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.

One sentence summaryCerebral dopamine neurotrophic factor (CDNF) was found to substantially attenuate amyotrophic lateral sclerosis (ALS)-associated pathology in three preclinical rodent models via modulation of the endoplasmic reticulum (ER) stress, highlighting its potential therapeutic use in this motor neuron disease. AbstractAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease primarily afflicting motor neurons (MNs) of the spinal cord, brainstem, and motor cortex, leading to paralysis and eventually death within 3 to 5 years of diagnosis. No cure or effective therapy to halt ALS progression is available. The role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of ALS, as well as a potential drug target, has received increasing attention. Here, we investigated the therapeutic effect of the ER resident protein cerebral dopamine neurotrophic factor (CDNF) in preclinical models of ALS harboring different genetic mutations. We identify that intracerebroventricular (i.c.v.) administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of ALS.CDNF rescues MNs in vitro and in vivo from ER stress associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for ALS.

The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling ( Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling

The signals that coordinate and control movement in vertebrates are transmitted from motoneurons (MNs) to their target muscle cells at neuromuscular junctions (NMJs). Human NMJs display unique structural and physiological features, which make them vulnerable to pathological processes. NMJs are an early target in the pathology of motoneuron diseases (MND). Synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is the starting point of the pathophysiological cascade leading to MN death. Therefore, the study of human MNs in health and disease requires cell culture systems that enable the connection to their target muscle cells for NMJ formation. Here, we present a human neuromuscular co-culture system consisting of induced pluripotent stem cell (iPSC)-derived MNs and 3D skeletal muscle tissue derived from myoblasts. We used self-microfabricated silicone dishes combined with Velcro hooks to support the formation of 3D muscle tissue in a defined extracellular matrix, which enhances NMJ function and maturity. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the function of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and found a decrease in neuromuscular coupling and muscle contraction in co-cultures with MNs harboring ALS-linked SOD1 mutation. In summary, the human 3D neuromuscular cell culture system presented here recapitulates aspects of human physiology in a controlled in vitro setting and is suitable for modeling of MND.

The adaptive significance of adjusting behavioural activities to the right time of the day is intuitive.Laboratory studies have implicated an important role of circadian clocks in behavioural timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under semi-natural Zeitgeber conditions.We here report evidence that proper timing of eclosion, a vital behaviour of the fruit fly Drosophila melanogaster, requires a functional molecular clock even under quasi-natural conditions. In contrast to wildtype flies, period 01 mutants with a defective molecular clock eclose mostly arrhythmically in a temperate environment even in the presence of a full complement of abiotic Zeitgebers. Moreover, period 01 mutants eclose during a much larger portion of the day, and peak eclosion time becomes more susceptible to variable day-to-day changes of light and temperature. Under the same conditions, flies with impaired peptidergic inter-clock signalling (pdf 01 and han 5304 mutants) stayed largely rhythmic with normal gate sizes. Our results suggest that the presence of natural Zeitgebers can mitigate a loss of peptide-mediated phasing between central clock neuron groups, but cannot substitute for the lack of a functional molecular clock under natural temperate conditions. BackgroundEndogenous timing via circadian clocks confers adaptive advantages as it allows organisms to anticipate daily changes in the environment (see [1][2][3]). In terms of behaviour, the fitness relevance of being able to schedule locomotor activity, feeding, mating or other actions at the right time of the day is intuitive as it may help maximize success and reduce risks. Many studies under constant laboratory conditions have revealed a key role of the central and peripheral clocks in timing of behaviours across taxa. However, the importance of circadian clocks in daily timing of behaviours under natural conditions or in ecological context has come under debate, as studies in the last decade have assessed the functional importance of endogenous clocks under (semi-) natural conditions in a variety of mostly vertebrate species (see [1,2,4]). One important conclusion derived from these studies is that diel activity rhythms can remarkably differ between seminatural and laboratory conditions, since the phase relationship between behavioural activity and a given Zeitgeber such as light is modulated by other abiotic Zeitgebers, particularly by temperature [5]. Furthermore, intraand interspecific interactions such as predation [6][7][8] or competition for food [9] determine ("mask") activity patterns in the wild. Most strikingly, under semi-natural conditions in an outdoor enclosure, Per2 BRDM1 mice carrying a mutation in a core clock gene showed the same activity pattern as controls, and both showed mostly diurnal feeding, although they are strongly nocturnal in laboratory conditions [10]. In the wild, chipmunks with a lesion in t...

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons (MNs) in the spinal cord, brainstem, and motor cortex, leading to paralysis and eventually to death within 3 to 5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the ER-resident protein cerebral dopamine neurotrophic factor (CDNF) in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and etiology: (i) the conditional choline acetyltransferase (ChAT)-tTA/TRE-hTDP43-M337V rat model previously described, (ii) the widely used SOD1-G93A mouse model, and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the ER stress response in MNs, we used three main methods: (i) primary culture of MNs derived from E13 days embryos, (ii) immunohistochemical analyses of spinal cord sections with ChAT as spinal MNs marker, and (iii) qPCR analyses of lumbar MNs isolated via laser microdissection. We show that intracerebroventricular administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. CDNF rescues motor neurons in vitro and in vivo from ER stress-associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.