Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.
Brain-Derived Neurotrophic Factor , Cerebral Cortex , Disease Models, Animal , Huntington Disease , Neurons , Synapses , Animals , Huntington Disease/metabolism , Huntington Disease/pathology , Brain-Derived Neurotrophic Factor/metabolism , Synapses/metabolism , Synapses/drug effects , Synapses/pathology , Cerebral Cortex/metabolism , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , Mice , Neurons/metabolism , Neurons/drug effects , Neurons/pathology , Mice, Transgenic , Cells, Cultured , Synapsins/metabolism , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Mice, Inbred C57BL
Top-down mass spectrometry (MS) is a versatile tool that has been employed to investigate both protein sequence and structure. Although a variety of different fragmentation methods are available in top-down MS that can potentially yield structural information, quantifying differences between spectra remains challenging. Herein, we show that subtle differences in spectra produced by a variety of fragmentation methods are surprisingly sensitive to protein structure and/or charge localization, even in highly unfolded proteins observed in high charge states. In addition to exposing information about the protein structure, differences in fragmentation also reveal insight into the mechanisms underlying the dissociation methods themselves. The results further reveal that small changes in experimental parameters (such as the addition of methanol instead of acetonitrile) lead to changes in structure that are reflected in statistically reproducible differences in dissociation. Collisional annealing of structurally dissimilar ions in the gas phase eventually leads to dissociation spectra that are indistinguishable, suggesting that structural differences can be erased by sufficient thermal activation. Additional experiments illustrate that identical charge states of the same protein can be distinguished if those produced directly by electrospray are compared to ions manipulated by in vacuo proton-transfer charge reduction. Overall, the results show that subtle differences in both three-dimensional structure and charge-site localization can influence the abundance of fragment ions produced by top-down MS, including dissociation methods not typically thought to be structurally sensitive.
Proteins , Protons , Proteins/chemistry , Ions/chemistry , Mass Spectrometry/methods , Amino Acid Sequence
Rab7 GTPase regulates mitochondrial morphology and function. Missense mutation(s) of Rab7 underlies the pathogenesis of Charcot Marie Tooth 2B (CMT2B) peripheral neuropathy. Herein, we investigate how mitochondrial morphology and function are impacted by the CMT2B associated Rab7V162M mutation. In contrast to recent studies of using heterologous overexpression systems, our results demonstrate significant mitochondrial fragmentation in both human CMT2B patient fibroblasts and CMT2B embryonic fibroblasts (MEFs). Primary cultured E18 dorsal root ganglion (DRG) sensory neurons also show mitochondrial fragmentation and altered axonal mitochondrial movement. In addition, we demonstrate that inhibitors to either the mitochondrial fission protein Drp1 or to the nucleotide binding to Rab7 normalize the mitochondrial deficits in both MEFs and E18 cultured DRG neurons. Our study reveals, for the first time, that expression of CMT2B Rab7 mutation at the physiological level enhances Drp1 activity to promote mitochondrial fission, potentially underlying selective vulnerability of peripheral sensory neurons in CMT2B pathogenesis.
Charcot-Marie-Tooth Disease , rab GTP-Binding Proteins , Charcot-Marie-Tooth Disease/genetics , Humans , Laminopathies , Mitochondria/metabolism , Sensory Receptor Cells/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding Proteins
