Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins.

The human apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found, by examining cells stably expressing each APOE isoform, that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-Golgi compartment is degraded through autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells, and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that might contribute to AD pathogenesis. This article has an associated First Person interview with the first author of the paper.

PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

IKKß slows Huntington's disease progression in R6/1 mice.

Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKß, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington's disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKß on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKß phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKß to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKß knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKß in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKß knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKß is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKß is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKß may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.

Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models.

Mammalian transglutaminases (TGs) catalyze calcium-dependent irreversible posttranslational modifications of proteins and their enzymatic activities contribute to the pathogenesis of several human neurodegenerative diseases. Although different transglutaminases are found in many different tissues, the TG6 isoform is mostly expressed in the CNS. The present study was embarked on/undertaken to investigate expression, distribution and activity of transglutaminases in Huntington disease transgenic rodent models, with a focus on analyzing the involvement of TG6 in the age- and genotype-specific pathological features relating to disease progression in HD transgenic mice and a tgHD transgenic rat model using biochemical, histological and functional assays. Our results demonstrate the physical interaction between TG6 and (mutant) huntingtin by co-immunoprecipitation analysis and the contribution of its enzymatic activity for the total aggregate load in SH-SY5Y cells. In addition, we identify that TG6 expression and activity are especially abundant in the olfactory tubercle and piriform cortex, the regions displaying the highest amount of mHTT aggregates in transgenic rodent models of HD. Furthermore, mHTT aggregates were colocalized within TG6-positive cells. These findings point towards a role of TG6 in disease pathogenesis via mHTT aggregate formation.

Potential function for the Huntingtin protein as a scaffold for selective autophagy.

Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51-like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.

Ganglioside GM1 induces phosphorylation of mutant huntingtin and restores normal motor behavior in Huntington disease mice.

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.

Selective histone deacetylase (HDAC) inhibition imparts beneficial effects in Huntington's disease mice: implications for the ubiquitin-proteasomal and autophagy systems.

We previously demonstrated that the histone deacetylase (HDAC) inhibitor, 4b, which preferentially targets HDAC1 and HDAC3, ameliorates Huntington's disease (HD)-related phenotypes in different HD model systems. In the current study, we investigated extensive behavioral and biological effects of 4b in N171-82Q transgenic mice and further explored potential molecular mechanisms of 4b action. We found that 4b significantly prevented body weight loss, improved several parameters of motor function and ameliorated Huntingtin (Htt)-elicited cognitive decline in N171-82Q transgenic mice. Pathways analysis of microarray data from the mouse brain revealed gene networks involving post-translational modification, including protein phosphorylation and ubiquitination pathways, associated with 4b drug treatment. Using real-time qPCR analysis, we validated differential regulation of several genes in these pathways by 4b, including Ube2K, Ubqln, Ube2e3, Usp28 and Sumo2, as well as several other related genes. Additionally, 4b elicited increases in the expression of genes encoding components of the inhibitor of kappaB kinase (IKK) complex. IKK activation has been linked to phosphorylation, acetylation and clearance of the Htt protein by the proteasome and the lysosome, and accordingly, we found elevated levels of phosphorylated endogenous wild-type (wt) Htt protein at serine 16 and threonine 3, and increased AcK9/pS13/pS16 immunoreactivity in cortical samples from 4b-treated mice. We further show that HDAC inhibitors prevent the formation of nuclear Htt aggregates in the brains of N171-82Q mice. Our findings suggest that one mechanism of 4b action is associated with the modulation of the ubiquitin-proteasomal and autophagy pathways, which could affect accumulation, stability and/or clearance of important disease-related proteins, such as Htt.

A bivalent Huntingtin binding peptide suppresses polyglutamine aggregation and pathogenesis in Drosophila.

Huntington disease is caused by the expansion of a polyglutamine repeat in the Huntingtin protein (Htt) that leads to degeneration of neurons in the central nervous system and the appearance of visible aggregates within neurons. We have developed and tested suppressor polypeptides that bind mutant Htt and interfere with the process of aggregation in cell culture. In a Drosophila model, the most potent suppressor inhibits both adult lethality and photoreceptor neuron degeneration. The appearance of aggregates in photoreceptor neurons correlates strongly with the occurrence of pathology, and expression of suppressor polypeptides delays and limits the appearance of aggregates and protects photoreceptor neurons. These results suggest that targeting the protein interactions leading to aggregate formation may be beneficial for the design and development of therapeutic agents for Huntington disease.

Aberrant splicing in Huntington's disease via disrupted TDP-43 activity accompanied by altered m6A RNA modification.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene encoding huntingtin. Prior reports have established a correlation between CAG expanded HTT and altered gene expression. However, the mechanisms leading to disruption of RNA processing in HD remain unclear. Here, our analysis of the reported HTT protein interactome identifies interactions with known RNA-binding proteins (RBPs). Total, long-read sequencing and targeted RASL-seq of RNAs from cortex and striatum of the HD mouse model R6/2 reveals increased exon skipping which is confirmed in Q150 and Q175 knock-in mice and in HD human brain. We identify the RBP TDP-43 and the N6-methyladenosine (m6A) writer protein methyltransferase 3 (METTL3) to be upstream regulators of exon skipping in HD. Along with this novel mechanistic insight, we observe decreased nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 in HD mice and human brain. In addition, TDP-43 co-localizes with HTT in human HD brain forming novel nuclear aggregate-like bodies distinct from mutant HTT inclusions or previously observed TDP-43 pathologies. Binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a novel mechanism underlying alternative splicing/unannotated exon usage in HD and highlights the critical nature of TDP-43 function across multiple neurodegenerative diseases.

APOE4 dysregulates autophagy in cultured cells.

Human APOE4 (apolipoprotein E4 isoform) is a powerful genetic risk factor for late-onset Alzheimer disease (AD). Many groups have investigated the effect of APOE4 on the degradation of amyloid ß (Aß), the main component of plaques found in the brains of AD patients. However, few studies have focused on the degradation of APOE itself. We investigated the lysosomal trafficking of APOE in cells and found that APOE from the post-Golgi compartment is degraded through an autophagic process requiring the lysosomal membrane protein LAMP2A. We found that APOE4 accumulates in enlarged lysosomes, alters autophagic flux, and changes the proteomic contents of lysosomes following internalization. This dysregulated lysosomal trafficking may represent one of the mechanisms that contributes to AD pathogenesis.

Diminished LC3-Associated Phagocytosis by Huntington's Disease Striatal Astrocytes.

BACKGROUND: In recent years the functions of astrocytes have shifted from conventional supportive roles to also include active roles in altering synapses and engulfment of cellular debris. Recent studies have implicated astrocytes in both protective and pathogenic roles impacting Huntington's disease (HD) progression. OBJECTIVE: The goal of this study is to determine if phagocytosis of cellular debris is compromised in HD striatal astrocytes. METHODS: Primary adult astrocytes were derived from two HD mouse models; the fast-progressing R6/2 and slower progressing Q175. With the use of laser nanosurgery, a single astrocyte was lysed within an astrocyte network. The phagocytic response of astrocytes was observed with phase contrast and by fluorescence microscopy for GFP-LC3 transiently transfected cells. RESULTS: Astrocyte phagocytosis was significantly diminished in primary astrocytes, consistent with the progression of HD in R6/2 and Q175 mouse models. This was defined by the number of astrocytes responding via phagocytosis and by the average number of vesicles formed per cell. GFP-LC3 was found to increasingly localize to phagocytic vesicles over a 20-min imaging period, but not in HD mice, suggesting the involvement of LC3 in astrocyte phagocytosis. CONCLUSION: We demonstrate a progressive decrease in LC3-associated phagocytosis in HD mouse striatal astrocytes.

Cooperation of cell adhesion and autophagy in the brain: Functional roles in development and neurodegenerative disease.

Cellular adhesive connections directed by the extracellular matrix (ECM) and maintenance of cellular homeostasis by autophagy are seemingly disparate functions that are molecularly intertwined, each regulating the other. This is an emerging field in the brain where the interplay between adhesion and autophagy functions at the intersection of neuroprotection and neurodegeneration. The ECM and adhesion proteins regulate autophagic responses to direct protein clearance and guide regenerative programs that go awry in brain disorders. Concomitantly, autophagic flux acts to regulate adhesion dynamics to mediate neurite outgrowth and synaptic plasticity with functional disruption contributed by neurodegenerative disease. This review highlights the cooperative exchange between cellular adhesion and autophagy in the brain during health and disease. As the mechanistic alliance between adhesion and autophagy has been leveraged therapeutically for metastatic disease, understanding overlapping molecular functions that direct the interplay between adhesion and autophagy might uncover therapeutic strategies to correct or compensate for neurodegeneration.

Serine residues 13 and 16 are key modulators of mutant huntingtin induced toxicity in Drosophila.

Poly-glutamine expansion near the N-terminus of the huntingtin protein (HTT) is the prime determinant of Huntington's disease (HD) pathology; however, post-translational modifications and protein context are also reported to influence poly-glutamine induced HD toxicity. The impact of phosphorylating serine 13/16 of mutant HTT (mHTT) on HD has been documented in cell culture and murine models. However, endogenous processing of the human protein in mammalian systems complicates the interpretations. Therefore, to study the impact of S13/16 phosphorylation on the subcellular behavior of HTT under a controlled genetic background with minimal proteolytic processing of the human protein, we employed Drosophila as the model system. We ectopically expressed full-length (FL) and exon1 fragment of human HTT with phosphomimetic and resistant mutations at serines 13 and 16 in different neuronal populations. Phosphomimetic mHTT aggravates and the phosphoresistant mutation ameliorates mHTT-induced toxicity in the context of both FL- and exon1- mHTT in Drosophila although in all cases FL appears less toxic than exon1. Our observations strongly indicate that the phosphorylation status of S13/16 can affect HD pathology in Drosophila and these residues can be potential targets for affecting HD pathogenesis.

Phosphorylation of threonine 3: implications for Huntingtin aggregation and neurotoxicity.

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH(2)-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.

Inhibition of specific HDACs and sirtuins suppresses pathogenesis in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is associated with transcriptional dysregulation, and multiple studies with histone deacetylase (HDAC) inhibitors suggest that global approaches for restoring transcriptional balance and appropriate protein acetylation are therapeutically promising. To determine whether more targeted approaches might be effective, we have tested the impact of all the HDACs in Drosophila on Huntingtin (Htt)-induced pathology. Among the zinc-dependent or 'classic' HDACs, we find that neurodegeneration is most sensitive to levels of Rpd3. We also find that among the NAD(+)-dependent class III deacetylases, genetic or pharmacological reduction of either Sir2 or Sirt2 provides neuroprotection to Htt-challenged animals and that even greater neuroprotection is achieved when Rpd3 and Sir2 are simultaneously reduced. Our experiments suggest that longevity promoting strategies may be distinct from those that protect against neurodegeneration in Drosophila challenged with mutant human Htt. These results highlight a novel therapeutic approach for HD in the form of Sir2 inhibition and possible combinatorial inhibition of Sir2 and Rpd3.

Gene expression and functional deficits underlie TREM2-knockout microglia responses in human models of Alzheimer's disease.

The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.

Nicotinamide restores cognition in Alzheimer's disease transgenic mice via a mechanism involving sirtuin inhibition and selective reduction of Thr231-phosphotau.

Memory loss is the signature feature of Alzheimer's disease, and therapies that prevent or delay its onset are urgently needed. Effective preventive strategies likely offer the greatest and most widespread benefits. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance memory and synaptic plasticity. We evaluated the efficacy of nicotinamide, a competitive inhibitor of the sirtuins or class III NAD(+)-dependent HDACs in 3xTg-AD mice, and found that it restored cognitive deficits associated with pathology. Nicotinamide selectively reduces a specific phospho-species of tau (Thr231) that is associated with microtubule depolymerization, in a manner similar to inhibition of SirT1. Nicotinamide also dramatically increased acetylated alpha-tubulin, a primary substrate of SirT2, and MAP2c, both of which are linked to increased microtubule stability. Reduced phosphoThr231-tau was related to a reduction of monoubiquitin-conjugated tau, suggesting that this posttranslationally modified form of tau may be rapidly degraded. Overexpression of a Thr231-phospho-mimic tau in vitro increased clearance and decreased accumulation of tau compared with wild-type tau. These preclinical findings suggest that oral nicotinamide may represent a safe treatment for AD and other tauopathies, and that phosphorylation of tau at Thr231 may regulate tau stability.

Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice.

BACKGROUND: Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions. OBJECTIVE: Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation. METHODS: Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers. RESULTS: Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods. CONCLUSIONS: Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.

Striatal Mutant Huntingtin Protein Levels Decline with Age in Homozygous Huntington's Disease Knock-In Mouse Models.

BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disorder associated with aging, caused by an expanded polyglutamine (polyQ) repeat within the Huntingtin (HTT) protein. In HD, degeneration of the striatum and atrophy of the cortex are observed while cerebellum is less affected. OBJECTIVE: To test the hypothesis that HTT protein levels decline with age, which together with HTT mutation could influence disease progression. METHODS: Using whole brain cell lysates, a unique method of SDS-PAGE and western analysis was used to quantitate HTT protein, which resolves as a monomer and as a high molecular weight species that is modulated by the presence of transglutaminase 2. HTT levels were measured in striatum, cortex and cerebellum in congenic homozygous Q140 and HdhQ150 knock-in mice and WT littermate controls. RESULTS: Mutant HTT in both homozygous knock-in HD mouse models and WT HTT in control striatal and cortical tissues significantly declined in a progressive manner over time. Levels of mutant HTT in HD cerebellum remained high during aging. CONCLUSIONS: A general decline in mutant HTT levels in striatum and cortex is observed that may contribute to disease progression in homozygous knock-in HD mouse models through reduction of HTT function. In cerebellum, sustained levels of mutant HTT with aging may be protective to this tissue which is less overtly affected in HD.