High-resolution respirometry of fine-needle muscle biopsies in pre-manifest Huntington's disease expansion mutation carriers shows normal mitochondrial respiratory function.

Alterations in mitochondrial respiration are an important hallmark of Huntington's disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.

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.

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.

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.

Serum Response Factor (SRF)-cofilin-actin signaling axis modulates mitochondrial dynamics.

Aberrant mitochondrial function, morphology, and transport are main features of neurodegenerative diseases. To date, mitochondrial transport within neurons is thought to rely mainly on microtubules, whereas actin might mediate short-range movements and mitochondrial anchoring. Here, we analyzed the impact of actin on neuronal mitochondrial size and localization. F-actin enhanced mitochondrial size and mitochondrial number in neurites and growth cones. In contrast, raising G-actin resulted in mitochondrial fragmentation and decreased mitochondrial abundance. Cellular F-actin/G-actin levels also regulate serum response factor (SRF)-mediated gene regulation, suggesting a possible link between SRF and mitochondrial dynamics. Indeed, SRF-deficient neurons display neurodegenerative hallmarks of mitochondria, including disrupted morphology, fragmentation, and impaired mitochondrial motility, as well as ATP energy metabolism. Conversely, constitutively active SRF-VP16 induced formation of mitochondrial networks and rescued huntingtin (HTT)-impaired mitochondrial dynamics. Finally, SRF and actin dynamics are connected via the actin severing protein cofilin and its slingshot phosphatase to modulate neuronal mitochondrial dynamics. In summary, our data suggest that the SRF-cofilin-actin signaling axis modulates neuronal mitochondrial function.

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

BACKGROUND: Neuronal degeneration, in particular in the striatum, and the formation of nuclear and cytoplasmic inclusions are characteristics of Huntington's 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. METHODS: We generated a new chronic in vitro model that is based on high-capacity adenovirus vector-mediated transduction of primary murine striatal and cortical neurons. Because the vector has a large capacity for transport of foreign DNA, it was possible to quantitatively express in these primary cells normal and mutant full-length htt (designed as fusion proteins with enhanced green fluorescent protein) in addition to its truncated versions. Pathological changes caused by mutant htt were characterized. RESULTS: The model mimicked several features observed in HD patients: prominent nuclear inclusions in cortical but not in striatal neurons, preferential neuronal degeneration of striatal neurons and neurofilament fragmentation in this cell type. Compared with expressed truncated mutant htt, the expression of full-length mutant htt in neurons resulted in a much slower appearance of pathological changes. Different from cortical neurons, the vast majority of nuclei in striatal cells contained only diffusely distributed N-terminal htt fragments. Cytoplasmic inclusions in both cell types contained full-length mutant htt. CONCLUSIONS: This model and the adenovirus vectors used will be valuable for studying the function of htt and the pathogenesis of HD at molecular and cellular levels in different neuronal cell types.

Myopathy as a first symptom of Huntington's disease in a Marathon runner.

A semi professional marathon runner at risk for Huntington's disease (HD) (43 CAG repeats) developed signs of a slowly progressive myopathy with exercise-induced muscle fatigue, pain, elevated creatine kinase level, and worsening of his running performance many years before first signs of chorea were detected. Muscle biopsy displayed a mild myopathy with mitochondrial pathology including a complex IV deficiency and analysis of the patient's fibroblast culture demonstrated deficits in mitochondrial function. Challenging skeletal muscle by excessive training might have disclosed myopathy in HD even years before the appearance of other neurological symptoms.

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.

Impairment of the ubiquitin-proteasome system by truncated cardiac myosin binding protein C mutants.

OBJECTIVE: Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHC patients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin-proteasome system (UPS). METHODS AND RESULTS: By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHC patients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70+/-4% and 11+/-5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS. CONCLUSION: Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC.

A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington's disease.

Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function. Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD). The network contains 186 PPIs among 35 bait and 51 prey proteins. It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments. The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles. Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions. Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis.

Protein surveillance machinery in brains with spinocerebellar ataxia type 3: redistribution and differential recruitment of 26S proteasome subunits and chaperones to neuronal intranuclear inclusions.

Intracellular aggregates commonly forming neuronal intranuclear inclusions are neuropathological hallmarks of spinocerebellar ataxia type 3 and of other disorders characterized by expanded polyglutamine-(poly-Q) tracts. To characterize cellular responses to these aggregates, we performed an immunohistochemical analysis of neuronal intranuclear inclusions in pontine neurons of patients affected by spinocerebellar ataxia type 3, using a panel of antibodies directed against chaperones and proteasome subunits. A subset of the neuronal intranuclear inclusions stained positively for the chaperones Hsp90alpha and HDJ-2, a member of the Hsp40 family. Most neuronal intranuclear inclusions were ubiquitin positive, suggesting degradation by ubiquitin-dependent proteasome pathways. Surprisingly, only a fraction of neuronal intranuclear inclusions were immunopositive for antibodies directed against subunits of the 20S proteolytic core, whereas most inclusions were stained by antibodies directed against subunits of the 11S and 19S regulatory particles. These results suggest that the proteosomal proteolytic machinery that actively degrades neuronal intranuclear inclusions is assembled in only a fraction of pontine neurons in end stage spinocerebellar ataxia type 3. The dissociation between regulatory subunits and the proteolytic core and the changes in subcellular subunit distribution suggest perturbations of the proteosomal machinery in spinocerebellar ataxia type 3 brains.