MA): Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034), Alexa Fluor 568- goat anti-rabbit IgG (A11036), Alexa Fluor 647- goat anti-rabbit (A21245), Alexa Fluor Plus 405- goat anti-rabbit (A48254), Alexa Fluor 568- goat anti-mouse IgG (A11031), Alexa Fluor 647- goat anti-mouse (A21235), and Alexa Fluor 647- goat anti-rat (A21247). HRP-linked secondary antibodies for immunoblotting were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA): Rabbit IgG (711-035-152), Mouse IgG (711-035-150), Rat IgG (712-035-150), and Goat IgG (705-035-003).

Immunoperoxidase and immunofluorescence IHC were performed according to the protocols previously described (Lee et al., 2019; Yang et al., 2009). Brain vibratome sections (40 μm) were blocked and incubated in primary antibody O/N (up to 3 d in some cases) at 4 °C. ABC detection method was used for immunoperoxidase labeling visualized with DAB while Alexa-Fluor conjugated secondary antibodies were used for immunofluorescence. Autofluorescence was quenched with 1% Sudan black (Sigma-Aldrich; St. Louis, MO) in 70% ethanol for 20 min. DAB-labeled sections was inspected on a Zeiss AxioSkop II equipped with a HrM digital camera (Carl Zeiss, Germany). Immunofluorescently labeled sections were examined on a Zeiss LSM880 confocal microscope. Independent investigator(s) coded animals to blind investigators when imaging and quantifying.

Confocal imaging was performed using a plan-Apochromat 20 x or 40 x/1.4 oil objective lens on a LSM880 laser scanning confocal microscope with the following parameters: eGFP (ex: 488, em: 490–560 with MBS 488), mRFP (ex: 561, em: 582–640 with MBS 458/561), Alexafluor 647 (ex: 633, em: 640–710 with MBS 488/561/633) and DAPI (ex: 405, em: 410–483), with the ‘best signal scanning mode’ which separates scanning tracks for each excitation and emission set to exclude crosstalk between each fluorophore signal. The resolution of 40 x images was 1024×1024 pixels (corresponding to an area of 212.34×212.34 μm²), and the resolution of 3 x digitally zoomed images was also 1024×1024 pixels (corresponding to an area of 70.78×70.78 μm²). Detailed settings for image collection were reported previously (Lee et al., 2019).

Hue angle-based vesicle analysis enables quantitative determination of AV/LY subtypes including AP, AL, poorly acidified AL (pa-AL), and pure LY and the method was described in detail previously (Lee et al., 2019). Briefly, high resolution confocal images containing the LC3 (red and green) and CTSD (blue) punctate signals were analyzed with the Zen Blue Image Analysis Module from Carl Zeiss Microscopy. Threshold for each of the three-color channels (red, green, blue; RGB) was set by taking the average of intensity value from 20 neuronal perikarya. The signal was segmented into discrete puncta by using the automatic watershed function to separate clumped vesicles into individual puncta. Background signal was eliminated using the size exclusion function of Zen Blue. The R, G, and B intensity values of each vesicle were calculated using the profile function of Zen Blue. The RGB ratio of each vesicle was converted into a hue angle and saturation range – which we assigned to each AV subtype and should match the desired AV color range as perceived visually (i.e. yellow for AP, blue for Ly, purple for AL and white for de-acidified AL) – by entering the values of R, G, and B for a given punctum into the formula, as follows: Hue°=IF(180/PI()*ATAN2(2*R-G-B,SQRT(3)*(G-B))<0,180/PI()*ATAN2(2*R-G-B,SQRT(3) *(G-B))+360,180/PI()*ATAN2(2*R-G-B,SQRT(3)*(G-B))). Saturation percent of the hue angle was calculated by entering the values of R, G, and B for a given punctum into the following formula = (MAX(RGB)-MIN(RGB))/SUM(MAX(RGB)+MIN(RGB))*100 (http://www.workwithcolor.com/), provided lightness is less than 1, which is the usual case for our data. The Hue angle was converted to color using the Hue color wheel. The data was pooled and categorized in Excel spreadsheets.

Vibratome brain sections (50 μm) were treated with 1% osmium tetroxide in 100 mM sodium cacodylate buffer pH 7.4 for 30 min, washed in distilled water four times (10 min/wash), and then treated with 2% aqueous uranyl acetate overnight at 4 °C in the dark. Samples were then washed and sequentially dehydrated with increasing concentrations of ethanol (20, 30, 50, 70, 90, and 100%) for 30 min each, followed by three additional treatments with 100% ethanol for 20 min each. Samples were then infiltrated with increasing concentrations of Spurr’s resin (25% for 1 hr, 50% for 1 hr, 75% for 1 hr, 100% for 1 hr, 100% overnight at room temperature), and then incubated overnight at 70 °C in a resin mold. For TEM ultrastructural analysis, 70 nm sections were cut using a Leica Reichert Ultracut S ultramicrotome and a Diatome diamond knife, placed onto grids and then post-stained with 2% uranyl acetate and lead citrate. Images were taken using a Ceta Camera on a ThermoFisher Talos L120C transmission electron microscope operating at 120kV.

For post-embedding IEM, ultrathin sections were placed onto carbon formvar 75 mesh nickel grids and etched using 4% sodium metaperiodate for 10 min before being washed twice in distilled water and then blocked for 1 hr. Grids were incubated in the primary antibodies at 4 °C overnight. Next day, grids underwent seven washes in 1xPBS and were then incubated in anti-mouse or anti-rabbit 10 nm gold secondary (1/50 dilution) for 1 hr. After this, the grid was washed seven times in 1x PBS and twice in distilled water. The grids were then silver enhanced for 5 min (Nanoprobes). Grids were finally post-stained with 1% uranyl acetate for 5 min followed by two washes in water and then stained with lead citrate for 5 min followed by a final two washes in distilled water. Samples were then imaged on a ThermoFisher Talos L120C operating at 120 kV.

Samples for WB were prepared by homogenizing brains in a tissue-homogenizing buffer (250 mM sucrose, 20 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA) containing protease and phosphatase inhibitors as previously described. (Schmidt et al., 2012) Following electrophoresis, proteins were transferred onto 0.2 µm-pore nitrocellulose membranes (Whatman, Florham Park, NJ) at 100 mA for 8–12 hr depending on the target protein. The blots were blocked for 1 hr in 5% non-fat milk in TBS, rinsed in TBST (TBS +0.1% Tween-20), then incubated with a primary antibody in 1% BSA/TBST overnight at 4 °C. The membrane was washed and incubated in a HRP-conjugated goat-anti-rabbit or mouse secondary antibody, diluted 1:5000 in 5% milk for 1 hr at room temperature. The membrane was again washed and then incubated in a Novex ECL (Invitrogen) for 1 min. The detection of the signals was achieved through either exposure to a film or scanning by a digital gel imager (Syngene G:Box XX9) as specified in the figure legends. Densitometry was performed with Image J and the results were normalized by the immunoblot(s) of given loading control protein(s) (usually GAPDH, unless otherwise noted).

Requests for resources and reagents should be directed to the lead contact, Dr. Ralph Nixon (nixon@nki.rfmh.org). The crossed TRGL/Q175 mice generated from this study are not available since the breeding had been stopped. However, Q175 mice are available from the Jackson Lab, and the TRGL mice are available from the lead contact and may require completion of a materials transfer agreement.