Vesicular glycolysis provides on-board energy for fast axonal transport.

Fast axonal transport (FAT) requires consistent energy over long distances to fuel the molecular motors that transport vesicles. We demonstrate that glycolysis provides ATP for the FAT of vesicles. Although inhibiting ATP production from mitochondria did not affect vesicles motility, pharmacological or genetic inhibition of the glycolytic enzyme GAPDH reduced transport in cultured neurons and in Drosophila larvae. GAPDH localizes on vesicles via a huntingtin-dependent mechanism and is transported on fast-moving vesicles within axons. Purified motile vesicles showed GAPDH enzymatic activity and produced ATP. Finally, we show that vesicular GAPDH is necessary and sufficient to provide on-board energy for fast vesicular transport. Although detaching GAPDH from vesicles reduced transport, targeting GAPDH to vesicles was sufficient to promote FAT in GAPDH deficient neurons. This specifically localized glycolytic machinery may supply constant energy, independent of mitochondria, for the processive movement of vesicles over long distances in axons.

The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo.

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson's disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) "cell-autonomous". Here, we explored whether ΔLRRK2G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2G2019S or its "dead" kinase form, ΔLRRK2DK, and human α-syn with the A53T mutation (AAV-α-synA53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-synA53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-synA53T with AAV-ΔLRRK2G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-synA53T alone or with AAV-ΔLRRK2DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2G2019 alone, through cell-autonomous mechanisms.

The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo.

The G2019S substitution in the kinase domain of LRRK2 (LRRK2G2019S) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2G2019S are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2G2019S is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2G2019S in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2WT or LV-ΔLRRK2G2019S, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2WT and ΔLRRK2G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2WT did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.

The unlikely partnership between LRRK2 and α-synuclein in Parkinson's disease.

Our understanding of the mechanisms underlying Parkinson's disease, the once archetypical nongenetic neurogenerative disorder, has dramatically increased with the identification of α-synuclein and LRRK2 pathogenic mutations. While α-synuclein protein composes the aggregates that can spread through much of the brain in disease, LRRK2 encodes a multidomain dual-enzyme distinct from any other protein linked to neurodegeneration. In this review, we discuss emergent datasets from multiple model systems that suggest these unlikely partners do interact in important ways in disease, both within cells that express both LRRK2 and α-synuclein as well as through more indirect pathways that might involve neuroinflammation. Although the link between LRRK2 and disease can be understood in part through LRRK2 kinase activity (phosphotransferase activity), α-synuclein toxicity is multilayered and plausibly interacts with LRRK2 kinase activity in several ways. We discuss common protein interactors like 14-3-3s that may regulate α-synuclein and LRRK2 in disease. Finally, we examine cellular pathways and outcomes common to both mutant α-synuclein expression and LRRK2 activity and points of intersection. Understanding the interplay between these two unlikely partners in disease may provide new therapeutic avenues for PD.

The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin.

The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington's disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington's disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington's disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington's disease could render the striatum highly vulnerable to neurodegeneration.

Energy defects in Huntington's disease: Why "in vivo" evidence matters.

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder associated with involuntary abnormal movements (chorea), cognitive deficits and psychiatric disturbances. The most striking neuropathological change in HD is the early atrophy of the striatum. While the disease progresses, other brain structures also degenerate, including the cerebral cortex. Changes are also seen outside the brain, in particular weight loss/cachexia despite high dietary intake. The disease is caused by an abnormal expansion of a CAG repeat in the gene encoding the huntingtin protein (Htt). This mutation leads to the expression of a poly-glutamine stretch that changes the biological functions of mutant Htt (mHtt). The mechanisms underlying neurodegeneration in HD are not totally elucidated. Here, we discuss recent results obtained in patients, animal and cellular models suggesting that early disturbance in energy metabolism at least in part associated with mitochondrial defects may play a central role, even though all data are not congruent, possibly because most findings were obtained in cell culture systems or using biochemical analyses of post mortem tissues from rodent models. Thus, we put a particular focus on brain imaging studies that could identify biomarkers of energy defects in vivo and would be of prime interest in preclinical and clinical trials testing the efficacy of new therapies targeting energy metabolism in HD.

Mutant Huntingtin alters retrograde transport of TrkB receptors in striatal dendrites.

Huntingtin (HTT), the protein mutated in Huntington's disease (HD), controls transport of the neurotrophin, brain-derived neurotrophic factor (BDNF), within corticostriatal neurons. Transport and delivery of BDNF to the striatum are reduced in disease, which contributes to striatal neuron degeneration. BDNF released by cortical neurons activates TrkB receptors at striatal dendrites to promote striatum survival. However, it remains to be determined whether transport of TrkB, the BDNF receptor, depends on HTT and whether such transport is altered in mutant situation. Here we show that TrkB binds to and colocalizes with HTT and dynein. Silencing HTT reduces vesicular transport of TrkB in striatal neurons. In HD, the polyQ expansion in HTT alters the binding of TrkB-containing vesicles to microtubules and reduces transport. Using a combination of microfluidic devices that isolate dendrites from cell bodies and BDNF coupled to quantum dots, we selectively analyzed TrkB retrograde transport in response to BDNF stimulation at dendrite terminals. We show that the retrograde transport of TrkB vesicles within striatal dendrites and the BDNF/TrkB-induced signaling through ERK phosphorylation and c-fos induction are decreased in neurons from an HD mouse model. Together, our findings demonstrate that HTT is a crucial regulator of TrkB trafficking. Transport defects in HD are not restricted to BDNF transport in cortical neurons but also affect trafficking of its ligand-bound receptor in the striatal neurons. This transport alteration may further impair BDNF-TrkB survival signaling within the corticostriatal connection that is most affected in HD.

Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma.

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons.

The transport of vesicles in neurons is a highly regulated process, with vesicles moving either anterogradely or retrogradely depending on the nature of the molecular motors, kinesins and dynein, respectively, which propel vesicles along microtubules (MTs). However, the mechanisms that determine the directionality of transport remain unclear. Huntingtin, the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport. Huntingtin is phosphorylated at serine 421 by the kinase Akt but the role of this modification is unknown. Here, we demonstrate that phosphorylation of wild-type huntingtin at S421 is crucial to control the direction of vesicles in neurons. When phosphorylated, huntingtin recruits kinesin-1 to the dynactin complex on vesicles and MTs. Using brain-derived neurotrophic factor as a marker of vesicular transport, we demonstrate that huntingtin phosphorylation promotes anterograde transport. Conversely, when huntingtin is not phosphorylated, kinesin-1 detaches and vesicles are more likely to undergo retrograde transport. This also applies to other vesicles suggesting an essential role for huntingtin in the control of vesicular directionality in neurons.

Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis.

Amyloid peptide (Aß) is generated by sequential cleavage of the amyloid precursor protein (APP) by ß-secretase (Bace1) and γ-secretase. Aß production increases after plasma membrane cholesterol loading through unknown mechanisms. To determine how APP-Bace1 proximity affects this phenomenon, we developed a fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET) technique for visualization of these molecules either by epifluorescence or at the plasma membrane only using total internal reflection fluorescence. Further, we used fluorescence correlation spectroscopy to determine the lipid rafts partition of APP-yellow fluorescent protein (YFP) and Bace1-green fluorescent protein (GFP) molecules at the plasma membrane of neurons. We show that less than 10 min after cholesterol exposure, Bace1-GFP/APP-mCherry proximity increases selectively at the membrane and APP relocalizes to raft domains, preceded by rapid endocytosis. After longer cholesterol exposures, APP and Bace1 are found in proximity intracellularly. We demonstrate that cholesterol loading does not increase Aß production by having a direct impact on Bace1 catalytic activity but rather by altering the accessibility of Bace1 to its substrate, APP. This change in accessibility is mediated by clustering in lipid rafts, followed by rapid endocytosis.

Zero Echo Time 17O-MRI Reveals Decreased Cerebral Metabolic Rate of Oxygen Consumption in a Murine Model of Amyloidosis.

The cerebral metabolic rate of oxygen consumption (CMRO2) is a key metric to investigate the mechanisms involved in neurodegeneration in animal models and evaluate potential new therapies. CMRO2 can be measured by direct 17O magnetic resonance imaging (17O-MRI) of H217O signal changes during inhalation of 17O-labeled oxygen gas. In this study, we built a simple gas distribution system and used 3D zero echo time (ZTE-)MRI at 11.7 T to measure CMRO2 in the APPswe/PS1dE9 mouse model of amyloidosis. We found that CMRO2 was significantly lower in the APPswe/PS1dE9 brain than in wild-type at 12-14 months. We also estimated cerebral blood flow (CBF) from the post-inhalation washout curve and found no difference between groups. These results suggest that the lower CMRO2 observed in APPswe/PS1dE9 is likely due to metabolism impairment rather than to reduced blood flow. Analysis of the 17O-MRI data using different quantification models (linear and 3-phase model) showed that the choice of the model does not affect group comparison results. However, the simplified linear model significantly underestimated the absolute CMRO2 values compared to a 3-phase model. This may become of importance when combining several metabolic fluxes measurements to study neuro-metabolic coupling.

Phosphorylation of mutant huntingtin at S421 restores anterograde and retrograde transport in neurons.

Huntingtin (htt), the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport whose function is lost in disease. Here, we demonstrate that phosphorylation of htt at serine 421 (S421) restores its function in axonal transport. Using a strategy involving RNA (ribonucleic acid) interference and re-expression of various constructs, we show that polyQ (polyglutamine)-htt is unable to promote transport of brain-derived neurotrophic factor (BDNF)-containing vesicles, but polyQ-htt constitutively phosphorylated at S421 is as effective as the wild-type (wt) as concerns transport of these vesicles. The S421 phosphorylated polyQ-htt displays the wt function of inducing BDNF release. Phosphorylation restores the interaction between htt and the p150(Glued) subunit of dynactin and their association with microtubules in vitro and in cells. We also show that the IGF-1 (insulin growth factor type I)/Akt pathway by promoting htt phosphorylation compensates for the transport defect. This is the first description of a mechanism that restores the htt function altered in disease.

The mTOR inhibitor Everolimus synergizes with the PI3K inhibitor GDC0941 to enhance anti-tumor efficacy in uveal melanoma.

Uveal melanoma (UM) is the most frequent malignant ocular tumor in adults. While the primary tumor is efficiently treated by surgery and/or radiotherapy, about one third of UM patients develop metastases, for which no effective treatment is currently available. The PKC, MAPK and PI3K/AKT/mTOR signaling cascades have been shown to be associated with tumor growth. However, none of the compounds against those pathways results in tumor regression when used as single agents. To identify more effective therapeutic strategies for UM patients, we performed a combination screen using seven targeted agents inhibiting PKC, MEK, AKT, PI3K and mTOR in a panel of ten UM cell lines, representative of the UM disease. We identified a strong synergy between the mTOR inhibitor Everolimus and the PI3K inhibitor GDC0941. This combination resulted in an increase in apoptosis in several UM cell lines compared to monotherapies and enhanced the anti-tumor effect of each single agent in two patient-derived xenografts. Furthermore, we showed that the synergism between the two drugs was associated with the relief by GDC0491 of a reactivation of AKT induced by Everolimus. Altogether, our results highlight a novel and effective combination strategy, which could be beneficial for UM patients.

Protein Tyrosine Phosphatase 4A3 (PTP4A3) Promotes Human Uveal Melanoma Aggressiveness Through Membrane Accumulation of Matrix Metalloproteinase 14 (MMP14).

PURPOSE: To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness. METHODS: Cell membrane localization of matrix metalloprotease 14 (MMP14) in uveal melanoma cells expressing protein tyrosine phosphatase A3 (PTP4A3) was assessed by flow cytometry or immunohistochemistry. The vesicular trafficking of MMP14 in the presence of PTP4A3 was evaluated in OCM-1 cells expressing either the wild-type or mutated phosphatase. Finally, MMP14 localization at the cell membrane of OCM-1 cells was impaired using RNA interference, and the PTP4A3-related migration in vitro and invasiveness in vivo of the treated cells were evaluated. RESULTS: We found that the membrane-anchored MMP14 is enriched at the cell surface of OCM-1 cells, patient-derived xenograft cells, and human primary uveal melanoma tumors expressing PTP4A3. Moreover, we show that PTP4A3 and MMP14 colocalize and that the vesicular trafficking of MMP14 is faster in the presence of active PTP4A3. Finally, we demonstrate that inhibition of MMP14 expression in uveal melanoma cells expressing PTP4A3 impairs their migration in vitro and invasiveness in vivo. CONCLUSIONS: Our observations indicate that PTP4A3 increases cell membrane accumulation of MMP14 as a result of increased cellular trafficking of the metalloprotease. We also show that downregulation of MMP14 expression reduced PTP4A3-induced cell migration and invasiveness. Taken together, our findings suggest that PTP4A3-related subcellular localization of MMP14 is an important event in metastasis induction.

Sulfheme formation during homocysteine S-oxygenation by catalase in cancers and neurodegenerative diseases.

Accumulating evidence suggests that abnormal levels of homocysteine are associated with vascular dysfunctions, cancer cell proliferation and various neurodegenerative diseases. With respect to the latter, a perturbation of transition metal homeostasis and an inhibition of catalase bioactivity have been reported. Herein, we report on some of the molecular bases for the cellular toxicity of homocysteine and demonstrate that it induces the formation of sulfcatalase, an irreversible inactive state of the enzyme, without the intervention of hydrogen sulfide. Initially, homocysteine reacts with native catalase and/or redox-active transition metal ions to generate thiyl radicals that mediate compound II formation, a temporarily inactive state of the enzyme. Then, the ferryl centre of compound II intervenes into the unprecedented S-oxygenation of homocysteine to engender the corresponding sulfenic acid species that further participates into the prosthetic heme modification through the formation of an unusual Fe(II) sulfonium. In addition, our ex cellulo studies performed on cancer cells, models of neurodegenerative diseases and ulcerative colitis suggest the likelihood of this scenario in a subset of cancer cells, as well as in a cellular model of Parkinson's disease. Our findings expand the repertoire of heme modifications promoted by biological compounds and point out another deleterious trait of disturbed homocysteine levels that could participate in the aetiology of these diseases.

2,7-Bis-(4-amidinobenzylidene)-cycloheptan-1-one dihydrochloride, tPA stop, prevents tPA-enhanced excitotoxicity both in vitro and in vivo.

Tissue-type plasminogen activator (tPA) is available for the treatment of thromboembolic stroke in humans. However, adverse effects of tPA have been observed in animal models of ischemic brain injuries. In the present study, we have used a synthetic tPA inhibitor, named 2,7-bis-(4-amidino-benzylidene)-cycloheptan-1-one dihydrochloride (tPA stop), to investigate the role of endogenous tPA in the cerebral parenchyma. In mouse cortical cell cultures, we observed that although tPA stop reduced N-methyl-D-aspartic acid (NMDA)-mediated excitotoxic neuronal death, it failed to modulate alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate-mediated necrosis. In addition, we found that tPA stop could prevent the deleterious effects of both endogenous and exogenous tPA during NMDA exposure. At the functional level, tPA stop was found to prevent tPA-dependent potentiation of NMDA receptor-evoked calcium influx. The relevance of those findings was strengthened by the observation of a massive reduction of NMDA-induced excitotoxic lesion in rats when tPA stop was co-injected. Altogether, these data demonstrate that the blockade of the endogenous proteolytic activity of tPA in the cerebral parenchyma could be a powerful neuroprotective strategy raised against brain pathologies associated with excitotoxicity.

Transforming growth factor-beta1-modulated cerebral gene expression.

Transforming growth factor-beta1 (TGF-beta1) plays a central role in the response of the brain to different types of injury. Increased TGF-beta1 has been found in the central nervous system of patients with acute or chronic disorders such as stroke or Alzheimer disease. To further define the molecular targets of TGF-beta1 in cerebral tissues, a selection of high-density cDNA arrays was used to characterize the mRNA expression profile of 7,000 genes in transgenic mice overexpressing TGF-beta1 from astrocytes as compared with the wild-type line. Selected findings were further evaluated by reverse transcription-polymerase chain reactions from independent transgenic and wild-type mice. Furthermore, the expression pattern of seven selected genes such as Delta-1, CRADD, PRSC-1, PAI-1, Apo-1/Fas, CTS-B, and TbetaR-II were confirmed in either cultured cortical neurons or astrocytes following TGF-beta1 treatment. The authors' observations enlarge the repertoire of known TGF-beta1-modulated genes and their possible involvement in neurodegenerative processes.

Matching gene expression with hypometabolism after cerebral ischemia in the nonhuman primate.

To correlate brain metabolic status with the molecular events during cerebral ischemia, a cDNA array was performed after positron emission tomography scanning in a model of focal cerebral ischemia in baboons. Cluster analysis for the expression of 74 genes allowed the identification of 4 groups of genes. In each of the distinct groups, the authors observed a marked inflection in the pattern of gene expression when the CMRo was reduced by 48% to 66%. These patterns of coordinated modifications in gene expression could define molecular checkpoints for the development of an ischemic infarct and a molecular definition of the penumbra.

Ciliogenesis is regulated by a huntingtin-HAP1-PCM1 pathway and is altered in Huntington disease.

Huntington disease (HD) is a devastating autosomal-dominant neurodegenerative disorder. It is caused by expansion of a CAG repeat in the first exon of the huntingtin (HTT) gene that encodes a mutant HTT protein with a polyglutamine (polyQ) expansion at the amino terminus. Here, we demonstrate that WT HTT regulates ciliogenesis by interacting through huntingtin-associated protein 1 (HAP1) with pericentriolar material 1 protein (PCM1). Loss of Htt in mouse cells impaired the retrograde trafficking of PCM1 and thereby reduced primary cilia formation. In mice, deletion of Htt in ependymal cells led to PCM1 mislocalization, alteration of the cilia layer, and hydrocephalus. Pathogenic polyQ expansion led to centrosomal accumulation of PCM1 and abnormally long primary cilia in mouse striatal cells. PCM1 accumulation in ependymal cells was associated with longer cilia and disorganized cilia layers in a mouse model of HD and in HD patients. Longer cilia resulted in alteration of the cerebrospinal fluid flow. Thus, our data indicate that WT HTT is essential for protein trafficking to the centrosome and normal ciliogenesis. In HD, hypermorphic ciliogenesis may affect signaling and neuroblast migration so as to dysregulate brain homeostasis and exacerbate disease progression.

Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity.

Huntington's disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington's disease pathogenesis. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington's disease in vitro and in a mouse model of Huntington's disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington's disease, which, in turn, stimulates its enzymatic activity. Mutant huntingtin-mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington's disease.