Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1 K38A ) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-b peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1 K38A . Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.

Huntington disease (HD) is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (HTT). PolyQ length determines disease onset and severity with a longer expansion causing earlier onset. The mechanisms of mutant HTT-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in HD pathogenesis1,2. Here we tested whether mutant HTT impairs the mitochondrial fission/fusion balance and thereby causes neuronal injury. We show that mutant HTT triggers mitochondrial fragmentation in neurons and fibroblasts of HD individuals in vitro and HD mice in vivo before the presence of neurological deficits and HTT aggregates. Interestingly, mutant HTT abnormally interacts with the mitochondrial fission GTPase dynamin-related protein 1 (DRP1) in HD mice and individuals which in turn stimulates its enzymatic activity. Importantly, mutant HTT-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 DRP1K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in HD.

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 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.

Mitochondrial respiratory complex II inhibition plays a central role in Huntington's disease (HD). Remarkably, 3-NP, a complex II inhibitor, recapitulates HD-like symptoms. Furthermore, decreases in mitochondrial fusion or increases in mitochondrial fission have been implicated in neurodegenerative diseases. However, the relationship between mitochondrial energy defects and mitochondrial dynamics has never been explored in detail. In addition, the mechanism of neuronal cell death by complex II inhibition remains unclear. Here, we tested the temporal and spatial relationship between energy decline, impairment of mitochondrial dynamics, and neuronal cell death in response to 3-NP using quantitative fluorescence time-lapse microscopy and cortical neurons. 3-NP caused an immediate drop in ATP. This event corresponded with a mild rise in reactive oxygen species (ROS), but mitochondrial morphology remained unaltered. Unexpectedly, several hours after this initial phase, a second dramatic rise in ROS occurred, associated with profound mitochondrial fission characterized by the conversion of filamentous to punctate mitochondria and neuronal cell death. Glutamate receptor antagonist AP5 abolishes the second peak in ROS, mitochondrial fission, and cell death. Thus, secondary excitotoxicity, mediated by glutamate receptor activation of the NMDA subtype, and consequent oxidative and nitrosative stress cause mitochondrial fission, rather than energy deficits per se. These results improve our understanding of the cellular mechanisms underlying HD pathogenesis. Huntington's disease (HD) is a fatal progressive neurodegenerative disorder with autosomal dominant inheritance. An abnormal CAG expansion coding for a polyglutamine stretch in the N-terminal region of the huntingtin gene causes HD. Disease results when the polyglutamine stretch contains 40 or more residues, and repeats of 36-39 residues have reduced penetrance. The clinical symptoms of HD include progressive motor, cognitive, and emotional deficits due to the changes in the cortex and striatum. How mutant huntingtin (mtHtt) triggers neurodegeneration is not clear. Among the proposed mechanisms are transcriptional dysregulation, axonal and dendritic transport defects, protein aggregation, and excitotoxic pathways mediated by glutamate receptors.In addition to these pathways, there is strong evidence that deficits in energy metabolism and mitochondria play a pivotal role in HD pathogenesis. [1][2][3][4][5][6] For example, brain tissue of HD patients 1,2 and transgenic mice expressing the mtHtt gene 5 exhibit reduced activity of the mitochondrial respiratory complexes II, III, and IV. In addition, striatal neuronal cultures expressing mtHtt exhibit decreased expression of respiratory complex II. 7 Furthermore, HD patients lose weight despite normal or increased calorie intake and their cortex and basal ganglia have increased lactate levels, indicative of a metabolic defect. 8 Moreover, mitochondria isolated from the lymphoblasts of HD patients, brain tissue of mtHtt mice,...

Amyloid peptide (Aβ) is generated by sequential cleavage of the amyloid precursor protein (APP) by β-secretase (Bace1) and γ-secretase. Aβ 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 Aβ 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.

IntroductionHuntington disease (HD) is an autosomal-dominant neurodegenerative disorder caused by the pathogenic expansion of the polyglutamine (polyQ) N-terminal stretch in the huntingtin protein (HTT; encoded by HTT). HD is characterized by the dysfunction and death of neurons in the brain, leading to devastating cognitive, psychiatric, and motor symptoms in patients. Studies in multiple cell and animal model systems support the notion that polyQ expansion in mutant Htt leads to the gain of new toxic functions and loss of the neuroprotective functions of the WT Htt (1).Although some of its functions are linked to transcriptional activities in health and disease, Htt is primarily a cytoplasmic protein that associates with microtubules (MTs) and vesicles. Htt regulates intracellular trafficking of various organelles, including vesicles, by interacting with the dynein/dynactin pathway (2-4). Htt facilitates MT-dependent transport by interacting directly with dynein (2) and indirectly via huntingtin-associated protein 1 (HAP1), which binds to the dynactin p150 Glued subunit (3,(5)(6)(7). In pathological situations, the Htt-HAP1-dynactin complex is altered, resulting in a reduction of vesicular transport in neurons (3). These findings show that Htt integrates vesicular transport by regulating the activity of specific protein complexes containing the dynein/dynactin and kinesin 1 complexes and adaptor proteins such as HAP1.With 2 patterns of axonemal MTs (9 + 0 in primary cilia and 9 + 2 in motile cilia), cilia are involved in sensory role, motility, and flow generation. Within the last 5 years, the importance of this

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.