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Elevated levels of neurofilament light chain (NF-L) in CSF and blood are linked to the presymptomatic and symptomatic phase of patients suffering from amyotrophic lateral sclerosis (ALS). However, whether the NF-L level in extracellular liquids like serum or CSF is a marker of destruction or NF-L is secreted actively outside the cell is not known so far. NF-L levels in CSF and blood clearly separate ALS patients and controls [9], serving as a prognostic biomarker for ALS [4]. Also in pre-symptomatic ALS gene mutation carriers NF-L levels are elevated thus allowing prediction for clinical phenoconversion [3, 4]. The picture of NF-L levels in ALS mouse models is less clear. Previous studies report on elevated NF-L plasma levels in SOD1G93Adl and TDP-43 (TAR6/6) mice [6], but the correlation to motor neuron (MN) loss has not been determined. We therefore employed a transgenic TDP-43G298S mouse model to study the interaction of motor neuron pathophysiology, muscle denervation and NF-L levels. TDP-43G298S mutant mice show decreased performance in grip strength and motor activity compared to controls [10]., publishedVersion

ImportanceThe practice of using medical writers to communicate scientific information has gained popularity, but it may affect how and what information is communicated.ObjectiveTo assess characteristics of oncology trials that use medical writers and whether there is an association between the use of medical writers and trial success or the primary outcome evaluated.Design, Setting, and ParticipantsThis cross-sectional study included oncology trials testing a tumor-targeting intervention that were published in The Lancet, The Lancet Oncology, JAMA, JAMA Oncology, Journal of Clinical Oncology, and The New England Journal of Medicine between May 1, 2021, and May 1, 2022.ExposuresAssistance of medical writers or no assistance.Main Outcomes and MeasuresThe main outcomes were the percentage of studies with medical writers, the percentage of trial successes reported with medical writers, the association between trial success and medical writer use, and the association between a primary end point and medical writer use.ResultsAmong 270 studies, 141 (52.2%) included a medical writer and 129 (47.8%) did not include a medical writer. Of the studies that included a medical writer, 83 (58.9%) were successful. Of the studies that did not include a medical writer, 64 (49.6%) were successful (P = .16 for difference). Studies with medical writers were less likely than studies without medical writers to have the end point of overall survival (15 [10.6%] vs 17 [13.2%]) and disease-free or event-free survival (16 [11.3%] vs 29 [22.5%]), whereas studies with a medical writer were more likely to have the end point of progression-free survival (32 [22.7%] vs 17 [13.2%]). Use of medical writer was associated with the conclusions being presented favorably in all studies (113 [80.1%] vs 89 [69.0%]; odds ratio [OR], 1.81 [95% CI, 1.04-3.19]), but when adjusted for other variables, there was no association (OR, 1.84 [95% CI, 0.92-3.72]).Conclusions and RelevanceIn this cross-sectional study, trials using medical writers were more likely to report surrogate end points, such as progression-free survival, and favorable conclusions, but when adjusted for trial phase, randomization, and study funding, there was no association with favorable conclusions. These findings suggest that journals need heightened scrutiny for studies with medical writers and that authorship should be properly acknowledged.

The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In complexes of these proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains rich in HEAT repeats, and a smaller bridge domain connecting the two. We show that HAP40 steady-state protein levels are directly dependent on HTT (both normal and mutant HTT) and that HAP40 is strongly stabilized by the interaction with HTT resulting in an at least 5-fold increase in HAP40's half-life when bound to HTT. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of Huntington Disease (HD) patients and in brain tissue of a full-length HTT mouse model of HD, concomitant with decreased soluble HTT levels in these cell types. This data and our previous demonstration of coevolution between HTT and HAP40 and evolutionary conservation of their interaction suggest that HAP40 is an obligate interaction partner of HTT. Our observation of reduced HAP40 levels in HD invites further studies, whether HAP40 loss-of-function contributes to the pathophysiology of HD.

Background The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In a complex of the two proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains, rich in HEAT repeats, and a smaller bridge domain that connects the two. When generating HEK293-based cell lines for producing the HTT-HAP40 complex for structural studies (Guo et al. 2020, Nature), enhancing HTT expression resulted in increased HAP40 steady-state levels. Aims/Methods By Western Blot analysis, we investigated whether HAP40 steady-state protein levels are directly dependent on HTT levels and whether HAP40 protein levels are decreased in samples (lymphocytes and fibroblasts) from Huntington’s disease (HD) patients and mouse models alongside HTT levels. Moreover, we assessed by cycloheximide-chase assay and qRT-PCR, respectively, if HAP40 protein stability or HAP40/F8A mRNA levels are dependent on HTT levels. Results We show that steady-state protein levels of HAP40 strongly correlated with and were directly dependent on HTT protein expression levels. While HAP40 alone had a very short half-life, HAP40 was stabilized upon HTT overexpression. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of HD patients and in brain tissue of a well-characterized HD mouse model, concomitantly with decreased HTT levels in these cell types. Conclusion This data, together with our previous demonstration of likely coevolution of the two proteins and evolutionary conservation of their interaction in metazoans (Seefelder et al. 2020, BMC Evolutionary Biology) suggests that HAP40 is an obligate and likely functionally important interaction partner of HTT. Our observation of reduced HAP40 levels in HD indicates that loss-of-function phenotypes due to lowered HAP40 levels might contribute to the pathology in HD.

Heterozygous loss-of-function mutations of TANK-binding kinase 1 ( TBK1 ) cause familial ALS, yet downstream mechanisms of TBK1 mutations remained elusive. TBK1 is a pleiotropic kinase involved in the regulation of selective autophagy and inflammation. We show that heterozygous Tbk1 deletion alone does not lead to signs of motoneuron degeneration or disturbed autophagy in mice during a 200-d observation period. Surprisingly, however, hemizygous deletion of Tbk1 inversely modulates early and late disease phases in mice additionally overexpressing ALS-linked SOD1 G93A , which represents a “second hit” that induces both neuroinflammation and proteostatic dysregulation. At the early stage, heterozygous Tbk1 deletion impairs autophagy in motoneurons and prepones both the clinical onset and muscular denervation in SOD1 G93A /Tbk1 +/− mice. At the late disease stage, however, it significantly alleviates microglial neuroinflammation, decelerates disease progression, and extends survival. Our results indicate a profound effect of TBK1 on brain inflammatory cells under pro-inflammatory conditions and point to a complex, two-edged role of TBK1 in SOD1 -linked ALS.

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.

This study shows that mice with an ALS-linked heterozygous Tbk1 deletion are normal until at least 200 d of age. However, proteostatic and neuroinflammatory challenge by additional expression of mutant SOD1 unmasks a two-edged role of TBK1 in motoneuron disease., Heterozygous loss-of-function mutations of TANK-binding kinase 1 (TBK1) cause familial ALS, yet downstream mechanisms of TBK1 mutations remained elusive. TBK1 is a pleiotropic kinase involved in the regulation of selective autophagy and inflammation. We show that heterozygous Tbk1 deletion alone does not lead to signs of motoneuron degeneration or disturbed autophagy in mice during a 200-d observation period. Surprisingly, however, hemizygous deletion of Tbk1 inversely modulates early and late disease phases in mice additionally overexpressing ALS-linked SOD1G93A, which represents a “second hit” that induces both neuroinflammation and proteostatic dysregulation. At the early stage, heterozygous Tbk1 deletion impairs autophagy in motoneurons and prepones both the clinical onset and muscular denervation in SOD1G93A/Tbk1+/− mice. At the late disease stage, however, it significantly alleviates microglial neuroinflammation, decelerates disease progression, and extends survival. Our results indicate a profound effect of TBK1 on brain inflammatory cells under pro-inflammatory conditions and point to a complex, two-edged role of TBK1 in SOD1-linked ALS., Graphical Abstract

The modulation of the brain endocannabinoid system has been identified as an option to treat neurodegenerative diseases including Parkinson's disease (PD). Especially the elevation of endocannabinoid levels by inhibition of hydrolytic degradation represents a valuable approach. To evaluate whether monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) inhibition could be beneficial for PD, we examined in parallel the therapeutic potential of the highly selective MAGL inhibitor KML29 elevating 2-arachidonoylglyerol (2-AG) levels and the highly selective FAAH inhibitor PF-3845 elevating anandamide (AEA) levels in a chronic methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid (MPTP/probenecid) mouse model of PD. Chronic administration of KML29 (10 mg/kg) but not PF-3845 (10 mg/kg) attenuated striatal MPTP/probenecid-induced dopamine depletion. Furthermore, KML29 induced an increase in Gdnf but not Bdnf expression, whereas PF-3845 decreased the MPTP/probenecid-induced Cnr2 expression without any effects on neurotrophin expression. Investigation of treatment-naive striatal mRNA levels revealed a high presence of Gdnf and Mgll in contrast to Bdnf and Faah. Treatment of primary mouse microglia with 2-AG increased Gdnf but not Bdnf expression, suggesting that microglia might mediate the observed KML29-induced increase in Gdnf. In summary, pharmacological MAGL but not FAAH inhibition in the chronic MPTP/probenecid model attenuated the MPTP/probenecid-induced effects on striatal dopamine levels which were accompanied by an increase in 2-AG levels.

International audience; 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.

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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system with limited therapeutic options. While an increasing number of ALS patients can be linked to a small number of autosomal-dominantly inherited cases, most cases are termed sporadic. Both forms are clinically and histopathologically indistinguishable, raising the prospect that they share key pathogenic steps, including potential therapeutic intervention points. The endocannabinoid system is emerging as a versatile, druggable therapeutic target in the CNS and its dysregulation is an early hallmark of neurodegeneration. Whether this is a defense mechanism or part of the pathogenesis remains to be determined. The neuroprotective and anti-inflammatory endocannabinoid 2-arachidonoylglycerol (2-AG), which is degraded by monoacylglycerol lipase (MAGL), accumulates in the spinal cords of transgenic models of ALS. We tested the hypothesis that this 2-AG increase is a protective response in the low-copy SOD1 G93A mouse model of ALS. We show that oral application of the MAGL inhibitor KML29 delays disease onset, progression and survival. Furthermore, we could demonstrate that KML29 reduced proinflammatory cytokines and increased brain-derived neurotrophic factor (BDNF) expression levels in the spinal cord, the major site of neurodegeneration in ALS. Moreover, treatment of primary mouse neurons and primary mousecroglia with 2-AG confirmed the neuroprotective and anti-inflammatory action by increasing BDNF and arginase-1 and decreasing proinflammatory cytokines in vitro . In summary, we show that elevating 2-AG levels by MAGL inhibition is a therapeutic target in ALS and demonstrate that the endocannabinoid defense mechanisms can be exploited therapeutically in neurodegenerative diseases. This article is part of the Special Issue entitled “A New Dawn in Cannabinoid Neurobiology”.

The neurodegenerative genetic disorder of Huntington’s disease (HD) is characterised by mitochondrial impairments of the respiratory chain. The ubiquitous expression of the disease causing mutant huntingtin gene raises the question to which extent changes in mitochondrial respiration are evident in the human skeletal muscle. In addition characterisation of mitochondrial respiration in the muscle might allow conclusions about the respiratory status in the brain. The integrated respiratory chain function of the human quadriceps vastus lateralis was measured by high-resolution respirometry in fine-needle biopsies of four pre-symptomatic HD mutation carriers and seven controls. The respiratory parameters indicated a trend towards a reduction in the respiratory control ratio (RCR) of the HD carriers. In parallel, murine cortex, liver, soleus muscle and heart of male HD knock-in mice (HdhQ111), were examined by the same method. Significant changes of the respiration were restricted to the liver and the cortex. In addition mitochondrial DNA copy number and citrate synthase activity were determined to quantify the mitochondrial mass, showing no differences. From the murine tissues mRNA levels of key enzymes characterised the mitochondrial metabolic pathways. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that differences in respiratory parameters of pre-symptomatic human muscle biopsies are rather limited, which is confirmed by the analysis of murine skeletal muscle tissue. The murine cortex and liver turned out to show respiratory changes in the HdhQ111 mouse model, which indicates that respiratory capacities are different between tissues. Acknowledgement The work is supported by the research training group cellular and molecular mechanisms in ageing (CEMMA, GRK1789) which is funded by the DFG and the seed fund grant of the EINSTEIN study no 584/14 from the EHDN.