By Lise Munsie, PhD
1 – Imaging in a Mouse Model
An ex vivo imaging study was performed on fixed brains from the R6/2 Huntington disease (HD) mouse model1. White matter integrity was analyzed by using diffusion imaging (DTI) and 17.6T high-field MRI, followed by fluorescent and electron microscopy to visualize associated cellular changes. The model used in this study was called continuous random walk and complements the standard DTI model. The group identified axonal degeneration in the corpus callosum (CC) and increased axonal density in both the MRI and microscopy imaging, consistent with the volume reduction of the CC in HD patient MRIs. The conclusions from this paper include early axonal degeneration as a biomarker for HD and the use of anomalous diffusion models in future clinical trials.
The valuable resource of MRI images from TRACK-HD continues to be utilized in studies globally. Bock’s group from Canada used this resource to investigate cortical microstructure in patients at different stage of disease2. Even though these images were not optimized for this type of analysis the researchers were still able to use T1W/T2W ratio images to map the changes in cortical composition. They identified an increase in intensity with HD progression starting in the insula and spreading as disease progressed. They hypothesize that the increased ratio could be due to an increase in myelin levels, decrease in neuronal density, or increase in level of cortical iron.
Following that, an MRI study was performed specifically investigating altered iron content in early-stage HD patients3. The group used quantitative susceptibility mapping (QSM) and R2 measures in a 1-year time-course study on early HD patients to elucidate the natural history of iron accumulation in the striatum and globus pallidus. Iron content accumulation and higher iron deposition rates were found in closer-to-onset premanifest and early HD patients but not in premanifest patients. This form of imaging and analysis tracking iron deposition in the striatum may act as a marker for clinical disease progression.
1Gatto. R.G. et al. (2019) Detection of Axonal Degeneration in a Mouse Model of Huntington’s Disease: Comparison Between Diffusions Tensor Imaging and Anomalous Diffusion Metrics. Magnetic Resonance Materials in Physics: Biology and Medicine https://doi.org/10.1007/s10334-019-00742-6.
2Rowley, C.D. et al. (2018) Altered Intracortical T1-weighted/T2-weighted Ration Signal in Huntington’s Disease. Frontiers in Neuroscience 12:805.
3Chen, L. et al. (2018) Altered Brain Iron Content and Deposition Rate in Huntington’s Disease as Indicated by Quantitative Susceptibility MRI. Journal of Neuroscience Research 97:467-479.
2 – Human Pluripotent Stem Cells
Neurons differentiated from human pluripotent stem cells serve as an important model for studying Huntington disease (HD).
Induced pluripotent stem cells (iPSC) from the HD iPSC consortium are available to the research community. The consortium recently published a study in which they differentiated the iPSC lines into neuronal cells and analyzed bioenergetic phenotypes1. They found that ATP levels were decreased in cells derived from HD patients in undifferentiated iPSC as well as in cells at different stages of neuronal differentiation. Treating the cells with glycolytic late pathway intermediates led to increased ATP production. The group performed LC-MS and identified a lower expression level of glycolytic enzymes in HD iPSC-derived neural cells. These data indicate that defects in glycolysis may contribute to HD bioenergetics and metabolism phenotypes.
The Kiselev lab uses a subset of the HD iPSC consortium lines in their studies on mitochondrial trafficking in differentiated neurons. Their recent manuscript examined mitochondrial density in neurites2. They found a decrease in the density of mitochondria in HD neurons. When the cells were artificially aged by inhibiting the proteasome, the mitochondrial density decreased proportionally in both HD and wild-type neurons, suggesting this impairment occurs before disease onset.
Another useful stem cell model is creating isogenic lines from a single donor. The Pouladi group used gene editing to create isogenic human embryonic stem cell lines with different CAG repeat lengths3. The group differentiated the lines into different cell types (neural precursor cells (NPC), neurons, hepatocytes, and muscle myotubes) and performed whole-transcriptome and/or whole-proteome analysis. They found mitochondrial alterations in NPCs recapitulated known HD mitochondrial phenotypes, including reduced ATP production. Mitochondrial defects were additionally noted in the myotubes and hepatocytes, meaning the bioenergetics phenotype is not limited to neuronal cells. Importantly, the group has provided a web portal to provide query-based access to the transcriptional and proteomic data generated in this study.
1The HD iPSC Consortium (2019) Bioenergetic deficits in Huntington’s Disease iPSC-derived Neural Cells and Rescue with Glycolytic Metabolites. Human Molecular Genetics http://dx.doi.org/10.1093/hmg/ddy430.
2Nekorovska E.D. and Kiselev S.L. (2018) Mitochondrial Distribution Violation and Nuclear Indentation in Neurons Differentiated from iPSCs of Huntington’s Disease Patients. Journal of Stem Cells and Regenerative Medicine (14:2) 80-85.
3Ooi, J. et al. (2019) Unbiased Profiling of Isogenic Huntington Disease hPSC-Derived CNS and Peripheral Cells Reveal Strong Cell-Type Specificity of CAG Length Effects. Cell Reports (26) 2494-2508.
3 – Normal Huntingtin Function
Cell and mouse models are an important tool for studying the normal function of huntingtin (htt) and the selective vulnerability of neurons due to the insult of mutant htt (mHtt).
An elegant study published in Cell Stem Cell used human embryonic stem cells (hESC) derived from embryos harboring mHtt, or wild-type sibling controls1. The group differentiated the hESCs into glial progenitor cells (GPC), performed RNA-seq, and identified a downregulation in transcription factors (TF) that regulate astroglial and oligodendroglial differentiation and downstream myelin synthesis in the mHtt GPC’s. These GPC’s were transplanted into mice that were both myelin-deficient and immunodeficient. The mHtt GPC resulted in slower and incomplete mylenation in vivo. The mHtt-induced hypomylenation was rescued by overexpression of the TF’s SOX10 and MYRF, demonstrating that the white matter failure in HD is a product of a mHtt-dependent block in differentiation by affected GPCs.
Early Huntington disease (HD) is hallmarked by altered neurotransmission. The Park group examined synaptic vesicle release in real time in primary cortical neurons isolated from the Q175 mouse model2. Neurons were loaded with FM1-43, a membrane-impermeable liphophilic styryl dye, and using electric field stimulation mHtt was observed to affect excitatory neurotransmission. Presynaptic terminals were shown to have an increased released probability which can be modulated through controlling voltage gated Ca2+ channels. This modulation may be a therapeutic avenue to explore to rescue the vulnerability of these excitatory neurons and stop subsequent neuronal loss.
There are many studies that support Htt involvement in cell trafficking and interaction with the cytoskeleton which may explain the selective vulnerability of neurons that have unique morphology and special trafficking requirements. To further probe Htt involvement in the cytoskeleton, a study in PLOS ONE features an imaging-based study in fibroblasts using growth factor cytoskeletal stimulation as a model3. Htt was required for proper cell morphology and adhesion, and when mHtt was present, cell remodeling was inhibited upon stimulation, further supporting the normal function of Htt in these processes.
1Osipovitch, M. et al. (2019) Human ESC-Derived Chimeric Mouse Models of Huntington’s Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation. Cell Stem Cell 24:107-122.
2Chen, S. et al. (2018) Altered Synaptic Vesicle Release and Ca2+ Influx at Single Presynaptic Terminals of Cortical Neurons in a Kock-in Mouse Model of Huntington’s Disease. Frontiers in Molecular Neuronscience 11:478.
3Tousely, A. et al. (2019) Huntingtin Associates with the Actin Cytoskeleton and a-actinin Isoforms to Influence Stimulus Dependent Morphology Changes. PLOS ONE https://doi.org/10.1371/journal.pone.0212337.