Highly Cited HD Research

In this edition of HD Insights, we take a look at some of the most impactful HD research articles of 2014. We searched Thomson Reuters’ “Web of Science” service in December 2015 and identified five papers as the most highly cited original research articles on HD published in 2014. Reviews and book chapters were excluded, as well as articles pertaining only tangentially to HD. We contacted the corresponding authors and requested articles on their work, and received the following responses. These pieces highlight several ongoing areas of exploration in clinical and basic science research in HD, and suggest new frontiers for clinical practice and potential therapeutic targets.

Astrocytes in HD

By: Baljit S. Khakh, PhD and Michael V. Sofroniew, MD, PhD

Original Article: Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington’s disease model mice (cited 45 times as of 2/8/2016)

Understanding the mechanisms that lead to neurological and psychiatric disorders remains a major goal of neuroscience. Considerable advances have been made in understanding the roles of neurons in brain function and disease. In contrast, astrocytes, which represent about half the cells in the human brain, have been less thoroughly investigated. Recent studies show that astrocytes are essential for the normal activity of neural circuits, but the possibility that astrocyte dysfunction may contribute to, or perhaps even drive disease mechanisms, remains incompletely explored.1 Broad analyses show that recent neuroscience drug development based on our improved understanding of neurons has resulted in many failures, suggesting that investigating astrocytes in diseases such as HD may be beneficial.2

Evidence suggests that astrocytes may be involved in HD. Brains from HD patients and from mouse models of HD show accumulation of mutant huntingtin protein (mHTT) in striatal astrocytes, which contributes to age-dependent HD-like pathology (see Figure, part a).3 However, it remains unknown whether and how astrocytes contribute to HD pathology or disease mechanisms. We therefore used HD mouse models R6/2 and Q175 to assess astrocyte contributions to HD pathophysiology.4 We found that concomitant with the onset of HD symptoms, significantly more astrocytes had mHTT inclusions and significant reductions in important functional proteins (including the potassium channel Kir4.1) without major phenotypic changes associated with astrocyte reactivity. These findings suggest that mHTT is associated with early disruption of the expression of important astrocyte functional proteins that alters astrocyte function (see Figure, part b) without triggering astrogliosis. Congruent with other studies of mouse models and human HD, we found progressively increasing astrogliosis at later disease stages that exhibit overt neurodegeneration.

Based on past studies, the loss of Kir4.1 currents in striatal astrocytes predicts reduced spatial K+ buffering, which, in the simplest interpretation, would lead to higher ambient K+ levels. We found that the extracellular K+ concentration was doubled in R6/2 mice, prompting us to explore the impact of increased K+ on the properties of striatal medium spiny neurons (MSNs).

Figure: Striatal astrocytes from R6/2 HD-model mice display nuclear mHTT inclusions and lower membrane conductances. Representative immunofluorescence images showing that GFAP, S100, GS and Aldh1L1−labeled astrocytes (green) contain nuclear mHTT inclusions. Nuclei were labeled blue with DAPI, and mHTT is shown in white. Representative traces of whole-cell voltage-clamp recordings from striatal astrocytes from WT and R6/2 mice at P60. The current waveforms show the response to a step depolarization, revealing clear differences in membrane conductance between WT and R6/2 astrocytes.

Figure: Striatal astrocytes from R6/2 HD-model mice display nuclear mHTT inclusions and lower membrane conductances.
Representative immunofluorescence images showing that GFAP, S100, GS and Aldh1L1−labeled astrocytes (green) contain nuclear mHTT inclusions. Nuclei were labeled blue with DAPI, and mHTT is shown in white.
Representative traces of whole-cell voltage-clamp recordings from striatal astrocytes from WT and R6/2 mice at P60. The current waveforms show the response to a step depolarization, revealing clear differences in membrane conductance between WT and R6/2 astrocytes.

To our surprise, we found that exposing wild-type mice to equivalent increases in K+ reproduced the elevated excitability features of MSNs described in a variety of HD mouse models. We then delivered Kir4.1-GFP channels to striatal astrocytes in HD-model mice by using adeno-associated viruses, and found that one motor symptom (stride length and width) was attenuated by this approach. We also found that MSN membrane properties were partly recovered by astrocyte expression of Kir4.1-GFP in R6/2 mice, strongly supporting the notion that some HD-like phenotypes derive from neuronal dysfunction that itself derives, in part, from astrocyte disturbances.

To date, research efforts have been focused almost exclusively on identifying neuronal cell-autonomous mechanisms to account for changes in MSN properties in HD models. Our findings provide evidence that key aspects of altered MSN excitability in HD are secondary to disturbance of astrocyte maintenance of extracellular K+. The precise cellular functions of HTT are not known, and it is not clear how mHTT impacts Kir4.1. Interestingly, transcriptome profiling of astrocyte responses to inflammatory mediators revealed HTT at the center of one of the top three most significantly altered gene networks.5 This intriguing finding warrants further exploration.

Overall, our findings show that aspects of altered neuronal excitability associated with HD may be secondary to changes in astrocyte function, thereby revealing striatal astrocytes as potential therapeutic targets for drug development. Interestingly, astrocytes display a distinctly different library of molecules compared to neurons. Further studies are warranted to determine whether astrocyte-specific molecular processes and pathways can be exploited to produce desirable effects, either directly or indirectly, on neural circuits in brain disease.

This study was supported by the CHDI Foundation (BSK, MVS) and partly by the NIH (NS060677, MH104069 to BSK).

1Khakh BS, Sofroniew MV. Diversity of astrocyte functions and phenotypes in neural circuits. Nat Neurosci. 2015;18:942-952.

2Ringel M, Tollman P, Hersch G, Schulze U. Does size matter in R&D productivity? If not, what does? Nat Rev Drug Discov. 2013;12:901-902.

3Bradford J, Shin JY, Roberts M, et al. Expression of mutant huntingtin in mouse brain astrocytes causes age-dependent neurological symptoms. Proc Natl Acad Sci U S A. 2009;106:22480-22485.

4Tong X, Ao Y, Faas GC, et al. Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington’s disease model mice. Nat Neurosci. 2014;17:694-703.

5Hamby ME, Coppola G, Ao Y, et al. Inflammatory mediators alter the astrocyte transcriptome and calcium signaling elicited by multiple G-protein-coupled receptors. J Neurosci. 2012;32:14489-14510.

C9orf72 expansions and HD phenocopies

By: Carolin A. M. Koriath, MD, Davina J. Hensman Moss, BA, MBBS, Sarah J. Tabrizi, MBChB, PhD

Original Article: C9orf72 expansions are the most common genetic cause of Huntington disease phenocopies (cited 28 times as of 2/8/2016)

HD is the most common genetically determined neurodegenerative disease.1 This autosomal dominant condition, caused by a CAG repeat expansion in the huntingtin gene, is typically defined by a triad of movement, cognitive, and psychiatric symptoms. However, while chorea is common and usually accompanied by cognitive decline, patients can also suffer from akinetic-rigid syndromes, dystonia, ataxia, as well as solely cognitive or psychiatric symptoms,2 which can complicate clinical diagnosis.

Approximately 1% of those with suspected HD do not carry the CAG expansion in the huntingtin gene.3,4 These patients suffer from so-called HD phenocopy syndromes; differential diagnoses include HD-like syndromes (HDL) 1, 2, and 3, spino-cerebellar atrophy 17, and dentatorubral-pallidoluysian atrophy.5 An intronic hexanucleotide repeat expansion in the C9orf72 gene6 was first described in 2011 as the most frequent cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS).7-10 The expansion has since been identified in additional syndromes, such as cerebellar ataxia.11 

In our study,12 514 patients, who had been referred for HD testing by an experienced specialist and tested negative for the HTT CAG expansion, were examined for the C9orf72 expansion to establish whether it should be included in the routine genetic assessment for this patient cohort. Ten patients (1.95%, 95% confidence interval) were found to carry the C9orf72 GGGGCC repeat expansion, as well as the associated risk allele (rs3849942 A), either homo- or heterozygously. The size of the expansion did not differ significantly from other C9orf72-mediated syndromes or between those with and without chorea/dystonia. At almost 2%, this is the most frequently identified cause of HD phenocopy syndromes in a United Kingdom cohort. While 70% of the C9orf72 positive cases had a family history of neurodegenerative disease, 30% did not, leaving sporadic cases a possibility. Furthermore, while the reported age of onset of C9orf72-caused disease is approximately 57 years of age, in this cohort it was 42.7 years, broadening not only the phenotype, but also the demographic in which this diagnosis should be considered.

True HD and HD phenocopy syndromes can present with a spectrum of cognitive, psychiatric, and movement symptoms, and without an obvious family history. C9orf72-caused disease can also present with a very heterogenous range of symptoms from FTLD/ALS to parkinsonism.13 We therefore propose that genetic testing for C9orf72 should be included in the clinical algorithm for the HD phenocopy work-up, following the test for the HTT expansion, and preceding the test for spino-cerebellar ataxia 17.

The function of C9orf72 remains unclear, but it resembles the DENN-like superfamily, suggesting a role in membrane traffic.14-16 An overview of the proposed disease mechanisms of C9orf72 expansion can be found in the accompanying Figure.

Figure: Overview of the proposed C9orf72-mediated toxicity mechanisms The expansion of intronic GGGGCC repeats in the C9orf72 gene from the normal less than 30 repeats to hundreds or thousands of GGGGCC repeats may lead to a loss of protein function,8 which normally is thought to regulate vesicular trafficking and autophagy because of its homology with the DENN-like superfamily.14-16 RNA-transcripts have been shown to form foci and quadruplex structures interacting with RNA- binding proteins and affecting transcription and RNA processing.17,18 Despite being intronic, the GGGGCC repeat expansion has been found to be translated in a mechanism called repeat-associated non-ATG (RAN) translation, to form characteristic neuronal inclusions,19 and to affect cell viability in vitro.20,21

Figure: Overview of the proposed C9orf72-mediated toxicity mechanisms
The expansion of intronic GGGGCC repeats in the C9orf72 gene from the normal less than 30 repeats to hundreds or thousands of GGGGCC repeats may lead to a loss of protein function,8 which normally is thought to regulate vesicular trafficking and autophagy because of its homology with the DENN-like superfamily.14-16 RNA-transcripts have been shown to form foci and quadruplex structures interacting with RNA- binding proteins and affecting transcription and RNA processing.17,18 Despite being intronic, the GGGGCC repeat expansion has been found to be translated in a mechanism called repeat-associated non-ATG (RAN) translation, to form characteristic neuronal inclusions,19 and to affect cell viability in vitro.20,21

Understanding the commonalities underlying neurodegenerative conditions, such as HD and C9orf72-mediated disease, and the interplay between pathological process and clinical presentation, may not only enrich understanding of the diseases themselves, but also improve patient management.

1Rawlins M. Huntington’s disease out of the closet? Lancet.376(9750):1372-1373.

2Huntington’s disease. 4th ed. New York, NY: Oxford University Press Inc.; 2014.

3Andrew SE, Goldberg YP, Kremer B, et al. Huntington disease without CAG expansion: phenocopies or errors in assignment? Am. J. Hum. Genet. 1994;54(5):852-863.

4Kremer B, Goldberg P, Andrew SE, et al. A Worldwide Study of the Huntington’s Disease Mutation: The Sensitivity and Specificity of Measuring CAG Repeats. N. Engl. J. Med. 1994;330(20):1401-1406.

5Wild EJ, Tabrizi SJ. Huntington’s disease phenocopy syndromes. Curr. Opin. Neurol. 2007;20(6):681-687.

6C9orf72 chromosome 9 open reading frame 72 [Homo sapiens (human)]. National Library of Medicine. http://www.ncbi.nlm.nih.gov/gene?linkname=protein_gene&from_uid=365906244. Accessed January 3, 2016

7Smith BN, Newhouse S, Shatunov A, et al. The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder. Eur. J. Hum. Genet. 2013;21(1):102-108.

8DeJesus-Hernandez M, Mackenzie Ian R, Boeve Bradley F, et al. Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS. Neuron. 2011;72(2):245-256.

9Mahoney CJ, Beck J, Rohrer JD, et al. Frontotemporal dementia with the C9ORF72 hexanucleotide repeat expansion: clinical, neuroanatomical and neuropathological features. Brain. 2012;135(3):736-750.

10Renton Alan E, Majounie E, Waite A, et al. A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD. Neuron. 2011;72(2):257-268.

11Corcia P, Vourc’h P, Guennoc AM, et al. Pure cerebellar ataxia linked to large C9orf72 repeat expansion. Amyotroph. Lateral Scler. Frontotemporal Degener. 2015:1-3.

12Moss DJH, Poulter M, Beck J, et al. C9orf72 expansions are the most common genetic cause of Huntington disease phenocopies. Neurol. 2014;82(4):292-299.

13Murray ME, DeJesus-Hernandez M, Rutherford NJ, et al. Clinical and neuropathologic heterogeneity of c9FTD/ALS associated with hexanucleotide repeat expansion in C9ORF72. Acta Neuropathol. 2011;122(6):673-690.

14Morris HR, Waite AJ, Williams NM, Neal JW, Blake DJ. Recent advances in the genetics of the ALS-FTLD complex. Curr. Neurol. Neurosci. Rep. 2012;12(3):243-250.

15Levine TP, Daniels RD, Gatta AT, Wong LH, Hayes MJ. The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs. Bioinformatics. 2013;29(4):499-503.

16Zhang D, Iyer LM, He F, Aravind L. Discovery of novel DENN proteins: implications for the evolution of eukaryotic intracellular membrane structures and human disease. Front. Genet. 2012;3.

17Reddy K, Zamiri B, Stanley SYR, Macgregor RB, Pearson CE. The Disease-associated r(GGGGCC)n Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA G-quadruplex Structures. J. Biol. Chem. 2013;288(14):9860-9866.

18Fratta P, Mizielinska S, Nicoll AJ, et al. C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes. Sci. Rep. 2012;2:1016.

19TLashley T, Hardy J, Isaacs AM. RANTing about C9orf72. Neuron. 2013;77(4):597-598.

20Kwon I, Xiang S, Kato M, et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science. 2014;345(6201):1139-1145.

21Mori K, Weng S-M, Arzberger T, et al. The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS. Science. 2013;339(6125):1335-1338.

Autophagosomes in HD

By: Yvette C. Wong, PhD and Erika L. F. Holzbaur, PhD

Original Article: The regulation of autophagosome dynamics by huntingtin and HAP1 is disrupted by expression of mutant huntingtin, leading to defective cargo degradation. (cited 30 times as of 2/8/2016)

Autophagy is an essential cellular pathway for degrading defective organelles and aggregated proteins, mediated by the formation of an autophagosome around its cargo and subsequent cargo degradation via lysosomal fusion.  In neurons, autophagosomes form constitutively at the axon tip and undergo robust retrograde axonal transport towards the cell body, during which they mature and gradually acidify via fusion with lysosomes along the axon. This retrograde transport is regulated by the retrograde motor protein dynein, but additional motor adaptors regulating autophagosome dynamics and maturation along the axon are also necessary to maintain this pathway.

Autophagy has previously been implicated in HD, as both soluble and aggregated mutant huntingtin (polyQ-Htt) are predominantly degraded via the autophagy pathway.

Huntingtin (Htt) and its adaptor protein huntingtin-associated protein-1 (HAP1) can act to scaffold motor proteins and regulate vesicular microtubule-based dynamics, as Htt binds dynein and HAP1 binds both the anterograde motor protein kinesin and the retrograde motor adaptor dynactin. We identified Htt and HAP1 as novel regulators of autophagosome axonal transport.1 Using live cell imaging of primary neurons from GFP-LC3 transgenic mice, we found that depleting either Htt or HAP1 in neurons resulted in decreased retrograde motility of autophagosomes and increased the stationary population of autophagosomes along the axon. In addition, Htt and HAP1 copurified and colocalized with neuronal autophagosomes along the axon. Interestingly, expression of Htt unable to bind dynein or HAP1 similarly disrupted autophagosome transport, suggesting that Htt forms a complex with both HAP1 and dynein to mediate the regulation of processive autophagosome transport in neurons.

We next examined whether autophagosome axonal transport might be disrupted by polyQ-Htt in HD and contribute to autophagy-related defects. We found that expression of polyQ-Htt in either primary neurons or striatal cells from HD homozygous knock-in mice was sufficient to disrupt the axonal transport of autophagosomes, leading to more stationary autophagosomes. Interestingly, we found that both wild-type and polyQ-Htt preferentially interacted with the neuronal-specific dynein isoform (DIC1A), rather than the ubiquitously expressed dynein isoform (DIC2C), suggesting that dynein-based transport of autophagosomes may be selectively impaired in neurons and contribute to the neuronal-selective degeneration observed in HD.

Finally, we investigated whether misregulation of autophagosome axonal transport might disrupt either upstream or downstream steps in the autophagy pathway. We found that Htt was not required for constitutive autophagosome formation at the axon tip, nor for cargo loading of ubiquitinated proteins or mitochondria into these constitutively-formed autophagosomes. However, defective autophagosome transport observed in both Htt-depleted neurons and polyQ-Htt-expressing neurons led to inefficient downstream degradation of autophagic cargo, such as engulfed mitochondrial fragments. Together, these results suggest that misregulation of the active transport of autophagosomes along the axon in HD may contribute to inefficient autophagosome maturation and cargo degradation, potentially due to inhibition of autophagosome-lysosome fusion along the axon (see Figure). The resulting defective clearance of cargo, including polyQ-Htt oligomers and aggregates, and dysfunctional mitochondria might further contribute to their neuronal accumulation, thus accelerating the progression towards cellular death in HD.

 Figure: Model of Htt’s regulation of autophagosome axonal transport in neurons. Htt and HAP1 normally regulate the motor activity of microtubule motors dynein, dynactin, and kinesin on autophagosomes, via Htt’s interactions with HAP1 and neuronal-specific dynein isoforms, to drive the robust retrograde transport of autophagosomes back to the cell body in neurons along microtubules (MT). Retrograde autophagosome transport is necessary for efficient fusion with lysosomes along the axon for degradation of autophagic cargo, such as mitochondria. In HD, pathogenic polyQ-Htt disrupts the Htt/HAP1 motor protein complex on autophagosomes, via altered polyQ-Htt/HAP1 association. This misregulation of motors leads to bidirectional/stationary autophagosome dynamics in HD neurons, thereby disrupting the retrograde transport of autophagosomes necessary for efficient degradation of dysfunctional mitochondria and polyQ-Htt.


Figure: Model of Htt’s regulation of autophagosome axonal transport in neurons.
Htt and HAP1 normally regulate the motor activity of microtubule motors dynein, dynactin, and kinesin on autophagosomes, via Htt’s interactions with HAP1 and neuronal-specific dynein isoforms, to drive the robust retrograde transport of autophagosomes back to the cell body in neurons along microtubules (MT). Retrograde autophagosome transport is necessary for efficient fusion with lysosomes along the axon for degradation of autophagic cargo, such as mitochondria. In HD, pathogenic polyQ-Htt disrupts the Htt/HAP1 motor protein complex on autophagosomes, via altered polyQ-Htt/HAP1 association. This misregulation of motors leads to bidirectional/stationary autophagosome dynamics in HD neurons, thereby disrupting the retrograde transport of autophagosomes necessary for efficient degradation of dysfunctional mitochondria and polyQ-Htt.

Our lab has subsequently shown that JNK-interacting protein-1 (JIP-1) also regulates autophagosome axonal transport,2 suggesting that autophagosome dynamics in neurons may be tightly regulated by several protein adaptors. We have also recently identified optineurin, a huntingtin interacting protein, as a novel autophagy receptor for damaged mitochondria in mitophagy.3 The latter observation is of particular interest given the link between mitochondrial dysfunction and HD pathogenesis,4 as well as the proposal that Htt functions as a scaffold for selective macroautophagy.5,6  Together, these observations suggest that further study of the mechanisms that regulate autophagosome dynamics in neurons will advance our understanding of how this essential degradative pathway is disrupted in HD.

1Wong YC, Holzbaur EL. The regulation of autophagosome dynamics by huntingtin and HAP1 is disrupted by expression of mutant huntingtin, leading to defective cargo degradation. J Neurosci. 2014;34(4):1293-1305.

2Fu MM, Nirschl JJ, Holzbaur EL. LC3 binding to the scaffolding protein JIP1 regulates processive dynein-driven transport of autophagosomes. Dev Cell. 2014;29(5):577-590.

3Wong YC, Holzbaur EL. Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation. Proc Natl Acad Sci U S A. 2014;111(42):E4439-4448.

4Guedes-Dias P, Pinho BR, Soares TR, de Proenca J, Duchen MR, Oliveira JM. Mitochondrial dynamics and quality control in Huntington’s disease. Neurobiol Dis. 2015.

5Rui YN, Xu Z, Patel B, et al. Huntingtin functions as a scaffold for selective macroautophagy. Nat Cell Biol. 2015;17(3):262-275.

6Ochaba J, Lukacsovich T, Csikos G, et al. Potential function for the Huntingtin protein as a scaffold for selective autophagy. Proc. Natl. Acad. Sci. U. S. A. 2014;111(47):16889-16894.