Involvement of CRMP2 in Regulation of Mitochondrial Morphology and Motility in Huntington’s Disease

Mitochondrial morphology and motility (mitochondrial dynamics) play a major role in the proper functioning of distant synapses. In Huntington’s disease (HD), mitochondria become fragmented and less motile, but the mechanisms leading to these changes are not clear. Here, we found that collapsin response mediator protein 2 (CRMP2) interacted with Drp1 and Miro 2, proteins involved in regulating mitochondrial dynamics. CRMP2 interaction with these proteins inversely correlated with CRMP2 phosphorylation. CRMP2 was hyperphosphorylated in postmortem brain tissues of HD patients, in human neurons derived from induced pluripotent stem cells from HD patients, and in cultured striatal neurons from HD mouse model YAC128. At the same time, CRMP2 interaction with Drp1 and Miro 2 was diminished in HD neurons. The CRMP2 hyperphosphorylation and dissociation from Drp1 and Miro 2 correlated with increased fission and suppressed motility. (S)-lacosamide ((S)-LCM), a small molecule that binds to CRMP2, decreased its phosphorylation at Thr 509/514 and Ser 522 and rescued CRMP2’s interaction with Drp1 and Miro 2. This was accompanied by reduced mitochondrial fission and enhanced mitochondrial motility. Additionally, (S)-LCM exerted a neuroprotective effect in YAC128 cultured neurons. Thus, our data suggest that CRMP2 may regulate mitochondrial dynamics in a phosphorylation-dependent manner and modulate neuronal survival in HD.

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In vivo progressive degeneration of Huntington’s disease patient-derived neurons reveals human-specific pathological phenotypes

10.1101/2020.10.21.347062 ◽

2020 ◽

Author(s):

Andrés Miguez ◽

Sara Fernández-García ◽

Marta Monguió-Tortajada ◽

Georgina Bombau ◽

Mireia Galofré ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Secretory Pathway ◽

Disease Patient ◽

Medium Spiny Neuron ◽

Host Cells ◽

Striatal Neurons ◽

Striatal Degeneration ◽

Human Neurons

AbstractResearch on neurodegenerative disorders has been hampered by the limited access to patients’ brain tissue and the absence of relevant physiological models with human neurons, accounting for the little success of clinical trials. Moreover, post-mortem samples cannot provide a detailed picture of the complex pathological mechanisms taking place throughout the course of the disease. This holds particularly true for Huntington’s disease (HD), an incurable inherited brain disorder marked by a massive striatal degeneration due to abnormal accumulation of misfolded huntingtin protein. To characterize progressive human neurodegeneration in vivo, we transplanted induced pluripotent stem cell-derived human neural progenitor cells (hNPCs) from control (CTR-hNPCs) and HD patients (HD-hNPCs) into the striatum of neonatal wild-type mice. Implanted human cells were examined by immunohistochemistry and electron microscopy, and chimeric mice were subjected to behavioral testing. Most grafted hNPCs differentiated into striatal neurons that sent axonal projections to their natural targets and established synaptic connections within the host basal ganglia circuitry. HD-hNPCs first showed developmental abnormalities characterized by an increased proliferation and accelerated medium spiny neuron (MSN) differentiation, mimicking the initial striatal hypertrophy of child mutant huntingtin (mHTT) carriers. HD human striatal neurons progressively developed mHTT oligomers and aggregates, which primarily targeted mitochondria, endoplasmic reticulum and nuclear membrane to cause structural alterations. Five months after transplantation, selective death of human MSNs and striatal degeneration altered mouse behavior, suggesting disease propagation to non-mutated host cells. Histological analysis and co-culture experiments revealed that HD-hNPCs secreted extracellular vesicles containing soluble mHTT oligomers, which were internalized by mouse striatal neurons triggering cell death. Finally, in vivo pharmacological inhibition of the exosomal secretory pathway through sphingosine-1 phosphate receptor functional antagonism, limited the spreading of apoptosis within the host striatum. Our findings cast new light on human neurodegeneration, unveiling cell and non-cell autonomous mechanisms that drive HD progression in patients.

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Neuroprotective Effects of Doxycycline in the R6/2 Mouse Model of Huntington’s Disease

Molecular Neurobiology ◽

10.1007/s12035-019-01847-8 ◽

2019 ◽

Vol 57 (4) ◽

pp. 1889-1903 ◽

Cited By ~ 6

Author(s):

Emanuela Paldino ◽

Claudia Balducci ◽

Pietro La Vitola ◽

Luisa Artioli ◽

Vincenza D’Angelo ◽

...

Keyword(s):

Mouse Model ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Neuroprotective Effect ◽

Neurological Dysfunction ◽

Striatal Neurons ◽

Neuroprotective Effects ◽

Beneficial Effects ◽

Doxycycline Treatment ◽

Immunohistochemical Studies

AbstractMechanisms of tissue damage in Huntington’s disease involve excitotoxicity, mitochondrial damage, and inflammation, including microglia activation. Immunomodulatory and anti-protein aggregation properties of tetracyclines were demonstrated in several disease models. In the present study, the neuroprotective and anti-inflammatory effects of the tetracycline doxycycline were investigated in the mouse model of HD disease R6/2. Transgenic mice were daily treated with doxycycline 20 mg/kg, starting from 4 weeks of age. After sacrifice, histological and immunohistochemical studies were performed. We found that doxycycline-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the saline-treated ones. Primary outcome measures such as striatal atrophy, neuronal intranuclear inclusions, and the negative modulation of microglial reaction revealed a neuroprotective effect of the compound. Doxycycline provided a significantly increase of activated CREB and BDNF in the striatal neurons, along with a down modulation of neuroinflammation, which, combined, might explain the beneficial effects observed in this model. Our findings show that doxycycline treatment could be considered as a valid therapeutic approach for HD.

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Essential role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, ROS generation and lung endothelial barrier loss

Scientific Reports ◽

10.1038/s41598-021-97006-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Panfeng Fu ◽

Yulia Epshtein ◽

Ramaswamy Ramchandran ◽

Joseph B. Mascarenhas ◽

Anne E. Cress ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Human Lung ◽

Mitochondrial Dynamics ◽

Dynamic Change ◽

Mitochondrial Fission ◽

Endothelial Permeability ◽

Ros Generation ◽

Mitochondrial Morphology ◽

Dependent Manner ◽

Lps Challenge

AbstractWe have shown that both reactive oxygen species (ROS) and paxillin tyrosine phosphorylation regulate LPS-induced human lung endothelial permeability. Mitochondrial ROS (mtROS) is known to increase endothelial cell (EC) permeability which requires dynamic change in mitochondrial morphology, events that are likely to be regulated by paxillin. Here, we investigated the role of paxillin and its tyrosine phosphorylation in regulating LPS-induced mitochondrial dynamics, mtROS production and human lung microvascular EC (HLMVEC) dysfunction. LPS, in a time-dependent manner, induced higher levels of ROS generation in the mitochondria compared to cytoplasm or nucleus. Down-regulation of paxillin expression with siRNA or ecto-expression of paxillin Y31F or Y118F mutant plasmids attenuated LPS-induced mtROS in HLMVECs. Pre-treatment with MitoTEMPO, a scavenger of mtROS, attenuated LPS-induced mtROS, endothelial permeability and VE-cadherin phosphorylation. Further, LPS-induced mitochondrial fission in HLMVECs was attenuated by both a paxillin siRNA, and paxillin Y31F/Y118F mutant. LPS stimulated phosphorylation of dynamin-related protein (DRP1) at S616, which was also attenuated by paxillin siRNA, and paxillinY31/Y118 mutants. Inhibition of DRP1 phosphorylation by P110 attenuated LPS-induced mtROS and endothelial permeability. LPS challenge of HLMVECs enhanced interaction between paxillin, ERK, and DRP1, and inhibition of ERK1/2 activation with PD98059 blocked mitochondrial fission. Taken together, these results suggest a key role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, mtROS generation and EC barrier dysfunction.

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Faculty Opinions recommendation of Mitochondrial fission and cristae disruption increase the response of cell models of Huntington's disease to apoptotic stimuli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.6516956.6647054 ◽

2010 ◽

Author(s):

Stefano Di Donato

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Mitochondrial Fission ◽

Cell Models

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Faculty Opinions recommendation of Striatal neurons directly converted from Huntington's disease patient fibroblasts recapitulate age-associated disease phenotypes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732609529.793548926 ◽

2018 ◽

Author(s):

David Schaffer ◽

Maroof Adil

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Disease Patient ◽

Striatal Neurons ◽

Disease Phenotypes

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Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Cell Death and Disease ◽

10.1038/s41419-021-03752-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Anthony R. Anzell ◽

Garrett M. Fogo ◽

Zoya Gurm ◽

Sarita Raghunayakula ◽

Joseph M. Wider ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Conditional Knockout ◽

Mitochondrial Morphology ◽

Primary Neurons ◽

Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy

Cell Death and Disease ◽

10.1038/s41419-021-03502-4 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Qing-Rui Wu ◽

Dan-Lin Zheng ◽

Pei-Ming Liu ◽

Hui Yang ◽

Lu-An Li ◽

...

Keyword(s):

Calcium Channel ◽

Mitochondrial Dysfunction ◽

High Glucose ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Calcium Handling ◽

Cardiomyocyte Hypertrophy ◽

Mitochondrial Morphology ◽

Calmodulin Binding ◽

Downstream Target

AbstractMitochondrial dysfunction and impaired Ca2+ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca2+ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca2+ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca2+ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca2+ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1–Drp1 axis is a novel target for treating DCM.

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Electrophysiological and Morphological Changes in Striatal Spiny Neurons in R6/2 Huntington's Disease Transgenic Mice

Journal of Neurophysiology ◽

10.1152/jn.2001.86.6.2667 ◽

2001 ◽

Vol 86 (6) ◽

pp. 2667-2677 ◽

Cited By ~ 209

Author(s):

Gloria J. Klapstein ◽

Robin S. Fisher ◽

Hadi Zanjani ◽

Carlos Cepeda ◽

Eve S. Jokel ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Action Potentials ◽

Morphological Changes ◽

Brain Slices ◽

Cag Repeats ◽

Membrane Properties ◽

Striatal Neurons ◽

Spiny Neurons ◽

Active Membrane

We examined passive and active membrane properties and synaptic responses of medium-sized spiny striatal neurons in brain slices from presymptomatic (∼40 days of age) and symptomatic (∼90 days of age) R6/2 transgenics, a mouse model of Huntington's disease (HD) and their age-matched wild-type (WT) controls. This transgenic expresses exon 1 of the human HD gene with ∼150 CAG repeats and displays a progressive behavioral phenotype associated with numerous neuronal alterations. Intracellular recordings were obtained using standard techniques from R6/2 and age-matched WT mice. Few electrophysiological changes occurred in striatal neurons from presymptomatic R6/2 mice. The changes in this age group were increased neuronal input resistance and lower stimulus intensity to evoke action potentials (rheobase). Symptomatic R6/2 mice exhibited numerous electrophysiological alterations, including depolarized resting membrane potentials, increased input resistances, decreased membrane time constants, and alterations in action potentials. Increased stimulus intensities were required to evoke excitatory postsynaptic potentials (EPSPs) in neurons from symptomatic R6/2 transgenics. These EPSPs had slower rise times and did not decay back to baseline by 45 ms, suggesting a more prominent component mediated by activation of N-methyl-d-aspartate receptors. Neurons from both pre- and symptomatic R6/2 mice exhibited reduced paired-pulse facilitation. Data from biocytin-filled or Golgi-impregnated neurons demonstrated decreased dendritic spine densities, smaller diameters of dendritic shafts, and smaller dendritic fields in symptomatic R6/2 mice. Taken together, these findings indicate that passive and active membrane and synaptic properties of medium-sized spiny neurons are altered in the R6/2 transgenic. These physiological and morphological alterations will affect communication in the basal ganglia circuitry. Furthermore, they suggest areas to target for pharmacotherapies to alleviate and reduce the symptoms of HD.

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