Cellular Analysis of Silencing the Huntington's Disease Gene Using AAV9 Mediated Delivery of Artificial Micro RNA into the Striatum of Q140/Q140 Mice.

J Huntingtons Dis.

2016 Sep 24

Keeler AM, Sapp E, Chase K, Sottosanti E, Danielson E, Pfister E, Stoica L, DiFiglia M, Aronin N, Sena-Esteves M.
PMID: 27689620 | DOI: 10.3233/JHD-160215

Abstract

BACKGROUND:

The genetic mutation in Huntington's disease (HD) is a CAG repeat expansion in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral vectors based on whole tissue assays, but the extent of htt mRNA lowering in individual neurons is unknown.

OBJECTIVE:

Here we characterize the effect of an AAV9-GFP-miRHtt vector on Htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice.

METHODS:

HD mice received bilateral striatal injections of AAV9-GFP-miRHtt or AAV9-GFP at 6 or 12 weeks and striata were evaluated at 6 months of age for levels of Htt mRNA and protein and for mRNA signal within striatal neurons using RNAscope multiplex fluorescence in situ hybridization.

RESULTS:

Compared to controls, the striatum of 6-month old mice treated at 6 or 12 weeks of age with AAV9-GFP-miRHtt showed a reduction of 40-50% in Htt mRNA and lowering of 25-40% in protein levels. The number of Htt mRNA foci in medium spiny neurons (MSNs) of untreated Q140/Q140 mice varied widely per cell (0 to 34 per cell), with ∼10% of MSNs devoid of foci. AAV9-GFP-miRHtt treatment shifted the distribution toward lower numbers and the percentage of cells without foci increased to 14-20%. The average number of Htt mRNA foci per MSN was reduced by 43%.

CONCLUSIONS:

The findings here show that intrastriatal infusion of an AAV9-GFP-miRHtt vector lowers mRNA expression of Htt in striatum by ∼50%, through a partial reduction in the number of copies of mutant Htt mRNAs per cell. These findings demonstrate at the neuronal level the variable levels of Htt mRNA expression in MSNs and the neuronal heterogeneity of RNAi dependent Htt mRNA knockdown.

Mutant huntingtin messenger RNA forms neuronal nuclear clusters in rodent and human brains

Brain Communications

2022 Oct 13

Ly, S;Didiot, M;Ferguson, C;Coles, A;Miller, R;Chase, K;Echeverria, D;Wang, F;Sadri-Vakili, G;Aronin, N;Khvorova, A;
| DOI: 10.1093/braincomms/fcac248

Mutant mRNA and protein contribute to the clinical manifestation of many repeat-associated neurological disorders, with the presence of nuclear RNA clusters being a common pathological feature. Yet, investigations into Huntington's disease - caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene - have primarily focused on toxic protein gain-of-function as the primary disease-causing feature. To date, mutant HTT mRNA has not been identified as an in vivo hallmark of Huntington’s disease. Here, we report that, in two Huntington’s disease mouse models (YAC128 and BACHD-97Q-ΔN17), mutant HTT mRNA is retained in the nucleus. Widespread formation of large mRNA clusters (∼0.6 to 5 µm3) occurred in 50-75% of striatal and cortical neurons. Cluster formation was independent of age and driven by expanded repeats. Clusters associate with chromosomal transcriptional sites and quantitatively co-localize with the aberrantly-processed N-terminal exon 1-intron 1 mRNA isoform, HTT1a. HTT1a mRNA clusters are observed in a subset of neurons from human Huntington’s disease post-mortem brain and are likely caused by somatic expansion of repeats. In YAC128 mice, clusters, but not individual HTT mRNA, are resistant to antisense oligonucleotide treatment. Our findings identify mutant HTT/HTT1a mRNA clustering as an early, robust molecular signature of Huntington’s disease, providing in vivo evidence that Huntington’s disease is a repeat expansion disease with mRNA involvement.

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin.

Cell Rep.

2018 Sep 04

Didiot MC, Ferguson CM, Ly S, Coles AH, Smith AO, Bicknell AA, Hall LM, Sapp E, Echeverria D, Pai AA, DiFiglia M, Moore MJ, Hayward LJ, Aronin N, Khvorova A.
PMID: 30184490 | DOI: 10.1016/j.celrep.2018.07.106

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.

Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington's disease model mice

Nature communications

2023 Mar 14

Pancani, T;Day, M;Tkatch, T;Wokosin, DL;González-Rodríguez, P;Kondapalli, J;Xie, Z;Chen, Y;Beaumont, V;Surmeier, DJ;
PMID: 36914640 | DOI: 10.1038/s41467-023-36556-3

Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

Uninterrupted CAG repeat drives striatum-selective transcriptionopathy and nuclear pathogenesis in human Huntingtin BAC mice

Neuron

2022 Jan 27

Gu, X;Richman, J;Langfelder, P;Wang, N;Zhang, S;Bañez-Coronel, M;Wang, HB;Yang, L;Ramanathan, L;Deng, L;Park, CS;Choi, CR;Cantle, JP;Gao, F;Gray, M;Coppola, G;Bates, GP;Ranum, LPW;Horvath, S;Colwell, CS;Yang, XW;
PMID: 35114102 | DOI: 10.1016/j.neuron.2022.01.006

In Huntington's disease (HD), the uninterrupted CAG repeat length, but not the polyglutamine length, predicts disease onset. However, the underlying pathobiology remains unclear. Here, we developed bacterial artificial chromosome (BAC) transgenic mice expressing human mutant huntingtin (mHTT) with uninterrupted, and somatically unstable, CAG repeats that exhibit progressive disease-related phenotypes. Unlike prior mHTT transgenic models with stable, CAA-interrupted, polyglutamine-encoding repeats, BAC-CAG mice show robust striatum-selective nuclear inclusions and transcriptional dysregulation resembling those in murine huntingtin knockin models and HD patients. Importantly, the striatal transcriptionopathy in HD models is significantly correlated with their uninterrupted CAG repeat length but not polyglutamine length. Finally, among the pathogenic entities originating from mHTT genomic transgenes and only present or enriched in the uninterrupted CAG repeat model, somatic CAG repeat instability and nuclear mHTT aggregation are best correlated with early-onset striatum-selective molecular pathogenesis and locomotor and sleep deficits, while repeat RNA-associated pathologies and repeat-associated non-AUG (RAN) translation may play less selective or late pathogenic roles, respectively.

Cerebellar granule neurons induce Cyclin D1 before the onset of motor symptoms in Huntington's disease mice

Acta neuropathologica communications

2023 Jan 20

Bauer, S;Chen, CY;Jonson, M;Kaczmarczyk, L;Magadi, SS;Jackson, WS;
PMID: 36670467 | DOI: 10.1186/s40478-022-01500-x

Although Huntington's disease (HD) is classically defined by the selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting primarily of granule cells, commonly considered disease resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected in these neurons. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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Probes for HTT

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