Oxidative Stress Research on Huntington's Disease Neurons Using Genetically Encoded Biosensors

Many neurodegenerative diseases, including Huntington’s disease (HD), are associated with oxidative stress in the neurons of the brain. Genetically encoded biosensorsare useful for studying these processesin vitro. Human cell cultures expressing the biosensors can serve as a cell model for developing and testing effective agents that reduce oxidative stress. In this work, transgenes encoding biosensors of glutathione oxidative potential(Grx1-roGFP2) with cytoplasmic and mitochondrial localization were introduced into human induced pluripotent stem cells of a healthy donor and an HD patient using CRISPR/Cas9-mediated genome editing. The cells were subsequently differentiated into medium spiny neurons of the striatum. The expression of the biosensors was detected in the iPSCs, neuronal precursors and mature neurons.The obtained cells could be used to study the redox potential of glutathione in HD neurons and to screen for new drug compounds aimed at reducing oxidative stress. Keywords: genetically encoded biosensors, Huntington’s disease, induced pluripotent stem cells, medium spiny neurons, oxidative stress, glutathione, Grx1-roGFP2

Error-related Signaling in Nucleus Accumbens D2 Receptor-expressing Neurons Guides Avoidance-based Goal-directed Behavior

Learnt associations between environmental cues and the outcomes they predict (cue-outcome associations) play a major role in behavioral control, guiding not only which responses we should perform, but also which we should avoid, in order to achieve a specific goal. The encoding of such cue-outcome associations, as well as the performance of cue-guided goal-directed behavior, is thought to involve dopamine D1 and D2 receptor-expressing medium spiny neurons (D1-/D2-MSNs) of the nucleus accumbens (NAc). Here, using a visual discrimination task in mice, we assessed the role of NAc D1-/D2-MSNs in cue-guided goal-directed avoidance of inappropriate responding. Cell-type specific neuronal silencing and in-vivo imaging revealed NAc D2-MSNs to selectively contribute to cue-guided avoidance behavior, with activation of NAc D2-MSNs following response error playing an important role in optimizing future goal-directed behavior. Our findings indicate that error-signaling by NAc D2-MSNs underlies the ability to use environmental cues to avoid inappropriate behavior.

Pyk2 Stabilizes Striatal Medium Spiny Neuron Structure and Striatal-Dependent Action

In day-to-day life, we often choose between pursuing familiar behaviors that have been rewarded in the past or adjusting behaviors when new strategies might be more fruitful. The dorsomedial striatum (DMS) is indispensable for flexibly arbitrating between old and new behavioral strategies. The way in which DMS neurons host stable connections necessary for sustained flexibility is still being defined. An entry point to addressing this question may be the structural scaffolds on DMS neurons that house synaptic connections. We find that the non-receptor tyrosine kinase Proline-rich tyrosine kinase 2 (Pyk2) stabilizes both dendrites and spines on striatal medium spiny neurons, such that Pyk2 loss causes dendrite arbor and spine loss. Viral-mediated Pyk2 silencing in the DMS obstructs the ability of mice to arbitrate between rewarded and non-rewarded behaviors. Meanwhile, the overexpression of Pyk2 or the closely related focal adhesion kinase (FAK) enhances this ability. Finally, experiments using combinatorial viral vector strategies suggest that flexible, Pyk2-dependent action involves inputs from the medial prefrontal cortex (mPFC), but not the ventrolateral orbitofrontal cortex (OFC). Thus, Pyk2 stabilizes the striatal medium spiny neuron structure, likely providing substrates for inputs, and supports the capacity of mice to arbitrate between novel and familiar behaviors, including via interactions with the medial-prefrontal cortex.

Input-specific control of interneuron numbers in nascent striatal networks

The assembly of functional neuronal circuits requires appropriate numbers of distinct classes of neurons, but the mechanisms through which their relative proportions are established remain poorly defined. Investigating the mouse striatum, here we found that the two most prominent subtypes of striatal interneurons, parvalbumin-expressing (PV+) GABAergic and cholinergic (ChAT+) interneurons, undergo extensive programmed cell death between the first and second postnatal weeks. Remarkably, the survival of PV+ and ChAT+ interneurons is regulated by distinct mechanisms mediated by their specific afferent connectivity. While long-range cortical inputs control PV+ interneuron survival, ChAT+ interneuron survival is regulated by local input from the medium spiny neurons. Our results identify input-specific circuit mechanisms that operate during the period of programmed cell death to establish the final number of interneurons in nascent striatal networks.

Striatal compartmentalization and clustering of different subtypes of astrocytes is altered in the zQ175 Huntington′s disease mouse model

Huntington′s disease (HD) is a devastating neurodegenerative disease that primarily affects the striatum, a brain region that controls movement and some forms of cognition. Dysfunction and loss of medium spiny neurons of the striatum is accompanied by astrogliosis (increased astrocyte density and pathology). For decades, astrocytes were considered a homogeneous cell type, but recent transcriptomic analyses revealed astrocytes are a heterogeneous population classified into multiple subtypes depending on the expression of different gene markers. Here, we studied whether three different striatal astrocyte subtypes expressing glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), or S100 calcium-binding protein B (S100B) are differentially altered in HD. We conducted a comparative immunofluorescence analysis in the striatum of WT and the heterozygous zQ175 HD mouse model and found that the expression and abundance of GFAP+ and S100B+ astrocytes increased in zQ175 mice, while GS+ astrocytes showed no alteration. We then explored whether there was a differential spatial distribution of any of these subtypes within the striatum. We developed a systematic brain compartmentalization approach and found that while GS+ and S100B+ astrocytes were more homogeneously distributed throughout the striatum in zQ175 mice, GFAP+ astrocytes preferentially accumulated in the dorsomedial and dorsolateral striatum, which are regions associated with goal-directed and habitual behaviors. Additionally, GFAP+ astrocytes in zQ175 mice showed increased clustering, a parameter that indicates increased proximity and that is associated with localized inflammation and/or neurodegeneration. Our data suggest a differential susceptibility in both increased density and striatal compartmentalization of different subtypes of astrocytes in zQ175. These results highlight new potential implications for our understanding of astrocyte pathology in HD.

A distinct D1-MSN subpopulation down-regulates dopamine to promote negative emotional state

Dopamine (DA) level in the nucleus accumbens (NAc) is critical for reward and aversion encoding. DA released from the ventral mesencephalon (VM) DAergic neurons increases the excitability of VM-projecting D1-dopamine receptor-expressing medium spiny neurons (D1-MSNs) in the NAc to enhance DA release and augment rewards. However, how such a DA positive feedback loop is regulated to maintain DA homeostasis and reward-aversion balance remains elusive. Here we report that the ventral pallidum (VP) projection of NAc D1-MSNs (D1NAc-VP) is inhibited by rewarding stimuli and activated by aversive stimuli. In contrast to the VM projection of D1-MSN (D1NAc-VM), activation of D1NAc-VP projection induces aversion, but not reward. D1NAc-VP MSNs are distinct from the D1NAc-VM MSNs, which exhibit conventional functions of D1-MSNs. Activation of D1NAc-VP projection stimulates VM GABAergic transmission, inhibits VM DAergic neurons, and reduces DA release into the NAc. Thus, D1NAc-VP and D1NAc-VM MSNs cooperatively control NAc dopamine balance and reward-aversion states.

Binge Alcohol Drinking Alters Synaptic Processing of Executive and Emotional Information in Core Nucleus Accumbens Medium Spiny Neurons

The nucleus accumbens (NAc) is a forebrain region mediating the positive-reinforcing properties of drugs of abuse, including alcohol. It receives glutamatergic projections from multiple forebrain and limbic regions such as the prefrontal cortex (PFCx) and basolateral amygdala (BLA), respectively. However, it is unknown how NAc medium spiny neurons (MSNs) integrate PFCx and BLA inputs, and how this integration is affected by alcohol exposure. Because progress has been hampered by the inability to independently stimulate different pathways, we implemented a dual wavelength optogenetic approach to selectively and independently stimulate PFCx and BLA NAc inputs within the same brain slice. This approach functionally demonstrates that PFCx and BLA inputs synapse onto the same MSNs where they reciprocally inhibit each other pre-synaptically in a strict time-dependent manner. In alcohol-naïve mice, this temporal gating of BLA-inputs by PFCx afferents is stronger than the reverse, revealing that MSNs prioritize high-order executive processes information from the PFCx. Importantly, binge alcohol drinking alters this reciprocal inhibition by unilaterally strengthening BLA inhibition of PFCx inputs. In line with this observation, we demonstrate that in vivo optogenetic stimulation of the BLA, but not PFCx, blocks binge alcohol drinking escalation in mice. Overall, our results identify NAc MSNs as a key integrator of executive and emotional information and show that this integration is dysregulated during binge alcohol drinking.

A Systematic Review of Transcriptional Dysregulation in Huntington’s Disease Studied by RNA Sequencing

Huntington’s disease (HD) is a chronic neurodegenerative disorder caused by an expansion of polyglutamine repeats in exon 1 of the Huntingtin gene. Transcriptional dysregulation accompanied by epigenetic alterations is an early and central disease mechanism in HD yet, the exact mechanisms and regulators, and their associated gene expression programs remain incompletely understood. This systematic review investigates genome-wide transcriptional studies that were conducted using RNA sequencing (RNA-seq) technology in HD patients and models. The review protocol was registered at the Open Science Framework (OSF). The biomedical literature and gene expression databases, PubMed and NCBI BioProject, Array Express, European Nucleotide Archive (ENA), European Genome-Phenome Archive (EGA), respectively, were searched using the defined terms specified in the protocol following the PRISMA guidelines. We conducted a complete literature and database search to retrieve all RNA-seq-based gene expression studies in HD published until August 2020, retrieving 288 articles and 237 datasets from PubMed and the databases, respectively. A total of 27 studies meeting the eligibility criteria were included in this review. Collectively, comparative analysis of the datasets revealed frequent genes that are consistently dysregulated in HD. In postmortem brains from HD patients, DNAJB1, HSPA1B and HSPB1 genes were commonly upregulated across all brain regions and cell types except for medium spiny neurons (MSNs) at symptomatic disease stage, and HSPH1 and SAT1 genes were altered in expression in all symptomatic brain datasets, indicating early and sustained changes in the expression of genes related to heat shock response as well as response to misfolded proteins. Specifically in indirect pathway medium spiny neurons (iMSNs), mitochondria related genes were among the top uniquely dysregulated genes. Interestingly, blood from HD patients showed commonly differentially expressed genes with a number of brain regions and cells, with the highest number of overlapping genes with MSNs and BA9 region at symptomatic stage. We also found the differential expression and predicted altered activity of a set of transcription factors and epigenetic regulators, including BCL6, EGR1, FOSL2 and CREBBP, HDAC1, KDM4C, respectively, which may underlie the observed transcriptional changes in HD. Altogether, our work provides a complete overview of the transcriptional studies in HD, and by data synthesis, reveals a number of common and unique gene expression and regulatory changes across different cell and tissue types in HD. These changes could elucidate new insights into molecular mechanisms of differential vulnerability in HD.Systematic Review Registration:https://osf.io/pm3wq