Research Round-Up: Insights of the Year 2014-15

In our final issue of 2015, HD Insights highlights influential HD research in the 2014-2015 academic year. The winners of last year’s Insights of the Year competition nominated nine papers in lab, clinical, and imaging and biomarkers research. The Editorial Board and other researchers voted for the most influential papers, selecting one each in basic and clinical science and two in imaging and biomarkers as most influential. Authors of the most influential papers will present their research in a special panel discussion at the 2015 HSG annual meeting. Congratulations to all the nominees and winners!

In the lab…
Most influential paper
Targeting ATM ameliorates mutant huntingtin toxicity in cell and animal models of HD
By: Lu XH, Mattis VB, Wang N, Al-Ramahi I, van den Berg N4, Fratantoni SA, Waldvogel H, Greiner E, Osmand A, Elzein K, Xiao J, Dijkstra S, de Pril R, Vinters HV, Faull R, Signer E, Kwak S, Marugan JJ, Botas J, Fischer DF, Svendsen CN, Munoz-Sanjuan I, Yang XW

(Summary by X. William Yang, MD, PhD)

Elevated ATM signaling in HD cells, mouse & patient brains

Figure: Diagram of TrkBR and p75NTR signaling in HD. TrkBR activation of PI3K and AKT leads to the induction of long-term potentiation (LTP) in striatal projection neurons (SPNs). This is attenuated in iSPNs in HD by downstream targets of p75NTR signaling.

Emerging evidence suggests that DNA damage and repair pathways can contribute to the pathogenesis of a number of neurodegenerative disorders. We investigated the pathogenic role in HD of ataxia-telangiectasia mutated (ATM), a protein kinase involved in DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been observed in Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and in polyglutamine disorders such as HD and spinocerebellar ataxia type 3 (SCA3). We showed that ATM signaling was consistently elevated in cells derived from the BACHD mouse model and in disease-vulnerable brain tissues from BACHD mice and HD patients (Figure).
ATM reduction mitigated toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we showed that genetic reduction of one copy of the Atm gene ameliorated multiple behavioral deficits, and partially improved neuropathology. Small-molecule ATM inhibitors, originally developed for radiosensitization in cancer therapy, reduced mHTT-induced cell death in a rat cortico-striatal neuronal co-culture assay and in induced pluripotent stem cells (iPSCs) derived from HD patients. Our study provides converging genetic and pharmacological evidence that partial reduction of ATM signaling could ameliorate mHTT toxicity in a number of cellular and animal models of HD, suggesting that ATM maybe a useful therapeutic target for HD.

 

 

 

Aberrant Astrocytes

Figure: Blood oxygen level-dependent magnetic resonance imaging with carbon challenge revealed impaired vascular reactivity in the cortical vessels of R6/2 mice (right) as compared to wild-type (left). The arrowheads represent the vessels with low vascular reactivity.

Aberrant astrocytes impair vascular reactivity in Huntington’s disease
By: Hsiao HY, Chen YC, Huang CH, Chen CC, Hsu YH, Chen HM, Chiu FL, Kuo HC, Chang C, Chern Y

(Summary by Chien-Hsiang Huang, PhD, Chen Chang, PhD, and Yijuang Chern, PhD)

Increased blood vessel density has been recognized in animals and patients with HD. Our study further reveals that a fraction of the cortical blood vessels in the brains of HD mice (R6/2) is nonreactive (Figure). Such impaired blood vessels in HD brains result from aberrant astrocytes that release too much vascular endothelial growth factor and inflammatory mediators. The impaired vascular reactivity causes abnormal regulation of cerebral blood flow and may be responsible for brain atrophy in R6/2 mice. Whether the formation of the nonreactive vessels is detrimental or beneficial to HD remains to be elucidated. The nonreactive vessels warrant future investigation in clinical settings, and the integrity of vascular reactivity may be used as a new biomarker to evaluate the progression of HD.

 

 

 

 

Figure: Diagram of TrkBR and p75NTR signaling in HD. TrkBR activation of PI3K and AKT leads to the induction of long-term potentiation (LTP) in striatal projection neurons (SPNs). This is attenuated in iSPNs in HD by downstream targets of p75NTR signaling.

Figure: Diagram of TrkBR and p75NTR signaling in HD. TrkBR activation of PI3K and AKT leads to the induction of long-term potentiation (LTP) in striatal projection neurons (SPNs). This is attenuated in iSPNs in HD by downstream targets of p75NTR signaling.

Impaired TrkB receptor signaling underlies corticostriatal dysfunction in Huntington’s disease
By: Plotkin JL, Day M, Peterson JD, Xie Z, Kress GJ, Rafalovich I, Kondapalli J, Gertler TS, Flajolet M, Greengard P, Stavarache M, Kaplitt MG, Rosinski, J, Chan CS, Surmeier DJ

(Summary by Joshua L. Plotkin, PhD)

Reduced trophic support of striatal projection neurons (SPNs) is thought to play a key role in the progression of HD. This has been widely attributed to diminished levels of brain-derived neurotrophic factor (BDNF). In a recent study, we showed that BDNF signaling through tyrosine-related kinase B receptors (TrkBRs) was indeed attenuated in the striatum of early symptomatic BACHD and Q175 mouse models of HD, but without changes in BDNF expression or delivery to the striatum. This attenuation led to a synapse-specific loss of corticostriatal long-term potentiation, and progressive weakening of cortical synapses to indirect pathway SPNs (iSPNs), a circuit pathology consistent with the early choreic symptoms of HD.

Although TrkBRs were activated normally, their signaling was blunted by engagement of another target of BDNF: p75 neurotrophin receptors (p75NTRs). Elevated p75NTR signaling occurred due to increased expression of its downstream target: phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is an inhibitor of TrkBR signaling, and was specifically up-regulated in iSPNs (Figure). Synaptic potentiation could be rescued in HD iSPNs by inhibiting 75NTRs, PTEN, or intermediate signaling partners. This study suggests that targeting postsynaptic p75NTR signaling, rather than presynaptic BDNF delivery, offers a promising therapeutic strategy for HD.

 

 

In the clinic…
Most influential paper

Figure: Levels of mHTT measured in cererebrospinal fluid from control, premanifest, and manifest HD patients

Figure: Levels of mHTT measured in cererebrospinal fluid from control, premanifest, and manifest HD patients

Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients
By: Wild EJ, Boggio R, Langbehn D, Robertson N, Haider S, Miller JR, Zetterberg H, Leavitt BR, Kuhn R, Tabrizi SJ, Macdonald D, Weiss A

(Summary by Edward J. Wild, PhD)

The genetic cause of HD – mHTT, encoded by the CAG-expanded mHTT gene – has been known since 1993. Clinical trials of “gene silencing” drugs that aim to reduce the production of mHTT in the brain are now underway in HD patients.

Quantifying mHTT in the central nervous system (CNS) would be helpful for understanding whether these therapies are having the desired effect, and would aid in studying the biology of mHTT in HD. However, mHTT accumulates inside cells, and its concentration in cerebrospinal fluid (CSF) is very low. Until now, mHTT could not be detected in the CNS.

We have developed a new, ultra-sensitive, “single molecule counting” immunoassay to detect and measure the concentration of mHTT in CSF. The immunoassay uses an antibody pair specific to mHTT over wild-type huntingtin. Fluorescence events corresponding to single molecules of antibody-bound protein are counted, enabling precise quantification at femtomolar concentrations.

We analyzed CSF from volunteers from two cohorts and for the first time, successfully quantified mHTT in mHTT carriers. We showed that mHTT levels in premanifest HD were intermediate between control and manifest HD levels (Figure). Moreover, the concentration of mHTT independently predicted disease burden as well as motor and cognitive scores. Associations with known neuronal proteins suggest the mHTT detectable in CSF is derived from dying neurons. We now plan to use this new assay to determine whether the level of mHTT in CSF can predict HD onset or rate of progression, and to study the first “gene silencing” drugs in clinical trials.

 

 

Figure: (Background) Pathological tau aggregates in an HD brain (Foreground left) George Huntington, MD (1850−1916), who first described the disease that now bears his name, in a report titled “On chorea” published in The Medical and Surgical Reporter of Philadelphia on April 13, 1872

Figure: (Background) Pathological tau aggregates in an HD brain (Foreground left) George Huntington, MD (1850−1916), who first described the disease that now bears his name, in a report titled “On chorea” published in The Medical and Surgical Reporter of Philadelphia on April 13, 1872

The role of tau in the pathological process and clinical expression of Huntington’s disease
By: Vuono R, Winder-Rhodes S, de Silva R, Cisbani G, Drouin-Ouellet J; REGISTRY Investigators of the European Huntington’s Disease Network,
Spillantini MG, Cicchetti F, Barker RA

(Summary by Romina Vuono, PhD)

More than twenty years after the discovery of the genetic defect responsible for HD, there are still many unanswered questions. For example, why do HD patients differ dramatically in their age at onset of manifest disease and disease progression, despite similar CAG repeat lengths? Recent studies have suggested that other genetic influences may play a role. So far, more than 24 genes have been linked to HD, but their relevance remains unclear as to whether, and how, they may drive the HD pathogenic cascade and resultant clinical features.

We have recently shown that the microtubule-associated protein tau gene (MAPT), known to be associated with many neurodegenerative diseases, can give rise to pathology in the brains of HD patients. We have reported pathological tau aggregates, as well as tau oligomers (thought to be the most toxic form of tau), in postmortem HD brain tissues (Figure). These findings were common to young-onset as well as older HD patients, confirming that tau pathology was related to the disease process, rather than related to age. We highlighted the clinical significance of this pathology by demonstrating that a genetic variation of the MAPT gene (MAPT H2 haplotype) affected the rate of cognitive decline in a large cohort of HD patients. Our findings highlight a novel and important role for tau in the pathogenic process and clinical expression of HD, which in turn may open up new therapeutic approaches.

 

 

Figure: Tau nuclear rods (TNRs), a newly identified kind of tau deposit in the brains of individuals with HD

Figure: Tau nuclear rods (TNRs), a newly identified kind of tau deposit in the brains of individuals with HD

Huntington’s disease is a four-repeat tauopathy with tau nuclear rods
By: Fernández-Nogales M, Cabrera JR, Santos-Galindo M, Hoozemans JJ, Ferrer I, Rozemuller AJ, Hernández F, Avila J, Lucas JJ

(Summary by Marta Fernández-Nogales, PhD)

Intronic mutations that produce an alteration in exon 10 alternative splicing of the MAPT (Tau) gene in frontotemporal dementia with parkinsonism associated with chromosome 17 (FTDP-17) result in an increase of tau isoforms with four repeats of microtubule binding domains. This is sufficient to cause neurodegeneration, though the reason is not yet known. It is possible that these tau binding domains increase the binding energy of tau to the microtubules.

We discovered an increase in tau isoforms with four microtubule binding domains in the cortex of HD patients and a concomitant decrease in isoforms with three microtubule binding domains, as well as an increase in total tau levels, similar to that observed in other dementias. This change is accompanied by the appearance of a new tau-histopathological hallmark in the brains of HD patients, consisting of deposits of rod-shaped tau protein that fill previously reported invaginations of the nuclei of some striatal and cortical neurons (Figure). We have named these inclusions “tau nuclear rods.”

Furthermore, we were able to demonstrate that tau protein plays a role in the pathogenesis of HD by verifying that the partial or total reduction of tau in an HD mouse model produces a significant improvement in motor coordination. This discovery opens the possibility of using emergent pharmacological tools under development for the treatment of other tauopathies in HD patients.

 

 

In imaging and biomarkers…
Most influential paper

Figure: The main cerebrovascular compromise in HD patients and in an HD mouse model of the disease, comprised of mhtt expression in cell types associated with blood vessels, the alteration of blood vessel morphology and an increase in blood-brain barrier permeability

Figure: The main cerebrovascular compromise in HD patients and in an HD mouse model of the disease, comprised of mhtt expression in cell types associated with blood vessels, the alteration of blood vessel morphology and an increase in blood-brain barrier permeability

Cerebrovascular and blood-brain barrier impairments in Huntington’s disease: potential implications for its pathophysiology
By: Drouin-Ouellet J, Sawiak SJ, Cisbani G, Lagacé M, Kuan WL, Saint-Pierre M, Dury RJ, Alata W, St-Amour I, Mason SL, Calon F, Lacroix S, Gowland, PA, Francis ST, Barker RA, Cicchetti F

(Summary by Janelle Drouin-Ouellet, PhD)

We report here on our work using a combination of approaches to show that the cerebrovasculature is compromised at four different levels in the context of HD. We show that:
1) mHTT protein aggregates are found in all compartments of the neurovascular unit
2) blood vessels are more dense but smaller in size
3) this change in size and density is accompanied by an increased blood-brain barrier permeability that leads to
peripheral blood cell infiltration, and
4) there are mHTT aggregates in transcytotic vesicles.

These observations appeared on MRI and in postmortem tissue in both the R6/2 mouse model and HD patients (Figure).
These findings raise critical questions regarding the impact of vascular alterations on neuronal network activity and degeneration in HD, and could indicate a more significant crosstalk between elements of the blood and the CNS than originally thought. This could in turn create an increased inflammatory response as a result of immune cell infiltration to the brain and facilitate mHTT accumulation due to the leaky blood-brain barrier. We aim to further elucidate the contribution of blood-brain barrier compromise, and the resulting presence of inflammatory elements, to the pathophysiology and progression of HD.

 

 

 

Most influential paper

Figure: Schematic of detection of mHTT in CSF by immunoprecipitation and flow cytometry

Figure: Schematic of detection of mHTT in CSF by immunoprecipitation and flow cytometry

Ultrasensitive measurement of huntingtin protein in cerebrospinal fluid demonstrates increase with Huntington disease stage and decrease following brain huntingtin suppression
By: Southwell AL, Smith SE, Davis TR, Caron NS, Villanueva EB, Xie Y, Collins JA, Li Ye M, Sturrock A, Leavitt BR, Schrum AG, Hayden MR

(Summary by Amber L. Southwell, PhD)

The lack of quantitative, robust, and reliable biomarkers for use in early subclinical diagnosis is a major obstacle for development of disease-modifying therapies for HD. Such markers are needed to assist in monitoring disease Progression, patient stratification, and evaluating efficacy of therapeutics in the clinic.

The Hayden lab at the University of British Columbia (UBC) has collaborated with colleagues at UBC and the
Mayo Clinic to develop an ultrasensitive method of measuring mHTT in the cerebrospinal fluid (CSF) of HD patients and model mice, called microbead-based immunoprecipitation and flow cytometry (IP-FCM), that could meet these needs. See Figure for a schematic that summarizes the method.

Using IP-FCM, we have shown that mHTT in the CSF originates in the brain and is released by injured or dying brain cells. Levels of mHTT in the CSF increase with worsening HD symptoms, suggesting that this approach will be useful as a measure of disease progression.

Additionally, lowering mHTT in the brain using gene-silencing treatments results in a measurable reduction of mHTT in the CSF, indicating that this method could be used to verify and quantify changes in brain mHTT levels in response to experimental therapies. This advance is clinically relevant and will enable more rigorous assessment of important endpoints in evaluating therapies for HD.

 

 

Figure: Schematic of mutant Huntingtin (mHTT) aggregation assays using whole-cell lysates (supernatants, middle of figure) and enhanced aggregation in whole cells prepared from the PC-12 cell model of HD

Figure: Schematic of mutant Huntingtin (mHTT) aggregation assays using whole-cell lysates (supernatants, middle of figure) and enhanced aggregation in whole cells prepared from the PC-12 cell model of HD

Huntington’s disease cerebrospinal fluid seeds aggregation of mutant huntingtin

By: Tan Z, Dai W, van Erp TG, Overman J, Demuro A, Digman MA, Hatami A, Albay R, Sontag EM, Potkin KT, Ling S, Macciardi F, Bunney WE, Long JD, Paulsen JS, Ringman JM, Parker I, Glabe C, Thompson LM, Chiu W, Potkin SG

(Summary by Zhiqun Tan, PhD, and Steven G. Potkin, MD)

The onset of symptoms in individuals who carry the mutant HD allele (mHTT) remains variable and unpredictable. To speed development of new therapies, biomarkers that reflect the development of the HD pathological process are quantitatively associated with the course of illness are needed. We reported that oligomeric peptides that contain the polyglutamine expansion coded by mHTT, and CSF fluid from individuals with HD, and BACHD transgenic rats, enhance seeded aggregation in a PC-12 cell model and its cell lysate. Oligomers derived from other proteins, including Aβ and α-synuclein, do not induce seeding. We demonstrate that seeding is mHTT template-specific and may reflect an underlying cell-to-cell mechanism of huntingtinopathy propagation.

Light and cryo-electron microscopy (cryo-EM) confirm that synthetic seeds nucleate and enhance mHTT aggregation in cell lysates. This seeding assay (Figure) distinguishes individuals with HD from healthy controls. Seeding measures in asymptomatic gene-positive individuals fall between HD patients and controls. This quantifiable seeding property in the CSF of individuals with HD may serve as a molecular biomarker assay to monitor HD progression, and to evaluate therapies that target mHTT. The automation of the assay is in process.