Full-length Huntingtin Research Articles

Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form

Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington’s disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.

Extensive Expression Analysis of Htt Transcripts in Brain Regions from the zQ175 HD Mouse Model Using a QuantiGene Multiplex Assay

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of the huntingtin (HTT) gene. HTT mRNA contains 67 exons and does not always splice between exon 1 and exon 2 leading to the production of a small polyadenylated HTTexon1 transcript, and the full-length HTT mRNA has three 3′UTR isoforms. We have developed a QuantiGene multiplex panel for the simultaneous detection of all of these mouse Htt transcripts directly from tissue lysates and demonstrate that this can replace the more work-intensive Taqman qPCR assays. We have applied this to the analysis of brain regions from the zQ175 HD mouse model and wild type littermates at two months of age. We show that the incomplete splicing of Htt occurs throughout the brain and confirm that this originates from the mutant and not endogenous Htt allele. Given that HTTexon1 encodes the highly pathogenic exon 1 HTT protein, it is essential that the levels of all Htt transcripts can be monitored when evaluating HTT lowering approaches. Our QuantiGene panel will allow the rapid comparative assessment of all Htt transcripts in cell lysates and mouse tissues without the need to first extract RNA.

Design and characterization of mutant and wildtype huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

The gene mutated in individuals with Huntington's disease (HD) encodes the 348-kDa huntingtin (HTT) protein. Pathogenic HD CAG-expansion mutations create a polyglutamine (polyQ) tract at the N terminus of HTT that expands above a critical threshold of ∼35 glutamine residues. The effect of these HD mutations on HTT is not well understood, in part because it is difficult to carry out biochemical, biophysical, and structural studies of this large protein. To facilitate such studies, here we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprised both N- and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, and juvenile HD patients, as well as the more extreme polyQ expansions used in some HD tissue and animal models. Our expression system yielded milligram quantities of pure recombinant HTT protein, including many of the previously mapped post-translational modifications. We characterized both apo and HTT-HTT-associated protein 40 (HAP40) complex samples produced with this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful HTT complexes. We further demonstrate that these resources can produce sufficient material for protein-intensive experiments, such as small-angle X-ray scattering, providing biochemical insight into full-length HTT protein structure. The work outlined and the tools generated here lay a foundation for further biochemical and structural work on the HTT protein and for studying its functional interactions with other biomolecules.

Mutant huntingtin reduction in astrocytes slows disease progression in the BACHD conditional Huntington's disease mouse model.

Neuronal and non-neuronal cells express the huntingtin (HTT) protein, yet neurodegeneration in Huntington's disease (HD) is largely selective, affecting most prominently striatal medium spiny neurons and cortical pyramidal neurons. Selective toxicity of full-length human mutant HTT (fl-mHTT) may be due in part to its expression in non-neuronal cells. While studies suggest neuronal-glial interactions are important in HD and fl-mHTT is expressed in astrocytes, it has not been determined whether the expression of fl-mHTT in astrocytes is necessary for HD pathogenesis. To directly assess the necessity of fl-mHTT in astrocytes for HD pathogenesis, we used a mouse genetic approach and bred the conditional mHTT-expressing BACHD mouse model with GFAP-CreERT2 mice. We show that GFAP-CreERT2 expression in these mice is highly selective for astrocytes, and we are able to significantly reduce the expression of fl-mHTT protein in the striatum and cortex of BACHD/GFAP-CreERT2-tam mice. We performed behavioral, electrophysiological and neuropathological analyses of BACHD and BACHD/GFAP-CreERT2-tam mice. Behavioral analyses of BACHD/GFAP-CreERT2-tam mice demonstrate significant improvements in motor and psychiatric-like phenotypes. We observe improvements in neuropathological and electrophysiological phenotypes in BACHD/GFAP-CreERT2-tam mice compared to BACHD mice. We observed a restoration of the normal level αB-crystallin in the striatum of the BACHD/GFAP-CreERT2 mice, indicating a cell autonomous effect of mHTT on its expression. Taken together, this work indicates that astrocytes are important contributors to the progression of the behavioral and neuropathological phenotypes observed in HD.

Altering cortical input unmasks synaptic phenotypes in the YAC128 cortico-striatal co-culture model of Huntington disease

BackgroundHuntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the huntingtin (HTT) gene, leading to selective and progressive neuronal death predominantly in the striatum. Mutant HTT expression causes dysfunctional cortico-striatal (CS) transmission, loss of CS synapses, and striatal medium spiny neuron (MSN) dendritic spine instability prior to neuronal death. Co-culturing cortical and striatal neurons in vitro promotes the formation of functional CS synapses and is a widely used approach to elucidate pathogenic mechanisms of HD and to validate potential synapto-protective therapies. A number of relevant in vivo synaptic phenotypes from the YAC128 HD mouse model, which expresses full-length transgenic human mutant HTT, are recapitulated in CS co-culture by 21 days in vitro (DIV). However, striatal spine loss, which occurs in HD patients and in vivo animal models, has been observed in YAC128 CS co-culture in some studies but not in others, leading to difficulties in reproducing and interpreting results. Here, we investigated whether differences in the relative proportion of cortical and striatal neurons alter YAC128 synaptic phenotypes in this model.ResultsYAC128 MSNs in 1:1 CS co-culture exhibited impaired dendritic length and complexity compared to wild-type, whereas reducing cortical input using a 1:3 CS ratio revealed a dramatic loss of YAC128 MSN dendritic spines. Chimeric experiments determined that this spine instability was primarily cell autonomous, depending largely on mutant HTT expression in striatal neurons. Moreover, we found that spontaneous electrophysiological MSN activity correlated closely with overall dendritic length, with no differences observed between genotypes in 1:3 co-cultures despite significant YAC128 spine loss. Finally, limiting cortical input with a 1:3 CS ratio impaired the basal survival of YAC128 neurons at DIV21, and this was partially selective for dopamine- and cAMP-regulated phosphoprotein 32-positive MSNs.ConclusionsOur findings reconcile previous discordant reports of spine loss in this model, and improve the utility and reliability of the CS co-culture for the development of novel therapeutic strategies for HD.

Artificial miRNAs Reduce Human Mutant Huntingtin Throughout the Striatum in a Transgenic Sheep Model of Huntington's Disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

The cryo-electron microscopy structure of huntingtin.

SummaryHuntingtin (Htt) is a large (348 kDa) protein, essential for embryonic development and involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and transcription regulation1,2. While an integrative understanding of Htt's biological functions is lacking, the large number of identified interactors suggests that Htt serves as a protein-protein interaction hub1,3,4. Furthermore, Huntington’s disease is caused by a mutation in the Htt gene, resulting in a pathogenic expansion of a polyglutamine (polyQ) repeat at the N-terminus of Htt5,6. However, only limited structural information on Htt is currently available. Here we employed cryo-electron microscopy (cryo-EM) to determine the structure of full-length human Htt in a complex with HAP40/F8A7 to 4 Å resolution. Htt is largely α-helical and consists of three major domains. The N- and C-terminal domains contain multiple HEAT repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat (TPR)-like organization. HAP40 binds in a cleft contacting the three Htt domains by hydrophobic and electrostatic interactions, thereby stabilizing Htt conformation. These data rationalize previous biochemical results and pave the way for an improved understanding of Htt’s diverse cellular functions.

Acute exposure to chlorpyrifos caused NADPH oxidase mediated oxidative stress and neurotoxicity in a striatal cell model of Huntington’s disease

We hypothesized that expression of mutant Huntingtin (HTT) would modulate the neurotoxicity of the commonly used organophosphate insecticide, chlorpyrifos (CPF), revealing cellular mechanisms underlying neurodegeneration. Using a mouse striatal cell model of HD, we report that mutant HD cells are more susceptible to CPF-induced cytotoxicity as compared to wild-type. This CPF-induced cytotoxicity caused increased production of reactive oxygen species, reduced glutathione levels, decreased superoxide dismutase activity, and increased malondialdehyde levels in mutant HD cells relative to wild-type. Furthermore, we show that co-treatment with antioxidant agents attenuated the CPF-induced ROS levels and cytotoxicity. Co-treatment with a NADPH oxidase (NOX) inhibitor, apocynin, also attenuated the CPF-induced ROS production and neurotoxicity. CPF caused increased NOX activity in mutant HD lines that was ameliorated following co-treatment with apocynin. Finally, CPF-induced neurotoxicity significantly increased the protein expression of nuclear factor erythroid 2-related factor (Nrf2) in mutant HD cells as compared to wild-type. This study is the first report of CPF-induced toxicity in HD pathophysiology and suggests that mutant HTT and CPF exhibit a disease-toxicant interaction wherein expression of mutant HTT enhances CPF-induced neurotoxicity via a NOX-mediated oxidative stress mechanism to cause neuronal loss in the full length HTT expressing striatal cells.

Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function.

The polyglutamine expansion in huntingtin protein causes Huntington's disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single-particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical α-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases α-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.

Nuclear retention of full-length HTT RNA is mediated by splicing factors MBNL1 and U2AF65.

Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Recent evidence suggests that HD is a consequence of multimodal, non-mutually exclusive mechanisms of pathogenesis that involve both HTT protein- and HTT RNA-triggered mechanisms. Here we provide further evidence for the role of expanded HTT (expHTT) RNA in HD by demonstrating that a fragment of expHTT is cytotoxic in the absence of any translation and that the extent of cytotoxicity is similar to the cytotoxicity of an expHTT protein fragment encoded by a transcript of similar length and with a similar repeat size. In addition, full-length (FL) expHTT is retained in the nucleus. Overexpression of the splicing factor muscleblind-like 1 (MBNL1) increases nuclear retention of expHTT and decreases the expression of expHTT protein in the cytosol. The splicing and nuclear export factor U2AF65 has the opposite effect, decreasing expHTT nuclear retention and increasing expression of expHTT protein. This suggests that MBNL1 and U2AF65 play a role in nuclear export of expHTT RNA.

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation.

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease.

Integration-independent Transgenic Huntington Disease Fragment Mouse Models Reveal Distinct Phenotypes and Life Span in Vivo

The cascade of events that lead to cognitive decline, motor deficits, and psychiatric symptoms in patients with Huntington disease (HD) is triggered by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein. A significant mechanism in HD is the generation of mutant HTT fragments, which are generally more toxic than the full-length HTT. The protein fragments observed in human HD tissue and mouse models of HD are formed by proteolysis or aberrant splicing of HTT. To systematically investigate the relative contribution of the various HTT protein proteolysis events observed in vivo, we generated transgenic mouse models of HD representing five distinct proteolysis fragments ending at amino acids 171, 463, 536, 552, and 586 with a polyglutamine length of 148. All lines contain a single integration at the ROSA26 locus, with expression of the fragments driven by the chicken β-actin promoter at nearly identical levels. The transgenic mice N171-Q148 and N552-Q148 display significantly accelerated phenotypes and a shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. We hypothesized that the accelerated phenotype was due to altered HTT protein interactions/complexes that accumulate with age. We found evidence for altered HTT complexes in caspase-2 fragment transgenic mice (N552-Q148) and a stronger interaction with the endogenous HTT protein. These findings correlate with an altered HTT molecular complex and distinct proteins in the HTT interactome set identified by mass spectrometry. In particular, we identified HSP90AA1 (HSP86) as a potential modulator of the distinct neurotoxicity of the caspase-2 fragment mice (N552-Q148) when compared with the caspase-6 transgenic mice (N586-Q148).

M04 Preventing Formation Of Toxic N-terminal Huntingtin Fragments Through Antisense Oligonucleotide-mediated Protein Modification

Several studies have implicated the importance of proteolytic cleavage of the mutant huntingtin protein in Huntington disease (HD) pathogenesis and it is generally accepted that N-terminal polyglutamine-containing huntingtin fragments are more toxic than full-length huntingtin protein. While recent publications have shown promising results reducing mutant huntingtin toxicity by lowering huntingtin levels using antisense oligonucleotides, there is some concern regarding unwanted side effects caused by lowering huntingtin protein levels too much. In our study we reduce mutant huntingtin toxicity by modifying the huntingtin protein to prevent proteolytic cleavage and subsequent formation of toxic N-terminal huntingtin fragments. Using antisense oligonucleotides, we induce skipping of huntingtin exon 12 that encodes important cleavage sites. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein, that is resistant to caspase 6 cleavage. Pilot studies in the YAC128 transgenic HD mouse model revealed skipping of human huntingtin exon 12 after a single intracerebroventricular injection of antisense oligonucleotides. We have recently started a follow-up study to assess the longitudinal phenotypical effect of antisense oligonucleotide-mediated exon skipping in YAC128 HD mice.

Aggregation behavior of chemically synthesized, full-length huntingtin exon1.

Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington’s disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued.

Role of Context in RNA Structure: Flanking Sequences Reconfigure CAG Motif Folding in Huntingtin Exon 1 Transcripts

The length of the CAG-repeat region in the huntingtin mRNA is predictive of Huntington's disease. Structural studies of CAG-repeat-containing RNAs suggest that these sequences form simple hairpin structures; however, in the context of the full-length huntingtin mRNA, CAG repeats may form complex structures that could be targeted for therapeutic intervention. We examined the structures of transcripts spanning the first exon of the huntingtin mRNA with both healthy and disease-prone repeat lengths. In transcripts with 17-70 repeats, the CAG sequences base paired extensively with nucleotides in the 5' UTR and with conserved downstream sequences including a CCG-repeat region. In huntingtin transcripts with healthy numbers of repeats, the previously observed CAG hairpin was either absent or short. In contrast, in transcripts with disease-associated numbers of repeats, a CAG hairpin was present and extended from a three-helix junction. Our findings demonstrate the profound importance of sequence context in RNA folding and identify specific structural differences between healthy and disease-inducing huntingtin alleles that may be targets for therapeutic intervention.

Large genetic animal models of Huntington's Disease.

The dominant nature of the Huntington's disease gene mutation has allowed genetic models to be developed in multiple species, with the mutation causing an abnormal neurological phenotype in all animals in which it is expressed. Many different rodent models have been generated. The most widely used of these, the transgenic R6/2 mouse, carries the mutation in a fragment of the human huntingtin gene and has a rapidly progressive and fatal neurological phenotype with many relevant pathological changes. Nevertheless, their rapid decline has been frequently questioned in the context of a disease that takes years to manifest in humans, and strenuous efforts have been made to make rodent models that are genetically more 'relevant' to the human condition, including full length huntingtin gene transgenic and knock-in mice. While there is no doubt that we have learned, and continue to learn much from rodent models, their usefulness is limited by two species constraints. First, the brains of rodents differ significantly from humans in both their small size and their neuroanatomical organization. Second, rodents have much shorter lifespans than humans. Here, we review new approaches taken to these challenges in the development of models of Huntington's disease in large brained, long-lived animals. We discuss the need for such models, and how they might be used to fill specific niches in preclinical Huntington's disease research, particularly in testing gene-based therapeutics. We discuss the advantages and disadvantages of animals in which the prodromal period of disease extends over a long time span. We suggest that there is considerable 'value added' for large animal models in preclinical Huntington's disease research.

Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington's disease

Huntington's disease (HD) is a hereditary neurodegenerative disease caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein and characterized by dysregulated calcium homeostasis. We investigated whether these disturbances are correlated with changes in the mRNA level of the genes that encode proteins involved in calcium homeostasis and signaling (i.e., the calciosome). Using custom-made TaqMan low-density arrays containing probes for 96 genes, we quantified mRNA in the striatum in YAC128 mice, a model of HD, and wildtype mice. HTT mutation caused the increased expression of some components of the calcium signalosome, including calretinin, presenilin 2, and calmyrin 1, and the increased expression of genes indirectly involved in calcium homeostasis, such as huntingtin-associated protein 1 and calcyclin-binding protein. To verify these findings in a different model, we used PC12 cells with an inducible expression of mutated full-length HTT. Using single-cell imaging with Fura-2AM, we found that store-operated Ca2+ entry but not endoplasmic reticulum (ER) store content was changed as a result of the expression of mutant HTT. Statistically significant downregulation of the Orai calcium channel subunit 2, calmodulin, and septin 4 was detected in cells that expressed mutated HTT. Our data indicate that the dysregulation of calcium homeostasis correlates with changes in the gene expression of members of the calciosome. These changes, however, differed in the two models of HD used in this study. Our results indicate that each HD model exhibits distinct features that may only partially resemble the human disease.

Transgenic rats expressing full-length huntingtin ( HTT ) as a model for Huntington's disease (HD)

Transgenic rats expressing full-length huntingtin ( HTT ) as a model for Huntington's disease (HD)

The dopaminergic stabilizer, (−)-OSU6162, rescues striatal neurons with normal and expanded polyglutamine chains in huntingtin protein from exposure to free radicals and mitochondrial toxins

Huntington's disease (HD) is a neurodegenerative disease characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and caused by a polyglutamine expansion in the huntingtin protein. There is so far neither cure nor approved disease-slowing therapy for HD, though recent clinical studies have shown a beneficial long-term effect of pridopidine in patients with HD. The nature of this effect, purely symptomatic or, in addition, neuroprotective, is difficult to elucidate in clinical trials. Pridopidine and (−)-OSU6162 are members of a new family of compounds referred to as dopaminergic stabilizers, which normalize abnormal dopamine neurotransmission.We investigated the effects of (−)-OSU6162 on huntingtin knocked-in striatal neurons in culture. Control neurons had normal full-length huntingtin with 7 glutamines while “mutant” neurons had large expansions (Q=111). We studied the dose–effect curves of (−)-OSU6162 on mitochondrial activity, LDH levels, necrosis and apoptosis in untreated Q7 and Q111 cells. In addition, we investigated the effects of (−)-OSU6162 on Q7 and Q111 neurons challenged with different neurotoxins such as sodium glutamate, H2O2, rotenone and 3-nitropropionic acid (3NP). As we found prevention of toxicity of some of these neurotoxins, we investigated the putative neuroprotective mechanisms of action of (−)-OSU6162 measuring the effects of this dopaminergic stabilizer on expression and release of BDNF, the ratios of Bcl2/Bax proteins and of p-ERK/ERK, the levels of chaperones and GSH, and the effects of (−)-OSU6162 on dopamine uptake and release.We found that (−)-OSU6162, 3–150μM, produces a dose dependent increase of mitochondrial activity and a reduction of cell death. (−)-OSU6162 does not change glutamate toxicity, but it partially prevents that of H2O2, rotenone and 3-nitropropionic acid. (−)-OSU6162 increases the intracellular levels of BDNF and Bcl2/Bax and decreases those of p-ERK/ERK and CHIP in Q111 cells. (−)-OSU6162 increased 3H-dopamine uptake and amphetamine-induced 3H-dopamine release in E13 mouse mid brain neurons.Our studies demonstrate that (−)-OSU6162 improves survival and mitochondrial function in striatal Q111 neurons and the resistance of these cells to several striatal neurotoxins, suggesting that (−)-OSU6162 and related compounds should be tested for neuroprotection in animal models and, eventually, in patients with HD.

Lysosomal function in neurodegenerative diseases

Protein aggregation, mitochondrial deficits, oxidative and autophagic stress are prominent pathologies in neurodegenerative diseases. Our laboratory has a long term interest in the regulation of autophagy-lysosomal activities in neurodegeneration. We previously found that deficiency in the lysosomal cathepsin D in a mouse knockout model led to accumulation of the key protein in Parkinson’s disease, α-synuclein. Overexpression of cathepsin D in human neuroblastoma cells as well as worms reduced α-synuclein aggregation and toxicity. Recent work found that enhanced expression of lysosomal cathepsin D reduced both full-length and fragmented huntingtin in transfected HEK cells, and protected against mutant huntingtin toxicity in primary neurons. Furthermore, as mitochondria are the major generators and targets of reactive species, and oxidative stress has been found in a wide range of neurodegenerative diseases, investigating the source and nature of oxidative stress in cathepsin deficient brains are of significant interest. It has been previously shown that α-synuclein modification by S-nitrosylation enhances its aggregation propensity. Furthermore, microglial produced nitrous oxide (NO) from an increase in inducible nitrous oxide synthase (iNOS) underlies some of the brain pathologies in CD knockout mice. Given that other lysosomal storage conditions have demonstrated mitochondrial involvement and that oxidative and nitrative changes have been associated with CD deficiency, we studied the effects of CD deficiency on mitochondrial morphology and function with the intent to understand how this lysosomal enzyme play a role in maintaining cellular bioenergetics and management of cellular oxidative states. We have found that although there are no changes in total mitochondrial biomass due to CD deficiency, there are enlarged mitochondria, and deficits in functional parameters of the electron transport chain. The changes demonstrated in this study contribute to the understanding of how mitochondrial/lysosomal interplay affects the pathogenesis of neurodegenerative diseases.