Rare GPR37L1 Variants Reveal Potential Association between GPR37L1 and Disorders of Anxiety and Migraine.

GPR37L1 is an orphan receptor that couples through heterotrimeric G-proteins to regulate physiological functions. Since its role in humans is not fully defined, we used an unbiased computational approach to assess the clinical significance of rare G-protein-coupled receptor 37-like 1 (GPR37L1) genetic variants found among 51,289 whole-exome sequences from the DiscovEHR cohort. Rare GPR37L1 coding variants were binned according to predicted pathogenicity and analyzed by sequence kernel association testing to reveal significant associations with disease diagnostic codes for epilepsy and migraine, among others. Since associations do not prove causality, rare GPR37L1 variants were functionally analyzed in SK-N-MC cells to evaluate potential signaling differences and pathogenicity. Notably, receptor variants exhibited varying abilities to reduce cAMP levels, activate mitogen-activated protein kinase (MAPK) signaling, and/or upregulate receptor expression in response to the agonist prosaptide (TX14(A)), as compared with the wild-type receptor. In addition to signaling changes, knock-out (KO) of GPR37L1 or expression of certain rare variants altered cellular cholesterol levels, which were also acutely regulated by administration of the agonist TX14(A) via activation of the MAPK pathway. Finally, to simulate the impact of rare nonsense variants found in the large patient cohort, a KO mouse line lacking Gpr37l1 was generated. Although KO animals did not recapitulate an acute migraine phenotype, the loss of this receptor produced sex-specific changes in anxiety-related disorders often seen in chronic migraineurs. Collectively, these observations define the existence of rare GPR37L1 variants associated with neuropsychiatric conditions in the human population and identify the signaling changes contributing to pathological processes.

Elevated SLC7A2 expression is associated with an abnormal neuroinflammatory response and nitrosative stress in Huntington's disease.

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for L-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients.

Characterization of huntingtin interactomes and their dynamic responses in living cells by proximity proteomics.

Huntingtin (Htt) is a large protein without clearly defined molecular functions. Mutation in this protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Identification of Htt-interacting proteins by the traditional approaches including yeast two-hybrid systems and affinity purifications has greatly facilitated the understanding of Htt function. However, these methods eliminated the intracellular spatial information of the Htt interactome during sample preparations. Moreover, the temporal changes of the Htt interactome in response to acute cellular stresses cannot be easily resolved with these approaches. Ascorbate peroxidase (APEX2)-based proximity labeling has been used to spatiotemporally investigate protein-protein interactions in living cells. In this study, we generated stable human SH-SY5Y cell lines expressing full-length Htt23Q and Htt145Q with N-terminus tagged Flag-APEX2 to quantitatively map the spatiotemporal changes of Htt interactome to a mild acute proteotoxic stress. Our data revealed that normal and mutant Htt (muHtt) are associated with distinct intracellular microenvironments. Specifically, mutant Htt is preferentially associated with intermediate filaments and myosin complexes. Furthermore, the dynamic changes of Htt interactomes in response to stress are different between normal and mutant Htt. Vimentin is identified as one of the most significant proteins that preferentially interacts with muHtt in situ. Further functional studies demonstrated that mutant Htt affects the vimentin's function of regulating proteostasis in healthy and HD human neural stem cells. Taken together, our data offer important insights into the molecular functions of normal and mutant Htt by providing a list of Htt-interacting proteins in their natural cellular context for further studies in different HD models.

Impaired Restoration of Global Protein Synthesis Contributes to Increased Vulnerability to Acute ER Stress Recovery in Huntington's Disease.

Neurons are susceptible to different cellular stresses and this vulnerability has been implicated in the pathogenesis of Huntington's disease (HD). Accumulating evidence suggest that acute or chronic stress, depending on its duration and severity, can cause irreversible cellular damages to HD neurons, which contributes to neurodegeneration. In contrast, how normal and HD neurons respond during the resolution of a cellular stress remain less explored. In this study, we challenged normal and HD cells with a low-level acute ER stress and examined the molecular and cellular responses after stress removal. Using both striatal cell lines and primary neurons, we first showed the temporal activation of p-eIF2α-ATF4-GADD34 pathway in response to the acute ER stress and during recovery between normal and HD cells. HD cells were more vulnerable to cell death during stress recovery and were associated with increased number of apoptotic/necrotic cells and decreased cell proliferation. This is also supported by the Gene Ontology analysis from the RNA-seq data which indicated that "apoptosis-related Biological Processes" were more enriched in HD cells during stress recovery. We further showed that HD cells were defective in restoring global protein synthesis during stress recovery and promoting protein synthesis by an integrated stress response inhibitor, ISRIB, could attenuate cell death in HD cells. Together, these data suggest that normal and HD cells undergo distinct mechanisms of transcriptional reprogramming, leading to different cell fate decisions during the stress recovery.

An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells.

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.

Dietary Chitin Particles Called Mimetic Fungi Ameliorate Colitis in Toll-Like Receptor 2/CD14- and Sex-Dependent Manners.

Chitin is a natural N-acetylglucosamine polymer and a major structural component of fungal cell walls. Dietary chitin is mucoadhesive; anti-inflammatory effects of chitin microparticles (CMPs; 1- to 10-µm diameters) have been demonstrated in models of inflammatory bowel disease (IBD). The goals of this study were to assess (i) whether CMPs among various chitin preparations are the most effective against colitis in male and female mice and (ii) whether host chitin-binding Toll-like receptor 2 (TLR2) and CD14 are required for the anti-inflammatory effect of chitin. We found that colitis in male mice was ameliorated by CMPs and large chitin beads (LCBs; 40 to 70 µm) but not by chitosan (deacetylated chitin) microparticles, oligosaccharide chitin, or glucosamine. In fact, LCBs were more effective than CMPs. In female colitis, on the other hand, CMPs and LCBs were equally and highly effective. Neither sex of TLR2-deficient mice showed anti-inflammatory effects when treated with LCBs. No anti-inflammatory effect of LCBs was seen in either CD14-deficient males or females. Furthermore, an in vitro study indicated that when LCBs and CMPs were digested with stomach acidic mammalian chitinase (AMC), their size-dependent macrophage activations were modified, at least in part, suggesting reduced particle sizes of dietary chitin in the stomach. Interestingly, stomach AMC activity was greater in males than females. Our results indicated that dietary LCBs were the most effective preparation for treating colitis in both sexes; these anti-inflammatory effects of LCBs were dependent on host TLR2 and CD14.

Aberrant subcellular localization of SQSTM1/p62 contributes to increased vulnerability to proteotoxic stress recovery in Huntington's disease.

Proteotoxic stress plays an important role in the pathogenesis of Huntington's disease (HD). Autophagy is proposed as a compensatory mechanism to remove protein aggregates under proteotoxic stress by up-regulating p62 expression. In the present study, we investigated the molecular action of p62 to proteotoxic stress in HD cells. Using two different HD cellular models, STHdhQ7 and STHdhQ111 cells derived from wild type and HD knock-in mice and human fibroblasts from healthy and HD patients, we found that HD cells are more vulnerable to cell death under proteotoxic stress and during stress recovery. We further showed that P62 was up-regulated in both STHdhQ7 and STHdhQ111 cells in response to the stress with distinct subcellular localization patterns. While dispersed p62 puncti were found in STHdhQ7 cells, p62 bodies were initially present in the lysosomes and accumulated to the juxtanuclear regions of STHdhQ111 cells as MG132 incubation continued. Unlike in STHdhQ7 cells, p62 puncti were not associated with K48-linked polyubiquitinated protein aggregates or proteasomal components in STHdhQ111. Interestingly, addition of cysteine during MG132 incubation rescued cell death in STHdhQ111 cells caused by stress recovery and altered the subcellular distribution of p62. Our data suggest that aberrant positioning of p62 affects the proteasomal clearance of protein aggregates and may contribute to the increased vulnerability to proteotoxic stress-induced cell death in HD cells.

Phagocytosis-mediated M1 activation by chitin but not by chitosan.

Chitin particles have been used to understand host response to chitin-containing pathogens and allergens and are known to induce a wide range of polarized macrophage activations, depending, at least in part, on particle size. Nonphagocytosable particles larger than a macrophage induce tissue repair M2 activation. In contrast, phagocytosable chitin microparticles (CMPs, 1-10 µm diameters) induce M1 macrophages that kill intracellular microbes and damage tissues. However, chitosan (deacetylated) microparticles (de-CMPs, 1-10 µm) induce poor M1 activation. Toll-like receptor 2 (TLR2) and associated coreceptors in macrophages appear to be required for the M1 activation. To understand the exact mechanism of phagocytosis-mediated M1 activation by chitin, we isolated macrophage proteins that bind to CMPs during early phagocytosis and determined that TLR1, TLR2, CD14, late endosomal/lysosomal adaptor MAPK and mechanistic target of rapamycin activator 1 (LAMTOR1), Lck/Yes novel tyrosine kinase (Lyn), and ß-actin formed phagosomal CMP-TLR2 clusters. These proteins were also detected in TLR2 phagosomal clusters in macrophages phagocytosing de-CMPs, but at relatively lower levels than in the CMP-TLR2 clusters. Importantly, CMP-TLR2 clusters further recruited myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1 receptor-containing adaptor protein (TIRAP) and phosphorylated Lyn, whereas neither the adaptors nor phosphorylated Lyn was detected in the de-CMP clusters. The results indicate that the acetyl group played an obligatory, phagocytosis-dependent role in the initiation of an integrated signal for TLR2-mediated M1 activation.

Altered lysosomal positioning affects lysosomal functions in a cellular model of Huntington's disease.

Huntington's disease (HD) is a hereditary and devastating neurodegenerative disorder caused by a mutation in the huntingtin protein. Understanding the functions of normal and mutant huntingtin protein is the key to revealing the pathogenesis of HD and developing therapeutic targets. Huntingtin plays an important role in vesicular and organelle trafficking. Lysosomes are dynamic organelles that integrate several degradative pathways and regulate the activity of mammalian target of rapamycin complex 1 (mTORC1). In the present study, we found that the perinuclear accumulation of lysosomes was increased in a cellular model of HD derived from HD knock-in mice and primary fibroblasts from an HD patient. This perinuclear lysosomal accumulation could be reversed when normal huntingtin was overexpressed in HD cells. When we further investigated the functional significance of the increased perinuclear lysosomal accumulation in HD cells, we demonstrated that basal mTORC1 activity was increased in HD cells. In addition, autophagic influx was also increased in HD cells in response to serum deprivation, which leads to premature fusion of lysosomes with autophagosomes. Taken together, our data suggest that the increased perinuclear accumulation of lysosomes may play an important role in HD pathogenesis by altering lysosomal-dependent functions.

Biohybrid Robotic Hand to Investigate Tactile Encoding and Sensorimotor Integration.

For people who have experienced a spinal cord injury or an amputation, the recovery of sensation and motor control could be incomplete despite noteworthy advances with invasive neural interfaces. Our objective is to explore the feasibility of a novel biohybrid robotic hand model to investigate aspects of tactile sensation and sensorimotor integration with a pre-clinical research platform. Our new biohybrid model couples an artificial hand with biological neural networks (BNN) cultured in a multichannel microelectrode array (MEA). We decoded neural activity to control a finger of the artificial hand that was outfitted with a tactile sensor. The fingertip sensations were encoded into rapidly adapting (RA) or slowly adapting (SA) mechanoreceptor firing patterns that were used to electrically stimulate the BNN. We classified the coherence between afferent and efferent electrodes in the MEA with a convolutional neural network (CNN) using a transfer learning approach. The BNN exhibited the capacity for functional specialization with the RA and SA patterns, represented by significantly different robotic behavior of the biohybrid hand with respect to the tactile encoding method. Furthermore, the CNN was able to distinguish between RA and SA encoding methods with 97.84% ± 0.65% accuracy when the BNN was provided tactile feedback, averaged across three days in vitro (DIV). This novel biohybrid research platform demonstrates that BNNs are sensitive to tactile encoding methods and can integrate robotic tactile sensations with the motor control of an artificial hand. This opens the possibility of using biohybrid research platforms in the future to study aspects of neural interfaces with minimal human risk.

αB-crystallin/sHSP protects cytochrome c and mitochondrial function against oxidative stress in lens and retinal cells.

BACKGROUND: αB-crystallin/sHSP protects cells against oxidative stress damage. Here, we mechanistically examined its ability to preserve mitochondrial function in lens and retinal cells and protect cytochrome c under oxidative stress conditions. METHODS: αB-crystallin/sHSP was localized in human lens (HLE-B3) and retinal (ARPE-19) cells. αB-crystallin/sHSP was stably over-expressed and its ability to preserve mitochondrial membrane potential under oxidative stress conditions was monitored. Interactions between αB-crystallin/sHSP and cytochrome c were examined by fluorescent resonance energy transfer (FRET) and by co-immune precipitation. The ability of αB-crystallin/sHSP to protect cytochrome c against methionine-80 oxidation was monitored. RESULTS: αB-crystallin/sHSP is present in the mitochondria of lens and retinal cells and is translocated to the mitochondria under oxidative conditions. αB-crystallin/sHSP specifically interacts with cytochrome c in vitro and in vivo and its overexpression preserves mitochondrial membrane potential under oxidative stress conditions. αB-crystallin/sHSP directly protects cytochrome c against oxidation. GENERAL SIGNIFICANCE: These data demonstrate that αB-crystallin/sHSP maintains lens and retinal cells under oxidative stress conditions at least in part by preserving mitochondrial function and by protecting cytochrome c against oxidation. Since oxidative stress and loss of mitochondrial function are associated with eye lens cataract and age-related macular degeneration, loss of these αB-crystallin/sHSP functions likely plays a key role in the development of these diseases. αB-crystallin/sHSP is expressed throughout the body and its ability to maintain mitochondrial function is likely important for the prevention of multiple degenerative diseases.

Chaperone-independent mitochondrial translocation and protection by αB-crystallin in RPE cells.

αB-crystallin is a small heat shock protein that exhibits chaperone activity and can protect multiple cell types against oxidative stress damage. Altered levels and specific mutations of αB-crystallin are associated with multiple degenerative diseases. We previously found that αB-crystallin translocates to lens and retinal cell mitochondria upon oxidative stress exposure where it provides protection against oxidative stress damage. To date, the role of the chaperone function of αB-crystallin in mitochondrial translocation and protection has not been established. Here, we sought to determine the relationship between the chaperone activity of αB-crystallin and its ability to translocate to and protect retinal cell mitochondria against oxidative stress damage. Our data provide evidence that three forms of αB-crystallin exhibiting different chaperone activity levels including wild-type, R120G (decreased chaperone activity) and M68A (increased chaperone activity) provide comparable levels of mitochondrial translocation and protection to retinal cells exposed to oxidative stress. The results provide evidence that mitochondrial translocation and protection by αB-crystallin is independent of its chaperone activity and that other functions of αB-crystallin may also be independent of its chaperone activity.

Palmitoylation and trafficking of GAD65 are impaired in a cellular model of Huntington's disease.

HD (Huntington's disease) is caused by an expanded polyQ (polyglutamine) repeat in the htt (huntingtin protein). GABAergic medium spiny neurons in the striatum are mostly affected in HD. However, mhtt (mutant huntingtin)-induced molecular changes in these neurons remain largely unknown. The present study focuses on the effect of mhtt on the subcellular localization of GAD (glutamic acid decarboxylase), the enzyme responsible for synthesizing GABA (γ-aminobutyric acid). We report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus of mhtt or full-length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal htt; however, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of HIP14 (huntingtin-interacting protein 14), the enzyme responsible for palmitoylating GAD65 in vivo, could rescue GAD65 palmitoylation and vesicle-associated GAD65 trafficking. Taken together, our data support the idea that GAD65 palmitoylation is important for the delivery of GAD65 to inhibitory synapses and suggest that impairment of GAD65 palmitoylation by mhtt may lead to altered inhibitory neurotransmission in HD.

Rare GPR37L1 variants reveal potential roles in anxiety and migraine disorders.

GPR37L1 is an orphan receptor that couples through heterotrimeric G-proteins to regulate physiological functions. Since its role in humans is not fully defined, we used an unbiased computational approach to assess the clinical significance of rare GPR37L1 genetic variants found among 51,289 whole exome sequences from the DiscovEHR cohort. Briefly, rare GPR37L1 coding variants were binned according to predicted pathogenicity, and analyzed by Sequence Kernel Association testing to reveal significant associations with disease diagnostic codes for epilepsy and migraine, among others. Since associations do not prove causality, rare GPR37L1 variants were then functionally analyzed in SK-N-MC cells to evaluate potential signaling differences and pathogenicity. Notably, receptor variants exhibited varying abilities to reduce cAMP levels, activate MAPK signaling, and/or upregulate receptor expression in response to the agonist prosaptide (TX14(A)), as compared to the wild-type receptor. In addition to signaling changes, knockout of GPR37L1 or expression of certain rare variants altered cellular cholesterol levels, which were also acutely regulated by administration of the agonist TX14(A) via activation of the MAPK pathway. Finally, to simulate the impact of rare nonsense variants found in the large patient cohort, a knockout (KO) mouse line lacking Gpr37L1 was generated, revealing loss of this receptor produced sex-specific changes implicated in migraine-related disorders. Collectively, these observations define the existence of rare GPR37L1 variants in the human population that are associated with neuropsychiatric conditions and identify the underlying signaling changes that are implicated in the in vivo actions of this receptor in pathological processes leading to anxiety and migraine. SIGNIFICANCE STATEMENT: G-protein coupled receptors (GPCRs) represent a diverse group of membrane receptors that contribute to a wide range of diseases and serve as effective drug targets. However, a number of these receptors have no identified ligands or functions, i.e., orphan receptors. Over the past decade, advances have been made, but there is a need for identifying new strategies to reveal their roles in health and disease. Our results highlight the utility of rare variant analyses of orphan receptors for identifying human disease associations, coupled with functional analyses in relevant cellular and animal systems, to ultimately reveal their roles as novel drug targets for treatment of neurological disorders that lack wide-spread efficacy.

Spatial expression patterns of autophagy genes in the eye lens and induction of autophagy in lens cells.

PURPOSE: Mutation of the autophagy gene FYVE (named after the four cysteine-rich proteins: Fab 1 [yeast orthologue of PIKfyve], YOTB, Vac 1 [vesicle transport protein], and EEA1) and coiled coil containing 1 (fyco1) causes human cataract suggesting a role for autophagy in lens function. Here, we analyzed the range and spatial expression patterns of lens autophagy genes and we evaluated whether autophagy could be induced in lens cells exposed to stress. METHODS: Autophagy gene expression levels and their spatial distribution patterns were evaluated between microdissected human lens epithelium and fibers at the mRNA and protein levels by microarray data analysis, real-time PCR and western blot analysis. Selected autophagy protein spatial expression patterns were also examined in newborn mouse lenses by immunohistochemistry. The autophagosomal content of cultured human lens epithelial cells was determined by counting the number of microtubule-associated protein 1 light chain 3B (LC3B)-positive puncta in cells cultured in the presence or absence of serum. RESULTS: A total of 42 autophagy genes were detected as being expressed by human lens epithelium and fibers. The autophagosomal markers LC3B and FYCO1 were detected throughout the newborn mouse lens. Consistently, the autophagy active form of LC3B (LC3B II) was detected in microdissected human lens fibers. An increased number of LC3B-positive puncta was detected in cultured lens cells upon serum starvation suggesting induction of autophagy in lens cells under stress conditions. CONCLUSIONS: The data provide evidence that autophagy is an important component for the function of lens epithelial and fiber cells. The data are consistent with the notion that disruption of lens autophagy through mutation or inactivation of specific autophagy proteins could lead to loss of lens resistance to stress and/or loss of lens differentiation resulting in cataract formation.

Robotically Embodied Biological Neural Networks to Investigate Haptic Restoration with Neuroprosthetic Hands.

Neuroprosthetic limbs reconnect severed neural pathways for control of (and increasingly sensation from) an artificial limb. However, the plastic interaction between robotic and biological components is poorly understood. To gain such insight, we developed a novel noninvasive neuroprosthetic research platform that enables bidirectional electrical communications (action, sensory perception) between a dexterous artificial hand and neuronal cultures living in a multichannel microelectrode array (MEA) chamber. Artificial tactile sensations from robotic fingertips were encoded to mimic slowly adapting (SA) or rapidly adapting (RA) mechanoreceptors. Afferent spike trains were used to stimulate neurons in a region of the neuronal culture. Electrical activity from neurons at another region in the MEA chamber was used as the motor control signal for the artificial hand. Results from artificial neural networks (ANNs) showed that the haptic model used to encode RA or SA fingertip sensations affected biological neural network (BNN) activity patterns, which in turn impacted the behavior of the artificial hand. That is, the exhibited finger tapping behavior of this closed-loop neurorobotic system showed statistical significance (p<0.01) between the haptic encoding methods across two different neuronal cultures and over multiple days. These findings suggest that our noninvasive neuroprosthetic research platform can be used to devise high-throughput experiments exploring how neural plasticity is affected by the mutual interactions between perception and action.

RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells.

BACKGROUND: Huntingtin (Htt) protein is the product of the gene mutated in Huntington's disease (HD), a fatal, autosomal dominant, neurodegenerative disorder. Normal Htt is essential for early embryogenesis and the development of the central nervous system. However, the role of Htt in adult tissues is less defined. Following the recent promising clinical trial in which both normal and mutant Htt mRNA were knocked down in HD patients, there is an urgent need to fully understand the molecular consequences of knocking out/down Htt in adult tissues. Htt has been identified as an important transcriptional regulator. Unbiased investigations of transcriptome changes with RNA-sequencing (RNA-Seq) have been done in multiple cell types in HD, further confirming that transcriptional dysregulation is a central pathogenic mechanism in HD. However, there is lack of direct understanding of the transcriptional regulation by normal Htt. METHODS: To investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on Htt-null and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion. RESULTS: In general, Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling. We further examined the transcription factors that may regulate these DEGs. Consistent with the disrupted pathways associated with cellular development, we showed that Htt-null cells exhibited slower cell proliferation than wild type cells. We finally validated some of the top DEGS with quantitative RT-PCR. CONCLUSIONS: The widespread transcriptome changes in Htt-null cells could be directly caused by the loss of Htt-mediated transcriptional regulation or due to the secondary consequences of disruption in the gene regulatory network. Our study therefore provides valuable information about key genes associated with Htt-mediated transcription and improves our understanding of the molecular mechanisms underlying the cellular functions of normal and mutant Htt.

BimEL as a possible molecular link between proteasome dysfunction and cell death induced by mutant huntingtin.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the N-terminus of the huntingtin protein. It is characterized by a selective loss of medium spiny neurons in the striatum. It has been suggested that impaired proteasome function and endoplasmic reticulum (ER) stress play important roles in mutant huntingtin (mHtt)-induced cell death. However, the molecular link involved is poorly understood. In the present study, we identified the essential role of the extra long form of Bim (Bcl-2 interacting mediator of cell death), BimEL, in mHtt-induced cell death. BimEL protein expression level was significantly increased in cell lines expressing the N-terminus of mHtt and in a mouse model of HD. Although quantitative RT-PCR analysis indicated that BimEL mRNA was increased in cells expressing mHtt, we provided evidence showing that, at the post-translational level, phosphorylation of BimEL played a more important role in regulating BimEL expression. Up-regulation of BimEL facilitated the translocation of Bax to the mitochondrial membrane, which further led to cytochrome c release and cell death. On the other hand, knocking down BimEL expression prevented mHtt-induced cell death. Taken together, these findings suggest that BimEL is a key element in regulating mHtt-induced cell death. A model depicting the role of BimEL in linking mHtt-induced ER stress and proteasome dysfunction to cell death is proposed.

Protective function of taurine in glutamate-induced apoptosis in cultured neurons.

Previously, we showed that taurine protects neurons against glutamate-induced excitotoxicity by inhibiting the glutamate-induced increase of [Ca2+](i). In this study, we report that taurine prevents glutamate-induced chromosomal condensation, indicating that taurine inhibits glutamate-induced apoptosis. We found that Bcl-2 was down-regulated while Bax was up-regulated by glutamate treatment, and these changes were prevented in the presence of taurine. We have also shown that taurine inhibits glutamate-induced activation of calpain. Furthermore, calpastatin, a specific calpain inhibitor, also prevented glutamate-induced cell death. Here we propose the mechanisms underlying glutamate-induced apoptosis and taurine's inhibition of glutamate-induced apoptosis to be as follows: glutamate stimulation induces [Ca2+](i) elevation, which in turn activates calpain; activation of calpain leads to a reduction of Bcl-2:Bax ratios; with decreased Bcl-2:Bax ratios Bax homodimers form, Bax homodimerization, and translocation to the mitochondria result in the release of cytochrome c; released cytochrome c in turn activates a downstream caspase cascade leading to apoptosis. The antiapoptotic function of taurine is due to its inhibition of glutamate-induced membrane depolarization.

Mechanism of neuroprotective function of taurine.

Taurine has potent protective function against glutamate-induced neuronal injury presumably through its function in regulation of intracellular free calcium level, [Ca2+]i. In this communication, we report that taurine exerts its protective function through one or more of the following mechanisms: 1. Inhibition of glutamate-induced calcium influx through L-, N- and P/Q-type voltage-gated calcium channels and NMDA receptor calcium channel; 2. Attenuation of glutamate-induced membrane depolarization; 3. Prevention of glutamate-induced apoptosis via preventing glutamate-mediated down-regulation of Bcl-2; 4. Prevention of cleavage of Bcl-2 by calpain. This action of taurine is due to its inhibition on glutamate induced calpain activation. Based on these observations, we propose that taurine protects neurons against glutamate-induced neurotoxicity in part, by preventing glutamate-induced membrane depolarization, elevation of [Ca2+]i, activation of calpain, reduction of Bcl-2 and apoptosis.