Huntingtin-associated Protein Research Articles

Dysregulation of Neuronal Calcium Signaling via Store-Operated Channels in Huntington's Disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder that is characterized by motor, cognitive, and psychiatric problems. It is caused by a polyglutamine expansion in the huntingtin protein that leads to striatal degeneration via the transcriptional dysregulation of several genes, including genes that are involved in the calcium (Ca2+) signalosome. Recent research has shown that one of the major Ca2+ signaling pathways, store-operated Ca2+ entry (SOCE), is significantly elevated in HD. SOCE refers to Ca2+ flow into cells in response to the depletion of endoplasmic reticulum Ca2+ stores. The dysregulation of Ca2+ homeostasis is postulated to be a cause of HD progression because the SOCE pathway is indirectly and abnormally activated by mutant huntingtin (HTT) in γ-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs) from the striatum in HD models before the first symptoms of the disease appear. The present review summarizes recent studies that revealed a relationship between HD pathology and elevations of SOCE in different models of HD, including YAC128 mice (a transgenic model of HD), cellular HD models, and induced pluripotent stem cell (iPSC)-based GABAergic medium spiny neurons (MSNs) that are obtained from adult HD patient fibroblasts. SOCE in MSNs was shown to be mediated by currents through at least two different channel groups, Ca2+ release-activated Ca2+ current (ICRAC) and store-operated Ca2+ current (ISOC), which are composed of stromal interaction molecule (STIM) proteins and Orai or transient receptor potential channel (TRPC) channels. Their role under physiological and pathological conditions in HD are discussed. The role of Huntingtin-associated protein 1 isoform A in elevations of SOCE in HD MSNs and potential compounds that may stabilize elevations of SOCE in HD are also summarized. Evidence is presented that shows that the dysregulation of molecular components of SOCE or pathways upstream of SOCE in HD MSN neurons is a hallmark of HD, and these changes could lead to HD pathology, making them potential therapeutic targets.

Brain-derived neurotrophic factor as a potential therapeutic tool in the treatment of nervous system disorders

Brain-derived neurotrophic factor (BDNF) plays an important role in the proper functioning of the nervous system. It regulates the growth and survival of nerve cells, and is crucial in processes related to the memory, learning and synaptic plasticity. Abnormalities related to the distribution and secretion of BDNF protein accompany many diseases of the nervous system, in the course of which a significant decrease in BDNF level in the brain is observed. Impairments of BDNF transport may occur, for example, in the event of a single nucleotide polymorphism in the Bdnf (Val66Met) coding gene or due to the dysfunctions of the proteins involved in intracellular transport, such as huntingtin (HTT), huntingtin-associated protein 1 (HAP1), carboxypeptidase E (CPE) or sortilin 1 (SORT1). One of the therapeutic goals in the treatment of diseases of the central nervous system may be the regulation of expression and secretion of BDNF protein by nerve cells. Potential therapeutic strategies are based on direct injection of the protein into the specific region of the brain, the use of viral vectors expressing the Bdnf gene, transplantation of BDNF-producing cells, the use of substances of natural origin that stimulate the cells of the central nervous system for BDNF production, or the use of molecules activating the main receptor for BDNF – tyrosine receptor kinase B (TrkB). In addition, an appropriate lifestyle that promotes physical activity helps to increase BDNF level in the body. This paper summarizes the current knowledge about the biological role of BDNF protein and proteins involved in intracellular transport of this neurotrophin. Moreover, it presents contemporary research trends to develop therapeutic methods, leading to an increase in the level of BDNF protein in the brain.

The evolution of the huntingtin-associated protein 40 (HAP40) in conjunction with huntingtin

BackgroundThe huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington’s disease (HD). Therefore, we analysed the evolution of HAP40 and its interaction with HTT.ResultsWe found that in amniotes HAP40 is encoded by a single-exon gene, whereas in all other organisms it is expressed from multi-exon genes. HAP40 co-occurs with HTT in unikonts, including filastereans such as Capsaspora owczarzaki and the amoebozoan Dictyostelium discoideum, but both proteins are absent from fungi. Outside unikonts, a few species, such as the free-living amoeboflagellate Naegleria gruberi, contain putative HTT and HAP40 orthologs.Biochemically we show that the interaction between HTT and HAP40 extends to fish, and bioinformatic analyses provide evidence for evolutionary conservation of this interaction. The closest homologue of HAP40 in current protein databases is the family of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs).ConclusionOur results indicate that the transition from a multi-exon to a single-exon gene appears to have taken place by retroposition during the divergence of amphibians and amniotes, followed by the loss of the parental multi-exon gene. Furthermore, it appears that the two proteins probably originated at the root of eukaryotes. Conservation of the interaction between HAP40 and HTT and their likely coevolution strongly indicate functional importance of this interaction.

Immunohistochemical relationships of huntingtin-associated protein 1 with enteroendocrine cells in the pyloric mucosa of the rat stomach

Huntingtin-associated protein 1 (HAP1) is a neuronal cytoplasmic protein that is predominantly expressed in the brain and spinal cord. In addition to the central nervous system, HAP1 is also expressed in the peripheral organs including endocrine system. Different types of enteroendocrine cells (EEC) are present in the digestive organs. To date, the characterization of HAP1-immunoreactive (ir) cells remains unreported there. In the present study, the expression of HAP1 in pyloric stomach in adult male rats and its relationships with different chemical markers for EEC [gastrin, marker of gastrin (G) cells; somatostatin, marker of delta (D) cells; 5-HT, marker of enterochromaffin (EC) cells; histamine, marker of enterochromaffin-like (ECL) cells] were examined employing single- or double-labelled immunohistochemistry and with light-, fluorescence- or electron-microscopy. HAP1-ir cells were abundantly expressed in the glandular mucosa but were very few or none in the surface epithelium. Double-labelled immunofluorescence staining for HAP1 and markers for EECs showed that almost all the G-cells expressed HAP1. In contrast, HAP1 was completely lacking in D-cells, EC-cells or ECL-cells. Our current study is the first to clarify that HAP1 is selectively expressed in G-cells in rat pyloric stomach, which probably reflects HAP1’s involvement in regulation of the secretion of gastrin.

Expression of huntingtin-associated protein 1 in adult mouse dorsal root ganglia and its neurochemical characterization in reference to sensory neuron subpopulations

Huntingtin-associated protein 1 (HAP1) is a polyglutamine (polyQ) length-dependent interactor with causal agents in several neurodegenerative diseases and has been regarded as a protective factor against neurodegeneration. In normal rodent brain and spinal cord, HAP1 is abundantly expressed in the areas that are spared from neurodegeneration while those areas with little HAP1 are frequent targets of neurodegeneration. We have recently showed that HAP1 is highly expressed in the spinal dorsal horn and may participate in modification/protection of certain sensory functions. Neurons in the dorsal root ganglia (DRG) transmits sensory stimuli from periphery to spinal cord/brain stem. Nevertheless, to date HAP1 expression in DRG remains unreported. In this study, the expression of HAP1 in cervical, thoracic, lumbar and sacral DRG in adult male mice and its relationships with different chemical markers for sensory neurons were examined using Western blot and immunohistochemistry. HAP1-immunoreactivity was detected in the cytoplasm of DRG neurons, and the percentage of HAP1-immunoreactive (ir) DRG neurons was ranged between 28–31 %. HAP1-immunoreactivity was comparatively more in the small cells (47–58 %) and medium cells (40–44 %) than that in the large cells (9–11 %). Double-immunostaining for HAP1 and markers for nociceptive or mechanoreceptive neurons showed that about 70–80 % of CGRP-, SP-, CB-, NOS-, TRPV1-, CR- and PV-ir neurons expressed HAP1. In contrast, HAP1 was completely lacking in TH-ir neurons. Our current study is the first to clarify that HAP1 is highly expressed in nociceptive/proprioceptive neurons but absent in light-touch-sensitive TH neurons, suggesting the potential importance of HAP1 in pain transduction and proprioception.

Huntingtin-associated protein 1 plays an essential role in the pathogenesis of type 2 diabetes by regulating the translocation of GLUT4 in mouse adipocytes

ObjectiveGlucose disposal by insulin-responsive tissues maintains the body glucose homeostasis and insulin resistance leads to a risk of developing type 2 diabetes (T2DM). Insulin stimulates the translocation of glucose transporter...

Loss of Hap1 selectively promotes striatal degeneration in Huntington disease mice

Huntington disease (HD) is an ideal model for investigating selective neurodegeneration, as expanded polyQ repeats in the ubiquitously expressed huntingtin (HTT) cause the preferential neurodegeneration in the striatum of the HD patient brains. Here we report that adeno-associated virus (AAV) transduction-mediated depletion of Hap1, the first identified huntingtin-associated protein, in adult HD knock-in (KI) mouse brains leads to selective neuronal loss in the striatum. Further, Hap1 depletion-mediated neuronal loss via AAV transduction requires the presence of mutant HTT. Rhes, a GTPase that is enriched in the striatum and sumoylates mutant HTT to mediate neurotoxicity, binds more N-terminal HTT when Hap1 is deficient. Consistently, more soluble and sumoylated N-terminal HTT is presented in HD KI mouse striatum when HAP1 is absent. Our findings suggest that both Rhes and Hap1 as well as cellular stress contribute to the preferential neurodegeneration in HD, highlighting the involvement of multiple factors in selective neurodegeneration.

Huntingtin-Associated Protein 1 in Mouse Hypothalamus Stabilizes Glucocorticoid Receptor in Stress Response.

Huntingtin-associated protein 1 (Hap1) was initially identified as a brain-enriched protein that binds to the Huntington’s disease protein, huntingtin. Unlike huntingtin that is ubiquitously expressed in the brain, Hap1 is enriched in the brain with the highest expression level in the hypothalamus. The selective enrichment of Hap1 in the hypothalamus suggests that Hap1 may play a specific role in hypothalamic function that can regulate metabolism and stress response. Here we report that Hap1 is colocalized and interacts with the glucocorticoid receptor (GR) in mouse hypothalamic neurons. Genetic depletion of Hap1 reduced the expression level of GR in the hypothalamus. Dexamethasone, a GR agonist, treatment or fasting of mice induced stress, resulting in increased expression of Hap1 in the hypothalamus. However, when Hap1 was absent, these treatments promoted GR reduction in the hypothalamus. In cultured cells, loss of Hap1 shortened the half-life of GR. These findings suggest that Hap1 stabilizes GR in the cytoplasm and that Hap1 dysfunction or deficiency may alter animal’s stress response.

HAP1 Modulates Epileptic Seizures by Regulating GABAAR Function in Patients with Temporal Lobe Epilepsy and in the PTZ-Induced Epileptic Model.

The number of γ-aminobutyric acid type A receptors (GABAARs) expressed on the surface membrane and at synaptic sites is implicated in the enhanced excitation of neuronal circuits and abnormal network oscillations in epilepsy. Huntingtin-associated protein 1 (HAP1), a key mediator of pathological alterations in protein trafficking, directly interacts with GABAARs and facilitates their recycling back to synapses after internalization from the surface; thus, HAP1 regulates the strength of inhibitory synaptic transmission. Here, we show that HAP1 modulates epileptic seizures by regulating GABAAR function in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We demonstrate that GABAARβ2/3 and HAP1 expression are decreased and that the HAP1-GABAARβ2/3 complex is disrupted in the epileptic rat brain. We found that HAP1 upregulation exerts antiepileptic activity in the PTZ-induced seizure and that these changes are associated with increased surface GABAARβ2/3 expression and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). This study provides evidence that hippocampal HAP1 is linked to GABAARs in evoking seizures and suggests that this mechanism is involved in epileptic seizures in the brain, representing a potential therapeutic target for epilepsy.

Alzheimer's Disease and Diabetes: Insulin Signaling as the Bridge Linking Two Pathologies.

Alzheimer's (or Alzheimer) disease (AD) is the most prevalent subset of dementia, affecting elderly populations worldwide. The cumulative costs of the AD care are rapidly accelerating as the average lifespan increases. Onset and risk factors for AD and AD-like dementias have been largely unknown until recently. Studies show that chronic type II diabetes mellitus (DM) is closely associated with neurodegeneration, especially AD. Type II DM is characterized by the cells' inability to take up insulin, as well as chronic hyperglycemia. In the central nervous system, insulin has crucial regulatory roles, while chronic hyperglycemia leads to formation and accumulation of advanced glycation end products (AGEs). AGEs are the major contributor to insulin resistance in diabetic cells, due to their regulatory role on sirtuin expression. Insulin activity in the central nervous system is known to interact with key proteins affected in neurodegenerative conditions, such as amyloid-β precursor protein (AβPP or APP), huntingtin-associated protein-1 (HAP1), Abelson helper integration site-1 (AHI1 or Jouberin), kinesin, and tau. Sirtuins have been theorized to be the mechanism for insulin resistance, and have been found to be affected in neurodegenerative conditions as well. There are hints that all these neuronal proteins may be closely related, although the mechanisms remain unclear. This review will gather existing research on these proteins and highlight the link between neurodegenerative conditions and diabetes mellitus.

HAP1 loss confers l-asparaginase resistance in ALL by downregulating the calpain-1-Bid-caspase-3/12 pathway

Abstractl-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3. Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase–induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.

Expression of AHI1 Rescues Amyloidogenic Pathology in Alzheimer's Disease Model Cells.

A hallmark of Alzheimer's disease (AD) pathogenesis is the accumulation of extracellular plaques mainly composed of amyloid-β (Aβ) derived from amyloid precursor protein (APP) cleavage. Recent reports suggest that transport of APP in vesicles with huntingtin-associated protein-1 (HAP1) negatively regulates Aβ production. In neurons, HAP1 forms a stable complex with Abelson helper integration site-1 (AHI1), in which mutations cause neurodevelopmental and psychiatric disorders. HAP1 and AHI1 interact with tropomyosin receptor kinases (Trks), which are also associated with APP and mediate neurotrophic signaling. In this study, we hypothesize that AHI1 participates in APP trafficking and processing to rescue AD pathology. Indeed, AHI1 was significantly reduced in mouse neuroblastoma N2a cells expressing human Swedish and Indiana APP (designed as AD model cells) and in 3xTg-AD mouse brain. The AD model cells as well as Ahi1-knockdown cells expressing wild-type APP-695 exhibited a significant reduction in viability. In addition, the AD model cells were reduced in neurite outgrowth. APP C-terminal fragment-β (CTFβ) and Aβ42 were increased in the AD cell lysates and the culture media, respectively. To investigate the mechanism how AHI1 alters APP activities, we overexpressed human AHI1 in the AD model cells. The results showed that AHI1 interacted with APP physically in mouse brain and transfected N2a cells despite APP genotypes. AHI1 expression facilitated intracellular translocation of APP and inhibited APP amyloidogenic process to reduce the level of APP-CTFβ in the total lysates of AD model cells as well as Aβ in the culture media. Consequently, AHI1-APP interactions enhanced neurotrophic signaling through Erk activation and led to restored cell survival and differentiation.

Chronic lead exposure decreases the expression of Huntingtin-associated protein 1 (HAP1) through Repressor element-1 silencing transcription (REST)

Chronic lead (Pb) exposure has been shown to reduce the expression of some synaptic proteins which are involved in vesicular trafficking and affect presynaptic neurotransmitter release. However, the precise mechanisms by Pb impairs neurotransmitter release are still not well defined. In the current study, we aimed to elucidate the changes of Huntingtin-associated protein 1 (HAP1) in Pb exposed rats and PC12 cells models and its molecular mechanism. Repressor element-1 silencing transcription (REST) modulates the expression of genes containing the repressor element 1 (RE-1) cis-regulatory DNA sequence. HAP1 promoter region contains a RE-1 binding motif. We also observed whether Pb exposure regulated the HAP1 transcription level through influencing the expression of REST. Mother rats were exposed to 0.5 and 2 g/L Pb acetate (PbAc) in drinking water from the first day of gestation until postnatal 21 days, then the offspring rats were continued to drink PbAc for 1 year, while the control groups received drinking water. PC12 cells were divided into 3 groups: 0 μM, 1 μM and 100 μM PbAc. The results revealed that Pb levels in blood and brain of Pb exposed groups were significantly higher than that of the control group. The ability of learning and memory in Pb exposed rats was decreased. Pb exposure reduced the expression of HAP1 and increased the REST expression. Silencing REST could reverse the decreasing of HAP1 in Pb exposed PC12 cells. Our findings raise a possibility that the decreasing of HAP1 expression by Pb exposure may affect neurotransmitter release and results in impairments in spatial learning and memory ability.

Evaluating the effect of BDNF Val66Met polymorphism on complex formation with HAP1 and Sortilin1 via structural modeling

Brain derived neurotrophic factor (BDNF) has a critical role in the neurogenesis, differentiation, survival of the neurons, regulation of the appetite, and energy homeostasis. Two key proteins, Huntingtin associated protein-1 (HAP1) and sortilin1, regulate the intracellular trafficking and stabilization of the precursor proBDNF through interaction with its prodomain region and mark it for secretion. Evidence suggests that the most frequent single nucleotide polymorphism (SNP) of BDNF gene (rs6265) has been associated with different psychiatric, neurodegenerative and eating disorders. In this study, structural bioinformatics and molecular dynamics (MD) simulations were applied, in order to get precise insights into the impact of Val66Met polymorphism on the proBDNF structure and its interaction with HAP1 and Sortilin1. Homology modeling, structure validation, refinement and also protein-protein docking were performed using appropriate servers. The stability, the fluctuations and the compactness of protein complexes were measured by MD simulation parameters including root mean square deviation (RMSD), root mean square fluctuation (RMSF) and Radius of gyration (Rg), respectively. The mutant proBDNF complexes with HAP1 and Sortilin1 revealed higher RMSD and RMSF values and also variable Rg over time compared with wild-type proBDNF. These computational results indicated that, wild-type proBDNF possessed more stable structure in binding with HAP1 and Sortilin1 compared with its mutant form. Therefore, Val66Met SNP could be deleterious due to making structural changes. It may cause a decrease in proBDNF secretion, which could possibly lead to different psychiatric, neurodegenerative and eating disorders. Further experimental lab studies are required for a more accurate conclusion.

Expression and Localization of Huntingtin-Associated Protein 1 (HAP1) in the Human Digestive System.

Huntingtin-associated protein 1 (HAP1) is a neuronal protein that is predominantly expressed in neurons in the brain. HAP1 is critical for maintenance of neuronal survival as well as regulation of food intake and body weight in animals. In addition to the critical role of HAP1 in the central nervous system, HAP1 is also found in endocrine cells, raising an interesting issue of whether HAP1 is expressed in the digestive system. To examine the expression and localization of HAP1 in the human gastrointestinal tract and to compare the differences of the HAP1 expression between benign and malignant tissues in the digestive system. We used Western blot and immunohistochemistry to examine the expression and distribution of HAP1 in the human gastrointestinal tract tissues. We observed that the presence of HAP1-positive cells in the gastrointestinal tract was not uniform with immunohistochemistry staining. Western blot revealed that only one isoform (75KD) HAP1 was present in the human gastrointestinal system. Interestingly, the expression of HAP1 was higher in the stomach than other regions of the gastrointestinal tract and was at the lowest level in the intestine. We also found that HAP1 was unlikely altered in benign gastric polyps, but was downregulated in pancreatic cancer. This is the first study showing the differential expression and location of HAP1 in the human digestive system. These findings suggested that HAP1 may have cell-type-dependent function in the gastrointestinal tract and may serve as a diagnostic marker for pancreatic cancer.

Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington's Disease.

Huntington’s disease (HD) is a hereditary neurodegenerative disease that is caused by polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular activities that is dysregulated in HD is store-operated calcium entry (SOCE), a process by which Ca2+ release from the endoplasmic reticulum (ER) induces Ca2+ influx from the extracellular space. HTT-associated protein-1 (HAP1) is a binding partner of HTT. The aim of the present study was to examine the role of HAP1A protein in regulating SOCE in YAC128 mice, a transgenic model of HD. After Ca2+ depletion from the ER by the activation of inositol-(1,4,5)triphosphate receptor type 1 (IP3R1), we detected an increase in the activity of SOC channels when HAP1 protein isoform HAP1A was overexpressed in medium spiny neurons (MSNs) from YAC128 mice. A decrease in the activity of SOC channels in YAC128 MSNs was observed when HAP1 protein was silenced. In YAC128 MSNs that overexpressed HAP1A, an increase in activity of IP3R1 was detected while the ionomycin-sensitive ER Ca2+ pool decreased. 6-Bromo-N-(2-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (C20H22BrClN2), identified in our previous studies as a SOCE inhibitor, restored the elevation of SOCE in YAC128 MSN cultures that overexpressed HAP1A. The IP3 sponge also restored the elevation of SOCE and increased the release of Ca2+ from the ER in YAC128 MSN cultures that overexpressed HAP1A. The overexpression of HAP1A in the human neuroblastoma cell line SK-N-SH (i.e., a cellular model of HD (SK-N-SH HTT138Q)) led to the appearance of a pool of constitutively active SOC channels and an increase in the expression of STIM2 protein. Our results showed that HAP1A causes the activation of SOC channels in HD models by affecting IP3R1 activity.

Distribution of HAP1-immunoreactive Cells in the Retrosplenial–retrohippocampal Area of Adult Rat Brain and Its Application to a Refined Neuroanatomical Understanding of the Region

Huntingtin-associated protein 1 (HAP1) is a neural interactor of huntingtin in Huntington’s disease and interacts with gene products in a number of other neurodegenerative diseases. In normal brains, HAP1 is expressed abundantly in the hypothalamus and limbic-associated regions. These areas tend to be spared from neurodegeneration while those with little HAP1 are frequently neurodegenerative targets, suggesting its role as a protective factor against apoptosis. In light of the relationship between neurodegenerative diseases and deterioration of higher nervous activity, it is important to definitively clarify HAP1 expression in a cognitively important brain region, the retrosplenial–retrohippocampal area. Here, HAP1 expression was evaluated immunohistochemically over the retrosplenial cortex, the subicular complex, and the entorhinal and perirhinal cortices. HAP1-immunoreactive (ir) cells were classified into five discrete groups: (1) a distinct retrosplenial cell cluster exclusive to the superficial layers of the granular cortex, (2) a conspicuous, thin line of cells in layers IV/V of the “subiculum-backing cortex,” (3) a group of highly immunoreactive cells associated with the medial entorhinal–subicular corner, (4) pericallosal cells just below layer VI and adjacent to the white matter, and (5) other sporadic, widely-disseminated HAP1-immunoreactive cells. HAP1 was found to be the first marker for the complex subiculum-backing cortex and a precise marker for several subfields in the retrosplenial–retrohippocampal area, verified through comparative staining with other neurochemicals. HAP1 may play an important role in protecting these cortical structures and functions for higher nervous activity by increasing the threshold to neurodegeneration and decreasing vulnerability to stress or aging.

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1−/− cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions.

The role of the kinesin superfamily proteins in the pathogenesis and progression of multiple diseases

Intracellular transportation serves as the basis of various cell functions. As the molecular motors responsible for the intracellular transportation, kinesin superfamily proteins (KIFs) recognize diverse molecular cargos, such as proteins, organelles, synaptic vesicles, and RNA granules, via their unique binding structures. KIFs have motor domain, which usually convey ATPase activity. KIFs gain energy from ATP hydrolysis, and transport their cargos moving along the microtubules, thus play important role in biological processes of those cargos. KIFs can be divided into 3 classes, according to the motor domain location on the N-terminal, the C-terminal or the middle of the proteins. Meanwhile, some KIFs lack motor function, which can still play important roles by regulating certain molecular pathways, through their binding proteins. Abundant evidence has shown that the KIFs are widely involved in the pathogenesis and progression of many diseases, such as neuronal diseases, metabolic diseases, kidney diseases, etc. In the present review, we summarize recent research findings which explore the relationship between the KIFs and the diseases. Amyloid β peptides (A β ) drives Alzheimer’s disease and is derived from the amyloid precursor protein (APP). APP is transported by binding with KIF5 through c-Jun N-terminal kinase-interacting protein 1 (JIP1) and kinesin light chain (KLC) in the axons. APP can also function as a receptor for the transportation of β -secretase (BACE1) and PS1. Thus, the dysfunction of KIF5 may impair the axonal transport of APP, induce the accumulation of A β , lead to Alzheimer’s disease. Huntingtin mutation can suppress the fast axonal transport by activating axonal c-Jun N-terminal kinase (JNK). Huntingtin-associated protein 1 (HAP1) is enriched in the neurons, which directly binds with KLC in vitro , and colocalizes with KLC in growth cones of the neurons. Knocking down HAP1 suppresses neurite outgrowth of PC12 cells, and inhibits the kinesin-dependent transport of APP vesicles. This may serve as a pathological mechanism of Huntington’s disease. KIF12 forms a complex with Hsc70 and Sp1, and maintains the proper expression of Hsc70 through a transcriptional mechanism in pancreatic beta cells. Defect in KIF12, either by knockout or high fat diet intake, can reduce the expression of Hsc70, and impair the peroxisomal targeting of anti-oxidative enzymes, lead to the increase of intracellular oxidative stress. Abnormal accumulation of oxidative stress eventually impairs the insulin secretion of the beta cells, and facilitates diabetes progression. In addition, KIF5B knockout mice shows glucose tolerance and hyperglycemia, which is caused by impaired insulin secretion. Mice, with tissue-specific knockout of KIF3A in renal tubular epithelial cells, begin to develop cysts in the kidney at postnatal day 5 and show renal failure by postnatal day 21, suggesting the association of KIF3A with polycystic kidney disease. Human kidney anion exchanger 1 (kAE1) binds with KIF3B through its C-terminal dileucine motif, and co-expresses with KIF3B in human kidney. Suppressing KIF3B in HEK293T cells results in the reduction of kAE1 on the cell membrane and the accumulation of kAE1 in cytoplasm. These findings suggest that KIF3B may be responsible for the transportation of kAE1 towards the membrane, thus involve in the pathogenesis of distal renal tubular acidosis. It is well accepted that KIFs serve as one of the bases for the intracellular transportation. Although they have been widely studied, their functions are still largely unknown. Usually multiple KIFs are expressed in one cell. Their functions may be distinct or overlapping with each other. Therefore, double, even triple, knockouts of these KIFs in specific tissues may help us to further explore their biological functions. In addition, next generation sequencing is highly developed in recent years. It must be informative using this novel technique to analyze the kinesin knockout mice for identifying more cargos.

DYRK1A regulates Hap1–Dcaf7/WDR68 binding with implication for delayed growth in Down syndrome

Huntingtin-associated protein 1 (Hap1) is known to be critical for postnatal hypothalamic function and growth. Hap1 forms stigmoid bodies (SBs), unique neuronal cytoplasmic inclusions of unknown function that are enriched in hypothalamic neurons. Here we developed a simple strategy to isolate the SB-enriched fraction from mouse brain. By analyzing Hap1 immunoprecipitants from this fraction, we identified a Hap1-interacting SB component, DDB1 and CUL4 associated factor 7 (Dcaf7)/WD40 repeat 68 (WDR68), whose protein level and nuclear translocation are regulated by Hap1. Moreover, we found that Hap1 bound Dcaf7 competitively in cytoplasm with dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), a protein implicated in Down syndrome (DS). Depleting Hap1 promoted the DYRK1A-Dcaf7 interaction and increased the DYRK1A protein level. Transgenic DS mice overexpressing DYRK1A showed reduced Hap1-Dcaf7 association in the hypothalamus. Furthermore, the overexpression of DYRK1A in the hypothalamus led to delayed growth in postnatal mice, suggesting that DYRK1A regulates the Hap1-Dcaf7 interaction and postnatal growth and that targeting Hap1 or Dcaf7 could ameliorate growth retardation in DS.