Katrin S. Lindenberg

Proteases Acting on Mutant Huntingtin Generate Cleaved Products that Differentially Build Up Cytoplasmic and Nuclear Inclusions

Proteolytic processing of mutant huntingtin (mhtt) is regarded as a key event in the pathogenesis of Huntingtons disease (HD). Mhtt fragments containing a polyglutamine expansion form intracellular inclusions and are more cytotoxic than full-length mhtt. Here, we report that two distinct mhtt fragments, termed cp-A and cp-B, differentially build up nuclear and cytoplasmic inclusions in HD brain and in a cellular model for HD. Cp-A is released by cleavage of htt in a 10 amino acid domain and is the major fragment that aggregates in the nucleus. Furthermore, we provide evidence that cp-A and cp-B are most likely generated by aspartic endopeptidases acting in concert with the proteasome to ensure the normal turnover of htt. These proteolytic processes are thus potential targets for therapeutic intervention in HD.

Mitochondrial impairment in patients and asymptomatic mutation carriers of Huntington's disease

Huntingtons disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the IT‐15 gene; however, it remains unknown how the mutation leads to selective neurodegeneration. Several lines of evidence suggest impaired mitochondrial function as a component of the neurodegenerative process in HD. We assessed energy metabolism in the skeletal muscle of 15 HD patients and 12 asymptomatic mutation carriers in vivo using 31P magnetic resonance spectroscopy. Phosphocreatine recovery after exercise is a direct measure of ATP synthesis and was slowed significantly in HD patients and mutation carriers in comparison to age‐ and gender‐matched healthy controls. We found that oxidative function is impaired to a similar extent in manifest HD patients and asymptomatic mutation carriers. Our findings suggest that mitochondrial dysfunction is an early and persistent component of the pathophysiology of HD.

Expanded polyglutamines impair synaptic transmission and ubiquitin–proteasome system in Caenorhabditis elegans

Polyglutamine (polyQ) expansion in many proteins, including huntingtin and ataxin‐3, is pathogenic and responsible for neuronal dysfunction and degeneration. Although at least nine neurodegenerative diseases are caused by expanded polyQ, the pathogenesis of these diseases is still not well understood. In the present study, we used Caenorhabditis elegans to study the molecular mechanism of polyQ‐mediated toxicity. We expressed full‐length and truncated ataxin‐3 with different lengths of polyQ in the nervous system of C. elegans. We show that expanded polyQ interrupts synaptic transmission, and induces swelling and aberrant branching of neuronal processes. Using an ubiquitinated fluorescence reporter construct, we also showed that polyQ aggregates impair the ubiquitin–proteasome system in C. elegans. These results may provide information for further understanding the pathogenesis of polyQ diseases.

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse, a model of Huntington’s disease

A mouse model of Huntington’s disease shows impaired calcium handling in skeletal muscle, potentially contributing to the pathology of the disease.

Two-Point Magnitude MRI for Rapid Mapping of Brown Adipose Tissue and Its Application to the R6/2 Mouse Model of Huntington Disease

The recent discovery of active brown fat in human adults has led to renewed interest in the role of this key metabolic tissue. This is particularly true for neurodegenerative conditions like Huntington disease (HD), an adult-onset heritable disorder with a prominent energy deficit phenotype. Current methods for imaging brown adipose tissue (BAT) are in limited use because they are equipment-wise demanding and often prohibitively expensive. This prompted us to explore how a standard MRI set-up can be modified to visualize BAT in situ by taking advantage of its characteristic fat/water content ratio to differentiate it from surrounding white fat. We present a modified MRI protocol for use on an 11.7 T small animal MRI scanner to visualize and quantify BAT in wild-type and disease model laboratory mice. In this application study using the R6/2 transgenic mouse model of HD we demonstrate a significantly reduced BAT volume in HD mice vs. matched controls (n = 5 per group). This finding provides a plausible structural explanation for the previously described temperature phenotype of HD mice and underscores the significance of peripheral tissue pathology for the HD phenotype. On a more general level, the results demonstrate the feasibility of MR-based BAT imaging in rodents and open the path towards transferring this imaging approach to human patients. Future studies are needed to determine if this method can be used to track disease progression in HD and other disease entities associated with BAT abnormalities, including metabolic conditions such as obesity, cachexia, and diabetes.

ALS-causing mutations differentially affect PGC-1α expression and function in the brain vs. peripheral tissues.

BACKGROUND Monogenetic forms of amyotrophic lateral sclerosis (ALS) offer an opportunity for unraveling the molecular mechanisms underlying this devastating neurodegenerative disorder. In order to identify a link between ALS-related metabolic changes and neurodegeneration, we investigated whether ALS-causing mutations interfere with the peripheral and brain-specific expression and signaling of the metabolic master regulator PGC (PPAR gamma coactivator)-1α (PGC-1α). METHODS We analyzed the expression of PGC-1α isoforms and target genes in two mouse models of familial ALS and validated the stimulated PGC-1α signaling in primary adipocytes and neurons of these animal models and in iPS derived motoneurons of two ALS patients harboring two different frame-shift FUS/TLS mutations. RESULTS Mutations in SOD1 and FUS/TLS decrease Ppargc1a levels in the CNS whereas in muscle and brown adipose tissue Ppargc1a mRNA levels were increased. Probing the underlying mechanism in neurons, we identified the monocarboxylate lactate as a previously unrecognized potent and selective inducer of the CNS-specific PGC-1α isoforms. Lactate also induced genes like brain-derived neurotrophic factor, transcription factor EB and superoxide dismutase 3 that are down-regulated in PGC-1α deficient neurons. The lactate-induced CNS-specific PGC-1α signaling system is completely silenced in motoneurons derived from induced pluripotent stem cells obtained from two ALS patients harboring two different frame-shift FUS/TLS mutations. CONCLUSION ALS mutations increase the canonical PGC-1α system in the periphery while inhibiting the CNS-specific isoforms. We identify lactate as an inducer of the neuronal PGC-1α system directly linking brain metabolism and neuroprotection. Changes in the PGC-1α system might be involved in the ALS accompanied metabolic changes and in neurodegeneration.

Comparison of Sirtuin 3 Levels in ALS and Huntington’s Disease—Differential Effects in Human Tissue Samples vs. Transgenic Mouse Models

Neurodegenerative diseases are characterized by distinct patterns of neuronal loss. In amyotrophic lateral sclerosis (ALS) upper and lower motoneurons degenerate whereas in Huntington’s disease (HD) medium spiny neurons in the striatum are preferentially affected. Despite these differences the pathophysiological mechanisms and risk factors are remarkably similar. In addition, non-neuronal features, such as weight loss implicate a dysregulation in energy metabolism. Mammalian sirtuins, especially the mitochondrial NAD+ dependent sirtuin 3 (SIRT3), regulate mitochondrial function and aging processes. SIRT3 expression depends on the activity of the metabolic master regulator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a modifier of ALS and HD in patients and model organisms. This prompted us to systematically probe Sirt3 mRNA and protein levels in mouse models of ALS and HD and to correlate these with patient tissue levels. We found a selective reduction of Sirt3 mRNA levels and function in the cervical spinal cord of end-stage ALS mice (superoxide dismutase 1, SOD1G93A). In sharp contrast, a tendency to increased Sirt3 mRNA levels was found in the striatum in HD mice (R6/2). Cultured primary neurons express the highest levels of Sirt3 mRNA. In primary cells from PGC-1α knock-out (KO) mice the Sirt3 mRNA levels were highest in astrocytes. In human post mortem tissue increased mRNA and protein levels of Sirt3 were found in the spinal cord in ALS, while Sirt3 levels were unchanged in the human HD striatum. Based on these findings we conclude that SIRT3 mediates the different effects of PGC-1α during the course of transgenic (tg) ALS and HD and in the human conditions only partial aspects Sirt3 dysregulation manifest.

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington’s Disease

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.

Adenovirus vector-based in vitro neuronal cell model for Huntington's disease with human disease-like differential aggregation and degeneration

Neuronal degeneration, in particular in the striatum, and the formation of nuclear and cytoplasmic inclusions are characteristics of Huntingtons disease (HD) as a result of the expansion of a polyglutamine tract located close to the N‐terminus of huntingtin (htt). Because of the large (10‐kb) size of the htt cDNA, expression of full‐length htt in primary neurons has proved difficult in the past.

A34 Mitochondrial respiration is limited by atp-production in the skeletal muscle of the R6/2 hd mouse model

Background Previous work has shown that mitochondrial respiration is not severely affected in the skeletal muscle of pre-symptomatic mHTT-mutation carriers.1 In contrast, patients with manifest HD exhibit a low anaerobic threshold and an increased skeletal muscle lactate production.2 This could result not only from a decreased capacity of the mitochondrial respiratory chain but also from a compromised ATP production process, which depends from the activity of the ATP-synthase and of the adenine nucleotide translocator. Aim The scope of the present study was therefore to compare ex-vivo the maximum respiratory activity in the coupled (OxPhos)-state and in the uncoupled (ETS)-state in skeletal muscle tissue samples from 12 weeks old R6/2 HD model male mice. Methods The mitochondrial respiratory activity in the homogenized tissue samples from 12 R6/2 mice and 11 wildtype controls were quantified according to previously published protocols.1 Results In the skeletal muscle from R6/2 mice we found a lower OxPhos-activity (118±47 [pmol O2/(s x mg tissue)]) compared to control (183±54 [pmol O2/(s x mg tissue)], p=0.006). The activity in the ETS-state did not show a statistically significant difference (148±59 [pmol O2/(s x mg tissue)] vs. 189±80 [pmol O2/(s x mg tissue)], p=0.21). The ratio of both capacities (OxPhos/ETS) was close to unity in the controls, but statistically significantly lower in the R6/2 mice (103±17 [%] vs. 82±6 [%], p=0.005) Conclusions We conclude that in HD, in addition to a possible reduction of the capacity of the mitochondrial respiratory chain, the ATP-production process may also assume a strong limiting role with regard to aerobic metabolism of the skeletal muscle. References . Buck, et al. PLoS One2017. https://doi.org/10.1371/journal.pone.0175248 . Ciammola, et al. Movement Disorders2011;26:130–7.